RESEARCH REPORT 2013 / 2014

Transkript

1 RESEARCH REPORT 2013 / 2014 LEIBNIZ-INSTITUT FÜR MOLEKULARE PHARMAKOLOGIE

2 SCIENTIFIC ADVISORY BOARD Prof. Dr. Karl-Heinz Altmann Institut für Pharmazeutische Wissenschaften ETH Zürich (seit 01 / 2012) Prof. Dr. Nils Brose (Chair) Max-Planck-Institut für Experimentelle Medizin Göttingen (seit 01 / 2012) Prof. Dr. Ulrike Eggert Randall Division of Cell and Molecular Biophysics King s College London (seit 01 / 2013) Prof. Dr. Beat Meier Laboratorium für Physikalische Chemie Eidgenössische Technische Hochschule Zürich (seit 01 / 2013) Prof. Dr. Eckhard Ottow Bayer Schering Pharma AG Bayer Health Care Berlin (seit 01 / 2008) Prof. Dr. Petra Schwille Max-Planck-Institut für Biochemie Martinsried (seit 01 / 2012) LEIBNIZ-INSTITUT FÜR MOLEKULARE PHARMAKOLOGIE RESEARCH REPORT 2013 / 2014 Prof. Dr. Michael Freissmuth Institut für Pharmakologie Universität Wien (seit 01 / 2008) Prof. Dr. Rebecca Wade Heidelberg Institute for Theoretical Studies HITS ggmbh Heidelberg (seit 01 / 2013) Prof. Dr. Thomas Gudermann Walter-Straub-Institut für Pharmakologie und Toxikologie Ludwig-Maximilians-Universität München (seit 01 / 2013) Prof. Dr. Eckart Gundelfinger Leibniz-Institut für Neurobiologie Magdeburg (seit 01 / 2013) Prof. Dr. Gerhard Klebe Institut für Pharmazeutische Chemie Universität Marburg (seit 01 / 2009) Stichtag

3 2 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CONTENT INHALT 3 CONTENT INHALT PREFACE VORWORT RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG Interview with Director Volker Haucke Bringing Two Worlds Together Zwei Welten verbinden 4 Thomas J. Jentsch The Pressure Relief Valve in the Cellular Membrane Decoded Das Druckventil in der Zellhülle entschlüsselt 8 Volker Haucke Cellular Transport: Like an Invisible Conductor Zelltransport: Wie ein unsichtbarer Dirigent 12 Adam Lange Bacteria with a Skeleton: The Structure of Bactofilin Has Been Elucidated Bakterienskelett: Struktur von Bactofilin aufgeklärt 16 Christian Hackenberger & Eberhard Krause Protein Phosphorylation: Hunting the Phantom Protein-Phosphorylierung: Die Jagd nach dem Phantom 22 The FMP Graduate School Die FMP Graduate School 26 Unique in Germany: The Chemical Biology Platform Einzigartig in Deutschland: Die Plattform der Chemischen Biologie 27 RESEARCH GROUPS FORSCHUNGSGRUPPEN Molecular Physiology and Cell Biology Section Bereich Molekulare Physiologie und Zellbiologie 30 Physiology and Pathology of Ion Transport Thomas J. Jentsch 34 Molecular Cell Physiology Ingolf E. Blasig 38 Molecular Pharmacology and Cell Biology Volker Haucke 42 Proteostasis in Aging and Disease Janine Kirstein 46 Behavioural Neurodynamics Tatiana Korotkova / Alexey Ponomarenko 50 Membrane Traffic and Cell Motility Tanja Maritzen 54 Molecular Neuroscience and Biophysics Andrew J.R. Plested 58 Protein Trafficking Ralf Schülein 62 Cellular Imaging Burkhard Wiesner / Dmytro Puchkov 66 Animal Facility Natali Wisbrun 70 Structural Biology Section Bereich Strukturbiologie 72 NMR-Supported Structural Biology Hartmut Oschkinat 76 Structural Bioinformatics and Protein Design Gerd Krause 80 Computational Chemistry / Drug Design Ronald Kühne 84 Molecular Biophysics Adam Lange 88 Solution NMR Peter Schmieder 92 Molecular Imaging Leif Schröder 96 In-Cell NMR Philipp Selenko 100 NMR Hartmut Oschkinat / Peter Schmieder 104 Chemical Biology Section Bereich Chemische Biologie 106 Chemical Biology II Christian P. R. Hackenberger 110 Peptide-Lipid Interaction / Peptide Transport Margitta Dathe 114 Mass Spectrometry Eberhard Krause 118 Medicinal Chemistry Marc Nazaré 122 Screening Unit Jens Peter Von Kries 126 Peptide Synthesis Rudolf Volkmer 130 APPENDIX ANHANG All Research Groups 136 Map Campus Berlin Buch 138 Administrative and Technical Services 140 Imprint

4 4 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 BRINGING TWO WORLDS TOGETHER ZWEI WELTEN VERBINDEN 5 BRINGING TWO WORLDS TOGETHER ZWEI WELTEN VERBINDEN of the shooting stars of solid state NMR. With him and Hartmut Oschkinat, the FMP has now become one of the world s leading centres in this technology. We have also considerably strengthened our promotion of new talent: there are now six junior groups at the FMP, and a gratifyingly large number of women in leading positions. Last July, Dorothea Fiedler came to the FMP from Princeton to take on a Director s post as one of very few women in such a position within the Leibniz Association. All vacant group leader positions at the Institute have now been filled, if not to say we re bursting at the seams. We were particularly glad that the MDC helped us considerably in implementing the project EU-OPENSCREEN, by making available an additional floor in the jointly used Timoféeff-Ressovsky-Haus. EU-OPENSCREEN is designed to coordinate the search for new active substances on a Europe-wide basis, and it has been classified by the BMBF as a major project particularly worthy of funding. However, it was not always easy to find the necessary compromise between the ambitious aims and the available funds. Thanks to the help of the MDC, we will not have to make concessions in quality, and the negotiations are so far advanced that we will be able to move into the operational phase in 2016 / FMP Director Volker Haucke about the Institute s mission and the successes achieved over the past two years Last year, the FMP went through its regular evaluation by the Leibniz-Association. What was the result? The international experts were unanimous in attesting the FMP a very high quality in basic molecular-pharmacological research. Not only were individual research groups rated as being excellent we were especially pleased to learn that the overall concept and the breadth of coverage of the Institute were given outstanding marks. What successes have been achieved at the FMP over the past two years? So many excellent papers have been published that I can only mention a few here as examples. The discovery of VRACs by Thomas Jentsch and his team was very exciting the abbreviation stands for volume-regulated anion channels. For many decades, it has been known that cells can regulate their volume depending on the osmotic conditions, but research groups throughout the world have for a long time searched unsuccessfully for corresponding membrane proteins that are permeable for anions, and Thomas and his group have at last closed this important physiological gap. The VRACs are highly relevant from a pharmacological point of view, since a whole range of different diseases are associated with a defect of volume regulation. Christian Hackenberger s group was also very successful: Christian and his team, for example, have found a way of monitoring lysine phosphorylation in cells, thereby enabling further research into this still largely unknown protein modification. Leif Schröder and his team produced equally exciting data: In a series of publications they demonstrated potential applications for the Xenon magnetic resonance imaging that they have developed. Using this method, it may one day be possible to detect pathological changes at an earlier stage than ever before. In co-operation with Christian Hackenberger, they have used MRI to identify cells that carry certain sugar compounds on their surfaces according to the same principle, it may now be possible to identify cancer cells. Moreover, I was very pleased that Adam Lange has succeeded in elucidating the structure of bactofilin so soon after joining us at the FMP. Bactofilin is an important building block of the bacterial cytoskeleton that only occurs in bacteria and is therefore an interesting starting point for antibacterial active substances. And, last but certainly not least, two junior groups led by Phil Selenko and Andrew Plested have been awarded the coveted and prestigious Consolidator Grant of the European Research Council. Your own group has also published a series of papers, including one in Nature. What was it about? With this work, we were successful in deciphering the coding system that enables cells to differentiate between packages with incoming and outgoing substances. The main switching elements are phosphoinositides, special membrane lipids that can be converted interchangeably and thus enable flexible reactions of the cell for example, switching from decay to growth. This is very interesting from a pharmacological point of view, because a cell can use this system to translate the messages of growth factors, for example, which in turn play a role in the development of cancer, or to recycle synaptic vesicles containing neurotransmitters in nerve cells, in order to sustain neurotransmission. What else has happened over the past two years, how has the Institute changed? When I came here three years ago, the FMP was in a phase of transition, with many important positions to be filled. Over the past two years, we have expanded the areas of structural biology and chemical biology in particular. We have closed a conceptional gap with the establishment of the group of Marc Nazaré. Marc is a medicinal chemist, he comes from industry and has brought great expertise in the development of active substances to the FMP. For structural biology, we managed to gain the services of Adam Lange, one Despite intensive biomedical research, there have been disappointingly few breakthroughs in the development of new therapies. What is the reason for this? The safety requirements placed on new medicinal products are extremely high today, meaning huge levels of investment and long lead-times. In order to minimise the high risks, pharmaceutical companies are forced to take decisions that are hostile to research. For truly new findings, what we need is high-risk basic research in which scientists can freely explore their curiosity and do not necessarily have to think about possible applications. There is a yawning gap between these two worlds, and it is part of the founding mission of the FMP to bring these two worlds together. The FMP conducts basic research in the run-up to drug development. We do not focus on specific diseases, but go one step further back we are initially interested in molecular mechanisms. Thus, we research into target structures that might be of interest to pharmaceutical companies. Success can never be predicted, of course, but it is hoped that the FMP and comparable institutes throughout the world will help to fill the pipeline for new medicinal products with new approaches and active substances. What will the future hold? Dorothea Fiedler will open up a completely new field of research her work involves small, sugar-like molecules that play a very important role in intracellular energy metabolism and about which little is known at present. Apart from this, beside our core objective of active substance research, we will be focusing on questions related to ageing research at different levels. Janine Kirstein and her junior group will be addressing the question of how cells manage to break down defective proteins and avoid aggregation this is a key subject in ageing research, because many problems of advanced age are determined by such processes. As a whole, the FMP is not only involved in the Leibniz-Forschungsverbund Healthy Ageing, but has also established a strategic alliance with other Leibniz institutes, in particular with the Fitz Lipmann Institute in Jena and the Leibniz Institute for Neurobiology in Magdeburg. This will involve taking various different approaches to research into cellular malfunctions in advanced age, such as loss of memory. I m already looking forward to the new findings we will gain here and, of course, to the close co-operation with our colleagues in the coming years! Prof. Dr. Volker Haucke Director of Leibniz-Institut für Molekulare Pharmakologie FMP-Direktor Volker Haucke über die Mission des Instituts und die Erfolge der letzten beiden Jahre Im vergangenen Jahr wurde das FMP durch die Leibniz- Gemeinschaft einem gründlichen Evaluierungsprozess unterzogen. Was ist dabei herausgekommen? Die internationalen Gutachter haben dem FMP unisono eine außerordentlich hohe Qualität in der molekular-pharmakologischen Grundlagenforschung attestiert. Nicht nur viele einzelne Arbeitsgruppen wurden als exzellent beurteilt besonders gefreut hat uns, dass auch das Gesamtkonzept und die Bandbreite des Instituts als herausragend bewertet wurde. Welche Erfolge hat es in den vergangenen zwei Jahren am FMP gegeben? Es wurden so viele exzellente Arbeiten veröffentlicht, dass ich hier nur einige beispielhaft nennen kann. Sehr spannend war die Entdeckung der VRACs durch Thomas Jentsch und sein Team die Abkürzung steht für Volumen-regulierte Anionen-Kanäle. Seit vielen Jahrzehnten ist bekannt, dass Zellen ihr Volumen in Abhängigkeit von den osmotischen Verhältnissen regulieren können, doch

5 6 RESEARCH REPORT FORSCHUNGSBERICHT 2013/2014 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 7 das entsprechende für Anionen durchlässige Membranprotein haben Arbeitsgruppen weltweit lange vergeblich gesucht. Thomas hat diese wichtige physiologische Lücke endlich geschlossen. Die VRACs sind dabei pharmakologisch hochrelevant, denn eine ganze Reihe von Krankheiten sind mit einer defekten Volumenregulation assoziiert. Auch die Gruppe Christian Hackenberger war sehr erfolgreich: So haben Christian und seine Mitarbeiter zum Beispiel einen Weg gefunden, um Lysin-Phosphorylierungen in Zellen nachzuweisen, so dass dieser noch weitgehend unbekannte zelluläre Signalweg nun erforscht werden kann. Erfreulich waren auch die Ergebnisse von Leif Schröder und seinem Team: In gleich mehreren Veröffentlichungen haben sie Anwendungsmöglichkeiten der von ihnen entwickelten Xenon-Kernspintomographie demonstriert. Mit dieser Methode könnte es einmal gelingen, krankhafte Veränderungen schon im Frühstadium zu entdecken. In Zusammenarbeit mit Christian Hackenberger haben sie im Kernspin Zellen identifiziert, die bestimmte Zuckerverbindungen auf ihren Oberflächen tragen nach dem gleichen Prinzip könnte man Krebszellen identifizieren. Gefreut habe ich mich auch, dass Adam Lange schon so bald nach seinem Start am FMP die Struktur von Bactofilin aufklären konnte das ist ein Baustein des Zellskeletts, der nur bei Bakterien vorkommt und der daher ein interessanter Ansatzpunkt für antibakterielle Wirkstoffe ist. Und gleich zwei Nachwuchsgruppen die von Phil Selenko und Andrew Plested wurden mit dem begehrten und angesehenen Consolidator Grant des Europäischen Forschungsrates ausgezeichnet. Auch Ihre eigene Gruppe hat eine Reihe von Arbeiten publiziert, darunter eine in Nature. Worum ging es darin? Mit dieser Arbeit ist es uns gelungen, das Kodierungssystem zu entschlüsseln, durch das Zellen zwischen Paketen mit eintreffenden und abgehenden Stoffen unterscheiden können. Das wesentliche Schaltelement dabei sind Phosphoinositide, das sind spezielle Membranlipide, die ineinander umgewandelt werden können und so flexible Reaktionen der Zelle ermöglichen zum Beispiel von Abbau auf Wachstum umzuschalten. Pharmakologisch ist das sehr interessant, denn durch dieses System kann eine Zelle zum Beispiel die Botschaften von Wachstumsfaktoren umsetzen, die wiederum bei der Entstehung von Krebs eine Rolle spielen oder Botenstoff enthaltende synaptische Vesikel in Nervenzellen recyceln, um so die Erregungsübertragung auf lange Sicht aufrecht zu erhalten. Was hat sich sonst in den letzten zwei Jahren getan, wie hat sich das Institut verändert? Als ich vor drei Jahren hierher kam, befand sich das FMP in einer Umbruchphase, viele wichtige Positionen waren neu zu besetzen. In den letzten beiden Jahren haben wir insbesondere die Bereiche Strukturbiologie und Chemische Biologie ausgebaut. Eine konzeptionelle Lücke haben wir mit der Gruppe von Marc Nazaré geschlossen Marc ist Medizinalchemiker, er kommt aus der Industrie und hat eine große Expertise in der Entwicklung von Wirkstoffen ans FMP gebracht. Für die Strukturbiologie konnten wir mit Adam Lange einen der Shooting-Stars der Festkörper-NMR gewinnen. Mit ihm und Hartmut Oschkinat ist das FMP nun zu einem der weltweit führenden Zentren in dieser Technologie geworden. Auch den Nachwuchsbereich haben wir deutlich verstärkt, es gibt am FMP mittlerweile sechs Junior-Gruppen, und außerdem erfreulich viele Frauen in Führungspositionen. Letzten Juli wechselte Dorothea Fiedler von Princeton ans FMP und übernahm einen Direktorenposten als eine von sehr wenigen Frauen innerhalb der Leibniz- Gemeinschaft auf einem solchen Posten. Das Institut ist nun vollbesetzt, um nicht zu sagen, wir platzen aus allen Nähten. Da waren wir besonders froh, dass uns das MDC bei der Verwirklichung des Projekts EU-OPENSCREEN erheblich geholfen hat, indem es uns eine zusätzliche Etage im gemeinsam genutzten Timoféeff- Ressovsky-Haus zur Verfügung stellen wird. Durch EU-OPEN- SCREEN soll die Suche nach neuen Wirkstoffen europaweit koordiniert werden. Es wurde vom BMBF als besonders förderungswürdiges Großvorhaben eingestuft. Es war allerdings nicht immer leicht, die nötigen Kompromisse zwischen dem ehrgeizigen Anspruch und den vorhandenen Mitteln zu finden. Dank der Hilfe des MDCs mussten wir nun aber keine Abstriche in der Qualität machen, und die Verhandlungen sind nun so weit fortgeschritten, dass wir 2016 / 2017 wohl in die operative Phase gehen können. Trotz der intensiven biomedizinischen Forschung gibt es enttäuschend wenig Durchbrüche bei der Entwicklung neuer Therapien. Wie kommt das? Die Anforderungen an die Sicherheit eines neuen Medikaments sind heute sehr groß, entsprechend sind auch die nötigen Investitionen und der zeitliche Vorlauf immens. Um die hohen Risiken zu minimieren, müssen pharmazeutische Unternehmen unweigerlich forschungsfeindliche Entscheidungen treffen. Für wirklich neue Erkenntnisse braucht es die risikoreiche Grundlagenforschung, bei der Wissenschaftler ihrer Neugierde folgen können und sich nicht unbedingt an möglichen Anwendungen orientieren. Zwischen diesen beiden Welten klafft eine Lücke, und es gehört zur Gründungsmission des FMP, diese beiden Welten zu verbinden. Das FMP betreibt Grundlagenforschung im Vorfeld der Arzneimittelentwicklung. Wir setzen dabei nicht bei bestimmten Krankheiten an, sondern gehen einen Schritt weiter zurück uns interessieren zunächst molekulare Mechanismen. Wir erforschen so Zielstrukturen, die für pharmazeutische Unternehmen interessant sein könnten. Vorhersagen kann man den Erfolg natürlich nie, doch die Hoffnung geht dahin, dass durch das FMP und vergleichbare Institute auf der ganzen Welt die Pipeline für neue Medikamente mit neuen Ansätzen und Wirkstoffen aufgefüllt wird. Was wird die Zukunft bringen? Dorothea Fiedler wird ein ganz neues Forschungsfeld eröffnen bei ihrer Arbeit geht es um kleine, zuckerähnliche Molekülen, die im intrazellulären Energiestoffwechsel eine sehr große Rolle zu spielen scheinen und über die man bislang kaum etwas weiß. Außerdem werden wir uns neben unserer Kernaufgabe, der Wirkstoffforschung, verstärkt mit Fragen der Alternsforschung beschäftigen. Janine Kirstein geht mit ihrer Nachwuchsgruppe der Frage nach, wie es Zellen gelingt, defekte Proteine abzubauen und Aggregationen zu vermeiden das ist für die Alternsforschung ein maßgebliches Thema, weil viele Probleme im Alter von solchen Prozessen bestimmt werden. Insgesamt engagiert sich das FMP nicht nur im Leibniz- Forschungsverbund Gesundes Altern, sondern hat auch eine strategische Allianz mit anderen Leibniz-Instituten, vor allem mit dem Fitz Lipmann Institut in Jena und dem Leibniz Institut für Neurobiologie in Magdeburg geschlossen. Es wird dabei in mehreren unterschiedlichen Ansätzen darum gehen, zelluläre Fehlfunktionen im Alter, wie zum Beispiel den Gedächtnisverlust, zu erforschen. Ich freue mich schon jetzt auf die Erkenntnisse, die wir hier gewinnen werden und natürlich die enge Zusammenarbeit mit den Kolleginnen und Kollegen in den kommenden Jahren! RESEARCH HIGHLIGHTS

6 8 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 9 THOMAS J. JENTSCH THE PRESSURE RELIEF VALVE IN THE CELLULAR MEMBRANE DECODED DAS DRUCKVENTIL IN DER ZELLHÜLLE ENTSCHLÜSSELT The ability of cells in our body to control their correct volume size is critical for several physiological processes, for example during normal cellular division, growth, and exposure to fluids of varying salt concentrations and also in diseases such as cancer, stroke and myocardial infarction. For a long time now, a certain chloride channel, a membrane protein that allows the passage of chloride ions, was known to sit on the surface of the cell safeguarding it from over-swelling, but the exact gene (or genes) in our DNA which encoded it was a mystery. The model for relief from swelling is simple: the cell activates this channel when it swells and to prevent the cell from becoming too big releases chloride ions and organic molecules (osmolytes) triggering water to exit the cell. Researchers from Berlin led by Professor Thomas J. Jentsch have now succeeded for the first time in elucidating the molecular identity of this volume-regulated anion channel (VRAC). The researchers from the Leibniz-Institut für Molekulare Pharmakologie (FMP) and from the Max-Delbrück-Centrum für Molekulare Medizin (MDC) in der Helmholtz-Gemeinschaft have identified a molecule called LRRC8A, which is an essential constituent of the volume-regulated anion channel (VRAC). This protein needs to be assembled with related proteins (LRRC8B to E) to form channels composed probably of six subunits. For the first time, they also showed that these chloride channels are permeable to small organic molecules such as taurine or amino acids. For over 20 years, research groups across the globe have been seeking to elucidate the molecular structure of the volume-regulated anion channel. It took Jentsch s team almost four years to achieve this breakthrough. The results have now been published in the renowned Science Journal. The regulation of cell volume is important for many functions in the organism. VRAC, which Thomas Jentsch and his coworkers Felizia Voss and Tobias Stauber have now decoded at the molecular level, is expressed in all vertebrate cells. If a particular cell volume is exceeded, the channel opens and permits the outflow of osmolytes such as chloride ions as well as small organic molecules such as taurine and amino acids. By contrast, cations such as potassium or sodium cannot permeate. Ion flux is a passive process, the channel s electrochemical properties only allowing anions and certain organic compounds to pass. Thus, the cell reduces the concentration of its osmotic active constituents to (or even below) that of the surrounding fluid. At the same time, the water content of the cell decreases as the water molecules flow out via aquaporins in the cell membrane. The volume of the cell then decreases. LRRC8A was discovered as a VRAC component using genome-wide RNA interference (sirna) in collaboration with Katina Lazarow and Jens von Kries from the FMP Screening Unit. They made use of small, synthetic RNA molecules that lead to enzymatic breakdown of messenger RNA in the cell, which matches its RNA sequence. As a result of the specific breakdown, no protein can be translated from this messenger RNA. The protein production comes to a halt, which is why synthetic RNA is also referred to as silencing or sirna. In a large-scale approach with the aid of complex pipetting robots and high-velocity measuring systems, the Berlin group individually suppressed the protein products of approximately all 20,000 human genes in cell culture. In an automated screening process the researchers investigated which genes are required for the swelling-activated anion flux across the cell membrane. The approximately 130,000 time-dependent ion flux measurements were statistically analysed by the team of Thomas Jentsch, with the support of Nancy Mah and Miguel Andrade-Navarro from the Bioinformatics Group of the MDC. After the needle (the gene LRRC8A) in the haystack of over 20,000 human genes had been found, a further year of intense work was necessary before the data were ripe for publication. This required thorough electrophysiological analysis by doctoral students Florian Ullrich and Jonas Münch. The essential role of LRRC8 proteins in the channel was initially verified using CRISPR / Cas technology, which has just become available during the past two years. With this method, specific genes on the chromosomes can be disrupted completely and permanently. In contrast to the only partial suppression of the swelling-activated chloride currents by sirnas, they disappeared entirely after complete inactivation of the gene. By restoring the current after reinsertion of the LRRC8A messenger RNA by over-expression, the group provided formal proof that LRRC8A is an essential component of the channel. However, this protein alone is not sufficient, as VRAC currents did not dramatically increase when LRRC8A by itself was over-expressed in cells. Therefore, besides LRRC8A, the researchers from Berlin suspected that the structurally related proteins LRRC8B E were also involved in forming the channel. While single individual elimination of each of these four genes did not suppress VRAC currents, simultaneous elimination of all four genes led to the complete loss of ion transport. More analysis revealed that combinations of at least two subunits were sufficient for VRAC currents. Thus, the combination of the essential A isoform with either the C, D or E enabled subtypes of VRAC, which they call heteromers. These heteromeric forms of VRAC had distinct electrophysiogical properties in the speed with which they closed (inactivation kinetics). This allows us to explain the behaviour of the channel in different tissues which until now has remained elusive, Thomas Jentsch said. Given the assumed structure of six subunits, the combination of five different isoforms can lead to the production of a large number of channels differing in biophysical detail. Cells can swell or in the worst case even burst. Water transport and its intracellular content must therefore be tightly regulated, added Thomas Jentsch. Water transport is always driven by the osmotic gradient defined by the concentration of molecules inside cells. Cells take up chloride from their surroundings, whereas organic substances such as taurine or amino acids are produced within the cells. Deciphering the molecular structure of this chloride channel is also important as this may pave the way for better medical treatments, for example, of excitotoxicity after stroke. In the case of damage in the brain, cells swell and release glutamate, which acts upon receptors on nerve cells. The subsequent inflow of calcium raises the intracellular concentration of this ion to toxic levels, Jentsch said. In other cases, the activation of VRAC is beneficial. With the onset of programmed cell death (apoptosis) during cancer chemotherapy, there is a strong reduction in cell volume implying an important role for the volumeregulated chloride channel. Für Körperzellen ist es lebenswichtig, ihr Volumen zu steuern. Das ist existenziell etwa beim Kontakt mit Flüssigkeiten verschiedener Salzkonzentration, bei der Zellteilung und beim Zellwachstum, aber auch bei Krankheiten wie Krebs, Schlaganfall und Herzinfarkt. Ein wesentlich an der Volumenregulation beteiligtes Protein ist ein bestimmter Chloridkanal, der durch das Anschwellen der Zelle aktiviert wird und durch den dann Chloridionen und organische Stoffe ( Osmolyte ) aus der Zelle ausgeschleust werden. Berliner Forschern um Prof. Thomas J. Jentsch ist es jetzt erstmals gelungen, die molekulare Identität dieses sogenannten Volumen-regulierten Anionen-Kanals (VRAC) aufzuklären. Die Wissenschaftler vom Leibniz-Institut für Molekulare Pharmakologie (FMP) und vom Max-Delbrück-Centrum für Molekulare Medizin (MDC) in der Helmholtz-Gemeinschaft identifizierten ein Molekül des Volumen-regulierten Anionen-Kanals namens LRRC8A. Dieses Molekül kann mit verwandten Proteinen (LRRC8B bis E) einen Kanal aus wahrscheinlich sechs Untereinheiten bilden. Außerdem konnten sie erstmals zeigen, dass diese Chloridkanäle gleichzeitig für kleine organische Moleküle wie Taurin oder Aminosäuren durchlässig sind. Nach dem molekularen Aufbau des Volumen-regulierten Anionen-Kanals (VRAC = volume-regulated anion channel) hatten Forschergruppen weltweit seit über 20 Jahren gesucht. Jentschs Team benötigte knapp vier Jahre für den Durchbruch. Die Ergebnisse wurden jetzt in der renommierten Zeitschrift Science veröffentlicht. Die Regulierung des Zellvolumens ist für viele Funktionen im Organismus bedeutsam. Der von Thomas Jentsch und seinen Mitarbeitern Felizia Voss und Tobias Stauber in seiner molekularen Struktur entschlüsselte VRAC ist bei allen Wirbeltieren in jeder Zelle vorhanden. Wenn ein bestimmtes Zellvolumen überschritten wird, dann öffnet sich der Kanal und lässt Osmolyte wie Chlorid- und organische Ionen wie Taurin und Aminosäuren austreten. Kationen wie Kalium oder Natrium werden hingegen nicht durchgelassen. Der Ionentransport verläuft passiv, der Kanal lässt durch seine elektrochemischen Eigenschaften nur Anionen und bestimmte organische Verbindungen passieren. Dadurch nimmt die Osmolarität, das heißt ihre osmotisch aktiven Bestandteile, in der Zelle ab, nähert sich der Umgebungsflüssigkeit an oder kann auch geringer werden. Gleichzeitig sinkt der Wassergehalt der Zelle, die Wassermoleküle wandern über sogenannte Aquaporine oder Wasserporen in der Zellmembran nach außen. Das Volumen der Zelle nimmt wieder ab. Entdeckt wurde LRRC8A als VRAC-Bestandteil durch Anwendung

7 10 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 11 Felizia Voss and Tobias Stauber ZELLEN KÖNNEN ANSCHWELLEN UND IM SCHLIMMSTEN FALL PLATZEN. DESHALB MÜSSEN DER WASSERTRANSPORT UND -GEHALT GENAU KONTROLLIERT WERDEN. genomweiter RNA-Interferenz, in Zusammenarbeit mit Katina Lazarow und Jens von Kries von der FMP-Screening Unit. Hierbei führen kleine, synthetische RNA-Moleküle nach Injektion in die Zelle zu einem enzymatischen Abbau von Boten-RNA, die zu ihrer RNA-Sequenz passt. Durch den spezifischen Abbau kann aus dieser Boten-RNA kein Protein übersetzt werden. Die Proteinproduktion wird stillgelegt und deshalb wird die synthetische RNA im englischen auch als silencer oder sirna bezeichnet (= Ruhigsteller). In einem großen Versuchsansatz mit Hilfe von komplexen Pipettier-Robotern und Hochgeschwindigkeits-Messsystemen unterdrückte die Berliner Gruppe in Zellkultur jeweils einzeln die Produkte aller zirka Gene des Menschen und untersuchte in einem automatisierten Verfahren, welche der Gene für den schwellungs-aktivierten Chloridstrom über die Zellmembran verantwortlich sind. Bei der statistischen Auswertung von ca zeitabhängigen Messungen des Ionenflusses wurde das Team von Thomas Jentsch durch Nancy Mah und Miguel Andrade-Navarro von der Bioinformatik-Gruppe des MDC unterstützt. Nachdem die Nadel (das Gen LRRC8A) im Heuhaufen der über menschlichen Gene gefunden war, benötigte es aber noch einmal ein Jahr intensiver Arbeit bis die Daten zur Publikation reif waren. Dies erforderte auch eine gründliche elektrophysiologische Analyse durch die Doktoranden Florian Ullrich und Jonas Münch. Mit der erst seit zwei Jahren zur Verfügung stehenden CRISPR / Cas- Technologie, mit der Gene auf den Chromosomen komplett und permanent ausgeschaltet werden können, wurde zunächst bestätigt, dass LRRC8A für den Kanal absolut notwendig ist anders als bei der nur teilweisen Unterdrückung der schwellaktivierten Chloridströme durch sirnas waren diese nach kompletter Ausschaltung des Gens völlig verschwunden. Durch Wiederherstellung des Stroms nach Wiedereinfügung der LRRC8A Boten-RNA führte die Gruppe den formalen Beweis, dass LRRC8A ein essenzieller Bestandteil des Kanals ist. Jedoch reicht dieses Protein alleine nicht aus, da die Kanalströme nach seiner Überproduktion nicht größer wurden. Die Berliner Forscher vermuteten daher, dass neben LRRC8A auch die strukturell verwandten Proteine LRRC8B E am Kanal beteiligt sind. Während die Zerstörung jedes einzelnen dieser vier Gene den Strom nicht unterdrückte, führte die gleichzeitige Elimination dieser vier Gene ebenfalls zum kompletten Verlust des Ionentransports. Zweierkombinationen reichten jedoch aus. So führte die Kombination der essenziellen A Isoform mit jeweils der C, D, oder E Form zu Strömen, die interessanterweise leicht unterschiedliche Eigenschaften hatten. Dadurch können wir das bisher rätselhafte unterschiedliche Verhalten des Kanals in verschiedenen Geweben erklären, erläutert Thomas Jentsch. Bei der angenommenen Struktur aus sechs Unterheiten können sich bei Kombination von fünf verschiedenen Isoformen eine große Anzahl im Detail verschiedener Kanäle bilden. Zellen können anschwellen und im schlimmsten Fall platzen. Deshalb müssen der Wassertransport und -gehalt genau kontrolliert werden, erklärt Thomas Jentsch. Der Wassertransport folge dabei immer dem osmotischen Gradienten. Die Zellen nehmen Chlorid aus der Umgebung auf, die organischen Stoffe wie Taurin oder Aminosäuren bilden die Zellen selbst. Die Entschlüsselung des molekularen Aufbaus dieses Chloridkanals ist auch deshalb bedeutsam, weil damit der Weg frei wird für bessere medizinische Behandlungen, beispielsweise nach einem Schlaganfall. Bei Schädigungen im Gehirn schwellen Zellen an, setzten Glutamat frei, das auf Rezeptoren in Nervenzellen wirkt. Dadurch strömt Calcium ein, das in der dann auftretenden hohen Konzentration toxisch wirkt, sagt Jentsch. Bei der chemotherapeutischen Behandlung von Krebs hingegen komme es mit dem Einsetzen des programmierten Zelltods (Apoptose) zu einer starken Volumenverminderung. Auch daran soll der Volumen-regulierte Chloridkanal beteiligt sein. Voss FK, Ullrich F, Münch J, Lazarow K, Lutter D, Mah N, Andrade-Navarro MA, von Kries JP, Stauber T, Jentsch TJ (2014) Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344, Components of the volume-regulated anion channel (VRAC) in the plasma membrane of the cell. The protein LRRC8A (stained red) together with at least one of the other five family members (here LRRC8E, stained green, and present as a complex in yellow). Bestandteile des volumenregulierten Anionenkanals (VRAC) in der Plasmamembran in der Zelle. Das Protein LRRC8A (rot gefärbt) zusammen mit mindestens einem anderen der fünf Familienmitglieder (hier LRRC8E, grün eingefärbt, als Komplex vorliegend in Gelb). (Tobias Stauber)

8 12 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 13 VOLKER HAUCKE INTRACELLULAR TRANSPORT: LIKE AN INVISIBLE CONDUCTOR ZELLTRANSPORT: WIE EIN UNSICHTBARER DIRIGENT (Dmytro Puchkov) During endocytosis, the cell membrane invaginates and a vesicle is pinched off. This is how nutrients, but also viruses, get into the cell. Während der Endozytose stülpt sich die Zellmembran ein und ein Vesikel schnürt sich ab. Auf diese Weise gelangen Nährstoffe, aber auch Viren, in die Zelle. The group led by Volker Haucke has succeeded in elucidating how a central transport process within cells is organised by simple biochemical reactions. Of decisive importance in this process are phosphoinositides, which serve as identification tags in the cell membrane and dictate the direction of transport. The intracellular transport investigated is important for numerous different body functions, such as the uptake of nutrients from the blood or neurotransmission in the brain, and also plays a role in the development of cancer and neurodegenerative disorders such as Alzheimer s disease. At first sight, the processes taking place in living cells appear to be totally chaotic: substances are permanently being synthesised and broken down again, three-dimensional structures are formed and decay. In order to take up substances from the surroundings and transport them, the cell invaginates its external membrane and pinches off tiny vesicles this process is called endocytosis. As if directed by an invisible conductor, the vesicles then migrate into the interior of the cell where they merge with internal structures. But where does the order come from in this apparent chaos? In their paper, the group led by Volker Haucke has shown how such a complicated process organises itself: The individual components, optimised over millions of years, interlock with each other like cogwheels. The paper was published in Nature in 2013 and also involved scientists from the NeuroCure Cluster of Excellence at the Charité and the Freie Universität Berlin. It was already known that certain components of the cell membrane are concentrated at spots where the cell membrane forms invaginations. These substances are phosphoinositides, called PIPs in the laboratory jargon: they consist of a fat-soluble tail, anchored in the lipid membrane, and a water-soluble head, which projects very slightly into the interior of the cell. These heads are particularly characteristic in their chemical properties, so that other cell components such as protein molecules recognise them and can bind to them. This is how the formation and transport of vesicles is driven. At the same time, the PIP heads can be easily modified, since perfectly fitting enzymes can detach the phosphate groups and reattach them in different orientations, thus giving the head a different face. In a complicated search for evidence, the group s leader Volker Haucke, his PhD student York Posor and other researchers involved show how a certain enzyme accumulates upon invagination and transforms the initial PIP within seconds into another, previously less characterised PIP. When York Posor blocked this enzyme using genetic-engineering methods, the system froze so to speak. In comparative movie sequences, he demonstrated how the invaginations remained suspended on the membrane. In the normal course of endocytotic vesicle transport, in contrast, the transformed PIP then attracts a special protein, which advances the further invagination and detachment of the vesicles. This in turn calls new enzymes into action, which further transform the PIPs. A chain of chemical reactions thus leads to a spatial-temporal dynamic with a specified direction. We can now quite precisely determine which molecules and how many of them are to be found at a particular time and place, explains Volker Haucke. This can even be expressed in mathematical models. The whole system runs under its own organisation, but also reacts to external influences. We suspect that the enzymes that produce or break down the PIPs also serve as sensors, in order to ensure the supply of nutrients to the cell and react appropriately. Among other things, this sensor function determines whether a cell grows and divides, which is of importance in the development of cancer. At the same time, the PIPs influence the communication between cells, for example in the brain, or the breakdown of clumped protein molecules, a central cause of neurodegenerative diseases such as Alzheimer s. WE CAN NOW QUITE PRECISELY DETERMINE WHICH MOLECULES AND HOW MANY OF THEM ARE TO BE FOUND AT A PARTICULAR TIME AND PLACE.

9 14 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 15 York Posor, Tanja Maritzen and Seong Joo Koo WIR KÖNNEN NUN ZIEMLICH PRÄZISE BESTIMMEN, WELCHE UND WIE VIELE MOLEKÜLE SICH WANN AN WELCHEM ORT BEFINDEN. Jan Schmoranzer Die Arbeitsgruppe von Volker Haucke konnte aufklären, wie ein zentraler Transportvorgang der Zelle durch einfache biochemische Reaktionen organisiert wird. Entscheidend sind dabei Phosphoinositide, die als Erkennungsmarker in der Zellmembran dienen und die Richtung des Transports vorgeben. Der untersuchte Zelltransport ist für eine Vielzahl von Körperfunktionen von Bedeutung, etwa bei der Aufnahme von Nährstoffen aus dem Blut oder bei der Erregungsübertragung im Gehirn, und spielt auch bei der Entstehung von Krebs und neurodegenerativen Erkrankungen wie Alzheimer eine Rolle. Die Vorgänge in lebenden Zellen erscheinen auf den ersten Blick wie ein undurchschaubares Gewimmel: Unablässig werden Stoffe synthetisiert und wieder abgebaut, dreidimensionale Strukturen entstehen und vergehen. Um Substanzen aus der Umgebung aufzunehmen und zu transportieren, stülpt die Zelle ihre Außenhaut ein und schnürt winzige Vesikel ab man nennt diesen Prozess Endozytose. Wie von einem unsichtbaren Dirigenten geleitet, wandern die Vesikel dann ins Innere der Zelle. Doch woher kommt die Ordnung in dem vermeintlichen Chaos? Die Arbeitsgruppe um Volker Haucke hat in ihrer Arbeit gezeigt, wie sich ein solch komplizierter Vorgang selbst organisiert: Die einzelnen Komponenten, in Jahrmillionen optimiert, greifen darin wie Zahnräder ineinander. Die Arbeit wurde 2013 im Fachmagazin Nature veröffentlich, beteiligt waren auch Wissenschaftler des Exzellenzcluster NeuroCure der Charité und der Freien Universität Berlin. Schon zuvor war bekannt, dass sich bestimmte Komponenten der Zellmembran dort ansammeln, wo sich die Zelle einstülpen wird. Es handelt sich dabei um Phosphoinositide, im Laborjargons PIPs genannt: Sie bestehen einerseits aus einem fettlöslichen Schwanz und sind damit in der Lipidmembran verankert, zum anderen aus einem wasserlöslichen Kopf, der ein klein wenig in das Innere der Zelle hineinragt. Diese Köpfe sind in ihren chemischen Eigenschaften besonders charakteristisch, so dass andere Zellkomponenten wie Eiweißmoleküle sie erkennen und daran binden können. So wird die Bildung oder der Transport der Vesikel vorangetrieben. Zugleich sind die PIP-Köpfe leicht wandelbar, denn passgenaue Enzyme können die Phosphatgruppen ablösen und in anderen Orientierungen wieder anbringen, der Kopf bekommt dadurch ein anderes Gesicht. In einer aufwändigen Indizienjagd konnten der Gruppenleiter Volker Haucke, sein Doktorand York Posor und andere beteiligte Forscher zeigen, wie sich ein bestimmtes Enzym bei der Einstülpung anlagert und das anfängliche PIP binnen Sekunden in ein anderes, bislang wenig charakterisiertes PIP umwandelt. Als York Posor dieses Enzym mit gentechnischen Methoden blockierte, fror das System gleichsam ein. Die Einstülpungen blieben an der Membran hängen, wie er in vergleichenden Filmsequenzen demonstrierte. Im normalen Verlauf des endozytotischen Vesikeltransports dagegen zieht das umgewandelte PIP dann ein spezielles Protein an, das die weitere Einstülpung und Ablösung der Vesikel befördert. Das wiederum ruft neue Enzyme auf den Plan, welche die PIPs weiter umwandeln. Aus einer Kette chemischer Reaktionen entsteht so eine räumlich-zeitliche Dynamik mit einer vorgegebenen Richtung. Wir können nun ziemlich präzise bestimmen, welche und wie viele Moleküle sich wann an welchem Ort befinden, erklärt Volker Haucke. Das kann man sogar in mathematischen Modellen ausdrücken. Das ganze System läuft zwar selbst organisiert, reagiert aber auch auf äußere Einflüsse. Wir vermuten, dass die Enzyme, welche die PIPs bilden oder abbauen, auch als Sensor dienen, um die Versorgung der Zelle mit Nährstoffen sicherzustellen und entsprechend zu reagieren. Diese Sensorfunktion bestimmt u.a. darüber, ob eine Zelle wächst und sich teilt, was bei der Entstehung von Krebs von Bedeutung ist. Zugleich beeinflussen die PIPs auch die Kommunikation zwischen Zellen, beispielsweise im Gehirn, oder den Abbau verklumpter Eiweißmoleküle, eine zentrale Ursache für neurodegenerative Krankheiten wie die Alzheimersche Krankheit. Dmytro Puchkov Michael Krauß Posor Y, Eichhorn-Gruenig M, Puchkov D, Schöneberg J, Ullrich A, Lampe A, Muller R, Zarbakhsh S, Gulluni F, Hirsch E, Krauss M, Schultz C, Schmoranzer J, Noé F, Haucke V (2013) Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499,

10 16 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 17 ADAM LANGE Songhwan Hwang and Chaowei Shi BACTERIA WITH A SKELETON: THE STRUCTURE OF BACTOFILIN HAS BEEN ELUCIDATED BAKTERIENSKELETT: STRUKTUR VON BACTOFILIN AUFGEKLÄRT The group led by Adam Lange has succeeded in elucidating the structure of bactofilin an important element of the bacterial cytoskeleton which has only recently been discovered. Bactofilin, for example, gives Helicobacter bacteria the screw shape that enables them to bore into the gastric mucosa, where they can cause inflammations and ulcers. The structural elucidation of bactofilin may provide a starting point for the development of urgently needed new antibiotics. Bacteria were long considered to be extremely primitive life forms compared with the cells of higher organisms, they are much smaller and appear to be largely unstructured under the standard light microscope. However, since cell biologists have been able to delve ever deeper into intricate structures using modern imaging procedures, it has become clear that these microorganisms are in fact full of complex architectures. For a long time, it was assumed that bacteria do not have any form of stabilising cytoskeleton, as can be found in the more modern cells of animals and plants. However, in the meantime, not only have elements analogous to the already known cytoskeleton building blocks been found, but even skeletal elements that occur exclusively in the realm of the bacteria. One of these new elements has now been investigated in more detail by Adam Lange and his team in cooperation with the group led by Martin Thanbichler at the Philipps University of Marburg. The subject of their research is the protein bactofilin, which was discovered by Martin Thanbichler in 2010 and occurs widely in various different species of bacteria. For example, with the aid of bactofilin, Helicobacter pylori develops its typical screw-shaped form. This enables the bacteria to bore into the protective mucous layer of the gastric mucosa where they are protected from the caustic effects of gastric acid and are responsible for the majority of the gastric and duodenal ulcers in humans. also takes place in the test tube, which makes structural elucidation a particular challenge, since proteins that can neither be dissolved in liquids nor form crystals are difficult to investigate using standard methods. For the structural elucidation of bactofilin, beside scanning transmission electron microscopy Adam Lange used the relatively new technique of solid-state NMR. NMR stands for nuclear magnetic resonance. The special thing about solid-state NMR is that the sample has to be rotated very rapidly in a strong magnetic field in order to simulate the movements of dissolved molecules. Lange s team discovered that the individual bactofilin molecules are wound up in a spiral shape to form a so-called beta helix and are then aligned molecule for molecule into filaments. This structural motif is then stabilised by recurrent hydrophobic areas, which were conserved in the bactofilin molecules by evolution. The extremely fine protofilaments can then accumulate further into thicker bundles or also tissue-like structures. Such a beta helix has yet to be found in any other cytoskeletal element but interestingly such a fold is adopted by the prion protein HET-s, which occurs in fungi. This work is just the starting point of our research into bactofilin, says Adam Lange. We now want to refine the structure down to atomic detail. Research into the bacterial cytoskeleton not only offers a fascinating insight into the structural diversity of these organisms, the most successful group from an evolutionary standpoint, but is also relevant from a medical point of view. Antibiotics are increasingly becoming ineffective as pathogens develop resistance this also applies to Helicobacter pylori, which is responsible for the development of gastric ulcers. Since bactofilin exclusively occurs in bacteria, it is an interesting starting point for the development of urgently needed novel active substances, says Adam Lange. Susanne Wojtke, Dagmar Michl and Claudia Bohg SINCE BACTOFILIN EXCLUSIVELY OCCURS IN BACTERIA, IT IS AN INTERESTING STARTING POINT FOR THE DEVELOPMENT OF URGENTLY NEEDED NOVEL ACTIVE SUBSTANCES. Generally, bactofilin molecules accumulate into filaments, from which higher ordered structures can then develop. Their polymerisation

11 18 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 19 (Barth van Rossum) In Caulobacter bacteria (blue), the bactofilin filaments (see zoom) play an important role in the development of the stalk, a thin protrusion of the cell body involved in cell attachment and nutrient acquisition. Bactofilins also give Helicobacter bacteria their typical screw shape that enables them to bore into the gastric mucosa. Helicobacter-bacteria can cause inflammations and ulcers there. The structural elucidation of bactofilin may provide a starting point for the development of urgently needed new antibiotics. In Caulobacter-Bakterien (blau) spielen die Bactofilinfilamente (siehe Zoom) eine wichtige Rolle bei der Ausprägung des Stiels, einer Ausstülpung mit der sich Caulobacter-Bakterien anheften können. Bactofiline verleihen außerdem Helicobacter-Bakterien ihre typische Schraubenform, mit der sie sich in die Magenschleimhaut bohren. Helicobacter-Bakterien können dort Entzündungen und Geschwüre auslösen. Die Strukturaufklärung von Bactofilin könnte hier einen Ansatzpunkt für die Entwicklung dringend benötigter neuer Antibiotika liefern.

12 20 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 21 Der Gruppe um Adam Lange ist es gelungen, die Struktur von Bactofilin aufzuklären einem wichtigen Element des bakteriellen Zytoskeletts, das erst vor kurzem entdeckt wurde. Bactofilin verleiht beispielsweise Helicobacter-Bakterien die Schraubenform, durch die sie sich in die Magenschleimhaut bohren und dort Entzündungen und Geschwüre auslösen können. Die Strukturaufklärung von Bactofilin könnte einen Ansatzpunkt für die Entwicklung dringend benötigter neuer Antibiotika liefern. Bakterien galten lange als äußerst primitive Lebensformen sie sind im Vergleich zu den Zellen höherer Lebewesen viel kleiner und erscheinen unter dem herkömmlichen Lichtmikroskop weitgehend unstrukturiert. Doch seit Zellbiologen mit modernen bildgebenden Verfahren zu immer feineren Strukturen vordringen können, wird klar, dass die Winzlinge in Wahrheit von komplexen Architekturen durchzogen sind. Lange Zeit ging man davon aus, dass Bakterien über keinerlei stabilisierendes Zytoskelett verfügen, wie man es bei den moderneren Zellen der Tiere und Pflanzen findet. Inzwischen aber hat man nicht nur die analogen Elemente zu den bereits bekannten Zytoskelett-Bausteinen gefunden, sondern sogar Skelettelemente, die exklusiv im Reich der Bakterien vorkommen. Eines dieser neuen Elemente hat nun Adam Lange mit seinem Team in Zusammenarbeit mit der Gruppe um Martin Thanbichler von der Philipps-Universität Marburg genauer erforscht. Es handelt sich um das Protein Bactofilin, das 2010 von Martin Thanbichler entdeckt wurde und das weit verbreitet in verschiedenen Bakterienarten vorkommt. Beispielsweise bildet Helicobacter pylori mit Hilfe von Bactofilin seine typische schraubenförmige Gestalt aus. Die Bakterien können sich so in die schützende Schleimschicht der Magenschleimhaut bohren sie sind hier vor der ätzenden Magensäure geschützt und verursachen einen Großteil der Magen- und Zwölffingerdarmgeschwüre beim Menschen. Das Besondere an der Festkörper-NMR besteht darin, dass die Probe in einem starken Magnetfeld sehr schnell rotiert werden muss, um die Bewegungen gelöster Moleküle zu simulieren. Langes Team fand heraus, dass die einzelnen Bactofilin-Moleküle sich spiralförmig zu einer sogenannten Beta-Helix aufdrehen und sich dann Molekül für Molekül zu Filamenten aneinanderreihen. Stabilisiert wird dieses Strukturmotiv durch wiederkehrende hydrophobe Bereiche, die in den Bactofilin-Molekülen evolutionär konserviert wurden. Die äußerst feinen Protofilamente können sich dann weiter zu dickeren Bündeln oder auch gewebeartigen Strukturen zusammenlagern. Eine solche Beta-Helix hat man bislang noch bei keinem anderen Zytoskelett-Element gefunden dafür aber interessanterweise bei dem Prionen-Protein HET-s, das in Pilzen vorkommt. Mit dieser Arbeit fängt für uns die Erforschung des Bactofilins erst an, sagt Adam Lange. Wir wollen nun die Struktur weiter bis ins atomare Detail verfeinern. Die Erforschung des bakteriellen Zytoskeletts bietet dabei nicht nur einen spannenden Einblick in die Formenvielfalt der evolutionär gesehen erfolgreichsten Gruppe der Organismen, sondern ist auch medizinisch relevant. Zunehmend werden Antibiotika gegen Krankheitserreger durch Resistenzen wirkungslos so auch bei Helicobacter pylori, dem Verursacher der Magengeschwüre. Da Bactofilin ausschließlich in Bakterien vorkommt, ist es ein interessanter Ansatzpunkt für die Entwicklung dringend benötigter neuartiger Wirkstoffe, sagt Adam Lange. Veniamin Chevelkov (photo above); Linda Ball (photo in the middle); Jean-Philippe Demers, Sascha Lange and Pascal Fricke Generell lagern sich Bactofilin-Moleküle zu Filamenten zusammen, aus denen dann wiederum unterschiedliche höher geordnete Strukturen entstehen können. Ihre Polymerisierung findet auch im Reagenzglas statt, was die Strukturaufklärung zu einer besonderen Herausforderung macht, denn Proteine, die sich weder in Flüssigkeiten lösen lassen noch Kristalle bilden, sind mit den gängigen Methoden nur schwer zu untersuchen. Für die Strukturaufklärung des Bactofilins wandte Adam Lange neben der Raster-Transmissionselektronenmikroskopie die noch relativ neue Technik der Festkörper-NMR an. NMR steht für Nuclear magnetic resonance, auf Deutsch Kernspinresonanz. Vasa S, Lin L, Shi C, Habenstein B, Riedel D, Kühn J, Thanbichler M, Lange A (2015) β-helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR. PNAS 112, E127 E136.

13 22 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 23 CHRISTIAN HACKENBERGER & EBERHARD KRAUSE PROTEIN PHOSPHORYLATION: HUNTING THE PHANTOM IN ANY EVENT, WE HAVE NOW DEVELOPED A TOOL FOR STUDYING SUCH PROCESSES. PROTEIN-PHOSPHORYLIERUNG: DIE JAGD NACH DEM PHANTOM Proteins are masters of metamorphosis: they are transformed, activated or deactivated in the cells as required. These vital modification processes have been the subject of research for many years, but some have only become accessible more recently as a result of the latest analytical methods. The group led by Christian Hackenberger at the FMP has now developed a method with which lysine phosphorylations can be demonstrated on proteins. This means that it is now possible to investigate undescribed regulatory mechanisms in the dynamic fine tuning of the cell. Whether as structural elements, enzymes, antibodies or receptors: proteins are the modelling clay from which living things are made. The human body is made up of around twenty thousand different proteins, the order of the amino acid chains being defined by our genes. However, the blueprint of the genes is just a basic framework to begin with: the form and function of the proteins are further defined by numerous molecular changes. A particularly prominent example is the phosphate group, which is attached to proteins by special enzymes and then detached again. The negative charge of the phosphate changes the structure and property of proteins, which makes it possible to turn enzymes on and off, for example. Phosphorylation, i.e. the addition of a phosphate group to a protein, thus takes on a central control function in the organism, and errors here can lead to various diseases such as cancer or Alzheimer s disease. Extensive research has been conducted on phosphates that are attached to neutral amino acids such as serine or tyrosine by so-called kinases. Apart from this, evidence has been available for decades that phosphate groups are also attached to basic amino acids. However, these compounds are so unstable that the search for them has been like hunting a phantom up to now. Consequently, for the past 30 years little more has been known about the phosphorylation of the amino acid lysine other than first rather imprecise evidence for its existence and that it probably plays an important biological role. In order to develop reliable proof, we first needed something to measure so we created peptides, i.e. short protein sequences, with phosphorylated lysine, explains Christian Hackenberger. In general, established methods exist for the synthesis of such peptides without the phosphate being attached to lysine. However, acid is always used, which inevitably counteracts the unstable lysine phosphorylation. Jordi Bertran-Vicente, the paper s first author, therefore had to create a completely new route of synthesis with his colleagues: instead of a phosphate group, he initially inserted an acid-resistant placeholder, which could then be substituted with phosphate at a later stage under milder conditions. Using this method, for the first time it is now possible to synthesise peptides in which only one lysine is phosphorylated selectively to a certain position. With the aid of the synthesised peptides, the group then cooperated with Eberhard Krause s research group, also present at the FMP, to develop a method for demonstrating such unstable phosphorylations, making use of the latest tandem mass spectrometry based on so-called electron-transfer dissociation (ETD). In this process, biomolecules are initially gently ionised and then isolated in an electrical field according to their mass. In a second step, the ETD mechanism breaks down the ions into fragments, whose mass is in turn detected. In the case of a protein or peptide, it is ultimately possible to conclude the sequence of the amino acids on the basis of the fragments. It s like stripping a bicycle down into its individual parts and then using the pieces to deduce the detailed construction, the positions and thus also the interplay of individual components, explains Krause. Transferred to peptides, this method reveals whether a phosphate group is attached to one of the amino acids, since the mass of a fragment is then larger and it is even possible to identify the position at which a phosphate group was attached. Using the new methodology, it is now possible to investigate the role of lysine phosphorylations in cells in molecular detail. For the first time, we were also able to test how stable this modification is at all, says Jordi Bertran-Vicente. It turns out that around one third of lysine phosphorylations decay within 18 hours under physiological conditions. This decay may even be biologically useful and wanted, adds Christian Hackenberger. Up to now, it had been assumed that phosphorylations of enzymes are actively detached again. However, not everything is driven enzymatically in nature, and it is becoming increasingly clear that the surface and binding behaviour of proteins are part of a very dynamic process. Perhaps, speculates Hackenberger, it is useful in certain cases for a mechanism to run for a while and then turn off automatically, like the snooze button on an alarm clock. In any event, we have now developed a tool for studying such processes. Proteine sind Verwandlungskünstler: Je nach Bedarf werden sie in den Zellen umgeformt, aktiviert oder abgeschaltet. Diese lebenswichtigen Modifikationsprozesse werden schon lange erforscht, manche werden allerdings erst in jüngerer Zeit durch neuere Analysemethoden zugänglich. Die Gruppe von Christian Hackenberger am FMP hat nun eine Methode entwickelt, mit der sich Lysin-Phosphorylierungen an Proteinen nachweisen lassen. Damit können nun potentiell neue Regulationsmechanismen im dynamischen Feintuning der Zelle erforscht werden Ob als Strukturelemente, Enzyme, Antikörper oder Rezeptoren: Proteine sind die Knetmasse des Lebendigen. Aus etwa zwanzigtausend verschiedenen Proteinen setzt sich ein menschlicher Körper zusammen, die Abfolge der Aminosäureketten ist im Erbgut festgeschrieben. Der Bauplan der Gene ist allerdings erst ein Grundgerüst: Durch eine Vielzahl molekularer Veränderungen werden Form und Funktion der Eiweißstoffe weiter präzisiert. Ein besonders prominentes Beispiel sind Phosphatgruppen, die von speziellen Enzymen an Proteine angehängt und wieder abgespalten werden. Durch die negative Ladung der Phosphatgruppe ändert sich die Struktur der Proteine, auf diese Weise lassen sich zum Beispiel Ion source of an Orbitrap mass spectrometer Ionenquelle eines Orbitrap-Massenspektrometers

14 24 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 25 Michael Schümann Enzyme an- und abschalten. Damit übernimmt die Phosphorylierung, d. h. die Verknüpfung der Phosphatgruppe mit dem Protein, eine zentrale Steuerfunktionen im Organismus, Fehler hierbei können zu verschiedenen Krankheiten wie Krebs oder Alzheimer führen. Gut erforscht sind Phosphatgruppen, die an neutrale Aminosäuren wie Tyrosin durch sogenannte Kinasen angehängt werden. Daneben gibt es schon seit Jahrzehnten Hinweise, dass Phosphatgruppen auch an die basischen Aminosäuren angeheftet werden. Diese Verbindungen sind allerdings so instabil, dass die Suche nach ihnen bislang der Jagd nach einem Phantom glich. So wusste man über die Phosphorylierung der Aminosäure Lysin seit dreißig Jahren nicht viel mehr, als dass es sie geben muss und dass sie womöglich eine wichtige biologische Rolle spielt. Um einen zuverlässigen Nachweis zu entwickeln, mussten wir erst einmal über das verfügen, was wir messen wollen wir mussten also Peptide, das sind kurze Proteinsequenzen, mit phosphoryliertem Lysin herstellen, erklärt Christian Hackenberger. Der Vorteil ist, dass für die Synthese solche Peptide etablierte Methoden existieren. Allerdings kommt dabei immer Säure zum Einsatz, was die labile Lysin-Phosphorylierung unweigerlich zunichtemacht. Jordi Bertran-Vicente, der Erstautor der Arbeit, musste daher zusammen mit seinen Kollegen einen ganz neuen Syntheseweg entwickeln: Anstelle der Phosphatgruppe fügte er zunächst einen säurefesten Platzhalter ein, der sich dann später unter milderen Bedingungen gegen Phosphat austauschen lässt. Mit dieser Methode ist es nun erstmals möglich, Peptide zu synthetisieren, bei denen selektiv jeweils nur ein Lysin an einer bestimmten Position phosphoryliert ist. Mit Hilfe der synthetischen Peptide konnte die Gruppe dann in Zusammenarbeit mit der Arbeitsgruppe von Eberhard Krause auch eine Methode zum Nachweis solcher labilen Phosphorylierungen entwickeln, bei der modernste Tandem- Massenspektrometrie basierend auf sogenannter electron-transfer dissociation (ETD) zum Einsatz kam. Dabei werden Biomoleküle zunächst schonend ionisiert, dann in einem elektrischen Feld anhand ihrer Masse isoliert. In einem zweiten Schritt werden die Ionen über den ETD Mechanismus in Fragmente zerlegt, deren Masse wiederum detektiert wird. Bei einem Protein oder Peptid kann man dann letztendlich anhand der Fragmente auf die Sequenz der Aminosäuren schließen. Das ist, als ob ich ein Fahrrad in Einzelteile zerlege und aus den Bruchstücken Hinweise auf die detaillierte Bauweise, die Positionen und damit auch auf das Zusammenspiel einzelner Komponenten herauslese, erklärt Krause. Auf unser Peptid übertragen offenbart diese Methode, ob an einer der Aminosäuren eine Phosphatgruppe hängt, da die Masse eines Fragments dann größer ist und man erkennt sogar, an welcher Position die Phosphatgruppe hing. Mit der neuen Methodik ist es nun möglich, die Rolle von Lysin- Phosphorylierungen in Zellen zu untersuchen. Erstmals konnten wir auch testen, wie stabil diese Modifikation überhaupt ist, sagt Jordi Bertran-Vicente. Es zeigte sich, dass Lysin-Phosphorylierungen bei physiologischen Bedingungen innerhalb 18 Stunden zu etwa einem Drittel zerfallen. Womöglich ist dieser Zerfall sogar biologisch sinnvoll und gewollt, fügt Christian Hackenberger hinzu. Bislang sei man davon ausgegangen, dass Phosphorylierungen von Enzymen aktiv wieder abgespalten werden. Doch in der Natur ist nicht immer alles enzymatisch getrieben, und zunehmend wird deutlich, dass Oberfläche und Bindungsverhalten von Proteinen Teil eines sehr dynamischen Prozesses sind. Vielleicht, so spekuliert Hackenberger, ist es in manchen Fällen sinnvoll, dass ein Mechanismus eine Weile läuft und sich dann von selbst abstellt, wie mit einer Snooze-Taste am Wecker. Auf jeden Fall haben wir nun ein Werkzeug entwickelt, um solche Prozesse zu studieren. Bertran-Vicente J, Serwa RA, Schümann M, Schmieder P, Krause E, Hackenberger CPR (2014) Site-Specifically Phosphorylated Lysine Peptides, J. Am. Chem. Soc., 136 (39), Jordi Bertran-Vicente and Anett Hauser

15 26 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 27 THE FMP GRADUATE SCHOOL DIE FMP GRADUATE SCHOOL UNIQUE IN GERMANY: THE CHEMICAL BIOLOGY PLATFORM EINZIGARTIG IN DEUTSCHLAND: DIE PLATTFORM DER CHEMISCHEN BIOLOGIE For postgraduates to go on to successfully complete their doctorate, they should not only be well supervised in their own project but should also become acquainted with the research questions and methods of other groups and have the opportunity to develop networks. In order to structure and improve postgraduate education and supervision, the FMP Graduate School was therefore founded in June 2013 and is open to all of the Institute s doctoral students. The Graduate School aims to provide a comprehensive overview of molecular pharmacological basic research and is orientated along the fields of research covered by the FMP these include structural biology, chemical biology, as well as molecular physiology and cell biology. The study programme is made up of the following elements: Annual meetings at which doctoral students present their latest work to a committee and have the opportunity to discuss it with them. Regular participation in seminars on one s own field of research and in the more general Marthe-Vogt seminars. Joint annual retreat with the Max Delbrück Centrum für Molekulare Medizin (MDC) with lectures held by the doctoral students and by invited scientists. The FMP Winter School with lectures and workshops. Several lectures that can be held within the context of seminars or during the FMP Winter School. Courses that mediate specific techniques or computer knowledge. Courses for soft skills, such as writing successful applications or improving time management. The Graduate School is directed by Christian Hackenberger, while its administration is in the hands of Katrin Wittig. In addition, every year four representatives are elected from among the doctoral students to participate in the organisation and composition of the programme. Representatives 2013 / 2014: Kristin Arnsburg, Oliver Reimann, Matthias Schnurr and Michel-Andreas Geiger. Für eine erfolgreiche Promotion sollten Doktoranden nicht nur in ihrem Projekt gut betreut werden, sondern auch die Fragestellungen und Methoden anderer Gruppen kennenlernen und Netzwerke bilden können. Um die Ausbildung und Betreuung zu strukturieren und zu verbessern, ist daher im Juni 2013 die Graduiertenschule des FMP eröffnet worden, die allen Doktoranden des Instituts offensteht. Die Graduiertenschule möchte zu einem umfassenden Überblick über die molekulare pharmakologische Grundlagenforschung verhelfen und orientiert sich dabei an den Forschungsbereichen des FMP dazu gehören die Strukturbiologie, die Chemische Biologie sowie Molekulare Physiologie und Zellbiologie. Zu den Elementen der Ausbildung gehören: Jährlich stattfindende Treffen, bei denen Doktoranden einem Komitee die Fortschritte ihrer Arbeit präsentieren und diese diskutieren. Regelmäßige Teilnahme an Seminaren zum eigenen Forschungsfeld und an den allgemeineren Marthe-Vogt-Seminaren. Gemeinsames jährliches Retreat mit dem Max Delbrück Centrum für Molekulare Medizin (MDC) mit Vorträgen der Doktoranden sowie von eingeladenen Wissenschaftlern. Die FMP Winter School mit Vorträgen und Workshops. Eigene Vorträge, die im Rahmen von Seminaren oder der FMP Winterschool gehalten werden können. Kurse, die bestimmte Techniken oder Computerkenntnisse vermitteln. Kurse für Softskills, wie zum Beispiel das Verfassen erfolgreicher Bewerbungen oder Zeitmanagement. Geleitet wird die Graduiertenschule von Christian Hackenberger, die Verwaltung liegt in den Händen von Katrin Wittig. Zudem werden jedes Jahr vier Repräsentanten unter den Doktoranden gewählt, die sich an der Organisation und Gestaltung des Programms beteiligen. Doktorandenvertreter 2013 / 2014: Kristin Arnsburg, Oliver Reimann, Matthias Schnurr und Michel-Andreas Geiger. The search for new active substances belongs to the core tasks of the FMP. Close collaboration between different working groups is vital for the success of an interdisciplinary technology platform. The Chemical Biology Platform works together with the Helmholtz Centres and Max Planck Institutes on many projects and is also networked in the European projects EU-OPENSCREEN and ANTIFLU. Small molecules act in organisms in numerous different ways for example, they can manipulate cell receptors or metabolic pathways and thus become medicinal substances or molecular tools for basic research. However, there are more than 70 million different chemical substances in existence today, on top of which molecules can be created virtually in computer simulations. In order to identify the truly suitable candidates within this broad diversity, scientists from different disciplines must work closely together using a wide array of high technology devices and systems. At the FMP, robots can test up to 40,000 substances in parallel on any given day, with a wide variety of different technologies being applied for example, microscopic images of cell cultures can be automatically analysed using image recognition software. However, this is only part of the challenge. Very often, the substances found (hits) are not active enough or they are maybe not specific enough. Over the past few years, the Screening Unit has therefore been expanded into an overall concept that is unique in German academic research, with vital contributions from the working groups Drug Design and Medicinal Chemistry. An additional enhancement was accomplished in 2013 with the arrival of the medicinal chemist Marc Nazaré, who was previously working in drug discovery research in the pharmaceutical industry. It is now possible to design and synthesise active substance candidates, to test them in repeated screens and in this way to further optimise them step by step. This method is used to obtain new, tailor-made molecules that can be used to investigate biological processes and potential active substances. (See Figure) The platform is open for scientific cooperation with groups from throughout Germany. It is not funded by the FMP alone, but receives financial support from the Max-Delbrück-Centrum (MDC) and the active substance initiative of the Helmholz-Gesellschaft. Since it was founded in 2003, the Screening Unit has carried out around 200 projects, its success stories including the cooperation with the cancer researcher Walter Birchmeier from the MDC, for example. This involved the development and further optimisation of a substance that selectively inhibits the enzyme SHP-2, which is overactive in many cases of metastatic breast cancer. Recently, animal experiments have shown that the active substance developed at the FMP inactivates cancer cells in mice. The possibilities opened up by the Chemical Biology Platform go far beyond the identification of small molecules. The facility has a collection of all sirna molecules that correspond to genes in humans, mice or threadworms and can block their translation into proteins. Using high-throughput procedures, it is thus possible to search genome-wide for an unknown gene and its corresponding protein. Last year, supported by this technology, Thomas Jentsch conducted a large-scale search in which he succeeded in identifying the long sought-after VRACs chloride channels in cell membranes through which cells regulate their volumes and which are linked to a range of different diseases (see page 8). The Chemical Biology Platform is part of the European project ANTIFLU, which develops innovative active substances for combating influenza viruses. It is also a central element of ChemBioNet, a network initiative of German biologists and chemists. However, it would be possible to considerably increase the efficiency of active substance searches if one could bring together the data from similar European institutions and make them available for general application. This provided the motivation that led to the idea for EU- OPENSCREEN under the auspices of the FMP, public institutions from twelve European countries want to join forces to create a unique high-technology research association. In 2013, the German Ministry of Education and Research considered this project to be particularly worthy of funding on the basis of its high potential for innovation. The negotiations with our European partners have now advanced to the stage that EU-OPENSCREEN is expected to enter into its operative phase as early as Die Suche nach neuen Wirkstoffen gehört zu den Kernaufgaben des FMP. Entscheidend für den Erfolg ist eine enge Verzahnung verschiedener Arbeitsgruppen zu einer effizienten interdisziplinären Technologie-Plattform. Die Plattform der Chemischen Biologie arbeitet in vielen Projekten auch mit Helmholtz-Zentren und Max-Planck-Instituten zusammen und ist darüber hinaus in den europäischen Projekten EU- OPENSCREEN und ANTIFLU vernetzt. Kleine Moleküle wirken in Organismen auf vielerlei Weise sie können beispielsweise Zellrezeptoren oder Stoffwechselwege manipulieren und so zu Arzneistoffen oder auch zu molekularen Wergzeugen für die Grundlagenforschung werden. Allerdings existieren heute mehr als 70 Millionen verschiedene chemische Substanzen, und darüber hinaus lassen sich Moleküle virtuell in Computersimulationen entwer-

16 28 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 29 The aim is to generate and optimize qualified probes. 28 members from three different research groups (Medicinal Chemistry, Screening Unit and Computational Chemistry/Drug Design) work at the Chemical Biology Plattform. The Prozess is devided in different steps. Ziel sind die Qualified probes, Substanzen, die gewünschte Wirkungen zeigen. Hieran arbeiten 28 Gruppenmitglieder aus drei Forschungsgruppen (Medizinische Chemie, Screening Unit und Wirkstoffdesign). Der Prozess ist in einzelnen Schritten dargestellt. CHEMINFORMATICS & DRUGDESIGN COMPOUND- MANAGEMENT & LIBRARY SYNTHESIS PROCESS- AUTOMATION SCREENING MEDICINAL CHEMISTRY ASSAY DEVELOPMENT & CELLBIOLOGY DATABASE / IT fen. Um in dieser Vielfalt die wirklich geeigneten Kandidaten zu identifizieren, benötigt man einen ganzen Apparat an Hochtechnologie, bei dem Wissenschaftler unterschiedlicher Disziplinen eng zusammenarbeiten müssen. Am FMP können Roboter bis zu Substanzen an einem Tag parallel testen, dabei kommen die unterschiedlichsten Technologien zum Einsatz beispielsweise lassen sich mikroskopische Aufnahmen von Zellkulturen mittels Bilderkennungssoftware automatisch analysieren. Das aber ist nur ein Teil der Herausforderung. Sehr häufig sind die gefundenen Substanzen (Hits) nicht aktiv genug oder aber unspezifisch. Die Screening Unit wurde daher seit einigen Jahren zu einem in der deutschen akademischen Forschung einzigartigen Gesamtkonzept erweitert, zu dem die Arbeitsgruppen Drug Design und die Medizinische Chemie essentiell beitragen. Eine wichtige Lücke wurde 2013 durch den Chemiker Marc Nazaré geschlossen, der zuvor in der Arzneimittelforschung der pharmazeutischen Industrie arbeitete. Nun ist es möglich, Wirkstoffkandidaten zu konzipieren und zu synthetisieren, in wiederholten Screens zu testen und auf diese Weise schrittweise immer weiter zu optimieren. Auf diese Weise erhält man neue, maßgeschneiderte kleine Moleküle zur Untersuchung biologischer Prozesse und potentielle Wirkstoffe. (siehe Grafik) Die Plattform ist offen für die wissenschaftliche Zusammenarbeit mit Gruppen aus ganz Deutschland. Sie wird nicht allein vom FMP, sondern auch vom Max-Delbrück-Centrum (MDC) und der Wirkstoffinitiative der Helmholz-Gesellschaft finanziell unterstützt. Seit ihrer Gründung 2003 hat die Screening Unit rund 200 Projekte durchgeführt, zu den Erfolgsgeschichten gehört zum Beispiel die Zusammenarbeit mit dem MDC-Krebsforscher Walter Birchmeier. Dabei wurde eine Substanz entwickelt und weiter optimiert, die selektiv das Enzym SHP-2 inhibiert, das in vielen Fällen von metastasierendem Brustkrebs überaktiv ist. Unlängst wurde im Tierversuch nachgewiesen, dass der am FMP entwickelte Wirkstoff Krebszellen in Mäusen lahm legt. verfügt über eine Sammlung aller sirna-moleküle, die mit Genen in Mensch, Maus oder Fadenwurm korrespondieren und deren Übersetzung in Proteine blockieren können. Im Hochdurchsatzverfahren kann man so genomweit nach einem unbekannten Gen und dem dazugehörigen Protein fahnden. Unterstützt von dieser Technologie gelang es im letzten Jahr Thomas Jentsch, in einer großangelegten Suche, die lang gesuchten VRACs zu identifizieren Chloridkanäle in Zellmembranen, durch die Zellen ihr Volumen regulieren, und die mit einer Reihe von Krankheiten in Verbindung stehen (siehe Seite 8). Die Plattform der Chemischen Biologie ist ein Teil des europäischen Projekts Antiflu, das innovative Wirkstoffe zur Bekämpfung von Grippeviren entwickelt. Sie ist außerdem ein zentraler Bestandteil von ChemBioNet, einer Netzwerk-Initiative deutscher Biologen und Chemiker. Die Effizienz der Wirkstoffsuche ließe sich allerdings erheblich steigern, wenn man die Daten ähnlicher europäischer Einrichtungen zusammenführen und für die Allgemeinheit zugänglich machen könnte. Aus dieser Motivation heraus entstand die Idee für EU-OPENSCREEN unter der Federführung des FMP wollen sich öffentliche Einrichtungen aus zwölf europäischen Ländern zu einem einzigartigen Hochtechnologie-Forschungsverbund zusammenschließen. Das Projekt wurde 2013 vom BMBF aufgrund seines hohen Innovationspotentials für besonders förderungswürdig erachtet. Die Verhandlungen mit den europäischen Partnern sind inzwischen so weit fortgeschritten, dass EU-OPENSCREEN wahrscheinlich schon in 2016 mit seiner operativen Phase starten. INHIBITOR ACTIVITY IN TARGETS HEMMSTOFFWIRKUNG IN ZIELPROTEINEN Regulation der Genaktivität (Demethylasen) Transportproteine (Protein-Protein Wechselwirkungen) Signaltransduktion (Transferasen) Die Möglichkeiten der Plattform der Chemischen Biologie gehen über das Auffinden kleiner Moleküle noch hinaus. Die Einrichtung Stoffwechsel von Signalmolekülen Signaltransduktion (Phosphatasen) Signaltransduktion (Kinasen)

17 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE Molecular Cell Physiology Molekulare Zellphysiologie Group leader PD Dr. Ingolf E. Blasig PAGE 38 Physiology and Pathology of Ion Transport Physiologie und Pathologie des Ionentransports Group leader Prof. Dr. Dr. Thomas J. Jentsch PAGE 34 Molecular Pharmacology and Cell Biology Molekulare Pharmakologie und Zellbiologie Group leader Prof. Dr. Volker Haucke PAGE 42 Proteostasis in Aging and Disease Die Rolle der Proteostase beim Altern und in Krankheit MOLECULAR PHYSIOLOGY AND CELL BIOLOGY SECTION BEREICH MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE Group leader Dr. Janine Kirstein PAGE 46 Behavioural Neurodynamics Verhaltensneurodynamik Group leaders Dr. Tatiana Korotkova Dr. Alexey Ponomarenko PAGE 50 Molecular Neuroscience and Biophysics Molekulare Neurowissenschaften und Biophysik Group leader Dr. Andrew J.R. Plested PAGE 58 Cellular Imaging Zelluläre Bildgebung Group leaders Dr. Burkhard Wiesner (Light Microscopy) Dr. Dmytro Puchkov (Electron Microscopy) PAGE 66 Animal Facility Tierhaltung Group leader Dr. Natali Wisbrun PAGE 70 Membrane Traffic and Cell Motility Membrantransport und Zellbeweglichkeit Group leader Dr. Tanja Maritzen PAGE 54 Protein Trafficking Group leader Prof. Dr. Ralf Schülein PAGE 62

18 32 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 33 SECTION MOLECULAR PHYSIOLOGY AND CELL BIOLOGY BEREICH MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE Life is based on complex cellular and physiological mechanisms and their well-orchestrated interplay. In disease, this interplay gets out of balance. Research in the section Molecular Physiology and Cell Biology aims at understanding such mechanisms in molecular detail, as well as their dysfunction in disease. Cellular targets for pharmaceutical intervention, many of them membrane proteins such as ion channels and G-protein-coupled receptors, are identified, studied in their physiological environment, and their modulation by bioactive compounds is explored. According to our mission to create a broader basis for pharmacology we focus on the study of less explored membrane proteins and of molecules of key importance for intracellular trafficking. To this end, we employ a broad arsenal of techniques, ranging from molecular and cellular biology over biochemistry and biophysics to whole-animal physiology using genetically modified mice, often with links to human disease. Our projects have benefitted greatly from interactions with other sections of the FMP, including those concerned with structural biology and modelling, drug and sirna screening, as well as chemical biology. Two main topics addressed in this section concern membrane proteins like ion channels and transporters, receptors, and junctional proteins, as well as cellular trafficking processes, in particular exo- and endocytosis. Strong links exist between these two topics: for instance, the interplay between exocytic membrane insertion of receptors into the plasma membrane and their endocytic retrieval determines the cellular activity of receptors; conversely, ion transport across endo- / lysosomal membranes regulates intracellular trafficking. During the reporting period, many exciting discoveries have been made by groups of the section, often in collaborations between FMP groups. For instance, the department Molecular Pharmacology and Cell Biology headed by Volker Haucke has identified phosphatidylinositol 3,4-bisphosphate and the enzyme that synthesizes this lipid as novel key regulators of endocytosis that are required for constriction of endocytic buds that emanate from the cell membrane (Posor et al., Nature 2013). In a collaboration with the FMP screening facility, the department Physiology and Pathology of Ion Transport led by Thomas Jentsch has used a genome-wide sirna screen to molecularly identify the long-sought volume-regulated anion channel VRAC which is not only important for cell volume regulation, but also for the transport of organic molecules like glutamate and for processes like apoptosis and stroke (Voss et al., Science 2014). The research group Molecular Cell Physiology headed by Ingolf Blasig discovered that the tight junction protein claudin-3 controls leucocyte infiltration at the choroid plexus during acute neurodegeneration, and the Protein Trafficking group of Ralf Schülein showed by fluorescence cross correlation spectroscopy that the monomer / dimer equilibrium of a G-protein-coupled receptor is established already in the endoplasmic reticulum. The four junior research groups of the section significantly contributed to the scientific output of the section. A novel role of KCNQ5 (Kv7.5) potassium channels in hippocampal synaptic inhibition, fast synchronization and spatial representation has been revealed in a close collaboration between the junior group Behavioral Neurodynamics headed by Tatiana Korotkova / Alexey Ponomarenko and the Jentsch laboratory (Fidzinski et al., Nature Commun. 2015). The group Molecular Neuroscience and Biophysics led by Andrew Plested, recipient of a prestigious ERC Consolidator Grant 2015, developed a novel genetically encoded approach for controlling glutamate receptors with light using unnatural amino acids. Tanja Maritzen s junior group Membrane Traffic and Cell Motility discovered that the so far uncharacterized endocytic adaptor protein stonin1 regulates focal adhesion dynamics, cell motility and tumor growth. Janine Kirstein, who since 2013 leads the new junior group Proteostasis in Aging and Disease, identified a cross-compartmental signaling that adjusts redox homeostasis to protein folding conditions. She found that cytosolic expression of neurotoxic aggregation-prone proteins alters the redox potential in the ER. Her group introduced C. elegans as a valuable model system into the FMP. The FMP is closely connected to the Berlin neuroscience community through the Cluster of Excellence NeuroCure, which is financed by the DFG. The heads of both departments (Jentsch, Haucke) are members of NeuroCure, as are the junior group leaders Alexey Ponomarenko, Tatiana Korotkova, Andrew Plested, and Janine Kirstein. These junior groups receive, in part substantial, co-financing from NeuroCure. Additional financing has also come from competitive intramural grants from the Leibniz Association. An SAW grant to Thomas Jentsch helped initially to establish the Korotkova / Ponomarenko group and Tanja Maritzen s group is largely financed by a competitive Leibniz program aimed at fostering female group leaders. In 2014 Volker Haucke and Thomas Jentsch, together with Hartmut Oschkinat and Jens von Kries from the Structural Biology and Chemical Biology sections, respectively, jointly obtained a SAW grant on the Role of protein homeostasis in cellular aging, in the framework of a program aimed at creating synergies with other Leibniz Institutes (Leibniz-Institut für Neurobiologie (LIN, Magdeburg) and the Fritz-Lipmann-Institut (FLI, Jena), who likewise received intramural grants for aging-related research. In the newest developments, a fifth junior group has joined the FMP. Alexander Walter heads the new group Molecular and Theoretical Neuroscience financed by the Emmy Noether program of the DFG. Like the group of Korotkova / Ponomarenko his Liaison Group is located at the Charité, further strengthening the ties of the institute to the university medicine and to the Berlin neuroscience community. Moreover, the section continues to invest heavily into top-notch cellular imaging techniques, a development that was initiated by Volker Haucke and is supervised by Burkhard Wiesner and Jan Schmoranzer. Super-resolution technologies such as PALM / STORM and STED, spinning disk microscopy and high pressure freezing for electron microscopy are now readily available to FMP scientists. Research of the section is well interconnected with research within the other sections of the FMP, in particular with the screening and chemical biology capacities provided by the Chemical Biology Platform including the Screening Unit as well as with the proteomics capabilities of the Mass Spectrometry group of the Chemical Biology section. The diversity of approaches and techniques, together with our common interest in cell biology and neurobiology, provides an excellent basis for advancing our knowledge of crucial mechanisms that may be amenable to pharmacological intervention. Leben gründet sich auf komplexe zelluläre und physiologische Mechanismen und ihr optimal abgestimmtes Zusammenspiel. Gerät dieses aus dem Gleichgewicht, entstehen Krankheiten. Das Verständnis dieser Mechanismen im molekularen Detail als auch deren Störung bei Krankheit ist Ziel der Forschung im Bereich Molekulare Physiologie und Zellbiologie. Zelluläre Zielmoleküle (Targets) für eine pharmakologische Einflussnahme, darunter viele Ionenkanäle und G-Protein-gekoppelte Rezeptoren, werden identifiziert und in ihrer physiologischen Umgebung untersucht. Zudem werden bioaktive Substanzen, die diese Targets modulieren können, gesucht. Im Sinne unserer Mission, die Basis für pharmakologische Einflussnahme zu vergrößern, ist unsere Forschung darauf ausgerichtet, wenig charakterisierte Membranproteine und Schlüsselmoleküle des intrazellulären Membrantransportes zu untersuchen. Dazu setzen wir eine breite Palette von Techniken und Methoden aus Molekular- und Zellbiologie, Biochemie, Biophysik und Physiologie an Tiermodellen, in der Regel genetisch veränderten Mäusen, ein. Die untersuchten Tiermodelle sind oft mit menschlichen Krankheiten verknüpft. Die Projekte des Bereiches profitieren hier sehr von der Zusammenarbeit mit den anderen Bereichen des FMP, insbesondere mit Strukturbiologie und Modelling, Wirkstoff-, sirna-screening und chemischer Biologie. Zwei wesentliche Forschungsschwerpunkte des Bereiches sind einerseits Membranproteine wie Ionenkanäle, Transporter, Rezeptoren und Tight Junction-Proteine, andererseits Vorgänge des zellulären Membrantransports. Zwischen diesen Schwerpunkten gibt es enge Verknüpfungen. Beispielsweise wird die zelluläre Aktivität von membranständigen Rezeptoren durch das Wechselspiel von Insertion der Rezeptoren in die Plasmamembran durch Exozytose und Entfernung aus dieser Membran durch Endozytose bestimmt. Im Gegenzug reguliert der Ionentransport über endo- / lysosomale Membranen den intrazellulären Membranfluss. Im Berichtszeitraum sind etliche hochinteressante Entdeckungen durch Mitglieder des Bereichs veröffentlicht worden, viele in enger Zusammenarbeit zwischen Arbeitsgruppen des FMP. So hat die Abteilung Molekulare Pharmakologie und Zellbiologie unter der Leitung von Volker Haucke Phosphatidyl 3,4-bisphosphat und das synthetisierende Enzym als neuartige Regulationspunkte der Endozytose identifiziert. Die Bildung dieses Lipides wird für die Ausbildung der endozytotischen Membranen, die sich von der Zellmembran abschnüren, benötigt (Posor et al., Nature 2013). In Zusammenarbeit mit der Screening Unit des FMP hat die Abteilung Physiologie und Pathologie des Ionentransports unter Leitung von Thomas Jentsch einen genomweiten sirna Screen zur Identifizierung des lange gesuchten volumenregulierten Anionenkanals VRAC durchgeführt. Dieser Kanal ist nicht nur für die zelluläre Volumenregulation wichtig, sondern auch für den Transport organischer Moleküle wie Glutamat und für Prozesse wie Apoptose und Schlaganfall (Voss et al., Science 2014). Die Arbeitsgruppe Molekulare Zellphysiologie, geleitet von Ingolf Blasig, hat herausgefunden, dass das Tight Junction-Protein Claudin-3 die Leukozyteninfiltration im Plexus choroideus während akuter Neurodegeneration kontrolliert. Die Arbeitsgruppe Protein Trafficking von Ralf Schülein konnte mittels Fluoreszenzkreuzkorrelationsspektroskopie zeigen, dass das Monomer- Dimer-Gleichgewicht G-Protein-gekoppelter Rezeptoren bereits im endoplasmatischen Retikulum etabliert wird. Die vier Nachwuchsgruppen des Bereichs haben ebenfalls signifikant zum wissenschaftlichen Erfolg des Bereichs beigetragen. Eine neuartige Rolle des KCNQ5 (Kv7.5) Kaliumkanals für die synaptische Hemmung, schnelle Synchronisierung und räumliche Repräsentation im Hippocampus wurde durch ein kollaboratives Projekt der Nachwuchsgruppe Verhaltensneurodynamik von Tatiana Korotkova, Alexey Ponomarenko und dem Jentsch-Labor aufgeklärt (Fidzinski et al., Nature Commun. 2015). Die Nachwuchsgruppe Molekulare Neurowissenschaft und Biophysik unter Leitung von Andrew Plested, der vor Kurzem einen der prestigereichen ERC Consolidator Grants 2015 eingeworben hat, hat einen neuartigen genetischen Ansatz unter Verwendung nicht-natürlicher Aminosäuren zur Kontrolle von Glutamatrezeptoren durch Licht entwickelt. Die Nachwuchsgruppe Membrantransport und Zellbeweglichkeit von Tanja Maritzen hat entdeckt, dass das endozytotische Adaptorprotein Stonin 1 die Dynamik von Focal Adhesions, Zellbeweglichkeit und Tumorwachstum reguliert. Janine Kirstein schließlich, die seit 2013 die neue Nachwuchsgruppe zur Rolle der Proteostase beim Altern und in Krankheit leitet, hat ein Signaling über Grenzen zellulärer Kompartimente hinweg entdeckt, durch das die Redox-Homöostase der Zelle an die Bedingungen der Proteinfaltung angepasst wird. Sie fand heraus, dass die Expression neurotoxischer Proteine mit Neigung zur Aggregation das Redoxpotential des endoplasmatischen Retikulum ändert. Ihre Gruppe hat den Fadenwurm C. elegans als einen wertvollen Modellorganismus am FMP etabliert. Das FMP ist über den Exzellenzcluster NeuroCure, der durch die DFG finanziert wird, intensiv in die Berliner neurowissenschaftliche Forschungsszene eingebunden. Die Abteilungsleiter beider Abteilungen des Bereiches (Thomas Jentsch, Volker Haucke) sowie die Nachwuchsgruppenleiterinnen und -leiter Janine Kirstein, Tatiana Korotkova, Andrew Plested und Alexey Ponomarenko sind Mitglieder dieses Exzellenzclusters. Ihre Nachwuchsgruppen erhalten eine zum Teil substanzielle finanzielle Co-Finanzierung durch NeuroCure. Zusätzliche Finanzmittel stammen von kompetitiv vergebenen Fördermitteln innerhalb der Leibniz-Gemeinschaft. Eine SAW-Förderung für Thomas Jentsch ermöglichte so die Einrichtung der Nachwuchsgruppe Korotkova / Ponomarenko und Tanja Maritzens Nachwuchsgruppe wird weitgehend aus einem kompetitiven Leibniz- Programm zur Förderung von Wissenschaftlerinnen in Leitungsfunktionen finanziert warben Volker Haucke und Thomas Jentsch zusammen mit Hartmut Oschkinat (Bereich Strukturbiologie) und Jens von Kries (Bereich Chemische Biologie) eine SAW-Förderung für das gemeinsame Projekt zur Rolle der Proteinhomöostase für das zelluläre Altern ein. Das Projekt ist Teil eines größeren Vernetzungsprojektes in einem Programm, das Synergien mit anderen Leibniz-Instituten schaffen soll, in diesem Fall mit dem Leibniz-Institut für Neurobiologie (LIN, Magdeburg) und dem Fritz-Lipmann-Institut (FLI, Jena), die gleichermaßen Fördermittel aus diesem Programm für Forschung zum Altern erhalten. Kürzlich hat zudem eine fünfte Nachwuchsgruppe unter Leitung von Alexander Walter am FMP ihre Arbeit aufgenommen. Die neue Gruppe Molekulare und theoretische Neurowissenschaft wird im Emmy- Noether-Programm der DFG gefördert. Wie die Nachwuchsgruppe Korotkova / Ponomarenko ist auch diese Liaisongruppe Neurowissenschaft in Räumen der Charité untergebracht, was die Verbindung des Instituts zur Berliner Universitätsmedizin und der Berliner Neurowissenschaft weiter festigt. Zudem investiert der Bereich kontinuierlich und substanziell in den Aufbau hochmoderner zellulärer Visualisierungstechniken, eine Entwicklung initiiert von Volker Haucke und geleitet von Burkhard Wiesner und Jan Schmoranzer. Hochauflösende Lichtmikroskopietechnologien wie PALM / STORM und STED, Spinning Disc- Mikroskopie und High Pressure Freezing für die Elektronenmikroskopie sind nunmehr für FMP-Wissenschaftlerinnen und -Wissenschaftler gut zugänglich. Die Forschung des Bereichs ist ausgezeichnet mit der Forschung der anderen Bereiche des FMP vernetzt. Das trifft insbesondere auf die Nutzung von Screening und Methoden der chemischen Biologie zu, die Screening Unit und die Chemical Biology Platform bereit halten, sowie auf das Proteomik-Portfolio der Arbeitsgruppe Massenspektrometrie im Bereich Chemische Biologie. Die Vielfalt der wissenschaftlichen Ansätze und Techniken zusammen mit unserem hohen Interesse an Zellbiologie und Neurobiologie bieten eine ausgezeichnete Grundlage um unser Wissen um grundlegende Mechanismen zu vergrößern, die pharmakologischer Intervention zugänglich sein könnten.

19 34 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 35 PHYSIOLOGY AND PATHOLOGY OF ION TRANSPORT Stefanie Weinert, Maja Hoegg-Beiler PHYSIOLOGIE UND PATHOLOGIE DES IONENTRANSPORTS GROUP LEADER PROF. DR. DR. THOMAS J. JENTSCH BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studied medicine at the FU Berlin Studied physics at the FU Berlin Staff scientist at the Institut für Klinische Physiologie (Prof. Wiederholt), FU Berlin 1982 Ph.D. in physics at the Fritz-Haber-Institute (Prof. Block), Berlin 1984 M.D. at the Institut für Klinische Physiologie (Prof. Wiederholt), FU Berlin Postdoctoral fellow at the Whitehead Institute (Harvey F. Lodish, MIT), Cambridge MA Research group leader at the Centre for Molecular Neurobiology Hamburg (ZMNH), Hamburg University 1991 Habilitation in Cell Biochemistry at the Medical School of Hamburg University Full professor (C4) of Molecular Neuropathology at the ZMNH, Hamburg University; Director of the Institut für Molekulare Neuropathobiologie and Director of the Centre for Molecular Neurobiology Hamburg (ZMNH) since 2006 Head of department at FMP and MDC, Berlin (joint appointment), Full Professor (W3) at Charité Universitätsmedizin Berlin since 2007 Member of NeuroCure Cluster of Excellence since 2009 Deputy Director of Leibniz- Institut für Molekulare Pharmakologie (FMP) We aim to understand ion transport processes from the molecular (structure-function analysis) to the subcellular and cellular levels, up to the level of the organism. The latter levels are addressed through an investigation of the phenotypes of knockout (KO) and knock-in (KI) mice and the analysis of human genetic diseases. Until now we have focused on CLC chloride channels and transporters, KCNQ potassium channels, KCC cation-chloride cotransporters, and anoctamin Ca 2+ -activated chloride channels. Key research areas are the endosomal / lysosomal system and the control of neuronal excitability. We study many organs, including the brain, inner ear, olfactory epithelium, skin mechanoreceptors, kidney, and bone. In 2014 we identified the long-sought volume-regulated anion channel VRAC. This has opened the door to an important new research area that we will explore extensively during the coming years. ZUSAMMENFASSUNG Unser Ziel ist es, Ionentransportprozesse von der molekularen Ebene (Struktur-Funktions- Beziehungen) über den subzellulären und zellulären Level bis auf die Ebene des Organismus zu verstehen. Letzteres versuchen wir durch Untersuchung der Phänotypen von knock-out (KO)- und knock-in (KI)-Mäusen und die Analyse humangenetischer Erkrankungen zu erreichen. Unser Schwerpunkt liegt dabei auf CLC-Chloridkanälen und -Transportern, KCNQ-Kaliumkanälen, KCC-Kation-Chlorid-Cotransportern und Anoctamin Ca 2+ - aktivierten Chloridkanälen. Im Zentrum stehen dabei das endosomal / lysosomale System und die Kontrolle neuronaler Erregbarkeit. Wir untersuchen eine Reihe von Organen, darunter Gehirn, Innenohr, Riechepithel, Mechanorezeptoren, Niere, und Knochen. Kürzlich (2014) gelang es uns, den seit langem gesuchten Volumen-regulierten Anionen- Kanal VRAC zu identifizieren. Dies ermöglicht uns den Zugang zu einem wichtigen neuen Forschungsgebiet, das wir in den kommenden Jahren intensiv bearbeiten werden. Ian Orozco CLC chloride channels and transporters Proteins of the CLC gene family, discovered by us in 1990, reside in the plasma membrane and intracellular vesicles. We have generated KO mouse models for most CLCs and have identified related human diseases, yielding insights into their diverse physiological roles. We have also identified ancillary β-subunits that are associated with human disease. Vesicular CLCs are Cl - / H + -exchangers, suggesting that they have functions beyond acidification of intracellular vesicles, as recently confirmed by mouse models in which we converted ClC-5 and ClC-7 into pure Cl - conductors. We are now analyzing similar models for ClC-3. Our new Clcn 7td / td mice, in which we abolished the transport activity of ClC-7, show that a lack of ClC-7 protein interactions is responsible for some of the phenotypes of ClC-7 KO mice. We functionally analyzed ClC-4 point mutations that were recently found in patients with epilepsy or mental retardation, a discovery that revealed an important CNS function of ClC-4, and have investigated the mechanism of slow gating of ClC-7 / Ostm1. We have previously shown that disruption of the plasma membrane Cl - channel ClC-2 leads to leukodystrophy in mice and that ClC-2 binds to the adhesion molecule GlialCAM, which in turn associates with MLC1. Mutations in all three genes cause leukodystrophy in humans. We have now investigated their interactions in vivo using several mouse models and concluded that loss of GlialCAM and MLC1 may cause leukodystrophy partially by a loss of ClC-2 function (Fig.1). Anoctamin (TMEM16) Ca 2+ -activated chloride channels We showed that Ano2 is the long-sought Ca 2+ -activated Cl - channel of olfactory sensory neurons, but surprisingly our Ano2 - / - mice showed that Ano2 is not essential for olfaction. Whereas the main olfactory epithelium expresses only Ano2, the vomeronasal organ (VNO), which is relevant for social interactions, also expresses Ano1. We are now studying conditional double KOs of both Ca 2+ activated Cl - channels in the VNO. KCNQ potassium channels We previously cloned and characterized the K + channels KCNQ2-5, showed that mutations in KCNQ2 and 3 cause neonatal epilepsy, and mutations in KCNQ4 a form of deafness, and have generated mouse models for KCNQ4 and KCNQ5. Both of the latter channels are expressed in the vestibular organ, but unlike KCNQ4 in the cochlea, they do not localize to sensory hair cells but rather to postsynaptic membranes of afferent vestibular neurons. Functional analysis of KCNQ4 KO mice revealed a mild vestibular phenotype. In collaboration dn / dn with A. Ponomarenko and T. Korotkova we analyzed Kcnq5 mice in which all heteromers containing KCNQ5 were functionally inactivated. These studies, which included slice electrophysiology and in vivo recordings from the hippocampus, showed that KCNQ5 is important for controlling synaptic inhibition and network activity. Potassium-chloride cotransporters We have previously generated and analyzed constitutive KOs of the KCC K + -Cl - -cotransporters KCC1 KCC4 and discovered unexpected functions in various tissues. The neuronal isoform KCC2 lowers intraneuronal Cl - concentration, a process required for the inhibitory response to the neurotransmitters GABA and glycine. We have previously investigated synaptic inhibition in cerebellar neuronal circuits in mice with granule- and Purkinje-cell specific KCC2 disruption. We have now explored the role of KCC2 and synaptic inhibition in odor discrimination in the olfactory bulb using a mitral cell-specific KO. Identification of the long-sought volume-regulated anion channel VRAC Cells need to regulate their volume, for instance during growth or when exposed to osmotic challenges. A key player in this process is the volume-regulated anion channel, VRAC (Fig. 2), that has been known biophysically for more than 20 years but whose molecular identity has remained obscure. Using a genome-wide sirna screen at the FMP screening facility, we have now identified heteromers of LRRC8 proteins, containing four transmembrane domains and C-terminal leucine-rich repeats, as crucial VRAC components. LRRC8A is essential for VRAC activity, but needs at least one other isoform (LRRC8B through LRRC8E) to form channels. Different heteromers differ in inactivation kinetics. We showed that VRAC is not only important for volume-regulation, but also conducts various organic compounds likely to be of crucial importance for extracellular signal transduction and pathologies such as stroke. This break through will allow us to address many important questions concerning the cell biological roles of VRAC and its structurefunction relationship over the coming years.

20 36 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 37 CIC-2 CIC-2 + GFAP Fig. 1: Immunohistochemistry of the Fig. 2: Role of VRAC in regulatory VRAC / VSOAC (LRRC8 heteromer) Glialcam - / - wild type cerebellum of WT and GlialCAM KO mice reveals that GlialCAM is crucial for the localization and stability of the ClC-2 chloride channel protein (see Hoegg-Beiler et al., Nature Commun. 5, 3475 (2014)). volume decrease (see Voss et al., Science 344, (2014)) osmotic swelling regulatory volume decrease GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Gwendolyn Billig International Christian Hübner Spitzmaul G, Tolosa L, Winkelman BHJ, Heidenreich M, Frens MA, Prix Louis Jeantet, , Dr. Pawel Fidzinski Dr. Maja Hoegg-Beiler Dr. Sabrina Jabs Dr. Ian Orozco Dr. Rosa Planells-Cases Dr. Guillermo Spitzmaul Dr. Tobias Stauber Dr. Janis Vogt Christian Chabbert Institut de Neurobiologie, Montpellier, France Carole Charlier Université de Liège, Belgium Raúl Estévez Universitat de Barcelona, Spain Yamuna Krishnan Universitätsklinikum Jena Vera Kalscheuer Max-Planck-Institut für Molekulare Genetik, Berlin Uwe Kornak Charité Universitätsmedizin Berlin and Max-Planck-Institut für Molekulare Genetik, Berlin Chabbert C, de Zeeuw CI, Jentsch TJ (2013). Vestibular role of KCNQ4 and KCNQ5 K + channels revealed by mouse models. J. Biol. Chem. 288, Ludwig CF, Ullrich F, Leisle L, Stauber T, Jentsch TJ (2013). Common gating of both CLC subunits underlies voltage-dependent activation of the 2Cl - / H + -exchanger ClC-7 / Ostm1. J. Biol. Chem. 288, Hoegg-Beiler MB, Sirisi S, Orozco IJ, Ferrer I, Hohensee S, Auberson Deutsche Forschungsgemeinschaft, Exzellenzinitiative an der Humboldt-Universität zu Berlin, Projekt NeuroCure: Towards a better outcome of central nervous system disorders, - Innovation Project 2013: Dynamics of exocytosis and cortical ER, , ; - Innovation Project 2014: CNS role of volume-stimulated organic osmolyte / anion channel VSOAC, , Dr. Stefanie Weinert Joanna Ziomkowska Dr. Norma Nitschke (research coordinator) Sebastian Albrecht (doctoral student) Anja Blessing (doctoral student) Andreia Cruz e Silva (doctoral student) Tony Daubitz (doctoral student) Deborah Elger (doctoral student) Kathrin Gödde (doctoral student) Corinna Göppner (doctoral student) Carmen Ludwig (doctoral student) Darius Lutter (doctoral student) University of Chicago, USA Michael F. Hammer University of Arizona, Tucson, USA Michael Pusch CNR, Genova, Italy Francisco Sepúlveda CECS, Valdivia, Chile Chris de Zeeuw Erasmus MC, Rotterdam and Netherlands Institute for Neuroscience, Amsterdam, The Netherlands Tatiana Korotkova Charité Universitätsmedizin Berlin and FMP, Berlin Eberhard Krause Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin Gerd Krause Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin Jens von Kries Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin M, Gödde K, Vilches C, López de Heredia M, Nunes V*, Estévez R*, Jentsch TJ* (2014). Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial Cl - channel dysfunction. Nature Communications 5, Voss FK, Ullrich F, Münch J, Lazarow K, Lutter D, Mah N, Andrade- Navarro MA, von Kries JP, Stauber T*, Jentsch TJ* (2014). Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344, Weinert S, Jabs S, Hohensee S, Chan WL, Kornak U, Jentsch TJ (2014). Transport activity and presence of ClC-7 / Ostm1 complex account for different cellular functions. EMBO Reports 15, Deutsche Forschungsgemeinschaft, SFB 740, C05, Protein modules involved in vesicular acidification and trafficking: focus of CIC-6, Continued as: SFB 740 / 2, C05, Funktionale Module in der endosomal-lysosomalen Ionenhomöostase und ihre Funktion , Continued as: SFB 740 / 3, C05, , Deutsche Forschungsgemeinschaft, Strukturelle Grundlagen und physiologische Funktion des Cl - / H + -Gegenaustausches bestimmter CLC-Chloridtransportproteine, ZD 58 / 1 1, with Zdebik A, Continued as: JE 164 / 9 2, , Jonas Münch (doctoral student) National Marc Nazaré Deutsche Forschungsgemeinschaft, Der CIC-7 / Ostm1 Chloridtransporter Karina Oberheide (doctoral student) Michael Amling Leibniz-Institut für Molekulare in Lysosomen und Osteoklasten, JE 164 / 7 1, Momsen Reincke (M.D. student) Universitätskrankenhaus Eppendorf, Pharmakologie (FMP), Berlin Continued as: JE 164 / 7 2, , Sebastian Schütze (doctoral student) Till Stuhlmann (doctoral student) Florian Ullrich (doctoral student) Felizia Voss (doctoral student) Hamburg Miguel Andrade-Navarro MDC, Berlin Maik Gollasch Alexey Ponomarenko Charité Universitätsmedizin Berlin and FMP, Berlin Dmytro Puchkov Deutsche Forschungsgemeinschaft, Funktionelle Charakterisierung ausgewählter Mitglieder der Anoctamin-Kanalfamilie, JE 164 / 10 1, , Carolin Backhaus (technical assistant) Anyess von Bock (technical assistant) Charité Universitätsmedizin Berlin Hans-Jürgen Holdt Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin Europäischer Forschungsrat (7. Forschungsrahmenprogramm), Ion homeostasis and volume regulation of cells and organelles (CYTOVO- Petra Göritz (animal care taker) Universität Potsdam Dietmar Schmitz LION), ERC-2011-ADG_ , , Nicole Krönke (technical assistant) Janet Liebold (technical assistant) Johanna Jedamzick (technical assistant) Ruth Pareja-Alcaraz (technical assistant) Charité Universitätsmedizin Berlin Björn Schroeder Max-Delbrück-Center for Molecular Medicine, Berlin Bundesministerium für Bildung und Forschung (ERA-NET: E-Rare), CLC chloride channels and Megalencephalic Leucoencephalopathy: molecular mechanisms and therapeutics, 01GM1403, , Katrin Räbel (technical assistant) Frank Zufall Leibniz-Gemeinschaft (Leibniz Wettbewerb 2014), Role of proteostasis Mario Ringler (lab coordinator) Patrick Seidler (technical assistant) Andrea Weidlich (technical assistant) Silke Zillmann (technical assistant) Universität des Saarlandes, Homburg / Saar Werner Zuschratter Leibniz-Institut für Neurobiologie (LIN), Magdeburg FMP authors Group members * corresponding authors in cellular aging, SAW-2014-FMP-2, with Haucke V, Oschkinat H, von Kries JP, , (pro rata)

21 38 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 39 MOLECULAR CELL PHYSIOLOGY Olga Breitkreutz-Korff and Heike Meyer MOLEKULARE ZELLPHYSIOLOGIE GROUP LEADER PD DR. INGOLF E. BLASIG BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studied biology and biochemistry in Leipzig, diploma thesis on cancer research at the Robert-Rössle-Hospital in Berlin-Buch 1984 Dissertation on the pharmacology of myocardial infarction at the Academy of Sciences, Berlin 1992 Habilitation for investigations on reactive species in myocardial dysfunction and venia legendi for biochemical pharmacology, University of Halle-Wittenberg since 1992 Head of the independent research group for Molecular Cell Physiology at the FMP, teaching of pharmacology, functional biochemistry, neurochemistry at the universities in Potsdam and Berlin Awarded project leader at the NIH, Washington, DC / USA The group focuses on the structure, function, and modulation of cell-cell contacts to explore tight junctions (TJs) in tissue barriers, in order to disclose pathological mechanisms and to improve therapies. One outcome of this work is to propose new strategies that specifically manipulate neurological and other barriers to improve drug delivery or to enhance their efficacy and to prevent barrier dysfunction (protection). Membrane proteins such as claudins, TAMPs (occludin, tricellulin) and associated scaffolding proteins (e.g. ZO-1) determine the paracellular tightness of endothelial and epithelial barriers. As the complex of the TJs is not well defined it is unclear how the structure, regulation, and interactions among these proteins control the barrier properties. Our work concentrates on the exploration of the TJ proteome and interactome, on the detailed processes that underlie the oligomerization of key proteins of tissue barriers, and on mechanisms to open and / or reconstitute the TJs after injury. We annually organize the international symposium Signal Transduction at the Blood-Brain Barriers to disseminate news of progress in the field, thus stimulating collaborations in order to create publications and new joint projects. ZUSAMMENFASSUNG Wir beschäftigen uns mit der Struktur, Funktion und Modulierung von Zell-Zellkontakten und erforschen hier Tight Junctions (TJs, permeationsdichte Zellzwischenräume) an Gewebsgrenzen. Ziel ist die Aufklärung von Pathomechanismen und eine Verbesserung von Therapiemöglichkeiten. So sollen neue Strategien erarbeitet werden, die es erlauben neurologische und andere Barrieren gezielt zu beeinflussen, um den Wirkstofftransport und / oder die Wirksamkeit zu verbessern und Störungen der Barrierefunktion zu verhindern (Protektion). Membranproteine wie z. B. Claudine, TAMPs (Occludin, Tricellulin) und assoziierte Gerüstproteine (z. B. ZO-1) bestimmen die parazelluläre Dichtheit endothelialer und epithelialer Barrieren. Da der Komplex der TJ noch nicht vollständig bekannt ist, ist auch unklar, wie Struktur, Regulationsmechanismen und Wechselwirkungen dieser Proteine die Barrierefunktion steuern. Wir konzentrieren uns auf die Aufklärung des Gesamtkomplexes der TJs, auf die Prozesse, die der Oligomerisierung von Schlüsselproteinen der Gewebsbarrieren zugrunde liegen, sowie auf Mechanismen, die nach Verletzungen die TJs öffnen und / oder wiederherstellen. Jedes Jahr organisieren wir das internationale Symposium Signal Transduction at the Blood-Brain Barriers zum Austausch wissenschaftlicher Ergebnisse und zur Anregung internationaler Kooperationen, um Publikationen und neue nationale wie internationale Projektkooperationen zu fördern. Elucidation of tightening mechanism at tight junctions: The interaction potential between main TJ proteins (Fig. 1) was analyzed in detail. The majority of the highly homologous classic claudins are able to associate homo- and heterophilically. In particular, extracellular loops are involved in the associations (Dabrowski et al., 2014), while to a lesser extent the TAMPs interact homo- and heterophilically except for occludin and tricellulin. Direct binding of TAMPs with classic claudins was demonstrated for the first time (Cording et al., 2013, Bellmann et al., 2014). The results show that claudins may determine binding properties, membrane localization or mobility of the TAMPs and, conversely, that TAMPs determine the strand morphology of the claudins. Claudin-1 and claudin-5 were identified as preferred interaction partners of other constituents of the TJs. At the blood-brain barrier (BBB), interactions found between the TJ proteins provide deeper insights into the principles of TJ assembly (Haseloff et al., 2014). As claudin-5 is essential for the tightness of the BBB and claudin-1 for the perineurium ensheathing peripheral nerves, both claudins were established as new pharmacological and diagnostic targets (Dabrowski et al., 2014). New regulatory role of tight junction proteins: The molecular function of the TAMPs is still unknown. A new concept was developed for how occludin, a unique TJ marker, is involved in redox-dependent signal-transduction mechanisms and how redoxsensitive domains of occludin are crucial in the redox regulation of protein interactions at the TJs. These data are highly relevant for diseases related to oxidative stress and corresponding pharmacological interventions (Bellmann et al., 2014, Dabrowski et al., 2014). In addition, the oligomerization status of occludin influences its association with other TJ proteins such as claudin-5 and ZO-1. ZO-1, the scaffolding protein of the TJs, was further investigated regarding its interaction with occludin. We identified casein kinase 2-dependent phosphorylation of occludin as an important regulatory mechanism (Dörfel et al., 2013). New modulators of tight junctions: Modulation of the TJs is a key topic of our studies. Claudin-1, in particular its first extracellular loop, was found to contribute to the interaction between TJ proteins. Peptidomimetics of this loop were generated with ß-sheet structural properties which transiently increased the paracellular permeability for ions, high and low molecular weight compounds in cell models (Fig. 2). Perineurial injection in rats facilitated the uptake of anaesthetics into the nerve (Dabrowski et al., 2014, Sauer et al., 2014). Alternatively, alkylglycerols were positively tested in a BBB cell culture model (Hülper et al., 2013). In conclusion, novel tools were developed to improve the delivery of pharmaceutical agents through neurological barriers. We further characterized the interacting structure of Clostridium perfringens enterotoxin, another modulator of barrier-forming claudins (Yelland et al., 2014). State-of-the-art barrier-opening approaches were summarized and discussed (Tscheik et al., 2013). Methodological advances: Protein interactions, in particular of tricellulin, the marker protein of tricellular junctions, were studied by application of powerful techniques. A new method was created to quantify the trans- and cis interaction at the tricellular contact. These studies were supplemented by novel super resolution microscopy experiments. Investigations for a better understanding of neuropathologies A collaborative study with the Chinese Academy of Sciences (Beijing) yielded evidence of a new role of interferon-γ signal transduction pathway at the BBB. We found that the interferon-γ receptor may have a protective effect at the BBB in neuroinflammation (Ni et al., 2014). In cooperation with the University Medical Center (Amsterdam / Netherlands), we discovered that claudin-3 is involved in the tightening of the blood-cerebrospinal fluid (CSF) barrier. This function is relevant for preventing the cerebral uptake of activated leukocytes and, hence, for the progress of neurodegenerative processes (Kooij et al., 2014). The identification of biomarkers is a field of growing importance for the diagnosis and therapy of neurological diseases. In collaboration with the Pasteur Institute (Paris / France), proteomic investigations aimed at disclosing potential biomarkers of lysosomal storage diseases were accomplished in dog models of the Hurler and Sanfilippo syndromes. A variety of biomarker candidates were identified in the CSF for both pathologies. In connection with the studies directed at the identification of potential biomarkers, methodological experiments were performed related to the transferability of data obtained from human and dog CSF (Günther et al., 2014).

22 40 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 41 Fig. 1: Proteins and morphology of tight junctions (TJs) at the blood brain barrier formed by capillary endothelial cells (CEC) with the focus on tetraspanning Fig. 2: Scheme of a peptidomimetic agent taken out of an extracellular loop of a classic claudin causing transient opening of the paracellular cleft intracellular TJ proteins. (A) Scheme of protein composition in TJs at the blood brain barrier in tissue barriers to improve drug delivery. Example: Peptide C1C2 from with special consideration of members of the claudin protein family and the murine claudin-1. TJ-associated marvel proteins (TAMPs) occludin and tricellulin (tetraspanning). Single membrane spanning JAMs (junctional adhesion molecules) as well as C1C2 extracellular membrane-associated zonula occludens proteins (ZO) or multi-pdz domain protein 1 (MUPP-1) are not directly involved in paraendothelial tightening. cell membrane Amongst others, signaling via membrane receptors, e.g., adenylyl cyclase (AC), second messengers, e.g., cyclic AMP or inositol-3-phosphate (IP 3 ), and protein kinases (PKs) may regulate TJs. (B) Ultrathin section transmission electron drug mclaudin-1 intracellular microscopy of two adhering CECs visualizing TJ elements sealing the intermembrane gap, arrows indicating TJ, and (C) freeze-fracture transmission electron microscopy of TJ strands in mouse BBB endothelial cells. Note that particles are associated nearly equally at E- and P-face. Arrows indicate TJ particles. EF / PF, exoplasmic and protoplasmic face of intramembranous TJ strands. no paracellular drug delivery pre-incubation with C1C2 paracellular drug delivery GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Rosel Blasig Dr. Reiner Haseloff International PEU FP7 collaborative project consortium National DFG Forschergruppe 721 Tight Junctions Günther R, Krause E, Schümann M, Ausseil J, Heard JM, Blasig IE, Haseloff RF (2014) Removal of albumin and immunoglobulins from Deutsche Forschungsgemeinschaft, Alterationen von Occludin bei oxidativem Stress und Aufklärung der Funktion von Claudin-12, FOR Dr. Christian Bellmann Dr. Lars Winkler Philipp Berndt (doctoral student) Olga Breitkreutz-Korff (doctoral student) Sophie Dithmer (doctoral student) Jimmi Cording (doctoral student) Nora Gehne (doctoral student) Sebastian Pfeil (Dabrowski) (doctoral student) Christian Staat (doctoral student) Christian Tscheik (doctoral student) Ulrike Borgmeier (technical assistent) JUSTBRAIN European consortium Brains4brain Anuska Andjelkovic University of Michigan Medical School, Ann Arbor, USA Ajit Basak University of London, UK Matthew Campbell Trinity College Dublin, Ireland Maria Deli Biological Research Centre, Szeged, Otmar Huber Universitätsklinikum Jena Petra Hülper Universität Göttingen Heike Rittner Universitätsklinikum Würzburg Hartwig Wolburg Eberhard-Karls-Universität Tübingen canine cerebrospinal fluid using depletion kits: a feasibility study. Fluids Barriers CNS 11, 1 5. Bellmann C, Schreivogel S, Günther R, Pfeil S, Schümann M, Wolburg H, Blasig IE (2014) Highly conserved cysteines are involved in the oligomerisation of occludin redox dependency of the second extracellular loop. Antioxid. Redox. Signal. 20, Kooij G, Kopplin K, Blasig R, Stuiver M, Koning N, Goverse G, Van Der Pol SMA, Van Het Hof B, Gollasch M, Drexhage J, Reijerkerk A, Meij IC, Mebius R, Willnow T, Müller D, Blasig IE, De Vries HE (2014) Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuroinflammation. Acta Neuropathol. 128, / 2, TP5 (BL 308 / 9 1), , Deutsche Forschungsgemeinschaft, Modulation der Claudinoligomerisierung zur Beeinflussung der Blut-Hirnschranke, BL 308 / 7 4, , (inklusive 1xE13 50 %) Deutsche Forschungsgemeinschaft, Molekulare Organisation von heteropolymeren Tight-Junction-Strängen, PI 837 / 2 1, with Piontek J, , (inklusive 1xE13) Europäische Kommission, 7. Framework Programme, Project: JUSTBRAIN, Blood-brain barrier junctions as targets for paracellular drug delivery to the brain, , Ramona Günther (technical assistent) Heike Meyer (technical assistent) Hungary Jean-Michel Heard Institut Pasteur, Paris, France Zhihai Qin Chinese Acad. Sci., Beijing, PR China Elga de Vries Neuroscience Campus, Cording J, Berg J, Käding N, Bellmann C, Tscheik C, Westphal JK, Milatz S, Günzel D, Wolburg H, Piontek J, Huber O, Blasig IE (2013) In tight junctions, claudins regulate the interactions between occludin, tricellulin and marveld3, which, inversely, modulate claudin oligomerization. J. Cell Sci. 162, Dörfel MJ, Westphal JK, Bellmann C, Krug SM, Cording J, Mittag S, B4B Foundation, Modifying arylsulphatase A with peptides to bypass the blood-brain barrier, , Else-Kröner-Fresenius-Stiftung, Neue molekulare Therapieansätze in der Schmerztherapie durch Öffnung der peripheren Nervenbarriere mittels Tight Junction modulierenden Peptiden, , Amsterdam, Netherlands Tauber R, Fromm M, Blasig IE, Hube O (2013) CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins Projekt Pasteur Paris, Definition and validation of surrogate biological markers of neuropathology in the cerebrospinal fluid for mucopolysac- and tight junction integrity. Cell Commun. Signal. 11, 40 charidosis III, , Manja Thiem (graduate student) and Nora Gehne (photo left), Sophie Dithmer VIP Projekt, BMBF, EASYPERM, Modulatoren der Blut-Hirnschranke als Drugenhancer für ZNS-Pharmaka, , ,00 FMP authors Group members

23 42 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 43 MOLECULAR PHARMACOLOGY AND CELL BIOLOGY Natalia Kononenko (photo left), Katrin Diesenberg and Michael Krauß MOLEKULARE PHARMAKOLOGIE UND ZELLBIOLOGIE GROUP LEADER PROF. DR. VOLKER HAUCKE BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studies in Biochemistry, Freie Universität Berlin and the University of Basel (Biozentrum) PhD student, Department of Biochemistry (Prof. G. Schatz), Biozentrum, University of Basel 1997 PhD (summa cum laude) Postdoctoral fellow, Yale University School of Medicine and HHMI (Prof. P. De Camilli), New Haven Independent group leader, Center for Biochemistry and Molecular Cell Biology, University of Göttingen Professor of Membrane Biochemistry, Freie Universität Berlin Full Professor and Chair (W3), Department of Membrane Biochemistry, Freie Universität Berlin since 2007 Member of Neurocure Cluster of Excellence Full Professor and Chair (W3), Membrane Biochemistry, Freie Universität and Charité Universitätsmedizin, Berlin Speaker, Collaborative Research Center (SFB) Speaker, Collaborative Research Center (SFB) 958 since 2012 Director of the FMP, Head of the Department of Molecular Pharmacology & Cell Biology at the FMP, Full Professor of Molecular Pharmacology (S-W3), Institute of Pharmacy, Freie Universität Berlin; co-optated to the Charité Universitätsmedizin, Berlin 2014 Elected Member of the European Molecular Biology Organization (EMBO) Membrane dynamics within the endocytic and endosomal system play crucial roles in cell physiology and membrane homeostasis, cell signaling and development, the functioning of the nervous system, and diseases such as cancer. Research within the department focuses on the molecular mechanisms of endocytic and endosomal membrane traffic using a wide arsenal of techniques that range from in vitro approaches to the in vivo analysis of cellular systems. We are particularly interested in the exo-endocytic cycling of synaptic vesicles at neuronal synapses and its role in brain function and disease. An important aspect of these studies is to determine how events at the molecular and subcellular levels translate into the functions of individual cells and of cellular networks within the context of an entire organism. To achieve this we are developing molecular tools to dissect and manipulate exo-endocytic cycling and endosomal membrane dynamics using genetic, biochemical, and pharmacological approaches, and by further developing high-resolution imaging techniques. The long-term goal of our work is to unravel the molecular basis of endocytic and endosomal function and dysfunction, thereby opening new avenues for pharmacological interference. ZUSAMMENFASSUNG Dynamische Membranprozesse des endozytotischen und endosomalen Systems spielen eine entscheidende Rolle in der Zellphysiologie und Membranhomöostase, bei der Signalübertragung zwischen Zellen und der Zellentwicklung, für die Aktivitäten des Nervensystems und bei Krankheiten wie Krebs. Der Forschungsschwerpunkt unserer Abteilung liegt auf den molekularen Mechanismen des endozytotischen und endosomalen Membrantransports, die wir mit einem breiten Spektrum an Techniken, von in vitro Ansätzen bis zur in vivo-analyse zellulärer Systeme, untersuchen. Besonders interessiert sind wir am exo-endozytotischen Zyklus synaptischer Vesikel an neuronalen Synapsen und dessen Rolle bei der Gehirnfunktion und Erkrankungen des Nervensystems. Ein wesentlicher Aspekt dieser Untersuchungen besteht darin, zu ermitteln, wie Ereignisse auf molekularer und subzellulärer Ebene in Funktionen einzelner Zellen und zellulärer Netzwerke innerhalb eines Gesamtorganismus übersetzt werden. Um dies zu erreichen, entwickeln wir mit Hilfe genetischer, biochemischer und pharmakologischer Ansätze und durch Weiterentwicklung hochauflösender bildgebender Verfahren molekulare Werkzeuge zur Analyse und Manipulation des Exo-Endozytose- Zyklus synaptischer Vesikel sowie der endosomalen Membrandynamik. Das langfristige Ziel unserer Arbeit ist es, die molekularen Grundlagen der endozytotischen und endosomalen Funktion und Dysfunktion zu enträtseln und dabei neue Wege für pharmakologische Eingriffe zu eröffnen. Research within the department is conducted within several subgroups (led by V. Haucke, M. Krauss and J. Schmoranzer) and covers five major areas: (i) the cycling of synaptic vesicle membranes at neuronal synapses; (ii) the mechanism of endocytosis; (iii) the regulation of the endolysosomal system, including autophagy, by spatiotemporallycontrolled synthesis and turnover of phosphoinositides; (iv) the role of the cytoskeleton in endosomal function, cell signalling, migration, and polarized secretion; and (v) the development and application of super-resolution light microscopy techniques for studying the above processes. Synaptic vesicle exo- and endocytosis Neurotransmission involves the calcium-triggered exocytic release of neurotransmitters from synaptic vesicles (SVs) at presynaptic active zones, followed by their endocytic recycling. Using mouse knockout technology, Drosophila mutants (with Stephan J. Sigrist, FU Berlin), and RNA interference in combination with optical imaging including optogenetics and electrophysiology, we aim to dissect the pathways and molecular mechanisms of SV recycling and regeneration. A key question is how exo- and endocytosis are coupled and how membrane homeostasis is maintained. We have identified endocytic adaptors (e.g. stonins, AP180, CALM) that facilitate endocytic sorting of select SV proteins during exoendocytic cycling of SVs. Furthermore, we have begun to unravel molecular links between the fusion machinery at active zones and the endocytic system. The consequences of loss of function of these factors on neurotransmission are currently being investigated at the cellular and organismic levels using genetic and optical-biophysical approaches. These studies bear important implications for the understanding of neurological disorders and for neurodegeneration. Mechanisms of endocytosis Eukaryotic cells internalize nutrients, antigens, growth factors, pathogens, ion channels and receptors via endocytosis. We are interested in determining the exact function of endocytic adaptors and scaffolds including clathrin, AP-2, intersectins, and Bin / Amphiphysin / Rvs homology (BAR) domain proteins in the spatiotemporal regulation of clathrin-mediated endocytosis using cell biological, chemical, and genetic techniques. Our lab has identified and characterized the first chemical inhibitors of clathrin function as well as endocytic adaptors for cargo sorting and their interaction with cargo proteins and lipids. Recent work has also dealt with the question of how membrane deformation in endocytosis is coupled to dynamin-mediated fission and how dynamin assembly is coupled to endocytic vesicle formation. Other projects aim to unravel factors mediating clathrin-independent endocytosis. Phosphoinositides within the endolysosomal system Phosphoinositides (PIs) serve as spatiotemporal landmarks within the endolysosomal system and for intracellular membrane traffic in general. We have discovered PI kinases and phosphatases with key roles in endocytosis and in endolysosomal membrane homeostasis, including autophagy. Genetics, RNA interference, and acute chemical and optogenetic perturbations are used to designate roles of specific PIs during progression of endocytosis and elucidate how PI conversion along the endolysosomal pathway is achieved. Furthermore, we use biochemical, lipidomic, and proteomic approaches to identify and characterize effectors and regulators of PI turnover and conversion. We also seek to identify novel pharmacological and chemical inhibitors of select PI-metabolizing enzymes. These studies bear implications for disease, as PI-metabolizing enzymes, e.g. PI 3-kinases or myotubularin PI phosphatases, are implicated in cancer as well as hereditary disorders such as Charcot-Marie-Tooth disease. Role of the cytoskeleton in endosomal function, cell signalling, migration, and polarized secretion Recent work from our lab has identified key connections between the endosomal system and the microtubule-based, as well as the actin cytoskeleton. Reciprocal relationships between the cytoskeleton (e.g. microtubules, septins) and the endosomal system regulate cellular functions ranging from nutrient uptake and signaling to cell motility. By employing cell biological and optical techniques in conjunction with mouse genetics we dissect the role of membrane scaffolds, small G proteins, and adaptors in regulating cytoskeletal dynamics, cell signaling and migration, and polarized secretion. Super-resolution light microscopy Recent advances have allowed us to break the diffraction barrier of light microscopy. We have further developed a multi-color STORM / PALM-based super-resolution microscopy platform for the imaging of fixed and live samples, e.g. the exo- / endocytic machineries at presynaptic terminals. Furthermore, we employ sensitive multicolor total internal reflection fluorescence (TIRF) and also, more recently, multicolor 3D structured illumination microscopy (SIM) to visualize the nanoscale organization of the exo-endocytic and endosomal systems.

24 44 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 45 PI(3,4)P 2 CHC merge Fig. 1: PI(3,4)P 2 regulates clathrin-mediated endocytosis. Partial colocalization of PI(3,4)P 2 with CCPs. Confocal images of Cos7 cells stained for PI(3,4)P 2 and clathrin heavy chain (CHC). Arrowheads, structures immunopositive for PI(3,4)P 2 and clathrin. Scale bar, 10 µm (inset: 2 µm). Fig. 2: Reduced SV density and accumulation of endosome-like vacuoles at cortical synapses of AP-2 conditional knockout mice. Representative electron tomograms and 3D reconstructions of presynaptic terminals from the somatosensory cortex of wild-type (A) and AP-2 knockout mice (B). GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS Prof. Dr. Michael Krauß (group leader) Dr. Jan Schmoranzer (group leader) Anela Vukoja (doctoral student) Uwe Fink (technical assistant / chemist) International Paolo Di Fiore Kononenko NL, Puchkov D, Classen GA, Walter A, Pechstein A, Sawade L, Kaempf N, Trimbuch T, Lorenz D, Rosenmund C, Maritzen Deutsche Forschungsgemeinschaft, SFB 958, A11, Structural and functional analysis of septin scaffolds mediating endosomal membrane trafficking, Dr. Caroline Bruns Sabine Hahn (technical assistant) FIRC Institute of Molecular Oncology M, Haucke V (2014) Clathrin / AP-2 mediate synaptic vesicle reformation to Krauss M, , Dr. Marielle Grünig Dr. Tania Lopez-Hernandez Dr. Gaga Kochlamazashvili Dr. Natalia Kononenko Dr. Seong Joo Koo Dr. Marijn Kuijpers Dr. Martin Lehmann Dr. Marta Maglione Delia Löwe (technical assistant) Maria Mühlbauer (technical assistant) Susanne Thomsen (technical assistant) Lena von Oertzen (technical assistant) Susanne Wojtke (technical assistant) Silke Zillmann (technical assistant) Foundation (IFOM) & European Institute of Oncology, Milano, Italy Emilio Hirsch University of Torino, Italy Adam McCluskey University of Newcastle, Australia Phillip J. Robinson Children s Medical Research Institute (CMRI), from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron, 82, Posor Y, Eichhorn-Grünig M, Puchkov D, Schöneberg J, Ullrich A, Lampe A, Müller R, Zarbakhsh, Gulluni F, Hirsch E, Krauss M, Schultz C, Noé F, Haucke V (2013) Spatiotemporal Control of Endocytosis by Phosphatidylinositol 3,4-Bisphosphate. Nature, 499, Kononenko N, Diril MK, Puchkov D, Kintscher M, Koo SJ, Pfuhl G, Deutsche Forschungsgemeinschaft, SFB 958, Z02, Super-resolution light microscopy to resolve nanoscale molecular structures, to Schmoranzer J, Deutsche Forschungsgemeinschaft, SFB 740, C08, Funktionelle Charakterisierung der Assemblierung und Dynamik PI4-Kinase-basierter Module wahrend der Proteinsortierung an endosomalen Membranen, with Krauss M, , (joint postdoc with S. J. Sigrist, FU Berlin) Dr. Andrea Lynn Marat Dr. Arndt Pechstein Dr. Dmytro Puchkov Sydney, Australia Takeshi Sakaba Kyoto University, Japan Oleg Shupliakov Winter Y, Wienisch M, Klingauf J, Breustedt J, Schmitz D, Maritzen T, Haucke V (2013) Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2. Proc Natl Acad Sci U S A, 110, E526 E535 Deutsche Forschungsgemeinschaft, SFB 740 / 3, C08, Funktionelle Organisation und Dynamik PI-Kinase-basierter Module für die Proteinsortierung an endosomalen Membranen, , Dr. Tolga Soykan Dr. Anna Wawrzyniak Dr. Mirjana Weimershaus Karolinska Institute, Stockholm, Sweden National Daumke O, Roux A, Haucke V (2014) BAR domain scaffolds in dynamin-mediated membrane fission. Cell 156, Deutsche Forschungsgemeinschaft, SFB 765, B04, Multivalente Modulation der Clathrin vermittelten Rezeptorendozytose, , Katharina Branz (doctoral student) Gala Claßen (doctoral student) Katrin Diesenberg (doctoral student) Fabian Feutlinske (doctoral student) Oliver Daumke Max-Delbrück-Center for Molecular Medicine, Berlin Christian Freund Wieffer M, Cibrián Uhalte E, Posor Y, Otten C, Branz K, Schütz I, Mössinger J, Schu P, Abdelilah-Seyfried S, Krauß M, Haucke V (2013) PI4K2β / AP-1-based TGN-endosomal sorting regulates Wnt signaling. Curr Biol., 23, Deutsche Forschungsgemeinschaft, Excellence Initiative, EXC-257 NeuroCure Towards a better outcome of central nervous system disorders, , Niclas Gimber (doctoral student) Claudia Gras (doctoral student) Burkhard Jakob (doctoral student) Freie Universität Berlin Jürgen Klingauf University of Münster EXTERNAL FUNDING Schram Foundation, Role of endocytic adaptors and accessory proteins synaptic vesicle protein sorting and recycling, T287 / / 2008, , Maria Jäpel (doctoral student) Natalie Kaempf (doctoral student) Christina Kath (doctoral student) André Lampe (doctoral student) Wen-Ting Lo (doctoral student) Tobias Moser Georg-August-Universität, Göttingen Christian Rosenmund Charité Universitätsmedizin Berlin Dietmar Schmitz Deutsche Forschungsgemeinschaft, Funktionelle Charakterisierung der Adaptorproteine Stonin 1 und γ-bar beim intrazellulären Membrantransport, HA 2686 / 3 2, with Maritzen T, , Deutsche Forschungsgemeinschaft, Functional characterization of the Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Forschung und Innovation, Role of proteostasis in cellular aging, SAW 2014-FMP-2, to Volker Haucke, Thomas Jentsch, and Hartmut Oschkinat, , York Posor (doctoral student) Linda Sawade (doctoral student) Charité Universitätsmedizin Berlin Stephan J. Sigrist SNARE adaptors AP180 and CALM in synaptic exo- and endocytosis in vivo, HA 2686 / 8 1, with Maritzen T, , European Molecular Biology Organization, EMBO Long-Term Fellowship, to Marielle C. Grünig, , Irene Schütz (doctoral student) Kyungyeun Song (doctoral student) Wiebke Stahlschmidt (doctoral student) Freie Universität Berlin Carsten Schultz EMBL, Heidelberg Deutsche Forschungsgemeinschaft, SFB 958, A01, Structural and functional organization of endocytic scaffolds within the periactive zone, with Maritzen T, , Banting Fellowship, Andrea Lynn Marat, , Alexander von Humboldt Foundation, Mirjana Leona Weimershaus, , FMP authors Group members Deutsche Forschungsgemeinschaft, SFB 958, A07, Regulation of SH3 domain-containing scaffolds in synaptic vesicle clustering, with Freund C, , Leibniz-DAAD-Postdoctoral Fellowship, Tania Lopez-Hernandez, , University of Newcastle, International Collaboration Award (ICA), ,

25 46 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 47 PROTEOSTASIS IN AGING AND DISEASE DIE ROLLE DER PROTEOSTASE BEIM ALTERN UND IN KRANKHEIT GROUP LEADER DR. JANINE KIRSTEIN DNJ-13-YFP DNJ-19 merge Fig. 1 BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS 2003 Diploma / MSc. University of Greifswald 2007 Ph.D. Free University Berlin (summa cum laude) Postdoc at Free University Berlin (supervisor: K. Turgay) Postdoc Northwestern University, IL, USA (supervisor: R. Morimoto) 2008 Postdoc fellowship of DFG and EMBO awarded, but declined Postdoc fellowship of Human Frontiers in Science (HFSP) Instructor at Northwestern University, IL, USA Since 2013 Group Leader in the Molecular Physiology and Cell Biology Section of the FMP and NeuroCure Our research goal is to advance our understanding of the mechanisms that maintain a functional proteome during the lifespan of a metazoan. We use a model organism that has a long-standing history as an excellent genetic model, in combination with recently available cell biology tools. In the literature of C. elegans research, biochemical or biophysical studies are few and far between, while our understanding of chaperones and proteolytic machines how they work, how they recognize a substrate and contribute to protein folding are almost entirely based on in vitro or ex vivo data. Our research approach will bridge this gap using C. elegans as an excellent model utilizing biochemical, cell biology and genetic techniques addressing important biological questions on the management of protein misfolding and aggregation in a metazoan in vivo. Specifically, we employ novel proteostasis sensors to analyze the chaperone and proteolytic capacity of distinct tissues and the whole organism during development and aging and upon chronic stress conditions in vivo in real-time. This extensive analysis will allow for an identification of the key chaperone and proteolytic complexes maintaining protein quality control and their interplay with imbalance of proteostasis during aging and in neurodegenerative disease models such as Huntington s disease, Alzheimer s and Parkinson s disease. ZUSAMMENFASSUNG Das Ziel unserer Forschung ein besseres Verständnis der Mechanismen, die es, einem Organismus erlauben, sein Proteom während der gesamten Lebenszeit in einem funktionellen Zustand zu halten. Wir nutzen den Nematoden C. elegans als ein Modellsystem, das eine lange Tradition für genetische Untersuchungen hat, und wenden auf diesen Modellorganismus zellbiologische Techniken an, die seit Kurzem verfügbar sind Biochemische oder biophysikalische Methoden waren für C. elegans bislang nur wenige bekannt. Die meisten Ergebnisse der Biologie von Chaperonen ihre Arbeitsweise, wie sie Substrate erkennen und zu deren Faltung beitragen beruhen fast ausschließlich auf in vitro oder ex vivo Analysen. Unser Forschungsansatz schließt diese Lücke. Wir nutzen C. elegans als ausgezeichntes Modell in Kombination mit biochemischen, zellbiologischen und genetischen Methoden, um wichtige biologische Fragen um die Behebung von Fehlfaltung und Aggregation von Proteinen an einem Vielzeller in vivo zu untersuchen. Insbesondere benutzen wir neuartige Proteinhomöostase-Sensoren, um die Proteinfaltungs- und Proteinabbau-Kapazität in bestimmten Geweben oder dem gesamten Organismus sowohl während der Entwicklung als auch im Alter und unter chronischem proteotoxischem Stress in vivo zu charakterisieren. Unsere umfassenden experimentelle Analyse wird es erlauben, Schlüsselkomponenten des Proteostase-Netzwerkes zu identifizieren und ihre Rolle während des Alterns und in den neurodegenerativen Krankeheiten wie z. B. Chorea-Huntington, Alzheimer und Parkinson zu verstehen. Our group uses the nematode Caenorhabditis elegans as its main model system to study proteotoxic challenges during aging and upon expression of disease-associated proteins and peptides. Protein aggregates are a hallmark for neurodegenerative diseases such as Alzheimer s, Huntington s and Parkinson s disease. We model these diseases in the nematode by cell- or tissue-specific expression of the pathogenic proteins and peptides, e.g. Aβ, tau, polyq and α-synuclein. Aging is a risk factor for the onset and manifestation of neurodegenerative diseases. The decline in chaperone and proteolytic capacity during aging is believed to be a strong contributing factor. However it is not known what exactly changes as aging progresses. We aim to unravel the key players of the cellular proteostasis network that is composed of molecular chaperones and proteolytic machines and how it combats protein misfolding and aggregation during aging and disease. Characterization of the disaggregase module in metazoans Very recently (and in cooperation with the Bukau lab) we identified the metazoan disaggregase module that has the capacity to disaggregate and subsequently refold amorphous model substrates such as luciferase and malate dehydrogenase. The disaggregase is composed of three molecular chaperones: Hsp110, Hsp70 and a J-protein (Hsp40). Apart from a single (C. elegans) or three (human) Hsp110 genes, the metazoan cell encodes numerous members of the Hsp70, and in particular of the J-protein family. We want to understand if an animal uses distinct disaggregase modules during its lifetime, in specific tissues and cell types, in response to a particular stress or type of protein aggregate (amorphous vs. amyloid). To address these questions we employ a wide range of in vivo and in vitro methods that complement each other. For instance, to gain insight into the composition of the disaggregase module we used an unbiased in vivo approach to study the regulation of all members of the respective families using qrt-pcr. The rationale behind this is the assumption that the same regulatory pattern ought to be a prerequisite for complex formation in an organism. The potential candidates were then analyzed in in vitro disaggregation assays. To analyze their capacity to disaggregate and refold proteins, this defined set of those co-regulated chaperones (members of the Hsp110, Hsp70 and J-proteins) was analyzed in any given combination. In this way we could identify distinct disaggregase modules that disaggregate luciferase to yield up to 80 % of the enzymatic activity of native protein. We have now expanded our assay to other aggregated protein species including amyloidogenic proteins such as HttpolyQ, α-synuclein, and Aβ, as well as endogenous proteins that are prone to aggregate with age. Moreover, we want to elucidate whether the distinct disaggregase modules identified exhibit a preference for tissues and cell types. The susceptibility of specific neuronal cells such as dopaminergic neurons for the occurrence of protein aggregates, e.g. those formed by α-synuclein, suggests a unique proteostatic balance in specific cell types and also suggests the possibility for therapeutic interference by, for example, induction of distinct disaggregase modules. Redox homeostasis and proteostasis Studies of the proteostatic balance and its changes during aging have so far mainly addressed the cytosolic compartment. To explore whether aging and proteotoxic stress in the cytosol also affects ER redox homeostasis, we have utilized two in vivo redox sensors, the redox-sensitive GFP and HyPer, to monitor the general redox state and the concentration of a specific reactive oxygen species, H 2 O 2, respectively. Using these endogenous sensors we can demonstrate that the progression of aging, the expression of neurotoxic aggregationprone proteins and proteasome inhibition lead to perturbation of the ER and cytosolic redox homeostasis in opposing directions. Whereas the ER becomes more reducing in response to proteotoxic challenges, the cytosolic redox state shifts towards more oxidizing conditions. Redox homeostasis in the cytosol and ER are coupled: proteotoxic stress in the cytosol leads to a shift in ER redox state towards reducing, whereas proteotoxic stress in the ER and the cytosol causes the cytosolic redox state to shift towards oxidizing conditions. Intriguingly, we also observed a trans-tissue signaling of the redox state between neuronal and muscle tissue. Imbalances of protein folding conditions in a particular tissue leads to a change in redox homeostasis not only in the affected cells, but also in a distal tissue of the organism. We are now aiming to understand the signaling pathway that adjusts the redox states with the folding requirements across organellar, cellular and tissue boundaries. Fig. 1: Co-localization of J-proteins of class A and class B in vivo. The image depicts the head region of C. elegans expressing DNJ-13-GFP (class B J-protein) under its endogenous promoter and is immostained with DNJ-19 (class A J-protein) antibodies and Alexa Fluor 594 conjugated secondary antibodies. The animal was subjected to heat shock prior to the fixation and imaging analysis.

26 48 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 49 Ellen Malovrh and Kristin Arnsberg Annika Scior and Kerstin Steinhagen Fig. 2: ER Redox homeostasis shifts towards reducing conditions during aging. The images depict the muscle cells of age-synchronized C. elegans at the indicated days. The animal expresses ER-targeted redox-gfp. The images are overlays of the emission spectra upon excitation with 405 nm (blue) and 488 nm (green). The change from blue to green during the course of aging reflects the change of the redox state towards reducing conditions. GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Annika Scior (postdoc) International National Kirstein-Miles J, Scior A, Deuerling E, Morimoto RI (2013) The nascent DFG EXC 257 NeuroCure ( ) Kristin Arnsburg (doctoral student) Kazuhiro Nagata Erich Wanker polypeptide associated complex is a key regulator of proteostasis. Ellen Malovrh (Erasmus student) Kyoto University, Kyoto, Japan Max-Delbrück-Center for EMBO J. 32, Kerstin Steinhagen (technical assistant) Richard Morimoto Northwestern University, Evanston, USA Claes Andreasson Molecular Medicine, Berlin Elke Krüger Charite, Berlin Kirstein-Miles J, Morimoto RI (2013) Ribosome-associated chaperones act as proteostasis sentinels. Cell Cycle 12, Stockholm University, Stockholm, Sweden Britta Eickholt Arnsburg K, Kirstein-Miles J (2014) Interrelation between Protein David Dougan Charite, Berlin Synthesis, Proteostasis and Life Span. Current Genomics 15, LaTrobe University, Melbourne, Australia Bernd Bukau ZMBH-DKFZ Alliance, Heidelberg Ulrich Hartl MPI, Martinsried Rampelt H, Kirstein-Miles J, Nillegoda NB, Kang C, Scholz SR, Morimoto RI, Bukau B (2012) Metazoan Hsp70 machines use Hsp110 to power protein disaggregation. EMBO J 31 (21): Eberhard Krause FMP, Berlin Hartmut Oschkinat FMP, Berlin Kirstein J*, Morito D, Kakihana T, Sugihara M, Minnen A, Hipp MS, Nussbaum-Krammer C, Hartl FU*, Nagata K* & Morimoto RI* (2015) Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments EMBO Journal in press Michael Krauss (* corresponding authors) FMP, Berlin Baris Tursun MDC, Berlin FMP authors Group members

27 50 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 51 BEHAVIOURAL NEURODYNAMICS VERHALTENSNEURODYNAMIK GROUP LEADERS DR. TATIANA KOROTKOVA DR. ALEXEY PONOMARENKO BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Tatiana Korotkova Studied biology and physiology at Lomonosov Moscow State University, Russia Ph.D., Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas and Prof. J.P. Huston) Post-Doc, Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas) Post-Doc, University Clinic for Neurology, Heidelberg (Prof. H. Monyer) Postdoc at the FMP (Dr. A. Ponomarenko), NeuroCure Fellow 2012 Junior group leader, FMP / NeuroCure Alexey Ponomarenko Studied biology and physiology at Lomonosov Moscow State University, Russia Ph.D., Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas and Prof. J.P Huston) Post-Doc, Heinrich-Heine- University, Düsseldorf (Prof. H.L. Haas) 2004 Internship at Rutgers University (Prof. G. Buzsaki), Newark, USA Post-Doc, University Clinic for Neurology, Heidelberg (Prof. H. Monyer) since 2009 Junior group leader, FMP / NeuroCure Our aim is to reveal the contribution of individual neuronal membrane conductancies to the excitability and synchronization of neuronal networks in vivo. Brain synchronization regimes are instrumental for cognitive functions such as learning and memory and play a key role in disorders such as epilepsy. We are focusing on the role of voltage-gated channels and GABAA-receptors in the operation of hippocampal networks using genetic mouse models. We also investigate how cortico-subcortical communication is organized through synchronization. A further goal is to establish how molecular metabolic signals sensed by the brain coordinate multiple vital functions, including food intake and sleep. We study the activity and interactions of specific neurons in the hypothalamus across vigilance states and homeostatic challenges using high-density electrophysiological recordings and optogenetics in freely behaving mice. We aim to gain insights into the neural basis and molecular determinants of vital functions and their pathologies, in particular of obesity and sleep. ZUSAMMENFASSUNG Unser Ziel ist es, den Beitrag, den die Leitfähigkeit einzelner neuronaler Membranen zur Erregbarkeit und Synchronisation neuronaler Netzwerke in vivo leistet, aufzudecken. Für kognitive Funktionen wie Lernen und Gedächtnis ist Synchronisation im Gehirn unabdingbar, Sie spielt auch bei Erkrankungen wie Epilepsie eine Schlüsselrolle. Der Fokus unserer Forschung liegt auf der Untersuchung der Rolle spannungsabhängiger Kanäle und GABAA-Rezeptoren auf die Arbeitsweise hippokampaler Netzwerke an genetische Mausmodelle. Außerdem untersuchen wir, wie die kortiko-subkortikale Kommunikation durch Synchronisation organisiert wird. Ein weiteres Ziel ist es, herauszufinden, wie das Gehirn molekulare metabolische Signale wahrnimmt und diese umsetzt, um verschiedene Vitalfunktionen wie das Essverhalten und den Schlaf / Wach-Rhythmus zu koordinieren. Mit Hilfe paralleler elektrophysiologischer Ableitungen und Optogenetik an sich frei bewegenden Mäusen untersuchen wir die Aktivität und Interaktionen spezifischer Neurone im Hypothalamus quer über Vigilanzzustände und homöostatische Herausforderungen. Wir wollen auf diese Weise Einblicke in die neuronalen Grundlagen und molekularen Determinanten von Vitalfunktionen und ihrer pathologischen Veränderungen, insbesondere bei Fettleibigkeit und Schlafstörungen, gewinnen. Control of hippocampal excitability in vivo by KCNQ3 and 5 channels We have investigated the role of KCNQ channels in the control of neuronal excitability in vivo in collaboration with Prof. T. J. Jentsch. This project is focused on channel proteins known as KCNQ3 and KCNQ5, which permit a passage of potassium ions in neurons. Mutations in KCNQ3 cause neonatal epilepsy. While these channels are expressed in the hippocampus and affect the excitability of neurons in vitro, their functions in vivo were not known. We found that the two channels play distinct and important roles in controlling neuronal excitability in the hippocampus of behaving mice. Their functions span from shaping the discharge mode of hippocampal neurons, through to determining efficacy of fast network synchronization and the precision of neural representations of space, to preventing hyperexcitability and pathological forms of neural synchronization. Our results (Fidzinski et al., Nature Communications, 2015) show that KCNQ5 controls excitability of hippocampal networks and influences cognitive processes. Optogenetic control of hippocampal theta oscillations reveals their function in spatial navigation The activity of large neuronal populations at various time scales is organized by hippocampal network oscillations that are important for cognitive processes. Hippocampal theta oscillations (5 12 Hz) in rodents occur during exploration and REM sleep, organize neuronal discharge and are implicated in spatial navigation and memory. Yet the role of theta oscillations in shaping spatial behavior remains elusive since until recently theta rhythm could not be manipulated in behaving animals. In this study we combined optogenetic control of theta rhythm generators, the medial septum and hippocampus, with electrophysiological monitoring of hippocampal network oscillations and neuronal activity. Excitatory (ChR2, ChETA) or inhibitory (halorhodopsin) opsins were expressed locally in the hippocampus or in the medial septum in wild-type and parvalbumin- Cre mice. Hippocampal theta oscillations were controlled by optostimulation of septo-hippocampal projections. Peak of power spectral density of optogenetically-paced theta oscillations matched stimulation frequency. This allowed us to interfere with the rhythm generation in vivo while monitoring mouse behavior as well as neuronal network and single cell activity. We use this approach to study the role of hippocampal theta oscillations in spatial navigation. We also study functions of theta-rhythmic coordination of the hippocampus and its output regions. (Bender et al., in revision). GABAergic cells in the lateral hypothalamus adaptively coordinate innate behaviors The lateral hypothalamus (LH) is crucial for the regulation of innate behaviors, including food intake and the sleep-wake cycle, yet temporal coordination of hypothalamic neuronal populations remains elusive. We used a combination of high-density electrophysiological recordings and optogenetics in behaving mice to study the function of GABAergic cells in LH. Excitatory (ChETA) or inhibitory (halorhodopsin, enphr3.0) opsins were expressed in the LH of VGAT-Cre mice to ensure selective targeting of GABAergic cells. Recordings of neuronal activity and optostimulation were performed in various behavioral paradigms for assessing innate behaviors. We found that optogenetic stimulation of GABAergic LH cells at various frequencies, as well as stimulation of projections of these neurons, changed transitions between innate behaviors (Gutierrez Herrera et al., in revision). Activation or inhibition of GABAergic neurons in the LH affected feeding behavior. Furthermore, neuronal activity of LH neurons was behavior- and state-dependent (Carus et al., FENS 2014). Marta Carus

28 52 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 53 Simultaneous recordings of multiple neurons in Ivy Xiaojie Gao, Tugba Özdogan and Maria Gorbati Franziska Bender and Marta Carus behaving mice, isolation of action potential trains emitted by individual neurons. (Bottom, right): optogenetic, cell-type specific, stimulation of neurons in lateral hypothalamus in vivo. GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Alexey Ponomarenko International National Fidzinski P*, Korotkova T*, Heidenreich M*, Maier N, Schuetze S, The Human Frontier Science Program, Neural basis of behavioural (principal investigator) Dr. Tatiana Korotkova (principal investigator) Franziska Bender (doctoral student) Marta Carus Cadavieco (doctoral student) Xiaojie Gao (doctoral student) Maria Gorbati (doctoral student) Tugba Ozdogan (doctoral student) Franziska Ramm (undergraduate student) Antoine Adamantidis McGill University, Montreal, Canada Christoph Börgers Tufts University, Boston, USA Denis Burdakov National Institute for Medical Research, London, UK Nancy Kopell Thomas J. Jentsch Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin Hannah Monyer DKFZ, Heidelberg Lisa Marshall University of Lübeck Dietmar Schmitz Kobler O, Zuschratter W, Schmitz D, Ponomarenko A, Jentsch TJ (2015). KCNQ5 K+ channels control hippocampal synaptic inhibition and fast network oscillations. Nature Communications 6, 6254 Maier N, Morris G, Schuchmann S, Korotkova T, Ponomarenko AA, Bohm C, Wozny C, Schmitz D (2012) Cannabinoids disrupt hippocampal sharp wave-ripples via inhibition of glutamate release. Hippocampus 22, multitasking and coordination by specific hypothalamic circuits, ; US $ Deutsche Forschungsgemeinschaft, Priority Program 1665 (Schwerpunktprogramm), Resolving and manipulating neuronal networks in the mammalian brain from correlative to causal analysis ; ; Deutsche Forschungsgemeinschaft, Cluster of Excellence NeuroCure: Suzanne van der Veldt (graduate student) Boston University, Boston, USA Charité Universitätsmedizin Berlin Korotkova T*, Fuchs EC*, Ponomarenko AA, von Engelhardt J, Monyer Towards a better outcome of central nervous system disorders, PI Achim Schweikard University of Lübeck H (2010) NMDA receptor ablation on parvalbumin-positive interneurons impairs hippocampal synchrony, spatial representations, and working memory. Neuron 68, position Ponomarenko A, ; Deutsche Forschungsgemeinschaft, Cluster of Excellence NeuroCure: Towards a better outcome of central nervous system disorders, Wulff P*, Ponomarenko AA*, Bartos M, Korotkova TM, Fuchs EC, Bahner F, Both M, Tort A B, Kopell N J, Wisden W, Monyer H (2009) Hippocampal theta rhythm and its coupling with gamma oscillations require fast inhibition onto parvalbumin-positive interneurons. Habilitationsgrant für Nachwuchswissen-schaftlerinnen (including PI position), Korotkova T, , Proc. Natl. Acad. Sci. USA 106, Racz A*, Ponomarenko AA*, Fuchs EC, Monyer H (2009) Augmented hippocampal ripple oscillations in mice with reduced fast excitation onto parvalbumin-positive cells. J. Neurosci. 29, FMP authors Group members * equal first author corresponding author

29 54 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 55 MEMBRANE TRAFFIC AND CELL MOTILITY Marietta Browarski and Claudia Schmid MEMBRANTRANSPORT UND ZELLBEWEGLICHKEIT GROUP LEADER DR. TANJA MARITZEN BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studies of Biochemistry and Molecular Biology, Universität Hamburg Ph.D. student, Zentrum für Molekulare Neurobiologie Hamburg (Prof. T.J. Jentsch), Dr. rer. nat. (summa cum laude) from Universität Hamburg Postdoctoral Research Associate, Zentrum für Molekulare Neurobiologie Hamburg (Prof. T.J. Jentsch) Postdoctoral Research Associate, Freie Universität Berlin (Prof. V. Haucke) since 2011 Co-Project Leader in the Collaborative Research Center SFB958 since 2012 Junior Group Leader at the FMP In order to interact with the extracellular environment cells rely to a large extent on cell surface-localized proteins. The binding of such cell surface receptors to extracellular ligands triggers diverse intracellular signaling cascades that determine, for instance, whether a cell starts to proliferate, to differentiate, to migrate or to initiate an immune response. Accordingly, the number of these signal receivers at the cell surface has to be tightly regulated to achieve in each situation the right level of responsiveness to a specific extracellular cue. Endocytosis, in conjunction with intracellular membrane transport, constitutes a powerful way to regulate the cell surface levels of diverse proteins and also to confine their localization to specific sites on the plasma membrane which is, for instance, essential for directed cell migration. However, for many cell surface proteins it is still unclear whether they are in fact internalized by endocytosis, how they interact with the endocytic machinery, and how their internalization is regulated. By employing cell biological and optical techniques, in conjunction with mouse genetics, our group dissects the role of endocytic and endosomal scaffold and adaptor proteins in the regulation of cell surface-localized proteins, especially in the context of cellular motility and immune functions. ZUSAMMENFASSUNG Zellen interagieren mit ihrer Umgebung im Wesentlichen über Proteine, die an der Zelloberfläche lokalisiert sind. Die Bindung solcher Zelloberflächenrezeptoren an extrazelluläre Liganden löst eine Vielzahl intrazellulärer Signalkaskaden aus, die beispielsweise Zellen zur Proliferation, Differenzierung oder Migration veranlassen oder eine Immunantwort auslösen. Um in jeder Situation angemessen auf extrazelluläre Signale antworten zu können, muss die Zelle die Anzahl ihrer Signalempfänger an der Zelloberfläche exakt regulieren. Endozytose, in Kombination mit intrazellulären Membrantransportprozessen, ermöglicht es Zellen, den Level verschiedenster Proteine an der Zelloberfläche genau einzustellen und sie an spezifischen Orten der Plasmamembran zu positionieren. Letzteres ist insbesondere für die zielgerichtete Bewegung von Zellen (Zellmigration) essentiell. Gegenwärtig ist für viele Zelloberflächenproteine noch nicht bekannt, ob sie tatsächlich durch Endozytose in die Zelle aufgenommen werden, wie sie gegebenenfalls mit der Endozytosemaschinerie interagieren und wie ihre Internalisierung in die Zelle gesteuert wird. Mit einer Kombination aus biologischen und optischen Methoden sowie Mausgenetik untersucht unsere Gruppe die Rolle von endozytotischen und endosomalen Gerüst- und Adaptorproteinen für die Regulation der Lokalisation von Zelloberflächenproteinen, v. a. im Kontext von Zellmigration und Immunfunktionen. Membrane transport in cell motility Cell motility is not only crucial during development, but also for immunity in the adult. Its dysregulation is involved in autoimmune syndromes as well as cancer, and cell migration inhibitors hold promise for the treatment of inflammatory conditions. Cell motility relies on coordinated membrane transport to regulate the cell surface levels of proteins mediating cell adhesion and migratory signaling such as integrins and proteoglycans. In addition, for cellular movement to occur membrane traffic has to be coordinated with actin cytoskeleton dynamics. Proteins that connect these processes are likely nodes of integration at which signals elicited by internal or external cues converge to regulate cell motility. One candidate for connecting membrane transport and actin dynamics in the context of cell migration is the protein Gadkin. We have shown that this AP-1 binding protein links, on the one hand, AP-1 positive endosomal vesicles to kinesin-1 for outward transport along microtubules and, on the other hand, functions as an inhibitor of the actin-nucleating ARP2 / 3 complex which plays a crucial role in lamellipodial-based cell migration. Therefore Gadkin seems ideally suited to coordinate membrane transport and cytoskeletal dynamics during cell migration. We could show that loss of Gadkin promotes migration in a melanoma cell model. Currently we are studying the physiological relevance of Gadkin for dendritic cells, which need to be able to efficiently migrate to fulfill their function as sentinels against pathogens, a role that is increasingly being exploited for cancer therapies. Diverse proteoglycans modulate cell motility by interacting with extracellular ligands and triggering intracellular signaling cascades that modulate the activity of actin regulatory proteins. However their endocytosis-based regulation is poorly understood. We have identified a proteoglycan as cargo of a so-far orphan endocytic adaptor and could show that its adaptor-dependent internalization is crucial for normal cell migration. As proteoglycans are known to be upregulated in tumors, we are currently investigating the potential relevance of this adaptor-proteoglycan interaction for tumorigenesis. The efficiency of this process depends on the accurate vesicular trafficking of MHCII molecules. We are currently investigating endocytic adaptor proteins that modulate the surface localization of MHCII molecules and might thereby be relevant to immune defense. Role of endocytic adaptors and scaffold proteins for brain function Neurons communicate by releasing neurotransmitters from synaptic vesicles. When these vesicles fuse with the presynaptic membrane, the synaptic vesicle proteins become stranded there. They have to be retrieved to regenerate synaptic vesicles for sustained neurotransmission. Thus the internalization of cell surface-localized proteins constitutes an essential step in the recycling of synaptic vesicles. In fact, loss-of-function mutants of main components of the endocytic machinery lead to premature death. In collaboration with the group of Volker Haucke we investigate, as part of the SFB958 and in a DFG-funded project, the importance of endocytic adaptor proteins for the high-fidelity retrieval of crucial synaptic vesicle proteins. In the past we have shown that the adaptor protein Stonin2 mediates the sorting of the synaptic vesicle Ca2+ sensor synaptotagmin1. Currently we are dissecting the role of the adaptor protein AP180 and the scaffold intersectin for the retrieval of synaptic vesicle proteins. To elucidate their physiological relevance in an organismic context we employ mouse genetics in conjunction with live cell imaging approaches, as well as behavioural studies. Role of membrane transport for immune cell functions Membrane transport is not only of relevance for immune cell migration, but affects many facets of immune cell life. The main function of dendritic cells is the sampling of antigens for presentation to T-cells to trigger effective immune responses against pathogens. To this end dendritic cells phagocytose pathogens and process their proteins into small peptides that are loaded onto MHCII molecules which are then presented at the dendritic cell membrane to T-cells.

30 56 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 57 Brain sections being aligned on glass slide for later immunohistochemical staining of synaptic proteins. Fig. 1: Endocytic adaptor protein (green) localizing Fig. 2: Tracking of cells that are moving along specifically to endocytic structures right behind the a chemotactic gradient to evaluate migration focal adhesions (red) at the leading edge of a mouse parameters such as speed and directionality of embryonic fibroblast (arrow indicating direction of movement. migration; cell nucleus stained in blue). GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Maritzen, Tanja (group leader) Dr. Schachtner, Hannah (postdoc) Browarski, Marietta (doctoral student) Fabian Feutlinske (doctoral student) Schmidt, Claudia (technical assistant) International Laura M. Machesky Beatson Institute for Cancer Research, Glasgow, UK Oleg Shupliakov Karolinska Institute, Stockholm, Sweden Daniel Legler Biotechnology Institute Thurgau, Kreuzlingen, Switzerland William B. Stallcup Sanford Burnham Medical Research National Jan Schmoranzer, Volker Haucke FMP, Germany Eberhard Krause FMP, Germany MinChi Ku, Sonia Waizcies, Thoralf Niendorf MDC, Berlin, Germany Uta Hoepken MDC, Berlin, Germany Matthias Selbach MDC, Berlin, Germany Seifert W, Kühnisch J, Maritzen T, Lommatzsch S, Hennies HC, Bachmann S, Horn D, Haucke V (2014) Cohen Syndrome-associated Protein COH1 Physically and Functionally Interacts with the Small GTPase RAB6 at the Golgi Complex and Directs Neurite Outgrowth. J Biol Chem. Epub 2014 Dec 9. Pechstein A, Gerth F, Milosevic I, Jäpel M, Eichhorn-Grünig M, Vorontsova O, Bacetic J, Maritzen T, Shupliakov O, Freund C, Haucke V (2014) Vesicle uncoating regulated by SH3-SH3 domainmediated complex formation between endophilin and intersectin at synapses. EMBO Rep. Epub 2014 Dec 17. Deutsche Forschungsgemeinschaft, Funktionelle Charakterisierung der SNARE Adaptoren AP180 und CALM bei der synaptischen Exound Endozytose in vivo, MA 4735 / 1 1, with Haucke V, , Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Forschung und Innovation, Regulation of cell motility by membraneassociated endosomal adaptors, SAW-2013-FMP-5, , Deutsche Forschungsgemeinschaft, SFB 958, A01, Structural and functional organization of endocytic scaffolds within the periactive Institute, San Diego, USA Christian Rosenmund Charité Universitätsmedizin Berlin, Germany Rainer Glass Ludwig-Maximilians-Universität München, Kononenko NL, Puchkov D, Classen GA, Walter AM, Pechstein A, Sawade L, Kaempf N, Trimbuch T, Lorenz D, Rosenmund C, Maritzen T, Haucke V (2014) Clathrin / AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron. 82(5):981 8 zone, with Haucke V, , München, Germany Tobias Moser Georg-August-Universität Göttingen, Göttingen, Germany Frank Schmitz Universität des Saarlandes, Homburg / Saar, Germany Kononenko N, Diril MK, Puchkov D, Kintscher M, Koo SJ, Pfuhl G, Winter Y, Wienisch M, Klingauf J, Breustedt J, Schmitz D, Maritzen T*, Haucke V* (2013) Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2. Proc Natl Acad Sci U S A. 110(6): FMP authors Group members * contributed equally

31 58 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 59 MOLECULAR NEUROSCIENCE AND BIOPHYSICS Superactivation At 14 Hz stimulation (red dots), GluA2 γ8 AMPA Receptor complexes undergo robust and indefatigable short-term facilitation. Superactivation, which readily reverses within a few hundred milliseconds, occurs because a proportion of the resting receptors (orange) convert into complexes with high open probability and conductance (green). Superactive complex 50 pa 500 ms GluA2 WT + γ8 MOLEKULARE NEUROWISSEN- SCHAFTEN UND BIOPHYSIK GROUP LEADER DR. ANDREW J.R. PLESTED Resting complex 14 Hz stimulation BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS M.Sci. 1st Class Hons, Physics, Imperial College, University of London, UK Ph.D. Imperial College (Prof. Franks and Prof. Lieb), University of London, UK Post-Doc, Dept. of Pharmacology (Prof. Colquhoun), University College London, UK Visiting Research Fellow, laboratory of Cellular and Molecular Neurophysiology (Dr. Mayer), NICHD, NIH, Bethesda, USA since 2008 Junior group leader, FMP, Berlin since 2008 Member, Cluster of Excellence NeuroCure Our main research interest is the glutamate receptors of excitatory synapses. These receptors are essential for brain function and play roles in diseases such as epilepsy, and cognitive and neurodegenerative disorders. We are particularly interested in how the composition and properties of receptor complexes determine features of synaptic transmission in the brain in health and disease. To achieve these goals, we manipulate receptors with molecular and chemical biology, including deploying unnatural amino acids in mammalian cells. We examine receptor gating with advanced electrophysiological methods such as ultra-rapid perfusion and single-channel recording. We complement these approaches with investigations of receptor structure and composition using X-ray crystallography, fluorescence microscopy and biochemistry. Through collaborations, we employ computational approaches to analyze and build novel insights into receptor activation. A second aspect of our research is to extend these studies to other important components of fast signaling in neurons, such as inhibitory neurotransmitter receptors and voltage-gated ion channels. ZUSAMMENFASSUNG Der Schwerpunkt unserer Forschung liegt auf Glutamatrezeptoren exzitatorischer Synapsen. Diese Rezeptoren sind für die Funktion unseres Gehirns essentiell und spielen eine wichtige Rolle bei Krankheiten wie Epilepsie sowie kognitiven und neurodegenerativen Störungen. Insbesondere interessiert uns, wie der strukturelle Aufbau und die Eigenschaften dieser Glutamatrezeptorkomplexe die Eigenschaften der synaptischen Transmission im Gehirn bei Krankheit und im gesunden Zustand bestimmen. Dafür verändern wir Rezeptoren mit molekularbiologischen und chemischen Methoden, wie z. B. durch den Einbau von unnatürlichen Aminosäuren und Expression in Säugerzellen. Wir untersuchen das Öffnen des Rezeptors, das sogenannte Rezeptor-Gating mittels fortschrittlicher elektrophysiologischer Methoden wie ultraschnelle Perfusion und Einzelkanalmessungen. Diese funktionellen Untersuchungen werden vervollständigt durch Untersuchungen der Rezeptorstruktur und -zusammensetzung mit Methoden der Röntgenstrukturanalyse, Fluoreszenzmikroskopie und Biochemie. In Zusammenarbeit mit anderen Gruppen nutzen wir computergestützte Methoden zur Analyse und zur Gewinnung neuer Einsichten Rezeptoraktivierung. Ein weiterer Aspekt unserer Forschung besteht in der Ausweitung dieser Studien auf weitere Bestandteile der schnellen Signalübertragung in Neuronen wie beispielsweise inhibitorische Neurotransmitterrezeptoren oder spannungsabhängige Ionenkanäle. Superactivation of glutamate receptor complexes To understand information processing in the nervous system, it is essential to know how one synaptic potential will affect the next. The textbook explanation is that plasticity that occurs in the Hz range reflects altered synaptic release, and desensitization is the only mechanism intrinsic to receptor complexes that can alter subsequent activity by reducing it. To date, no postsynaptic mechanisms for short-term activity-dependent increases in synaptic strength are reported. Auxiliary subunits of AMPA receptors control trafficking but also boost activity. We used a new method to isolate complexes of receptors with Stargazin and gamma-8, two auxiliary proteins important for nervous system function. Taking advantage of a family of mutant AMPA receptors with a spectrum of activities at equilibrium (Carbone and Plested, Neuron 2012), we built a model of auxiliary subunit action. This model predicted activity-dependent potentiation of receptor activity, which we confirmed in electrophysiological recordings (Carbone and Plested, in revision at Nature Neuroscience). We term this positive feedback mechanism superactivation. Such dynamic behavior in a single protein complex is, to our knowledge, unprecedented. We are actively working to detect superactivation at native synapses. Structural dynamics of glutamate receptor activation To better understand how agonists activate the AMPA receptor, we have solved multiple crystal structures of the ligand binding domains (LBDs) in complex with glutamate and partial agonists. Previous work identified active dimers of these domains but information about the tetrameric arrangement of the binding domains was lacking, even in the light of recent full-length receptor structures. A key element of our approach is extensive verification of the tetrameric arrangements with metal bridges and disulfide bonds. Fluorescence studies of receptor intracellular domains Information about the structural dynamics of intracellular domains during AMPA receptor (AMPAR) function is lacking. We used single and dual insertion of green fluorescent protein variants at various positions in AMPAR subunits to enable measurements of conformational changes using Förster Resonance Energy Transfer (FRET) in live cells. We produced dual CFP / YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. Patch clamp fluorimetry of the double and single insert constructs showed that both the C-terminus and the turret domain move during activation, and the C-terminus is detached from the membrane. Time-resolved measurements revealed unexpectedly complex fluorescence changes within the intracellular domains, providing clues as to how post-translational modifications at intracellular sites could influence receptor function. In turn, these receptors have considerable promise as fluorescent reporters of glutamate receptor activation in neurons. We previously identified an activation intermediate (Lau et al. Neuron 2013) that paradoxically crystallised with an antagonist bound. To extend this work, we crystallized the GluA2 LBDs with glutamate and the partial agonist fluorowillardine bound (Chebli et al., submitted); these structures revealed both compact and extended arrangements of the LBDs, respectively, suggesting dynamic interconversion between quite distinct arrangements during normal gating.

32 60 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 61 A Photoinactivation of glutamate receptors We have exploited highly selective and efficient genetic encoding of unnatural amino acids that are sensitive to light in order to build a library of glutamate receptors that can be inactivated by UV illumination (Klippenstein et al. JNS, 2014). These receptors can be shut down with innocuous levels of UV light in a matter of seconds. These mutants take advantage of our knowledge of subunit interface motions during activation and desensitization, and provide complementary information about movements within the transmembrane domains. Importantly, we could show using Western blots that subunits can be crosslinked by covalent bonds formed by UVinduced recombination in live cells. These receptors prove the principle of photocontrol in mammalian cells and should prove useful tools for optogenetic applications in vivo. Miriam Chebli and Jelena Baranovic B Photoinactivation A Kymogram of an outside out patch response for an AMPA receptor mutant harboring benzoylphenylalanine (BzF) at a key functional interface. B Exposure to UV light (violet) pregressively reduces the glutamate activated current, eventually to negligible levels (green). The same UV exposure for 10s has little effect on wild-type receptors (WT). Marcus Wietstruk Viktoria Klippenstein GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Anna Carbone (postdoc) Dr. Hector Salazar (postdoc) Dr. Jelena Baranovic (postdoc) Dr Mette Homann Poulsen (postdoc) Dr Clarissa Eibl (postdoc) Dr. Vera Martos (postdoc) Dr Valentina Ghisi (postdoc) Viktoria Klippenstein (doctoral student) Miriam Chebli (doctoral student) Ljudmila Katchan (doctoral student) Marcus Wietstruk (Technician) International Chris Ahern University of Iowa, Iowa City, IA, USA Anders Kristensen University of Copenhagen, Denmark Albert Lau Johns Hopkins University, Baltimore, MD, USA Daniel Choquet Interdisciplinary Institute for Neuroscience, Bordeaux, France Eric Gouaux Howard Hughes Medical Institute & Oregon Health Sciences University, USA National Oliver Daumke Max-Delbrück-Center for Molecular Medicine, Berlin Dietmar Schmitz Charité, Berlin James Poulet Max-Delbrück-Center for Molecular Medicine, Berlin Philip Selenko FMP-Berlin Petzoldt AG, Lee Y-H, Khorramshahi O, Reynolds E, Plested AJR, Herzel H and Sigrist SJ (2014) Gating characteristics control Glutamate Receptor distribution and trafficking in vivo. Current Biology 24, Klippenstein V, Ghisi V, Wietstruk M, Plested AJR (2014) Photoinactivation of glutamate receptors by a genetically encoded unnatural amino acid. J. Neurosci. 34, Lau A Y*, Salazar H*, Blachowitz L, Ghisi V, Plested AJR, Roux B (2013) A conformational intermediate in glutamate receptor activation. Neuron 79, Miranda P, Contreras J, Plested AJR, Sigworth F J, Holmgren M, Giraldez T (2013) State-dependent FRET reports calcium and voltage-dependent gating-ring motions in whole BK channels. Proc. Natl. Acad. Sci. 110, FMP authors Group members These authors contributed jointly * Corresponding authors Deutsche Forschungsgemeinschaft, Trapping the activation mechanism of glutamate receptors, PL 619 / 1 1, , Deutsche Forschungsgemeinschaft, Excellence Initiative, EXC-257 NeuroCure Towards a better outcome of central nervous system disorders, , Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Forschung und Innovation, Illuminating glutamate receptors, SAW, , Carlsberg Foundation Travel Grant to Mette Poulsen Control of receptor gating by light: development and functional characterization of ionotropic glutamate receptors with genetically incorporated photo-activated cross-linkers Boehringer-Ingelheim Fonds, PhD stipend to Ljudmila Katchan, , Europäischer Forschungsrat (8. Forschungsrahmenprogramm), Activation Mechanism of a Glutamate Receptor gluactive, ERC-CoG-2015, ,

33 62 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 63 PROTEIN TRAFFICKING GROUP LEADER PROF. DR. RALF SCHÜLEIN Wolfgang Klein and Anita Kinne BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Biology studies at the University of Würzburg 1989 Diploma in Biology PhD thesis on toxin transport in E. coli in the laboratory of Werner Goebel (Department of Microbiology, University of Würzburg) Postdoc in the laboratory of Walter Rosenthal (Department of Pharmacology, University of Gießen); work on the vasopressin V2 receptor since 1997 Group leader at the FMP; work on the trafficking mechanisms of GPCRs 2004 Habilitation in Pharmacology and Toxicology (Charité University Medicine Berlin) 2014 Adjunct professorship (Charité University Medicine Berlin) G protein-coupled receptors (GPCRs) are the most important drug targets. The receptors must reach their correct subcellular locations, usually the plasma membrane, in order to function. Transport is enabled by the secretory pathway and starts with a signal sequence-mediated insertion of the receptors into the membrane of the endoplasmic reticulum (ER). GPCRs possess different types of signal sequences: N-terminal signal peptides (SP) which are cleaved off following ER insertion; signal anchor sequences (SAS, usually transmembrane domain 1) which form part of the mature protein; signal peptides which are not cleaved off represent a third but rare type (pseudo signal peptides, PSP). The aim of the Protein Trafficking group is to determine why GPCRs possess different types of signal sequences. For these studies, we take the subtypes of the corticotropin-releasing factor (CRF) receptors as models. A further aim is to find novel substances that influence ER insertion of GPCRs.. ZUSAMMENFASSUNG G-Protein-gekoppelte Rezeptoren (GPCRs) sind die wichtigsten Zielstrukturen für Arznei mittel. Um korrekt zu funktionieren, müssen die Rezeptoren in das richtige Zielkompartiment der Zelle transportiert werden, normalerweise in die Plasmamembran. Dieser Transport wird über den sekretorischen Weg ermöglicht und startet mit einem Signalsequenz-vermittelten Einbau des Rezeptors in die Membran des endoplasmatischen Retikulums (ER). GPCRs verfügen über zwei Arten von Signalsequenzen: N-terminale Signalpeptide, die nach dem Einfädeln in die ER-Membran abgespalten werden, und Signalankersequenzen (üblicherweise die erste Transmembrandomäne), die Teil des reifen Proteins sind. Nicht abspaltbare Signalpeptide stellen einen seltenen, dritten Typ von Signalsequenzen dar (Pseudo-Signalpeptide). Ziel der Arbeitsgruppe Protein Trafficking ist es, am Beispiel der Cortictropin-Releasing-Factor (CRF)-Rezeptoren zu klären, warum GPCRs verschiedene Signalsequenzen haben. Ein weiteres Ziel ist, neue Wirkstoffe zu finden, die den Einbau spezifischer GPCRs in die ER-Membran beeinflussen. Wolfgang Klein and Arthur Gibert Functional significance of the signal peptides of CRF receptors. The ER insertion of GPCRs and other integral membrane proteins is mediated by signal sequences (SPs or SASs) and the proteinconducting Sec61 channel. The CRF receptors possess different types of signal sequences. The CRF1R possesses a conventional cleaved SP, while the CRF 2(a) R, in contrast, possesses an uncleaved PSP which represents a novel and so far unique protein domain within the GPCR protein family. We have analyzed whether the N-terminal PSP has functions beyond the ER insertion level and found that it causes a low plasma membrane expression of the CRF 2(a) R and prevents receptor dimerization, and thereby coupling to the Gi protein. The CRF 2(a) R is consequently expressed as a monomer and is only able to couple to Gs. The homologous CRF 1 R with its cleaved signal peptide, on the other hand, is highly expressed in the plasma membrane and is able to couple to both Gs and Gi. By using fluorescence cross-correlation spectroscopy (FCCS), we recently showed that the CRF 1 R is able to form dimers and is expressed in a specific monomer / dimer equilibrium at the plasma membrane and in the ER. The PSP thus represents the first example of a signal sequence influencing receptor expression, dimerization and signal transduction processes. These results are summarized schematically in Fig. 1. CRF receptors play an important role for the stress response of the body and for the development of anxiety and depression. It was shown by others in rats that the CRF 1 R is highly expressed in the plasma membrane of the serotonergic neurons of the dorsal raphe nucleus. Its continued activation decreases serotonin release and favours the development of anxiety and depression. The CRF 2(a) R, in contrast, is expressed in only small quantities in the plasma membrane. Its activation increases serotonin release and helps to cope with stress. Our data for the molecular pharmacology of the CRF receptors suggest that the differences in cell surface expression of the receptors are imposed by their different signal sequences. Our results may thus help to shed new light on the development of anxiety and depressive disorders. nascent chains into the Sec61 channel at the ER membrane. Cotransin is thus a selective, but not a specific inhibitor of signal peptides. Actual cotransin selectivity, i.e. how many proteins are indeed sensitive, was unknown. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture (SILAC) technique in combination with quantitative mass spectrometry (cooperation with the mass spectrometry group of the FMP). We also used a saturating concentration of cotransin (30 micromolar) to identify less sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin is thus less selective than previously thought. Using the dataset of the proteomic study, we identified in the few sensitive SASs of membrane proteins the first conformational consensus motif mediating cotransin sensitivity (Fig. 2) and could confirm these results by site-directed mutagenesis experiments. We also performed a screen to identify novel inhibitors of Sec61 and its associated proteins. Twenty-nine novel compounds were found; their targets and mechanism of action are currently being analyzed. Other results of the group. Results obtained for the structure-function relationships of the glycoprotein hormone receptors (FMP-integrated project) are summarized in the report of the Structural Bioinformatics group. Substances influencing ER insertion of GPCRs. Signal peptides do not have sequence homologies and may thus represent good novel drug targets. The idea is to find substances targeting specific signal peptides and consequently prevent synthesis of specific proteins. A substance pointing in this direction is the cyclodepsipeptide cotransin. Originally, cotransin was shown to inhibit the biosynthesis of a small subset of proteins in a signal peptide discriminatory manner by preventing stable insertion of the

34 64 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 65 A CRF 1 R CRF 2(a) R SP2-CRF 1 R SP1-CRF 2(a) R Expression at the PM High Low Low High Dimerization Monomer / dimer Monomer Monomer Monomer / dimer ratio ratio G protein coupling Gs > Gi Gs Gs Gs > Gi B Fig. 1: Functional significance of the signal peptides of CRF receptors. The CRF 1 R (grey) possesses a conventional cleaved signal peptide and is expressed in high amounts at the plasma membrane. It is able to form dimers and is expressed in a specific monomer / dimer ratio. The receptor couples to both Gs and Gi. The CRF 2(a) R (black) instead possesses an uncleaved PSP and is expressed only in low amounts at the plasma membrane. The receptor forms exclusively monomers and couples only to Gs. These properties can be transferred in signal peptide swap experiments (constructs SP2-CRF 1 R and SP1-CRF 2(a) R). Fig. 2: Identification of a putative conformational consensus motif in cotransin sensitive SASs. A. Sequence alignment of the 12 sensitive SASs (grey) identified by a proteomic analysis. The consensus motif consists of two groups of small amino acids in the central part (black), which are separated by two or three bulky amino acid residues and flanked by large charged, large polar or aromatic amino acids residues (white with black frame). The abbreviations are: IMP2C, integral membrane protein 2 C; IMP2B, integral membrane protein 2 B; HLA II, HLA class II histocompatibility antigen gamma chain; TMEM230, transmembrane protein 230; MHC I, MHC class I antigen; ECE-1, endothelin-converting enzyme 1; Acyl-CoA, Acyl-CoA desaturase; TM Prot2, transmembrane protein 2; Erlin1, Erlin-1; LIMP2, lysosome membrane protein 2; Erlin2, Erlin-2; AGPR1, asialoglycoprotein receptor 1; TNF-α, tumor necrosis factor-α. B. Exemplary α-helical (left) and solvent-reachable surface projection (right) of the SAS of IMP2B. Green colour represents the small amino acid residues, red colour the separating residues and blue colour the flanking residues. The motif causes the formation of two cavities in the helical surface (arrows) which are responsible for cotransin sensitivity (site-directed mutagenesis data not shown). GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Claudia Rutz International National Hoyer I, Haas AK, Kreuchwig A, Schülein R, Krause G (2013) Molecular Deutsche Forschungsgemeinschaft, Ableitung von Struktur- / Dr. Jens Furkert Ulrike Steckelings Ulrike Alexiev sampling of the allosteric binding pocket of the TSH receptor provides Funktionsbeziehungen spezifischer Hemmstoffe der Biosynthese Wolfgang Klein (doctoral student) University of Southern Denmark Freie Universität Berlin discriminative pharmacophores for antagonist and agonists. Biochem. G-Protein-gekoppelter Rezeptoren, SCHU 1116 / 2 1, Arthur Gibert (doctoral student) Odense, Denmark Duska Dragun Soc. Trans. 41, , Bettina Kahlich (technical assistant) Nikos Tsopanoglou, University of Patras, Greece Giovanna Valenti University of Bari, Italy Charité University Medicine Berlin Mathias Dreger Caprotec Bioanalytics GmbH, Berlin Gunnar Kleinau Charité University Medicine Berlin Enno Klußmann Teichmann A, Gibert A, Lampe A, Grzesik P, Rutz C, Furkert J, Schmoranzer J, Krause G, Wiesner B, Schülein R (2014) The specific monomer / dimer equilibrium of the corticotropin-releasing factor receptor type 1 is established in the endoplasmic reticulum. J. Biol. Chem. 289, Max-Delbrück Center for Molecular Schmidt A, Wiesner B, Schülein R, Teichmann A (2014) Use of Kaede Medicine, Berlin and Kikume green-red fusions for live cell imaging of G protein-coupled Walter Rosenthal receptors. Methods Mol. Biol. 1174, Max-Delbrück Center for Molecular Medicine, Berlin Klaus Weißhart Carl Zeiss MicroImaging GmbH, Jena Grzesik P, Teichmann A, Furkert J, Rutz C, Wiesner B, Kleinau G, Schülein R, Gromoll J, Krause G (2014) Differences between lutropinmediated and choriogonadotropin-mediated receptor activation. FEBS J. 281, Klein W, Westendorf C, Schmidt A, Conill-Cortés M, Rutz C, Blohs M, Beyermann M, Protze J, Krause G, Krause E, Schülein R (2014) Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study. PLoS One, in press. FMP authors Group members

35 66 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 67 CORE FACILITY CELLULAR IMAGING ZELLULÄRE BILDGEBUNG Transmission electron microscopy images of perinuclear area of the cell obtained by alternative tissue preparation technics. Left ultrathin section of epoxy resin embedded cell. Right Tokuyasu ultrathin section of frozen cell. Golgi stacks are marked by arrows. GROUP LEADERS DR. BURKHARD WIESNER LIGHT MICROSCOPY DR. DMYTRO PUCHKOV ELECTRON MICROSCOPY SUMMARY Since 2006 our group has functioned as a core facility. It is split into two sub-units to provide optimal technical support: laser scanning microscopy and electron microscopy. We are open to collaborations with all research groups within the FMP. We complete sub-projects independently and this leads to joint publications with the research groups. The light microscopy core facility The light microscopy facility supports all research groups within the FMP with technology and expertise to study biological samples including living cells, fixed cells, tissue, and solutions. Our central role is to establish single cell techniques and apply diverse microscopic methods to describe cellular signal transduction. Microscopic methods such as FRET (Fluorescence Resonance Energy Transfer), FRAP (Fluorescence Recovery After Photobleaching) or FCS (Fluorescence Correlation Spectroscopy) are well established in the institute. Additionally, we examine the use of a range of caging compounds in connection with intracellular Ca2+ measurements that employ UV- and / or IR-irradiation for the process of uncaging. Furthermore, we are developing opportunities for data analysis by developing novel algorithms for biophysical issues (e.g. we create macros for the analysis). We also test and create protocols for the use of new dyes, such as the photoconversion of Kaede. The electron microscopy core facility The EM facility provides support in the visualisation of cellular ultrastructure and localising individual proteins at the subcellular level. The lab provides standard and advanced specimen preparation techniques, sampling, imaging, quantitative analysis and interpretation for all biological applications. Tomographical 3D reconstruction with a FEI Tecnai G2 FEG 200kV electron microscope is possible, if required. In addition to our main focus on cell biology, we can assist with visualization of in vitro structures such as proteins, fibril structures and liposomes with negative staining or as cryosamples. Korrelations-Spektroskopie) sind bei uns bestens eingeführt. Daneben untersuchen wir im Zusammenhang mit intrazellulären Ca2+-Messungen den Einsatz einer ganzen Reihe von caged-verbindungen (Verbindungen, die durch das Einbringen einer sogenannten Schutzgruppe, Käfig, biologisch inaktiv sind), bei denen für das Uncaging (Abspaltung der Schutzgruppe) UV- und / oder IR-Bestrahlung eingesetzt wird. Außerdem entwickeln wir neue Möglichkeiten der Datenanalyse, indem wir neuartige Algorithmen für biophysikalische Themen entwickeln (z. B. Makros für die Analyse) und Protokolle für die Anwendung neuer Farbstoffe wie beispielsweise die Photokonvertierung des Proteins Kaede testen und entwickeln. Elektronenmikroskopie Die FMP-Service-Gruppe Elektronenmikroskopie unterstützt die Visualisierung zellulärer Ultrastrukturen und die Lokalisierung einzelner Proteine auf subzellulärer Ebene. Das Labor stellt Probenpräparationstechniken in Standard- und gehobener Ausführung, mikroskopische Aufnahmen, quantitative Analysen und Interpretationshilfen für alle biologische Applikationen bereit. Tomographische 3D-Rekonstruktionen mit dem FEI Tecnai G2 FEG 200kV Elektronenmikroskope sind möglich. Zusätzlich zu unserem Hauptfokus in der Zellbiologie, helfen wir bei der Visualisierung von in vitro Strukturen bzw. Proteinen, fibrillären Strukturen und Liposomen mit Negativfärbung sowie als Kryoprobe. Svea Hohensee (photo left), Jenny Eichhorst ZUSAMMENFASSUNG Seit 2006 fungiert unsere Gruppe als Technologieplattform des FMP und ist zur Bereitstellung einer besseren technischen Unterstützung in die beiden Service-Gruppen Laser-Scanning-Mikroskopie und Elektronenmikroskopie unterteilt. Wir bieten allen Forschungsgruppen am FMP unsere Mitarbeit an. Darüber hinaus bearbeiten wir aber auch als unabhängige Arbeitsgruppe eigene Forschungsprojekte, die dann zu gemeinsamen Publika tionen mit den Forschungsgruppen führen. Lichtmikroskopie Die zentrale Service-Gruppe Lichtmikroskopie unterstützt alle Forschungsgruppen des FMP mit ihrer Technologie und Kompetenz beim Studium biologischer Proben, einschließlich lebender Zellen, fixierter Zellen, Geweben und Lösungen. Unsere zentrale Rolle dabei ist es, Einzelzelltechniken zu etablieren und verschiedene mikroskopische Methoden für die Darstellung der zellulären Signaltransduktion anzuwenden. Mikroskopische Methoden wie FRET (Fluoreszenz-Resonanz-Energie-Transfer), FRAP (Fluorescence Recovery After Photobleaching, Fluoreszenz-Rückgewinnung nach Photobleichung) oder FCS (Fluoreszenz-

36 68 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 69 DESCRIPTION OF PROJECTS The light microscopy core facility The heptahelical G protein-coupled receptors (GPCRs) are important drug targets. Following activation by their ligands, they exert their function via the binding of G proteins and activation of specific signal transduction pathways. Our focus is on the analysis of GPCR oligomerization, whose functional significance is not completely understood. For some GPCRs it is known that oligomerization modulates receptor transport and / or the dynamics of receptor activation. Most importantly, it is not clear for most of the GPCRs whether they exist exclusively as oligomers or in a certain monomer-dimer ratio (M / D), or whether a given ratio is dynamic. In our group, the oligomerization of GPCRs is analysed using the following biophysical methods: fluorescence resonance energy transfer (FRET), fluorescence-lifetime imaging microscopy (FLIM) and fluorescence cross-correlation spectroscopy (FCCS). Using these techniques, we are able to determine the ratio of monomers and dimers in the plasma membrane of living cells. In cooperation with the group of Ralf Schülein (Protein-Trafficking, FMP) and Gerd Krause (Structural Bioinformatics, FMP) we have shown that the corticotropin-releasing factor receptors type 1 (CRF 1 R) and type 2a (CRF 2(a) R) exhibit differences in their homo-dimerization, despite their very homologous sequence (Teichmann et al. 2012). Whereas 23 % of the CRF 1 R exist as homodimers, the CRF 2(a) R shows no receptor interactions, explained by its so-called pseudo signal peptide. The electron microscopy core facility The major interest of the group is the regulation of membrane trafficking that underlies or governs the majority of physiological functions in eukaryotic cells. In cooperation with groups of Volker Haucke (Department Molecular Pharmacology and Cell Biology, FMP) and Tanja Maritzen (Membrane Traffic and Cell Motility, FMP) we investigate the contribution of various adaptor proteins such as AP2, AP180, Stonin 2, scaffolding and accessory proteins such as clathrin, GIT, intersectin or different phosphatidylinositol kinases or phosphatases to endocytosis, endosomal sorting and organelle maturation, generally in cells and in neuronal synapses in particular. In cooperation with Thomas Jentsch (Department Physiology and Pathology of Ion Transport, FMP) we investigate a number of mice lacking various ion transporters that also exhibit significant alterations in membrane trafficking and organelle dynamics in a variety of tissues and organs. In cooperation with Ingolf Blasig (Molecular cell Physiology, FMP) we investigate cell adhesion molecules maintaining the blood-brain barrier that have exciting potential for therapeutic intervention We cooperate closely with Jan Schmoranzer (Department Molecular Pharmacology and Cell Biology, FMP) on optimal specimen preparation protocols for superresolution light microscopy. Martin Lehmann and Jan Schmoranzer Martina Ringling GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS Light Microscopy Anke Teichmann (Postdoc) Jenny Eichhorst (technical assistant) International Peter Pohl Johannes Kepler University, Linz, Austria Grazzia Tamma University of Bari, Bari, Italy Thomas Walther Hull University, Hull, UK Pavel Nedvetsky Catholic University Leuven, Leuven, Belgium Max-Delbrück Center for Molecular Medicine, Berlin: Oliver Daumke Iduna Fichtner Enno Klussmann Dominique Müller Matthew Poy Bettina Purfürst Salim Seyfried Anje Sporbert Reiner Zeisig Bogum J, Faust D, Zühlke K, Eichhorst J, Moutty MC, Furkert J, Eldahshan A, Neuenschwander M, von Kries JP, Wiesner B, Trimpert C, Deen PM, Valenti G, Rosenthal W, Klussmann E (2013) Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking. J. Am. Soc. Nephrol. 24, Teichmann A, Gibert A, Lampé A, Grzesik P, Rutz C, Furkert J, Schmoranzer J, Krause G, Wiesner B, Schülein R (2014) The specific monomer / dimer equilibrium of the corticotropin releasing factor receptor type 1 is established in the endoplasmic reticulum. J. Biol. Chem. 289, Electron Microscopy Dr. Dorothea Lorenz (senior scientist) Svea Hohensee (research assistant) Martina Ringling (technical assistant) Schwefel D, Arasu BS, Marino SF, Lamprecht B, Köchert K, Rosenbaum E, Eichhorst J, Wiesner B, Behlke J, Rocks O, Mathas S, Daumke O (2013) Structural Insights into the Mechanism of GTPase Activation in the GIMAP Family. Structure 21, Balletta A, Lorenz D, Rummel A, Gerhard R, Bigalke H, Wegner F (2013) Human mast cell line-1 (HMC-1) cells exhibit a membrane capacitance increase when dialysed with high free-ca2+ and GTPγS containing intracellular solution. Eur. J. Pharmacol. 720, Kononenko NL, Puchkov D, Classen GA, Walter AM, Pechstein A, Sawade L, Kaempf N, Trimbuch T, Lorenz D, Rosenmund C, Maritzen T, Haucke V (2014) Clathrin / AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron 82, National Dorothea Eisenhardt Freie Universität Berlin Karin Müller Leibniz Institute for Zoo and Wildlife Research, Berlin Alexander Wenig Charité Universitätsmedizin Berlin Leibniz-Institut für Molekulare Pharmakologie (FMP): Ingolf Blasig Margitta Dathe Volker Haucke Thomas Jentsch Gerd Krause Michael Krauß Ronald Kühne Tanja Maritzen Jan Schmoranzer Ralf Schülein Kononenko NL, Diril MK, Puchkov D, Kintscher M, Koo SJ, Pfuhl G, Winter Y, Wienisch M, Klingauf J, Breustedt J, Schmitz D, Maritzen T, Haucke V (2013) Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2. Proc. Natl. Acad. Sci. USA 110, E526 E535. Posor Y, Eichhorn-Gruenig M, Puchkov D, Schöneberg J, Ullrich A, Lampe A, Müller R, Zarbakhsh S, Gulluni F, Hirsch E, Krauss M, Schultz C, Schmoranzer J, Noé F, Haucke V (2013) Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499, Podufall J, Tian R, Knoche E, Puchkov D, Walter AM, Rosa S, Quentin C, Vukoja A, Jung N, Lampe A, Wichmann C, Böhme M, Depner H, Zhang YQ, Schmoranzer J, Sigrist SJ, Haucke V (2014) A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling. Cell Rep. 7, Hoegg-Beiler MB, Sirisi S, Orozco IJ, Ferrer I, Hohensee S, Auberson M, Gödde K, Vilches C, de Heredia ML, Nunes V, Estévez R, Jentsch TJ (2014) Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction. Nat. Commun. 5, Grzesik P, Teichmann A, Furkert J, Rutz C, Wiesner B, Kleinau G, Schülein R, Gromoll J, Krause G (2014) Differences Between Lutropin- And Choriogonadotropin-Mediated Receptor Activation. FEBS J. 281, FMP authors Group members

37 70 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 71 CORE FACILITY ANIMAL FACILITY TIERHALTUNG GROUP LEADERS DR. NATALI WISBRUN BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS INTERESTS AND FOCUS Until 1999 Study of Veterinary Medicine at University Zagreb, Croatia and at FU Berlin, Germany 1999 Veterinary degree from the Faculty of Veterinary Medicine at the FU Berlin, Germany 2006 Dissertation: Cloning and Expression of Enzymes involved in the salvage pathway of L-fucose 2009 Graduate in veterinary, specialized in laboratory animal science PROFESSIONAL BACKGROUND Since 2013 Group Leader at the FMP Berlin Head of animal experimental facilities at Philipps University of Marburg, Marburg The animal facility takes care of management and organization of breeding and keeping laboratory animals for use in scientific projects. Animal welfare legislation an the highest scientific standards are enforced to obtain highly relevant scientific results. We support and give advice to scientists in all questions of planning and performing experiments involving animals. We also provide practical support, e.g. by taking samples and in keeping proper documentation. Furthermore, we organize world-wide export and import of laboratory animals as well as embryonal stem cells. ZUSAMMENFASSUNG Die Tierhaltung beschäftigt sich mit dem Management und der Organisation der Zucht und Haltung von Versuchstieren für deren Einsatz in der Bearbeitung wissenschaftlicher Fragestellungen. Dies erfolgt unter Berücksichtigung der Anforderungen des Tierschutzgesetzes zur Erzielung von relevanten wissenschaftlichen Ergebnissen. Wissenschaftler werden bei allen Fragen zur Planung und Durchführung von Tierversuchen unterstützt und beraten. Sie erhalten zudem praktische Unterstützung, z. B. bei der Probengewinnung und der Führung der Dokumentation. Zudem organisiert die Tierhaltung den Export und Import von Tieren und Embryonalzellen weltweit. The team of the laboratory animal facility supports the scientists using animals for experiments in compliance with the Animal Welfare Act and with an animal welfare officer. It is a service supplying Animal Environment housing and management of genetically modified mice, and also frogs and rats under performance standards, veterinary care, qualified animal care staff, using a standard operation procedure and related documentation. The service includes also education and training of personnel responsible for animal care and of personnel carrying out experimental procedures (research technicians) and also for scientists involved in experimental trials with animals. The aim of the team is to establish a well-planned, well-designed, well- constructed, and properly maintained laboratory animal facility that is responsible for animal care in compliance with applicable laws and regulations. The Animal Facility helps oversee all research at the Institute that involves animals, including: Management and organization of breeding and care of a variety of genetically modified mice and frogs, in compliance with animal welfare regulations. Organization of veterinary care and health monitoring in accordance with FELASA guidelines. Implementation of European guidelines on animal welfare in high-quality research. Organization of imports and exports of animals, embryos, stem cells, and related materials. Supporting and consulting scientists in the design of experiments that use animals. Supporting scientists in implementing experiments using animals. Participation in training courses for scientists and other staff involved in animal experimentation Head of an animal experimental facility and Animal Welfare Officer at the Max Delbrück Center for Molecular Medicine, Berlin Buch Research Fellow and Animal Welfare Officer at the Central Animal Laboratory of the University Clinic Essen, Essen GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS Eva Lojek (animal keeper) Jannette Unnasch (animal keeper) Lena Braun (studentische Hilfskraft / student of Veterinärmedizin) Steffi Wolf (studentische Hilfskraft / student of Veterinärmedizin) National Animal Facility Max-Delbrück-Center for Molecular Medicine, Berlin Animal Facilities Charité, Berlin AG Blasig AG Haucke AG Jentsch AG Maritzen AG Schröder Pohlmann A, Karczewski P, Ku MC, Dieringe B, Waiczies H, Wisbrun N, Kox S, Palatnik I, Reimann HM, Eichhorn C, Waiczies S, Hempel P, Lemke B, Niendorf T, Bimmler M (2014) Cerebral blood volume estimation by ferumoxytol-enhanced steady-state MRI at 9.4 T reveals microvascular impact of α1 -adrenergic receptor antibodies. NMR Biomed. 27, FMP authors Group members

38 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE Molecular Biophysics Molekulare Biophysik Group leader Prof. Dr. Adam Lange PAGE 88 NMR-Supported Structural Biology NMR-Unterstützte Strukturbiologie Group leader Prof. Dr. Hartmut Oschkinat PAGE 76 Computational Chemistry / Drug Design Wirkstoff-Design Group leader Dr. Ronald Kühne PAGE 84 Behavioural Neurodynamics Struktur-Basierte Bioinformatik und Proteindesign Group leader Dr. Gerd Krause PAGE 80 STRUCTURAL BIOLOGY SECTION BEREICH STRUKTURBIOLOGIE In-Cell NMR NMR in Zellen Group leader Dr. Philipp Selenko PAGE 100 Solution NMR Lösungs-NMR Group leader Dr. Peter Schmieder PAGE 92 Molecular Imaging Molekulare Bildgebung Group leader Dr. Leif Schröder PAGE 96 NMR Group leaders Prof. Dr. Hartmut Oschkinat Dr. Peter Schmieder PAGE 104

39 74 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 75 SECTION STRUCTURAL BIOLOGY BEREICH STRUKTURBIOLOGIE Molecular pharmacology requires three-dimensional representations of super-molecular arrangements within the cell, which are controlled in vivo by temporal and spatial coordination of protein expression, degradation, and post-translational modification. The dynamic nature of these phenomena challenges static structure determination techniques and implies a strong incentive to employ Nuclear Magnetic Resonance spectroscopy (NMR). In this spirit, the Structural Biology section develops and applies solution and solid-state NMR techniques to investigate pharmacologically relevant proteins in native environments or even in intact biological systems such as cells or functional modules. The departments of Adam Lange and Hartmut Oschkinat apply solid-state NMR to membrane proteins in native lipid bilayers, for example, and Philipp Selenko detects proteins and their modifications in cells. Leif Schröder images live cells or organisms by means of molecule-specific contrast agents. In concert with molecular modelling and structure function studies provided by the groups of Gerd Krause and Ronald Kühne, atomic-resolution structural data is derived that is indispensable on the path to pharmacological interference, typically supported by the Solution NMR group of Peter Schmieder. With regards to the design of bioactive molecules, the development of protein interaction inhibitors is a major theme of the section. During the reporting period, the section welcomed a new member, Adam Lange, who arrived from the Max-Planck-Institut für biophysikalische Chemie in Göttingen to start a department with research on solid-state NMR applied to structural problems of the field of infection biology. He replaced Bernd Reif, who took a position at the Technische Universität München. His recent work covers high-resolution structural investigations of the Shigella type-iii secretion needle by solid-state NMR combined with cryo-electron microscopy and the voltage-dependent anion channel from mitochondria as well as NMR-technical contributions. In the department of Hartmut Oschkinat, the application of microwave-based hyperpolarisation (DNP) yielded a picture of the nascent polypeptide chain growing inside the ribosome. Further applications of the technique yielded resolved spectra of proteins at temperatures around 200 K. The Solution NMR group of Peter Schmieder studied protein dynamics in MHC complexes and investigated the structural basis of auto-inflammatory disorders. The In-cell NMR junior group headed by Philipp Selenko explores novel NMR-based methodologies that allow monitoring of proteins inside cells. Projects include profiling of kinase activities via peptide-based Kinase Activity Reporters (KARs). These allow measuring dynamic changes of cellular kinase activities under different physiological and pathophysiological conditions. Investigation of the structure of alpha-synuclein within cells aims at a molecular understanding of Parkinson s disease. The Molecular Imaging junior group of Leif Schröder works at the physiological level using MRI as a major technique and explores new grounds in developing a biosensor-based NMR imaging approach, employing hyperpolarisation to enhance the signal-to-noise ratio. In the course of this work, Leif Schröder and his co-workers devised a highly efficient technique for hyperpolarizing xenon, which is then used to provide hyperpolarised nuclei to membrane-embedded and receptor-attached probes. In this way, magnetic resnonance imaging (MRI) enhanced by hyperpolarisation detects specific cells, opening avenues to tissue- or molecule-specific contrast that shall be used for non-invasive detection of cancer at its earliest stages. The cheminformatics / bioinformatics groups utilize structural information to derive protein interaction inhibitors, and to understand protein function, especially of G-protein-coupled receptors (GPCRs) via pharmacological interference. The Drug Design group led by Ronald Kühne accomplished a very demanding task in developing a set of small-molecule fragments that inhibit protein-protein interactions involving proline-rich motifs, yielding efficient inhibitors of EVH1 and WW protein domain interactions. The Structural Bioinformatics and Protein Design group headed by Gerd Krause generated a new web-based system that assists in evaluating the molecular effects of genetic variations in GPCRs. The structure-function relationship of two receptors, LHR and TSHR, were deciphered to derive molecular details of activation and inactivation patterns, and to understand the activity of agonists and antagonists. Both computational groups test the compounds developed in their biological laboratories, thereby closing the loop between structural studies and computational modelling, as well as biochemical and cell physiological analyses. In der molekulare Pharmakologie ist die Kenntnis der dreidimensionalen Struktur super-molekularer Komplexe innerhalb der Zelle, die in vivo durch die zeitliche und räumliche Koordination von Proteinexpression, -abbau und posttranslationaler Modifikation kontrolliert wird, von entscheidender Bedeutung. Die diesen Prozessen innewohnende Dynamik ist eine große Herausforderung für statische Strukturbestimmungsmethoden und legt es nahe, Kernspinresonanzmethoden (NMR) anzuwenden. Daher entwickeln und nutzen die Wissenschaftler des Bereichs Strukturbiologie Lösungs- und Festkörper-NMR-Techniken, um pharmakologisch relevante Proteine in ihrer natürlichen Umgebung oder sogar in intakten biologischen Systemen wie Zellen oder funktionellen Modulen zu untersuchen. Die Abteilungen von Hartmut Oschkinat und von Adam Lange nutzen beispielsweise Festkörper-NMR für Membranproteine in nativen Lipidmembranen, und Phil Selenko detektiert Proteine und Proteinmodifikationen in lebenden Zellen. Leif Schröder arbeitet an der Bildgebung von lebenden Zellen und Organismen mittels spezifischer Kontrastmittel. Zusammen mit molekularem Modelling und Struktur-Funktions-Untersuchungen der Arbeitsgruppen von Gerd Krause und Ronald Kühne werden Strukturdaten mit atomarer Auflösung erarbeitet, die unverzichtbar sind, um Wege zu einer pharmakologischer Einflussnahme auf Zielproteine zu finden. Diese Untersuchungen werden gewöhnlich von der Lösungs-NMR -Gruppe unter der Leitung von Peter Schmieder durchgeführt. Hinsichtlich des Designs von biologisch aktiven Wirkstoffen ist insbesondere die Entwicklung von Inhibitoren von Protein-Wechselwirkungen ein Leitthema des Bereichs. Während des Berichtszeitraumes nahm Adam Lange vom Max-Planck-Institut für Biophysikalische Chemie in Göttingen den Ruf auf eine S-W3-Professur an der Humboldt-Universität in Verbindung mit der Leitung einer Abteilung am FMP an. Seine Abteilung wendet Festkörper-NMR auf strukturbiologische Fragestellungen im Bereich der Infektionsbiologie an. Er ist Nachfolger von Bernd Reif, der an die Technische Universität München gegangen ist. Jüngste Arbeiten aus seiner Gruppe beschäftigen sich mit hochauflösenden Strukturuntersuchungen mittels Festkörper-NMR der Shigella Typ-III Sekretionsnadel in Kombination mit Kryo- Elektronenmikroskopie, des spannungsabhängigen Anionenkanals von Mitochondrien sowie mit technischen Beiträgen zur Weiterentwicklung NMR-basierter Strukturuntersuchungsmethoden. Durch die Abteilung von Hartmut Oschkinat wurde die Struktur der naszierenden Polypeptidkette innerhalb des Ribosoms aufgeklärt. Dies geschah mittels Mikrowellen-gestützter Hyperpolarisation (DNP). Weitere Anwendungen dieser Technik erbrachten aufgelöste Spektra von Proteinen bei Temperaturen um 200 K. Die Arbeitsgruppe Lösungs-NMR von Peter Schmieder untersuchte die Proteindynamik in MHC-Komplexen und die strukturellen Grundlagen autoinflammatorischer Erkrankungen. Die Nachwuchsgruppe NMR in Zellen von Phil Selenko erforscht neuartige NMR- Methoden, die eine Beobachtung von Proteinen in lebenden Zellen ermöglichen. So erarbeitet seine Gruppe beispielsweise ein Profil von Proteinkinaseaktivitäten mit Peptiden als Kinaseaktivitätsreportern (KARs). Diese ermöglichen es, dynamische Änderungen zellulärer Kinaseaktivitäten unter verschiedenen physiologischen und pathophysiologischen Bedingungen zu messen. Ein weiteres Projekt, die Untersuchung der Struktur von Alpha-Synuclein, zielt auf ein molekulares Verständnis der Parkinson-Krankheit. Die Nachwuchsgruppe Molekulare Bildgebung von Leif Schröder nutzt die Magnetresonanztomographie (MRI) zu Bildgebung auf einer physiologischen Ebene. Die Arbeitsgruppe entwickelt einen neuartigen, auf Biosensoren beruhenden Ansatz zur Bildgebung, der Hyperpolarisation nutzt um das Signal-Rausch-Verhältnis zu verbessern. Es gelang den Wissenschaftlern, eine sehr effiziente Technik zur Hyperpolarisation von Xenon zu entwickeln, das dann benutzt wird, um membranständigen oder Rezeptor-gebundenen Sondenmolekülen hyperpolarisierte Atomkerne zuzuführen. So gelang es mit durch Hyperpolarisation verbesserter MRI Zellen spezifisch darzustellen. Damit steht der Weg für eine Weiterentwicklung in Richtung auf gewebe- oder molekülspezifische Kontrastmittel offen, die zum Beispiel für eine nicht-invasive Früherkennung von Krebs eingesetzt werden könnten. Die Cheminformatik- / Bioinformatik-Gruppen nutzen Strukturdaten um Inhibitoren für Proteinwechselwirkungen abzuleiten und um Proteinfunktionen, insbesondere von G-Protein-gekoppelten Rezeptoren (GPCRs), mittels pharmakologischer Beeinflussung zu verstehen. Der Arbeitsgruppe Wirkstoff-Design von Ronald Kühne gelang die Lösung einer ausgesprochen anspruchsvollen Aufgabe. Sie entwickelte eine Gruppe von Fragmenten kleiner Moleküle, die Protein-Protein-Wechselwirkungen von Prolinreichen Motiven hemmen. Resultat waren effiziente Inhibitoren für die Interaktion von EVH1 und WW-Proteindomänen. Die Gruppe Strukturelle Bioinformatik und Proteindesign unter Gerd Krause entwickelte ein neues Web-basiertes System, das Wissenschaftler darin unterstützt, molekulare Effekte genetischer Unterschiede bei GPCRs zu analysieren. Die Struktur-Funktions-Beziehungen zweier Rezeptoren, LHR und TSHR, wurden entschlüsselt und so die Muster für Aktivierung oder Inaktivierung dieser Rezeptoren durch Liganden im molekularen Detail aufgeklärt. Dies ermöglicht es, die Wirkungsweise von Agonisten und Antagonisten zu verstehen. Beide Gruppen testen die cheminformatisch / bioinformatisch als aktiv vorhergesagten Substanzen in biologischen Experimenten. Sie schließen damit den Kreis zwischen Strukturuntersuchungen, computergestütztem Modelling, biochemischen und zellphysiologischen Ansätzen.

40 76 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 77 NMR-SUPPORTED STRUCTURAL BIOLOGY Lilo Handel and Anja Voreck (photo left), Barth van Rossum, Sascha Lange and Trent Franks NMR-UNTERSTÜTZTE STRUKTURBIOLOGIE GROUP LEADER PROF. DR. HARTMUT OSCHKINAT BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Chemistry degree at the University of Frankfurt and Chemistry degree at the University of Frankfurt; Diploma Visit to the laboratory of Prof. Dr. Ray Freeman, Oxford, England Completion of dissertation in Prof. Kessler s Laboratory at the University of Frankfurt 1986 Graduate thesis: Analysis of the conformation of Cyclosporin in solution using NMR-spectroscopy: development and use of new methods Postdoctoral work with Prof. Dr. Bodenhausen at the University of Lausanne, Switzerland Position as NMR-spectroscopist at the Max-Planck-Institute for Biochemistry (Martinsried, Germany), first in the Clore / Gronenborn group, later independently in the department of Prof. Huber 1992 Habilitation in Biophysical Chemistry at the Technical University of Munich Group leader at the EMBL, Heidelberg Since 1998 Head of the department NMR-supported Structural Biology at the Leibniz-Institut für Molekulare Pharmakologie, Professor of Structural Chemistry at the Free University in Berlin Magic-angle-spinning (MAS) solid state NMR delivers high-resolution structural information on complex samples of heterogeneous molecules, independent of their molecular weight and without depending on crystallization. It is an attractive method for structural investigations of difficult systems such as proteins embedded in lipid bilayers or attached to the cytoskeleton. We aim, in the long run, to carry out structural investigations within the real space of a cell, capitalizing on a fold increase in signal-to-noise through dynamic nuclear polarization (DNP). For this purpose, we are improving and testing DNP methods on biological samples in investigations on the nascent chain emerging from the ribosome and of membrane proteins in native lipid environments. Our short-term aims include determining the structures of membrane-integrated or cytoskeleton-attached proteins to unravel their mechanisms of action. In particular we are investigating the transport cycle of an ABC-transporter embedded in lipid bilayers, proton transport in channel rhodopsin, and bacterial membrane proteins such as OmpG and YadA. Furthermore, we investigate protein systems involved in protein homeostasis, including small heat shock proteins and initial folding events of the nascent chain on the ribosome. ZUSAMMENFASSUNG Die Magic-Angle-Spinning (MAS)-Festkörper-NMR-Spektroskopie liefert strukturelle Informationen zu komplexen Proben heterogener Moleküle in hoher Auflösung, und zwar unabhängig von ihrem Molekulargewicht und ohne vorherige Kristallisation. Es handelt sich damit um eine attraktive Methode, die Struktur von schwierig zu untersuchenden Systemen wie beispielsweise in Lipid-Bilayer eingebauten oder mit dem Zytoskelett verbundenen Proteinen zu untersuchen. Unser langfristiges Ziel ist es, Strukturuntersuchungen innerhalb des realen Raums einer Zelle durchzuführen und außerdem durch dynamische Kernpolarisation (dynamic nuclear polarization, DNP) eine fache Verbesserung des Signal-Rausch-Verhältnisses zu erzielen. Zu diesem Zweck optimieren und testen wir DNP-Methoden an biologischen Proben durch Untersuchungen von am Ribosom wachsenden Proteinketten und von Membranproteinen in ihrer nativen Lipidumgebung. Zu den kurzfristigen Zielen gehört die Strukturaufklärung membranintegrierter oder zytoskelettassoziierter Proteine, um ihre Wirkungsmechanismen aufzuklären. Insbesondere untersuchen wir den Transportzyklus eines in den Lipid-Bilayer integrierten ABC- Transporters, den Protonentransport in Kanalrho-dopsin und bakterielle Membranproteine wie OmpG und YadA. Weiterhin untersuchen wir Proteinsysteme, die an der Proteinhomöostase beteiligt sind, u. a. kleine Hitzeschockproteine und die anfänglichen Faltungsereignisse wachsender Proteinketten am Ribosom. Dynamic Nuclear Polarization Methods enabling structural studies of membrane-integrated receptor systems without the necessity of protein purification or the structure determination of proteins bound to the cyto-skeleton offer attractive prospects in structural biology. Dynamic nuclear polarization (DNP) magic angle spinning NMR allows the investigation of such systems, delivering the required sensitivity. With this method, the very strong polarization of electron spins is transferred to nuclear spins, which can then be detected at much higher signal-to-noise. We have a number of ongoing studies using DNP and involving crystalline preparations of soluble proteins (SH3 domain), membrane proteins (neurotoxin II bound to the nicotinic acetyl choline receptor, OmpG, mistic), functional amyloid fibrils (Het-s, curli) and even selectively labeled ribo-somes. Initial results from these studies include the enhancement of sensitivity through additional application of protein deuteration, studies of the effects of low temperatures on protein structures, and first assignments of residues in the nascent protein chain emanating from the ribosome. Strong emphasis was put on making dynamic nuclear polarization a routine method for structural investigations of proteins by adopting a special approach that is termed high temperature DNP. Although higher enhancements of signals are observed at a measurement temperature of 100 K, spectra are much better resolved when measuring at 200 K. Following the preparation of standard protein samples we developed a methodology that enabled us to record high resolution protein spectra while retaining an appreciable enhancement around 20 to 40 in the temperature range of 180 to 200 K, respectively. This approach is now tested in structure determination projects. Future applications of DNP comprise the interaction of ribo-some-bound signal peptides with the signal recognition particle and chromophore states of channel rhodopsin. Structures of membrane proteins in native lipids by solid-state NMR Several membrane protein systems are investigated in our lab. We prepared the ABC-transporter ArtMP from geobacillus stearothermophilus to investigate in detail the structural changes upon ATP hydrolysis during the transport cycle in a native lipid environment. Deuterated samples of ArtMP have been prepared, and CP- and J-coupling-based HN-correlations were showing either the signals of the whole transporter or selectively the ATP binding cassette protein, respectively. This offers tremendous potential for functional studies by allowing for the editing of subunits on a simple spectroscopic level. As a first result, the effects of nucleotide binding were investigated. The membrane-integrated transporter showed expected chemical shift changes around the nucleotide binding side, but also towards the coupling helices (Fig. 1). This suggests that a considerable strain is employed onto the helices in the membrane portion ArtM, which is responsible for the closing movement upon transporter activation. As a pilot project for developing a structure determination concept suitable for medium-sized membrane proteins, we investigate the structure of OmpG in lipid bilayers. A number of studies concerning labeling strategies, flexibility of the loop forming the lid of the porin, and of suitable pulse sequences, are ongoing. Further structural work concerns the trimeric autotransporter adhesin A (YadA) from Yersinia enterocolitica Adhesin A. Many members of this family are important pathogenicity factors that mediate adhesion to host cells and tissues in such diverse diseases as diarrhea, urinary tract infections, or airway infections. Solid-state NMR provided information on flexibility and mobility of parts of the structure, which in combination with evolutionary conservation information allowed for new insights into the autotransport mechanism of YadA. In future, considerable focus will be placed on the investigation of proton transport in Channelrhodopsin, an important protein used in neurobiology for light-induced activation of nerve cells. The application of DNP in investigations of the ground state lead to the surprising finding that it is solely composed of the all-trans form (Fig. 2). Chaperone systems and their functional complexes Small heat shock proteins (Hsp) such as αb-crystallin or larger chaperone systems such as Hsp40 / 70 / 110 are attractive but yet not well explored drug targets. Following structural work on αb-crystallin we investigate its mechanism of activation and interaction with substrates such as β- and γ-crystallins. Initial investigations aimed at the characterization of the binding site for γs-crystallin and a mutant of βb-crystallin. Interfering with protein-protein interactions In a third line of projects, we search for small-molecule inhibitors of protein-protein interac-tions, using PDZ (PSD-95, Dlg, ZO-1) domains as an example. They play important roles in cellular signaling pathways and are structurally characterised by a hydrophobic pocket sur-rounded by a conserved sequence motif, G-L-G-F. This pocket binds the C-termini of target proteins, in most cases receptors and ion channels. Their functional diversity and characteristic,

41 78 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 79 relatively small binding pocket, make them attractive targets for the design of small-molecule inhibitors which may, in the long run, allow for the treatment of several PDZ-related human disorders such as neuropathic pain, congenital diseases, psychiatric disorders, and cancer. A large number of high-resolution structures of PDZ-ligand complexes provide an excellent basis for rational design. Inhibitors with low to medium affinity for several PDZ domains were identified and members of the respective substance classes were collected in a PDZ-library. Improvements of the AF6 PDZ domain inhibitors by modeling-chemistry cycles resulted in compounds with ten to twenty micromolar dissociation constants, disrupting the AF6-Bcr interaction in cell lysates. We intend to exploit our results further by targeting three PDZ domains (AF6, DVL, Shank3), as we seek an understanding of the biology of the respective proteins. Martin Ballschk, Anne Diehl and Martina Leidert Fig. 1: Model of the ABC-transporter ArtMP with its ATP-binding cassette ArtP showing selected side chains of residues color-coded from white, yellow to red according to the response of their chemical shifts towards nucleotide binding. Surprisingly, strong responses are found around the coupling helices (top) whereas the symmetric binding site is silent (white), suggesting an induced strain on the membrane protein prior to the formation of a closed dimer. Fig. 2: DNP MAS NMR spectra of channelrhodopsin labelled with 13C-retinal according to the pattern shown on top. The size ratio of the cross peaks indicates an all-trans configurati-on, which was corroborated with measurements at short mixing times. Signals due to the 13-cis form were not obeserved Secondary Binding Site Primary Binding Site 13 C (ppm) 13 C (ppm) GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Umit Akbey Dr. Linda Ball Dr. Anne Diehl Dr. Frank Eisenmenger Dr. Trent Franks Dr. Matthias Hiller Dr. Nestor Kamdem Dr. Madhu Nagaraj Dr. Andrew Nieuwkoop Dr. Marcella Orwick-Rydmark Dr. Shakeel Ahmad Shahid Dr. Barth van Rossum Nils Cremer (bioengineer) Anup Chowdhury (doctoral student) Gregorio Giuseppe de Palma (doctoral student) Michael Andreas Geiger (doctoral student) Johanna Münkemer (doctoral student) Joren Retel (doctoral student) Florian Seiter (doctoral student) Mahsheed Sohrabi (doctoral student) Daniel Stöppler (doctoral student) International Burkhard Bechinger University of Strasbourg / CNRS, France Lyndon Emsley ENS Lyon, France Lucio Frydman The Weizmann Institute of Science, Rehovot, Israel Daniela Goldfarb The Weizmann Institute of Science, Rehovot, Israel Robert G. Griffin Massachusetts Institute of Technology, Cambridge, USA Victoria A. Higman University of Oxford, Oxford, UK Rachel Klevit University of Washington, Seattle, USA Rachel W. Martin UC Irvine, Irvine, USA Niels Chr. Nielsen Center for Insoluble Protein Structures National Bernd Bukau Universität Heidelberg Michael Habeck Max Planck Institut für Intelligente Systeme, Tübingen Sandro Keller Technische Universität Kaiserslautern Dirk Linke Max-Planck-Institut für Entwicklungsbiologie, Tübingen Thomas Müller Universität Würzburg Thomas Prisner Johann-Wolfgang-Goethe Universität, Frankfurt Bernd Reif Technische Universität München Hans-Günther Schmalz Universität zu Köln Akbey Ü, Nieuwkoop AJ, Wegner S, Voreck A, Kunert B, Bandara P, Engelke F, Nielsen NC, Oschkinat H (2014) Quadruple-resonance magic-angle spinning NMR spectroscopy of deuterated solid proteins. Angew Chem Int Ed Engl., 53(9), Moura-Alves P, Faé K, Houthuys E, Dorhoi A, Kreuchwig A, Furkert J, Barison N, Diehl A, Munder A, Constant P, Skrahina T, Guhlich-Bornhof U, Klemm M, Koehler AB, Bandermann S, Goosmann C, Mollenkopf HJ, Hurwitz R, Brinkmann V, Fillatreau S, Daffe M, Tümmler B, Kolbe M, Oschkinat H, Krause G, Kaufmann SH (2014) AhR sensing of bacterial pigments regulates anibacterial defence. Nature, 512(7515), Vargas C, Radziwill G, Krause G, Diehl A, Keller S, Kamdem N, Czekelius C, Kreuchwig A, Schmieder P, Doyle D, Moelling K, Hagen V, Schade M, Oschkinat H (2014) Small-molecule inhibi-tors of AF6 PDZ-mediated protein-protein interactions. ChemMedChem, 9(7), Muench F, Retel J, Jeuthe S, van Rossum B, Oh-Ici D, Berger F, Kuehne T, Oschkinat H, Mess-roghli D (2014) Metabolic profiling in experimental autoimmune myocarditis rat model using ex-vivo proton magic angle spinning magnetic resonance spectroscopy (1H-MAS-MRS). Eur Heart J, 35, Deutsche Forschungsgemeinschaft, SFB 765 / C4, Multivalente Protein- Protein-Interaktionen zwischen WW-Domänen und Prolin-reichen Segmenten, with Christian Freund, , Deutsche Forschungsgemeinschaft, SFB 740 / B07 1, Untersuchungen an Komplexen kleiner Hitzeschockproteine mit Substraten mittels Festkörper-NMR-Spektroskopie und dynamischer Kernpolarisation, with van Rossum B, , Deutsche Forschungsgemeinschaft, SFB 1078 / B1, Strukturelle Dynamik von Kanalrhodop-sinen, , Deutsche Forschungsgemeinschaft, DIP Dynamic Nuclear Polarization: Integrating fundamentals and new applications, OS 106 / 12 1, , Bruker, Entwicklung von Invers-1H-Bio-MAS-Probenköpfen, Einrichtung einer β-test-site für das DNP Spektrometer und Generierung von Metabonomics-Daten und Entwicklung von neuen NMR-MAS-Probenköpfen, Festkörper-NMR-Techniken und DNP-NMR für die Anwendung auf dem Gebiet BioSolids, , Alexander on Humboldt-Stiftung, Forschungskostenzuschuss als Gastgeber von Dr. Andrew Nieuwkoop, , Anja Voreck (doctoral student) (inspin), Interdisciplinary Nanoscience Center Akbey Ü, Altin B, Linden A, Özçelik, Gradzielski M, Oschkinat H (2013) European Union, 7. Framework Programme, project within Bio-NMR Arndt Wallmann (doctoral student) (inano) and Aarhus University, Denmark Dynamic nuclear polarization of spherical nanoparticles. NMR for Structural Bio-logy, FP7-Infrastructures Anne Wartenberg (doctoral student) Michael Nilges PhysChemChemPhys, 15(47), , Natalja Erdmann (technical assistant) Institut Pasteur, Paris, France Liselotte Handel (technical assistant) Melanie Rosay Martina Leidert (technical assistant) Bruker BioSpin, Billerica, USA Dagmar Michl (technical assistant) Paul Tordo Kristina Rehbein (technical assistant) Aix-Marseille Université, France Shimon Vega The Weizmann Institute of Science, Rehovot, Israel FMP authors Group members

42 80 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 81 STRUCTURAL BIOINFORMATICS AND PROTEIN DESIGN Jonas Protze, Dominik Neef and Katrin M. Hinz STRUKTUR-BASIERTE BIOINFOR- MATIK UND PROTEINDESIGN GROUP LEADER DR. GERD KRAUSE BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studied chemistry at the University in Leipzig 1982 Ph.D. in biochemistry at the Martin Luther University, Halle Researcher at the Institute of Drug Design, Berlin on behalf of pharmaceutical industry Research Position at the Institute of Drug Design, Berlin Visiting Scientist at the Washington University (Prof. Marshall) in St. Louis, MO, USA Project leader at research institute of molecular pharmacology (FMP), Berlin since 1998 Group Leader of Structural Bioinformatics and Protein Design at the FMP The group focuses on analyzing the relationship between sequences and structures of membrane proteins using structural bioinformatics, combined with experimental studies of the functions of altered sequence(s). Our aim is to reveal the structure-function relationships of proteins and potential interaction partners. One major activity is to develop bioinformatic tools for investigating such structure-function relationships; another is to apply them to particular molecular biological projects of protein-ligand, protein-substrate or proteinprotein interactions. To verify our structure-function hypotheses we perform experimentally site-directed mutagenesis of model guided specific residues and analyze available mutation data. Bioinformatic tool / database development and molecular biology applications mutually support one another. The main aims of the group are to achieve: (1) A detailed understanding of intramolecular mechanisms of membrane proteins; (2) The rational discovery of molecular mechanisms and sites for protein-protein interactions and protein-ligand or protein-substrate interactions; and (3) A narrowing down of locations to the amino acid and atomic level of the target protein and prediction of small molecules for potential pharmacological interventions. ZUSAMMENFASSUNG Die Gruppe beschäftigt sich mit der Analyse der Beziehung zwischen Sequenz und Struktur von Membranproteinen; dies erfolgt mittels struktureller Bioinformatik in Kombination mit experimentellen Funktionsuntersuchungen gezielt veränderter Proteinsequenzen. Unser Ziel ist es, Struktur-Funktionsbeziehungen von Proteinen und potenziellen Interaktionspartnern aufzuklären. Einerseits werden bioinformatische Werkzeuge und Datenbanken zur Untersuchung solcher Struktur-Funktionsbeziehungen entwickelt; andererseits werden diese bei spezifischen molekularbiologischen Projekten wie z. B. allosterische Ligandenbindung an G-Protein-gekoppelte Rezeptoren, und der Wechselwirkung von Tight junction Proteinen wie Claudinen eingesetzt. Zur Überprüfung unserer Struktur-Funktions-Hypothese führen wir zielgerichtete Mutagenesen spezifischer Reste experimentell durch und analysieren die verfügbaren Mutationsdaten. Die Entwicklung von Bioinformatikwerkzeugen / Datenbanken und experimentelle Molekularbiologie unterstützen sich dabei gegenseitig. Die Hauptziele, die wir mit unserer Gruppe erreichen wollen, sind (1) ein detailliertes Verständnis der intramolekularen Mechanismen von Membranproteinen, (2) die rationale Aufklärung der molekularen Mechanismen und Bindungsstellen von Protein-Protein-Interaktionen und Protein-Ligand- bzw. Protein-Substrat-Interaktionen und (3) eine Eingrenzung der Bindungsstellen am Zielprotein auf Aminosäure- und atomarer Ebene und die Vorhersage von kleinen Molekülen mit dem Potenzial für eine pharmakologische Intervention. AhR supports the immune system The aryl hydrocarbon receptor (AhR), a transcription factor, has been shown to be sensible to environmental pollutants, such as 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). To prove the hypothesis that it also plays an important role in the immune defense system against bacteria, structurally similar microbial insults, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, were tested as ligands for this receptor (cooperation with S. Kaufmann, MPI and H. Oschkinat, FMP). To derive an interaction model of AhR with these bacterial virulence factors, a receptor model was generated and subsequent docking studies were performed (Figure 1). The in silico predictions were verified in a radioactive ligand binding assay (Moura-Alves et al. Nature 2014). Inhibitor design for the AKAP-PKA interaction Interactions between A-kinase anchor proteins (AKAPs) and protein kinase A (PKA) play a key role in many physiologically relevant processes. We aimed to derive inhibitors that compete with this interaction. Helical AKAP18 peptides interact with the dimerization and docking (D / D) domain of PKA. In silico studies led to the prediction of small molecule helix-mimetics (terpyridines, Figure 2) and subsequent synthesis and functional characterization (cooperation E. Klussmann, MDC and P. Schmieder, FMP) confirmed the inhibitory function (Schäfer et al. Angewandte Chem. Intl Ed. 2013). Suggested folding models of claudins Claudins (Cld) are the major constituents in tight junctions (TJ). The mechanism of TJ assembly is unclear. To identify determinants of assembly of Cld3 and Cld5, chimeric mutants were analyzed by cellular reconstitution of TJ strands and live cell imaging (cooperation with J. Piontek, Charité). Evolutionary sequence couplings and comparative modelling of intramolecular interfaces in the transmembrane region of claudins led to a complete claudin-5 model (Rossa & Protze et al. Biochem J. 2014). Our suggested claudin subtype-specific intra- and intermolecular interfaces were confirmed by the very recently released crystal structure of claudin-15 (PDB-ID: 4P79). pharmacophores for small molecule antagonists and agonists (Hoyer I. et al, Biochem. Soc.Trans. 2013), ii) the differences between lutropin and choriogonadotropin-mediated receptor activation (Grzesik et al. FEBS J in cooperation with R. Schülein and B. Wiesner FMP), and iii) a description of the functional plasticity of the luteinizing hormone / choriogonadotropin receptor (Troppmann et al. Reproduction 2013 in cooperation with J. Gromoll, Münster). GPHRs belong to the G-protein coupled receptors that play an important role in pharmacology. The recently completed renewal and extension of a previous version of a data resource for naturally occurring and designed mutations at GPHRs ( included further development of novel bioinformatic tools. Integrating the structural template for the hinge region with its second hormone binding site allowed us to assign functional data to the new structural features between hormone and receptor of GPHRs (Kreuchwig et al. Mol. Endocrinology, 2013). Determinants at thyroid hormone transporter MCT8 identified Thyroid hormones are transported across the cell membrane via transport-proteins comprising 12 transmembrane helices; one of these is the Monocarboxylate transporter 8 (MCT8). The fact that the thyroid hormone (TH) triiodothyronine (T3) is bound between a His-Arg clamp in the crystal structure of the T3-receptor / T3 complex prompted us to likewise dock the thyroid hormone T3 in our previously generated MCT8 model. Subsequently two histidines (His192 and His415) of MCT8 were confirmed as interaction partners by side-directed mutagenesis and thus define a substrate channel in MCT8 (Braun et al. Endocrinology 2013, cooperation with U. Schweizer, Bonn). Structure function relationships at glycoprotein hormone receptors (GPHRs) Such relationships were deciphered for i) the allosteric binding pocket of the thyrotropin receptor providing discriminative

43 82 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 83 Fig. 1: Homology model of the aryl hydrocarbon receptor (AHR). Docking of bacterial toxins identified the phenazines from P. aeruginosa that fit perfectly in the ligand binding pocket of the AHR model (sliced view) and have similar interactions as environmental pollutants such as TCDD. Fig. 1 Fig. 2: Interaction models for PKA (yellow surface) with AKAP-helix and predicted helix-mimetica. The peptide AKAP18δ- L314E is shown in magenta, its interacting side chains are depicted in stick representation with hydrogen bonds shown. The main interactions either for A) with the helical peptide or B) with the designed terpyridine (cyan) are of hydrophobic nature (yellow ) or by hydrophilic interactions (blue and red) as depicted in the figures A) and B). Patrick Marcinkowski Katrin M. Hinz and Anna Piontek Fig. 2 GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS Dr. Anna Piontek (Veshnyakova) Dr. Anita Kinne (doctoral student) Paul Grzesik (doctoral student) Annika Kreuchwig (doctoral student) Inna Hoyer (doctoral student) Jonas Protze (doctoral student) Katrin M. Hinz (doctoral student) Franziska Kreuchwig (student) Stefan Dinter (student) Katja Meyer (student) Miriam Eichner (doctoral student, #) International M. Gershengorn and S. Neumann NIH Bethesda, MD, USA A. Ijzerman and L Heitman Uni Leiden, The Netherlands T. Visser Uni Rotterdam, The Netherlands National S. Kaufmann MPI für Infektionsbiologie, Berlin M. Fromm and H. Biebermann, J Köhrle Charité-Universitätsmedizin Berlin U. Schweizer Wilhelm Friedrich Universität, Bonn E. Klussmann MDC, Berlin J. Gromoll Universität Münster R. Schülein, B. Wiesner, P. Schmieder, A. Diehl, H. Oschkinat FMP Moura-Alves P, Fae K, Houthuys E, Dorhoi A, Kreuchwig A, Furkert J, Barison N, Diehl A, Munder A, Constant P, Guhlich-Bornhof U, Klemm M, Koehler A-B, Bandermann S, Goosmann C, Mollenkopf HJ, Hurwitz R, Brinkmann V, Fallatreau S, Daffe M, Tümmler B, Kolbe M, Oschkinat H, Krause G, Kaufmann SHE (2014), AhR sensing of bacterial pigments regulates anti-bacterial defences. Nature 512, Schäfer G, Milić J, Eldahshan A, Götz F, Zühlke K, Schillinger C, Kreuchwig A, Elkins JM, Abdul Azeez KR, Oder A, Moutty MC, Masada N, Beerbaum M, Schlegel B, Niquet S, Schmieder P, Krause G, von Kries JP, Cooper DMF, Knapp S, Rademann J, Rosenthal W, Klussmann E (2013) Highly functionalised terpyridines as a competitive inhibitors of PKA-AKAP interaction. Angew. Chem. Intl Ed Engl. 52, Grzesik P, Teichmann A, Furkert J, Rutz C, Wiesner B, Kleinau G, Schülein R, Gromoll J, Krause G (2014) Differences between lutropin and choriogonadotropin- mediated receptor activation. Braun D, Lelios I, Krause G, Schweizer U (2013) Histidines in Potential Substrate Binding Sites Affect Thyroid Hormone Transport by Monocarboxylate Transporter 8 (MCT8). Endocrinology 154, Hoyer I, Haas A-K, Kreuchwig A, Schülein R, Krause G (2013) Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists. Biochem. Soc. Trans. 41, EXTERNAL FUNDING Deutsche Forschungsgemeinschaft, SPP 1629, - Thyroid Trans Act-Role of L-type amino acid transporter Lat2 in transport of thyroid hormones, KR 1273 / 5 1, , KI 1751 / 1 1, , Deutsche Forschungsgemeinschaft Modulatoren für den Thyrotropin- FEBS J. 281, Rezeptor: Molekulare Mechanismen allosterischer Bindung und Kreuchwig A, Kleinau G, Krause G (2013) Research Resource: Novel structural insights bridge gaps in glycoprotein receptors. Wirkungsweise kleiner Moleküle, KR 1273 / 4 1, verlangert , Mol Endocrinology 27, Deutsche Forschungsgemeinschaft, Molekuare und strukturelle Muster parazellulärer Poren durch subtypabhängige Claudin-Claudin-Wechselwirkungen in tight junction, FOR 721 KR 1273 / 3-2, with Jörg Pontek, , FMP authors # supervision also by J. Piontek Group members

44 84 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 85 COMPUTATIONAL CHEMISTRY / DRUG DESIGN WIRKSTOFF-DESIGN GROUP LEADER DR. RONALD KÜHNE BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Study of Biochemistry at the Martin-Luther-Universität Halle Witttenberg 1973 Diploma in Biochemistry Research associate at the Zentralinstitut für Molekularbiologie und Medizin der Akademie der Wissenschaften Research associate at the Institut für Wirkstofforschung der Akademie der Wissenschaften 1980 Doctorate degree since 1993 Group leader at the Leibniz- Institut für Molekulare Pharmakologie The development of compounds that bind to biologically important target proteins is the major task of the Drug Design group. It requires strong interdisciplinary collaborations, integrating in silico ligand design and bioinformatics, as well as experimentally driven disciplines like structure biology, biophysics, molecular biology, and cell biology. During recent years we have focussed our interests on targeting protein-protein interactions mediated by protein domains specifically recognizing proline-rich motifs. These domains are involved in many disease-relevant signal transduction cascades and in cytoskeleton remodelling. ZUSAMMENFASSUNG Die Entwicklung von Substanzen, die an biologisch wichtige Zielproteine binden, ist das zentrale Forschungsziel der Arbeitsgruppe Wirkstoffdesign. Forschungen auf diesem Gebiet sind vor allem durch starke Vernetzung unterschiedlicher wissenschaftlicher Disziplinen wie computergestützter Chemie und Bioinformatik, Biophysik, Struktur- und Zellbiologie gekennzeichnet. Zentrales Forschungsthema der Gruppe ist die Entwicklung neuartiger Inhibitoren von Protein-Protein-Wechselwirkungen, die durch Prolin-reiche Motive vermittelt werden. Solche Wechselwirkungen finden sich in etlichen, mit Krankheiten assoziierten Signaltransduktionswegen sowie in der strukturellen Organisation des Zytoskeletts. Development of inhibitors to block proline-rich mediated protein-protein interactions Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialised in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena) / vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. We developed a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. These rationally designed chemical fragments mimic dipeptide motifs (e.g. PP, xp, Px), adopting backbone angles typical of a left-handed polyproline II helix (PPII). A modular stereoselective synthesis of these peptidomimetic fragments was developed at the Universität zu Köln (Inst. Organische Synthese, Prof. H.-G. Schmalz). We expected combinations of such fragments to allow complete replacement of the proline-rich core motifs. As a proof of concept we were able to develop, for the first time, low molecular weight inhibitors of Ena / VASP EVH1 domains. Starting with the ActA-derived peptide Ac-SFEFPPPPTEDEL-NH2 (K d, EnaH-EVH1 20 µm), introduction of ProM-fragments, in combination with optimization of flanking epitops, yielded a 700 Da peptidomimetic EVH1 inhibitor (K d, EnaH-EVH1 0.2 µm). As compared to the core motif peptide Ac-FPPPP-OH (K d, EnaH-EVH1 460 µm), a substantial increase in affinity of at least three magnitudes was achieved by introduction of only six heavy atoms. Furthermore we used our ProM toolkit to successfully replace proline-rich core motifs in peptides binding to the WW domains of Yes-associated protein (Yap) and the formin binding protein 28 (FBP28). Structural biology of the new EVH1 inhibitors The optimization of EVH1 inhibitors was supported by detailed insights into their binding modes. We were able to solve more than 10 X-ray structures (resolutions in the range of 1.0 Å-2.5Å) of different Ligand-EnaH- / EVL-EVH1 complexes. Importantly, it was also possible to crystallize EnaH-EVH1 in complex with two peptides containing the C-terminal part of the ActA-peptide which comprises a second flanking epitop responsible for a ten-fold increase in affinity. Based on these structures we could identify the position of the second flanking epitop and then use this information to optimize our low molecular weight EVH1 inhibitors. All high resolution structures confirmed the canonical binding mode of our new EVH1 inhibitors. In the case of VASP-EVH1, which did not crystallize, we used NMR to confirm the binding modes. Functional studies using the new EVH1 inhibitors The Ena / VASP protein family is involved in modulation of the actin cytoskeleton, a complex and highly regulated process that is the driving force of directed cell migration and plays important roles in disease-relevant processes like tumour metastasis. It is known that Ena / VASP proteins are located at focal adhesions and the leading edge of migrating cells, as well as at the tips of filopodia, but their functional role at these cellular locations is still unclear. Our new inhibitors open novel ways to study the specific function of the Ena / VASP EVH1 domain. We found that inhibition of Ena / VASP EVH1 domains disrupts co-localisation of Ena / VASP proteins with the focal adhesion protein zyxin and lamellipodin localized at the leading edge (Fig. 1). SILAC pulldowns with and without our inhibitors additionally indicate interaction with proteins involved in the activation of Arp2 / 3, a key protein involved in F-actin branching and with proteins of the formin-family which are responsible for fast elongation of actin filaments. Both Arp2 / 3 and proteins of the formin family play an important role in remodelling of the cytoskeleton during cell migration. Our results suggest that Ena / VASP proteins regulate activity of these proteins. We found that delocalization of Ena / VASP caused by inhibition of the EVH1 domain strongly reduces cell migration of invasive tumor cells. Cheminformatics, Library design, and Molecular Modelling Cheminformatics, bioinformatics, and molecular modelling are important disciplines supporting rational design of chemical probes. In combination with experimental chemical biology, such in silico tools improve the probability of success for ligand development. We have established a wide range of methods covering library design, ligand optimization, and calculation of ADMET parameters. Our in-house library design toolbox utilizes an innovative fragment-based concept to select chemically diverse screening compounds with sufficient solubility, low toxicity, and low chemical reactivity. Our library design toolbox provides a core module to select the EU- OPENSCREEN screening library. For compound selection we developed within the Helmholtz-initiative Wirkstoffforschung a biannually updated Web-based database of commercially available compounds (DACS) containing more than 80 million compounds. Such a combination of toolbox and DACS allows fast design of hitor target-focussed libraries as well as of new screening libraries. Target-based ligand optimization is another focus of our work. In particular, we support selected screening projects of the FMP Screening Unit.

45 86 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 87 contr. DMSO comp. 4b contr. DMSO comp. 4b Matthias Müller and Matthias Barone 4a, R=H 4b, R=ethyl (cell permeable) DMSO comp. 4b DMSO comp. 4b Fig. 1: Left, upper panel: Chemical structure of the EVH1 inhibitor 4a and its cell- permeable derivate 4b. Left, lower panel: Pull-down experiments with lysates from MDA MB 231 cells and GST-tagged EnaH-EVH1 and VASP-EVH1 immobilized on glutathione sepharose beads (upper frame: Lpd and zyxin in western blot analysis; lower frame: loading control). With GST alone no zyxin / Lpd was pulled. GST-EVH1 pulled zyxin / Lpd. Zyxin / Lpd displacement from GST-EVH1 domains was performed by adding different concentrations of comp. 4a to the lysate. Right, upper panel: Immunofluorescent stain of MDA MB 231 cells. Control 0.5 % DMSO (column 1 and 3) and 100 µm compound 4b (column 2 and 4) incubated for 3h. Upper row: Zyxin (ZYX,) and Lpd, middle: VASP and lower row: merge (ZYX, blue; VASP, red; co-local. in purple; Lpd, green, co-local. in yellow). Right, lower panel: Compound 4b treated cells showed a reduction of VASP location to focal adhesions (FA) by approx. 40 % and to the leading edge by 30 %. FA are marked by Zyxin, the leading edge by Lpd. The localisation of Zyxin and Lpd remains unchanged. The white bar represents 5 µm. Hafiza Nayab Omer and Michael Lisurek (photo left), Raed Al-Yamori and Bernd Rupp GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Michael Lisurek International National Horvath D, Lisurek M, Rupp B, Kühne R, Specker E, von Kries J, Design and Synthesis of low-molecular weight proline-rich motif (PRM) Dr. Bernd Rupp Prof. M. Hibert Prof. H.-G. Schmalz Rognan D, Andersson CD, Almqvist F, Elofsson M, Enqvist PA, mimetics recognized by PRM binding domains, DFG, KU 845 / 2 / 2, Dr. Daniela Müller (PostDoc upto 05 / 2014) Univ. Strasbourg, France Inst. Organische Chemie, Universität zu Köln Gustavsson AL, Remez N, Mestres J, Marcou G, Varnek A, Hibert M, For806 TP 03, 03 / / 2013, Förderumfang: 36 Mon. BATII / E13 Dr. Kiril Piotoukh (PostDoc) Dr. J. Quintana Prof. Chr. Freund Quintana J, Frank R (2014) Design of a general-purpose European 50 %; Sachmittel +Programmpauschale Dr. Robert Opitz (PostDoc) Matthias Müller (PhD student) Matthias Barone (PhD student) Raed Al-Yamori (Techn. Infomatics) Kathrin Motzny (Techn. Ass. since 01 / 2014) Katy Franke (Chem.-techn. Ass. since 06 / 2014) Barcelona Science Park, Spain FU Berlin Prof. M. Wahl FU Berlin Prof. J. Rademann FU Berlin Prof. U.Stein MDC Berlin compound screening library for EU-OPENSCREEN. ChemMedChem 9, Soicke A. Reuter C; Winter M, Neudorfl JM, Schlorer N, Kühne R, Schmalz H-G (2014) Stereoselective Synthesis of Tricyclic Diproline Analogues that Mimic a PPII Helix: Structural Consequences of Ring-Size Variation. Eur. J. Org. Chem. 29, Mechanismus des durch HLA-DM und kleine Molküle vermittelten MHCII: Peptid-Austauschs, DFG KU 845 / 3 1, 02 / / 2014, Förderumfang: 36 Mon. BATII / E13 60 %; Sachmittel+Programmpauschale Innovative Inhibitoren Polyprolin-Motiv-erkennender Protein-Protein- Interaktionsdomänen als Ausgangspunkt zur Validierung neuer pharmakologisch relevanter Zielproteine und zur Entwicklung neuartiger Prof. Heinemann Reuter C, Kleczka M, de Mazancourt S, Neudorfl JM, Kühne R, Schmalz Ansätze in der Krebstherapie, BMBF 03V0475, 07 / / 2016, MDC Berlin H-G (2014) Stereoselective Synthesis of Proline- Derived Dipeptide Förderumfang: Bayer AG Scaffolds ( ProM-3 and ProM-7) Rigidified in a PPII Helix Conformation. Eur. J. Org. Chem. 13, Preidl JJ, Gnanapragassam VS, Lisurek M, Saupe J, Horstkorte R, Rademann J (2014) Fluorescent mimetics of CMP-Neu5Ac are highly potent, cell-permeable polarization probes of eukaryotic and bacterial sialyltransferases and inhibit cellular sialylation. Angew. Chem. Int. Ed. Engl. 53, Hack V, Reuter C, Opitz R, Schmieder P, Beyermann M, Neudörfl JM, Kühne R, Schmalz HG (2013) Efficient α-helix induction in a linear peptide chain by N-capping with a bridged-tricyclic diproline analogue. Angew. Chem. Int. Ed. Engl. 52, FMP authors Group members

46 88 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 89 MOLECULAR BIOPHYSICS MOLEKULARE BIOPHYSIK GROUP LEADER PROF. DR. ADAM LANGE BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studies of Physics at the Georg-August-University Göttingen PhD / Postdoctoral studies at the Max Planck Institute for Biophysical Chemistry, Göttingen Research visit at the National Institutes of Health, Bethesda, Maryland, USA 2006 Ernst Award of the German Chemical Society 2006 Otto Hahn Medal of the Max Planck Society Postdoctoral fellow at the Laboratory of Physical Chemistry, ETH Zürich, Switzerland, EMBO long-term fellowship Research Group Leader at the Max Planck Institute for Biophysical Chemistry, Emmy Noether fellowship 2013 ERC Starting Grant 3D structures of bacterial supramolecular assemblies by solid-state NMR Since 2014 Department Head at the Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin and W3-S Professor at the Humboldt-University of Berlin We study protein structure and dynamics using nuclear magnetic resonance in the solid state (solid-state NMR). In the last decade, solid-state NMR has emerged as a powerful technique in structural biology as it gives access to structural information for systems which are insoluble or do not crystallize easily. For instance, membrane proteins in a lipid bilayer environment or supramolecular assemblies such as the needle of the type three secretion system (T3SS) composed of multiple copies of a single small protein can be readily studied. For solid-state NMR investigations, samples are placed in a strong superconducting magnet (external field up to 20 T, i.e. ~400,000 times as strong as the earth s magnetic field) and spun rapidly (ca. 10,000 rotations per second). This rotation around an axis that is inclined to the magnetic field by a magic angle of 54.7 emulates the situation of fast and freely tumbling molecules in solution. By means of magic-angle-spinning, high resolution and sensitivity can be reached in the NMR spectra of solid proteins. A focus of our group is on bacterial supramolecular protein assemblies including T3SS needles, the type I pilus and on cytoskeletal bactofilin filaments. Furthermore, we are also interested in solid-state NMR method development. ZUSAMMENFASSUNG Wir untersuchen mittels Festkörper-Kernspinresonanzmethoden (Festkörper-NMR) die Struktur und Dynamik von Proteinen. In den vergangenen zehn Jahren hat sich die Festkörper-NMR zu einer mächtigen Technik in der Strukturbiologie entwickelt, die Zugang zu Strukturinformationen an solchen Systemen ermöglicht, die unlöslich oder nicht einfach zu kristallisieren sind. Membranproteine in ihrer Lipidbilayer-Umgebung beispielsweise oder supramolekulare Strukturen wie die Nadeln des Typ-3-Sekretionssystems (T3SS), die aus vielen Kopien eines einzelnen kleinen Proteins aufgebaut sind, lassen sich so vergleichsweise einfach untersuchen. Dazu werden die Proben in einen starken, supraleitenden Magneten gebracht (mit Feldstärken bis 20 T, d. h. ca mal so stark wie das Magnetfeld der Erde) und in eine schnelle Rotation versetzt (ca Umdrehungen pro Sekunde). Die Rotationsachse der Probe ist gegenüber dem Magnetfeld um den magischen Winkel von 54,7 gekippt, was die Situation von sich frei und schnell bewegenden Molekülen in Lösung simuliert. Dieses sogenannte magic-angle-spinning erlaubt eine hohe Auflösung und Sensitivität bei NMR-Spektren von Proteinen in der festen Phase. Ein Schwerpunkt der Forschung unserer Arbeitsgruppe liegt hier auf bakteriellen supramolekularen Strukturen, die unter anderem T3SS-Nadeln, den Typ I-Pilus und Bactofilinfilamente des bakteriellen Zytoskeletts umfassen. Gleichzeitig entwickeln wir Festkörper-NMR-Methoden weiter. The type III secretion system needle Homo-oligomeric supramolecular protein assemblies such as the needle of the type III secretion system (T3SS) are ideal targets for solid-state NMR investigations. The T3SS is a nanomachine used by many gram-negative bacteria to inject effector proteins into host cells. There, these substances manipulate essential metabolic processes and disable the immune defence of the infected cells. A schematic representation of the T3SS is provided in Figure 1. The base of the T3SS is firmly anchored in the inner and outer bacterial membranes. A channel through the base ends in a hollow extracellular needle. This needle is formed by the self-assembly of around copies of a single, small-sized protein in the case of Salmonella typhimurium the 80-residue protein PrgI. Recently, we could show that T3SS needles can be produced in the lab by self-association of recombinantly expressed PrgI and that solid-state NMR spectra of such in vitro needles exhibit exceptionally high spectral resolution [Loquet et al., Nature, 2012]. As a result of the excellent data quality we could determine an atomic model of the needle. For this purpose solid-state NMR data were combined with results from electron microscopy (EM) and computer modelling. The resulting atomic model [Loquet et al., Nature, 2012] represents one of the largest and most complex protein structures determined to date by solid-state NMR. The structure reveals an inner needle diameter of only 2 nm. The secreted proteins thus have to pass the needle in an unfolded state and need to refold again in the host cell before performing their tasks. The solid-state NMR structure also showed that the conserved C-terminus of the needle protein faces the lumen and that the more variable N-terminus is found on the outer needle surface. This variability might reflect a strategy of the bacteria to evade immune recognition by the host. High-resolution solid-state NMR structures of secretion needles More recently, we have determined high-resolution solid-state NMR structures of needles from Salmonella [Loquet et al., J Am Chem Soc, 2013] and from Shigella [Demers et al., Nat Commun, 2014], which allowed us to determine the handedness of the needle. This parameter was not unambiguously defined in our previous study [Loquet et al., Nature, 2012]. We found that the T3SS needle adopts a right-handed helical structure with approximately eleven PrgI subunits per two turns, similar to the arrangement observed in the related flagellar filament. Solid-state NMR method development In terms of solid-state NMR method development, the focus in our lab is on new experiments for efficient sequential resonance assignment. For instance, we have introduced a set of proton-detected 3D experiments based on dipolar out-and-back transfers and applied it to deuterated T3SS needles [Chevelkov et al., J Magn Reson, 2014]. By means of BSH-CP for efficient CO-CA transfer [Chevelkov et al., J Magn Reson, 2013], we also assembled a set of carbon-detected 3D experiments for sequential backbone assignment [Shi et al., J Biomol NMR, 2014]. Furthermore, we exploit sparse isotope-labeling schemes that enhance spectral resolution and facilitate the detection of long-range distance restraints [Loquet et al., J Am Chem Soc, 2011]. Pascal Fricke and Chaowei Shi (photo above), Sascha Lange

47 90 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 91 Menschl. Zelle Bakterielle Proteine Fig. 1: Schematic representation of the type III secretion system. The structure of the needle was recently determined by our group using a combination of solid-state NMR, electron A C microscopy and computer modelling [Loquet et al., Acc Chem Res, 2013]. B D T3SS NADEL E Basis F Bakt. Zelle Bakterielle Proteine Fig. 2: A new methodological approach to determine the structure of the T3SS needle and other supramolecular assemblies. (A) Side view and (B) top view of the S. typhimurium needle assembly. (C) Transmission electron micrograph of in vitro preparation of T3SS needles. (D) solid-state NMR rotor and (E) carbon-carbon correlation spectrum. (F) Intermolecular interface between two PrgI subunits in the needle assembly [Loquet et al., Med Sci (Paris), 2012]. GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Andrea Steuer (Secretary) International National Loquet A, Habenstein B, Chevelkov V, Vasa SK, Giller K, Becker S, Emmy Noether Grant (DFG) Solid-state NMR characterization of tau in Dr. Matthias Hiller (Staff Scientist) David Baker Martin Thanbichler Lange A (2013) Atomic structure and handedness of the building block paired helical filaments and bound to microtubules as well as of toxic Dr. Sascha Lange (Staff Scientist) University of Washington, Seattle, USA Philipps-Universität Marburg of a biological assembly. J. Am. Chem. Soc. 135, and non-toxic oligomers of a-synuclein and tau. (DFG GZ.: Dr. Veniamin Chevelkov (Postdoc) Dr. Jean-Philippe Demers (Postdoc) Dr. Chaowei Shi (Postdoc) Hannes Fasshuber (Ph.D. Student) Pascal Fricke (Ph.D. Student) Songhwan Hwang (Ph.D. Student) Nikolaos Sgourakis NIH, Bethesda, USA Yusuke Nishiyama RIKEN, Kobe, Japan Stefan Becker Max Planck Institute for Biophysical Chemistry, Göttingen Michael Kolbe Max Planck Institute for Infection Biology, Berlin Demers J-P, Sgourakis NG, Gupta R, Loquet A, Giller K, Riedel D, Laube B, Kolbe M, Baker D, Becker S, Lange A (2013) The common structural architecture of Shigella flexneri and Salmonella typhimurium type three secretion needles. PLOS Pathogens 9, e Loquet A, Habenstein B, Lange A (2013) Structural investigations LA 2705 / 1 1); Duration: 10 / / 2014; Total amount: ERC Starting Grant 3D structures of bacterial supramolecular assemblies by solid-state NMR (Project acronym: assemblynmr; Grant agreement no.: ); Duration: 60 months, starting date: May 1, 2014; Total amount: Dagmar Michl (Technical Assistant) Michael Habeck of molecular machines by solid-state NMR. Accounts Chem. Kristina Rehbein (Technical Assistant) Georg-August-Universität Göttingen Res. 46, Roland Benz Julius-Maximilians-Universität Würzburg Bernd Reif Technische Universität München Markus Zweckstetter Demers J-P, Habenstein B, Loquet A, Vasa SK, Giller K, Becker S, Baker D, Lange A*, Sgourakis NG* (2014) High-resolution structure of the Shigella type-iii secretion needle determined by solid-state NMR and cryo-electron microscopy. Nature Communications 5, Max Planck Institute for Biophysical Fricke P, Demers J-P, Becker S, Lange A (2014) Studies on the MxiH Chemistry, Göttingen protein in T3SS needles using DNP-enhanced ssnmr spectroscopy. Bert de Groot Max Planck Institute for Biophysical Chemistry, Göttingen ChemPhysChem, 15, FMP authors Group members

48 92 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 93 SOLUTION NMR LÖSUNGS-NMR GROUP LEADER DR. PETER SCHMIEDER Brigitte Schlegel Peter Schmieder and Monika Beerbaum Martin Ballaschk BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Study of Chemistry at the University of Frankfurt and Study of Chemistry at the University of Frankfurt 1988 Diploma thesis in Prof. Kessler s group Ph.D. at the TU Munich (Prof. Kessler) Post-Doc at Harvard Medical School (Prof. Wagner) since 1995 Group leader Solution NMR spectroscopy at the FMP The group focuses on the application of solution state NMR-spectroscopic techniques to the investigation of the structure and dynamics of biomolecules at atomic resolution. The full repertoire of multidimensional NMR techniques is used in combination with appropriate labelling schemes and other biophysical techniques. Currently we are interested in a quantitative determination of the role of dynamics in biomolecules. We are exploring the role of mobility in the presentation of peptides by MHC class one molecules and their recognition by T-cell receptors, exploiting the ability of NMR spectroscopy to provide such information with atomic resolution. Since the MHC-TCR interaction cannot be rationalized based on static structures alone this work will help in understanding immunological processes and in applications like vaccine design. In addition, a variety of projects have been pursued with other research groups, either studying the interaction of biomolecules with binding partners or the constitution of small molecules and biologically relevant natural products. One example is the determination of the phosphorylation pattern of glycogen in the context of Lafora disease. ZUSAMMENFASSUNG Der Fokus unserer Gruppe liegt auf der Anwendung Lösungs-NMR-spektroskopischer Techniken zur Untersuchung der Struktur und Dynamik von Biomolekülen in atomarer Auflösung. In Kombination mit geeigneten Markierungsmethoden und anderen biophysikalischen Techniken einschließlich analytischer Ultrazentrifugation (AUC), isothermischer Titrationskalorimetrie (ITC) und Kleinwinkel-Röntgenbeugung (SAXS) wird das gesamte Spektrum an mehrdimensionalen NMR-Techniken eingesetzt. In den letzten Jahren hat sich die Gruppe mit der Untersuchung photoaktiver Peptide und Proteine beschäftigt; jüngstes Projekt war die Aufklärung der strukturellen Grundlagen des Aktivierungsmechanimus des blaulicht-sensitiven Photorezeptors YtvA. Derzeit versuchen wir die die Rolle der Dynamik von Biomolekülen quantitativ zu erfassen. In diesem Zusammenhang erforschen wir die Rolle der Mobilität bei der Präsentation von Peptiden durch MHC Klasse I-Moleküle und ihrer Erkennung durch T-Zell-Rezeptoren und nutzen dazu die Fähigkeit der NMR- Spektroskopie, solche Informationen in atomarer Auflösung zu liefern. NMR-spectroscopic investigation of micropolymorphism-dependent dynamics of human major histocompatibility antigens MHC class I molecules accommodate small peptide fragments of 8 to 12 amino acids within a binding groove and present them to T-cell receptors on cytotoxic T-cells. T-cells constitute a necessary component of normal adaptive immune responses, but can also be involved in autoimmunity. Over the years it has become clear that the static picture resulting from crystallographic studies will not be able to fully explain the interaction between MHCs and T-cell receptors. NMR spectroscopy is able to provide information on dynamic features and we employ heteronuclear techniques to investigate the dynamics of MHC complexes of two HLA-B27 subtypes that differ only by a single amino acid, but which are differentially associated with an autoimmune disease. Figure 1a shows the structure of one of the MHC molecules with the amino acid (116) highlighted. We plan to determine differences between the dynamics of the two subtypes when complexed with different peptides. Labeling of all components of the complex has been established and the NMR experiments to determine the relaxation times have been carefully optimized. Assignments of β2m and of both heavy chains in four complexes have been obtained and evaluated in a qualitative manner, inspecting the line shape of amino protons. For the heavy chain we found differences within the molecule (the α3 domain is more rigid than the α1 / α2 domain with the peptide binding groove) and between subtypes; the latter is also true for the peptides, which appear to be extremely mobile. β2m has been investigated more extensively and Figure 1b shows an overlay of two 1 H, 15 N-HSQCs. While most of the peaks overlay perfectly some show shifts and reduced intensity, indicating mobility. They can be mapped onto the structure as shown in the lower part of Figure 1b; interestingly only the interface to the heavy chain is affected. A key player in the mobility is Trp-60 of β2m, which forms a hydrogen bond to Asp-122 in the heavy chain. While the indole peak of Trp-95 (the only other Trp in β2m, see Figure 1a) does not exhibit changes between subtypes and peptides, the peak of Trp-60 changes dramatically (Figure 1c), again indicating mobility at the interface between β2m and the heavy chain. stores are chemically similar but differ in some important structural features. In glycogen and starch, glucosyl residues are linked by only two types of glucosidic bonds, α-1,4- and α-1,6-linkages. The former are quantitatively dominant, giving rise to helical chains, while the latter constitute branching points. Native starch is a hydro-insoluble particle that typically consists of two types of α-polyglucans, amylopectin and amylose. By contrast, glycogen is a hydro-soluble and relatively homogeneous biomolecule. The carbohydrate stores starch and glycogen typically contain monophosphate esters. The glucose-based steady-state levels vary between species and organs but are generally low (<1 %). In glycogen metabolism in humans, the topic of phosphorylation of glycosyl residues is quite relevant since it is known that deficiency in a functional carbohydrate phosphatase (designated as Laforin) causes a severe type of epilepsy (Lafora disease). Given its significance for cellular processes and even disease, precise knowledge about the phosphorylation sites in starch and glycogen is crucial for an analysis of the enzymology of starch and glycogen phosphorylation. The determination of the phosphorylation site, even for complex carbohydrates, using NMR spectroscopy is in principle straightforward, although a complete resonance assignment may not be a trivial task due to the low dispersion of the 1H spectrum. Samples used for the determination of the phosphorylation sites in starch or glycogen, however, are usually obtained by enzymatic digestion followed by an enrichment of the phosphorylated glucans and due to the resulting heterogeneity an unambiguous assignment is impossible. We are using NMR spectroscopy to determine the phosphorylation sites and have developed a strategy that uses 1 H, 13 C heteronuclear correlation with extremely high resolution in combination with 1 H, 13 C, 31 P triple-resonance experiments. Figure 2a shows a multiplicity edited 1 H, 13 C-HSQC of a glycogen sample. The spectrum resulting from the triple-resonance experiment is shown in Figure 2b. Regions from the spectrum in (a), extracted at those points where there are peaks in (b), are shown in (c). The pattern of the peaks indicates the presence of phosphorylation and allows us to distinguish whether it is a site of direct phosphorylation or a neighboring position. Using this strategy the sites of phosphorylation in glycogen could be unambiguously determined. NMR-spectroscopic determination of glucan phosphorylation Almost all plants synthesize starch while most heterotrophic organisms rely on glycogen for carbohydrate storage. Both polyglucan

49 94 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 95 A B C A B C Fig. 1: Investigation of the dynamic of β2-microglubulin (β2m) in MHC class I complexes. (a) X-ray structure of HLA-B27*09 in complex with the peptide pvipr, β2m is shown in grey, the heavy chain in green and the peptide in a stick representation. Residue 116 (that is His in the 09-subtype while it is Asp in 05) as well as the two Trp residues of β2m are highlighted, in addition the region of β2m that is flexible in the complex is coloured in orange. (b) An overlay of two 1 H, 15 N-HSQCs from HLA-B27*09 in complex with two different peptides is shown in the upper part. While most of the peaks have similar intensity and overlay perfectly some are shifted and reduced in intensity. They can be mapped to the interface between β2m and the heavy chain (see lower part of the figure and (a)) (c) Region of the indole side chain H N peaks of the two Trp residues of β2m from several complexes of either B27*05 or B27*09 with a set of four peptides. While the peak from Trp-95 remains unchanged in all spectra, the peak of Trp-60 changes in a subtype and peptide dependent manner, indicating flexibility in the protein-protein interface. Fig. 2: Investigation of the phosphorylation of glycogen using heteronuclear triple resonance NMR spectroscopy. (a) multiplicity-edited 1 H, 13 C-HSQC of glycogen from mouse, methylene groups are shown in red, other peaks in black. (b) 31 P-edited 1 H, 13 C-HSQC of glycogen from mouse, the positions in which glycogen is phosphorylated (2, 3, 6) can be determined by comparison with model compounds and the spectrum shown in (c). (c) Peak pattern of a high-resolution 1 H, 13 C HSQC of glycogen from mouse, the pattern and the size of the coupling constants (given in Hz) allows to determine whether the peaks that appear in the spectrum shown in (b) result from a direct phosphorylation or a phosphorylation of the neighbouring position. GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Martin Ballaschk (doctoral student), EF, PT Monika Beerbaum (technical assistant), PT National A. Ziegler und B. Uchanska-Ziegler FMP-intern Anne Diehl Bertran-Vicente J, Serwa RA, Schumann M, Schmieder P, Krause E, Hackenberger CPR (2014) Site-specifically phosphorylated lysine peptides. Deutsche Forschungsgemeinschaft NMR spectroscopic investigation of micropolymorphism-dependent dynamics of human major histocompatibility Marcel Jurk (doctoral student), EF, PT Charité Universitätsmedizin Berlin Ronald Kühne J. Am. Chem. Soc. 136, antigens, SCHM 880 / 9-1, Peter Schmieder, Brigitte Schlegel (technical assistant) Dr. Peter Schmieder (group leader) E. Klussmann MDC Berlin U. Curth MH Hannover M. Steup Gerd Krause Michael Beyermann Christian Hackenberger Marc Nazaré Schmieder P, Nitschke F, Steup M, Mallow K, Specker E (2013). Determination of glucan phosphorylation using heteronuclear 1 H, 13 C double and 1 H, 13 C, 31 P triple-resonance NMR spectra. Magnetic Resonance in Chemistry 51, , (davon 1xE13 67 %) U Potsdam T. Niedermeyer U Tübingen Thomas Müller U Würzburg Nitschke F, Wang P, Schmieder P, Girard J-M, Awrey DE, Wang T, Israelian J, Zhao X, Turnbull J, Heydenreich M, Kleinpeter E, Steup M, Minassian BA (2013) Hyperphosphorylation of Glucosyl C6 Carbons and Altered Structure of Glycogen in the Neurodegenerative Epilepsy Lafora Disease. Cell metabolism 17, Hee C-S, Beerbaum M, Loll B, Ballaschk M, Schmieder P, Uchanska- Ziegler B, Ziegler A (2013) Dynamics of free versus complexed β2- microglobulin and the evolution of interfaces in MHC class I molecules. Immunogenetics 65, Beerbaum M, Ballaschk M, Erdmann N, Schnick C, Diehl A, Ziegler B, Ziegler A, Schmieder P (2013). NMR spectroscopy reveals unexpected structural variation at the protein protein interface in MHC class I molecules. J. Biomol. NMR 57, FMP authors Group members

50 96 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 97 MOLECULAR IMAGING Stefan Klippel and Leif Schröder (photo left) MOLEKULARE BILDGEBUNG Jörg Döpfert and Martin Kunth (photo right) GROUP LEADER DR. LEIF SCHRÖDER BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studies of Physics and Chemistry, Georg-August Universität Göttingen Studies of Physics and Astronomy, Ruprecht-Karls Universität Heidelberg, Diploma in Physics PhD student, Deutsches Krebsforschungszentrum and Ruprecht-Karls Universität Heidelberg, Dr. rer. nat Research Assistant, Deutsches Krebsforschungszentrum, Heidelberg Emmy Noether Fellow of the DFG, University of California, Berkeley Research Fellow, Lawrence Berkeley National Laboratory 2009 Emmy Noether Fellow of the DFG, Group Leader at FMP ERC Starting Grantee at FMP Basic research has made significant progress in understanding many diseases on a molecular level and opened up the field of molecular imaging and novel targeted drug delivery approaches. Both can be summarized in the concept of personalized medicine, based on the identification of multiple molecular markers and the delivery of functionalised drug carriers. Techniques for imaging the spatial distribution of such markers and monitoring drug delivery in a living organism are therefore highly sought-after in order to enable early diagnosis and individualized therapy. Limitations in sensitivity and / or specificity often restrict conventional diagnostic imaging to the search for mesoscopic changes in morphology. Drug development and therapeutic monitoring would therefore benefit significantly from specific contrast agents and sensors that work in opaque cellular environments and which are non-invasive to allow for repetitive studies. This encourages the use of NMR as the detection modality. Improving the intrinsic low sensitivity of NMR is the central task of our work. The ERC Project BiosensorImaging aims to establish a novel approach to magnetic resonance imaging (MRI). Xenon biosensors have an outstanding potential to contribute to this field and advance diagnostic imaging and NMR sensing. Our group develops methodologies to specifically detect dilute molecular targets within a few minutes that would otherwise require hundreds of years. ZUSAMMENFASSUNG Der Fortschritt der Grundlagenforschung ermöglicht es heute, viele Krankheiten im molekularen Detail zu verstehen. Diese Entwicklung eröffnete gleichermaßen das Feld der molekularen Bildgebung und machte neuartige Methoden zugänglich, Wirkstoffe gezielt an ihren Wirkort zu bringen. Beides macht nunmehr die Entwicklung des Konzeptes einer personalisierten Medizin möglich, die auf einer Identifizierung multipler molekularer Marker und auf der Nutzung funktionalisierter Wirkstoffcarrier fußt. Techniken zur Visualisierung der räumlichen Verteilung solcher Marker und zur Verfolgung der Wirkstoffaufnahme in einem Organismus sind für eine frühzeitige Diagnostik und eine individualisierte Therapie äußerst erstrebenswert. Grenzen bei der Sensitivität und / oder Spezifität schränken oft die Möglichkeiten konventioneller diagnostischer Bildgebungsverfahren ein, nach mesoskopischen Veränderungen in der Morphologie zu suchen. Die Wirkstoffentwicklung und das Monitoring von Therapien würde daher erheblich von spezifischen Kontrastmitteln und Sensoren profitieren, die nicht-invasiv im opaken zellulären Umfeld funktionieren und daher wiederholte Untersuchungen möglich machen. Diese Randbedingungen legen die Nutzung der Kernspinresonanz (NMR) als Detektionsmethode nahe. Unsere Arbeit auf diesem Feld beschäftigt sich vor allem ganz zentral mit einer signifikanten Verbesserung der niedrigen intrinsischen Sensitivität von NMR. Das ERC-Projekt BiosensorImaging beschäftigt sich mit der Etablierung eines neuartigen Ansatz der Bildgebung mittels Magnetresonanz (MRT). Xenon-Biosensoren haben hier ein außergewöhnliches Potential, die Entwicklung der diagnostischen Bildgebung durch NMR voranzutreiben. Unsere Arbeitsgruppe entwickelt in diesem Zusammenhang Methoden, die es möglich machen, verdünnte molekulare Ziele in wenigen Minuten spezifisch zu detektieren, obwohl deren Messung mit konventionellen Methoden Hunderte von Jahren benötigen würde. Quantitative Hyper-CEST The reversible binding of xenon to host structures is the key element of the detection technique Hyper-CEST (chemical exchange saturation transfer with hyperpolarized nuclei). It provides unprecedented sensitivity to diag nostic imaging. Quantitative characterization of the exchange dynamics is important for understanding and optimizing the physicochemical behaviour of such xenon hosts. With qhyper-cest we have established a sensitive quantification technique for this purpose. The method yields accurate and precise results and is robust in the presence of large amounts of noise (10 %), an aspect that is of particular importance for conditions with completely unknown exchange rates. Taken together, qhyper-cest facilitates sensitive quantification of Xe exchange dynamics and makes it an indispensable tool for the efficient design of highly specific biosensors. Cellular Labelling Despite its great potential in overcoming sensitivity limitations for solution-state NMR detection, MRI with hyperpolarized xenon as a functionalized contrast agent was not performed with live cells until We demonstrated the first MRI localization of cells labelled with caged xenon in an NMR-compatible bioreactor. This was made possible through a comprehensive approach working on aspects of physics, chemistry, and cell biology. We were able to overcome limitations such as the required high sensor concentration, long image acquisition times, narrow design approaches for functionalised Xe hosts, and very limited experience with caged Xe in cellular environments. Our NMR / MRI experiments come with switchable contrast and selectivity for cell-associated versus unbound cages. This allowed us to obtain MR images with fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence / MRI) xenon hosts at low micromolar concentrations. This project served as a stepping stone for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications. Progress for live cell studies was in general enabled by various fast acquisition techniques that our group established for NMR of dissolved xenon. Modular, antibody-based design of bimodal contrast agents Subsequent to the initial work of unspecific cellular labelling, a suitable approach for the design of targeted sensors was required since the field of xenon MRI is moving closer to in vivo applications. In this project, we capitalize on our previous improvements regarding efficient production of hyperpolarized xenon and fast MRI techniques by demonstrating targeted xenon imaging of cells using a modular xenon biosensor. Building blocks containing reporters for both MRI and fluorescence readout were coupled to antibodies designed for easy cross-validation and adaptation to novel targets. With this method, we can detect target cells with as little as 20 nm of our xenon contrast agent. Imaging of such low levels of cell-specific xenon hosts is unprecedented and reinforces the potential of xenoncryptophane biosensors for molecular imaging applications. Labelled liposomal systems in Xe NMR We also extended the use of functionalized xenon to initial studies of tracking targeted liposomal carriers. In general, delivery of therapeutic and diagnostic agents to the brain is hampered by the highly selective blood brain barrier (BBB). Targeting the BBB with MRI contrast agents is of significant interest for mapping the BBB and for enabling the control and imaging of drug delivery to the brain. We demonstrated selective imaging of microvascular endothelial cells of the BBB in vitro. A carrier system based on functionalized large unilamellar vesicles (LUVs) was used to deliver cryptophane as a xenon host in a biocompatible and selective way into these cells. An LUV concentration of M generates sufficient contrast in Hyper-CEST MRI to distinguish target cells from control cells. The approach transfers the challenge of direct chemical modification of cryptophane for targeting purposes to well-studied nanoparticulate carrier systems for drug delivery. Furthermore, we used Xe NMR to investigate biomembranes possessing different fluidities. By varying the Hyper-CEST saturation parameters and utilizing an inverse Laplace transform we can determine depolarization times for the noble gas in different local environments. We also extended this technique to magnetic resonance imaging, mapping the spatial distribution of the different biomembranes. This approach will provide further insights into saturation transfer dynamics of reversibly bound Xe, with applications extending into biomedical diagnostics such as the testing of pore formation through cytolytic peptides. Leif Schröder and Federica Rossella

51 98 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 99 Fig. 1: Summary of selected achievements during Fig. 2: Specific detection of cell surface proteins by xenon magnetic resonance Matthias Schnurr, Honor Rose and Jabadurai the ERC project to advance xenon biosensor MRI imaging (MRI). Illustrated is the use of hyperpolarized xenon gas (purple), in Jayapaul (photo above), Leif Schröder and Chris Witte on all experimental levels with the ultimate goal to combination with cryptophane cages (pale blue) which are attached to cells via achieve sufficient in vivo sensitivity. antibodies. During the MRI experiment a unique radio frequency pulse (red) is used to selectively image and light up the surface of macrophage cells. (Image courtesy of Barth-Jan van Rossum, FMP). GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Jabadurai Jayapaul International Salim Seyfried Kunth M, Witte C, Schröder L (2014) Quantitative Chemical Exchange European Union, 7. Framework Programme, ERC, Biosensor-Imaging : Dr. Honor Rose Alexander Pines Max Delbrück Center for Molecular Saturation Transfer with Hyperpolarized Nuclei (qhyper-cest): Hyperpolarized Biosensors in Molecular Imaging, ERC Starting Grant, Dr. Christopher Witte Lawrence Berkeley National Laboratory and Medicine, Berlin Sensing Xenon-Host Exchange Dynamics and Binding Affinities by ; Jörg Döpfert (doctoral student) Stefan Klippel (doctoral student) Martin Kunth (doctoral student) Federica Rossella (doctoral student) Matthias Schnurr (doctoral student) University of California at Berkeley, USA David Wemmer University of California at Berkeley, USA Matthew Francis University of California at Berkeley, USA Hartmut Kühn Institute of Biochemistry, Charité, Berlin Peter Bachert Medical Physics in Radiology, German Cancer Research Center (DKFZ), Heidelberg Daniel Messroghli NMR. J. Chem. Phys. 141, Schnurr M, Sydow K, Rose HM, Dathe M, Schröder L (2014) Brain Endothelial Cell Targeting via a Peptide-functionalised Liposomal Carrier for Xenon Hyper-CEST MRI. Adv. Healthcare Mat. 4, (cover article). Leibniz-Gemeinschaft, Vorhaben im Rahmen des Pakts für Forschung und Innovation, Development of Novel NMR Probes: Improving Cell Profiling for Early Diagnosis, SAW, with C. Freund, ; International Human Frontiers Science Program Organization, Cell Pro- National Christian Freund Protein Biochemistry, Freie Universität Berlin Rainer Haag Macromolecular Chemistry, Freie Universität Berlin Andreas Hennig / Ute Resch-Genger Biophotonics, Federal Institute for Materials Department of Congenital Heart Disease / Pediatric Cardiology, Deutsches Herzzentrum Gerhard Wenz Organische Makromolekulare Chemie, Universität des Saarlandes Bettina Kracke / Thorsten Hugel Zentralinstitut für Medizintecknik IMETUM, Technische Universität München Rose HM, Witte C, Rossella F, Klippel S, Freund C, Schröder L (2014) Development of an antibody-based, modular 129 Xe NMR biosensor for molecular imaging applications of cells at nanomolar concentrations. Proc. Natl. Acad. Sci. USA 111, Klippel S, Döpfert J, Jayapaul J, Kunth M, Rossella F, Schnurr M, Witte C, Freund C, Schröder L (2014) Cell Tracking with Caged Xenon: Using Cryptophanes as MRI Reporters upon Cellular Internalization. Angew. Chem. Int. Ed. 53, (back cover article). filing with Xenon Biosensors, Long-Term Postdoctoral Fellowship for Christopher Witte, ; Various conference travel grants for the Experimental Nuclear Magnetic Resonance Conference 2013, the EUROMAR 2013 conference, the World Molecular Imaging Congress 2013 and 2014, DAAD Fellowships, and Fellowships by the Foundation Lindau Nobel Laureate Meetings Research and Testing (BAM), Berlin Schnurr M, Witte C, Schröder L (2013) Functionalized 129 Xenon as a Potential Biosensor for Membrane Fluidity. Phys. Chem. Chem. Phys. 15, (inside front cover article). FMP authors Group members

52 100 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 101 IN-CELL NMR NMR IN ZELLEN GROUP LEADER DR. PHILIPP SELENKO BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS 2002 Ph.D. at the European Molecular Biology Laboratory (EMBL), Heidelberg Post-Doc at Harvard Medical School EMBO fellowship Human Frontiers in Science (HFSP) fellowship Max Kade fellowship by the Austrian Academy of Science Since 2007 Group Leader in the Structural Biology Section of the FMP, Emmy Noether fellowship by the Deutsche Forschungsgemeinschaft (DFG) We employ high-resolution in-cell NMR spectroscopy to study the structural and functional properties of proteins in intact cells. This tool allows us to directly visualize proteins in their native intracellular environments as they carry out their individual biological functions. By doing so, we obtain novel mechanistic insights into previously unknown cellular aspects of protein functions in health and disease, thereby opening new routes for drug discovery and therapeutic intervention. ZUSAMMENFASSUNG Wir untersuchen strukturelle und funktionelle Eigenschaften von Proteinen in lebenden Zellen mittels hochauflösender NMR-Spektroskopie an lebenden Zellen. Diese Methode erlaubt eine unmittelbare Visualisierung von Proteinen bei der Arbeit in ihren natürlichen zellulären Umgebungen. Dadurch gewinnen wir neuartige mechanistische Einsichten in bisher unbekannte zelluläre Aspekte von Proteinfunktionen. Unsere Untersuchungen an gesunden und an kranken Zellen erlauben so die Entwicklung neuer Ansätze für das Aufspüren potentieller neuer Pharmaka und neue pharmakologische Therapien. One question that we address with in-cell NMR spectroscopy is how Intrinsically Disordered Proteins, or IDPs, behave in cells. This class of proteins does not exhibit any of the classical features of folded proteins when studied in isolation; however, their intracellular structures are not known. Because many IDPs participate in key biological processes, knowledge about their three-dimensional in vivo conformations is of fundamental importance. One such protein is the human tumorsuppressor and oncoprotein p53, which is mutated in over 90 % of all cancers. We recently demonstrated that intrinsic disorder and residual helicity of p53 is critically required for its physiological role in response to DNA damage, namely to induce cell cycle arrest and to facilitate DNA repair (Figure 1). We were able to show that by making p53 more structured and less disordered in cells, we completely abolished its ability to stop cells from dividing, which meant that damaged DNA was no longer repaired and, instead, passed on to newborn daughter cells. This enabled the spreading of genetic mutations, one of the key aspects of cancer development. These results showed, for the first time, that defined levels of structural disorder are important determinants of signaling fidelity in cells and that altering these levels of structural disorder can have deleterious consequences for cell viability. Other projects in the lab deal with IDPs that play important roles in human neurodegenerative disorders. In this regard, we are particularly interested in a neuronal IDP called alpha-synuclein, which aggregates in the course of Parkinson s disease (PD) and forms insoluble amyloid fibrils in dopaminergic neurons of PD patients. Using in-cell NMR spectroscopy, we investigate how different neuronal and non-neuronal intracellular environments affect the structure of alpha-synuclein (Figure 2) and how they promote aggregation under certain cellular conditions. Here, our goal is to derive a comprehensive structural understanding of intracellular amyloid formation. Induced DNA Damage Altered p53 Dynamics Fig. 1: Role of disorder and residual helicity for the biological function of p53. Disorder of the N-terminal transactivation domain (TAD) of human p53 ensures oscillatory p53 expression, target gene expression and cell cycle arrest in cells upon DNA damage (top row). Increasing p53 TAD helicity by site-directed mutations leads to altered intracellular p53 dynamics, reduced target gene expression and failure to induce cell cycle arrest (bottom row). wt p53 TAD Efficient Target Gene Expression Cell Cycle Arrest Helical p53 TAD Mutants Reduced Target Gene Expression No Cell Cycle Arrest

53 102 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 103 Marchel Stuiver (photo left), Francois-Xavier Theillet Fig. 2: alpha-synuclein structure and dynamics in cells and crowded in vitro environments. In-cell NMR relaxation measurements in human A2780 cells (top row) and in Ficoll-, BSA- and Lysozyme-crowded solutions (below), reveal site-specific, weak interactions with intracellular components of N- and C-terminal alpha-synuclein (αsyn) residues (expressed as Exchange Contributions). Paramagnetic relaxation enhancement (PRE) measurements delineate volume dimensions and overall structural features of intracellular αsyn (right columns). Marleen van Rossum and Akis Liokatis GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Andres Binolfi International National Borcherds W, Theillet FX, Katzer A, Finzel A, Mishall KM, Powell A, Deutsche Forschungsgemeinschaft, The protein disorder paradox: Dr. Stamatios Liokatis Gary Daughdrill Wolfgang Fischle Wu H, Manieri W, Dieterich C, Selenko P, Loewer A, Daughdrill G What do natively unfolded proteins look like inside living cells?, Dr. Honor May Rose University of South Florida, USA Max Planck Institute of Biophysical (2014) Disorder and residual helicity alter p53-mdm2 binding affinity SE 1794 / 1 1, III., , Dr. Francois-Xavier Theillet Beata Bekei (doctoral student) Jonas Kosten (doctoral student) Silvia Verzini (doctoral student) Marleen van Rossum (technical assistant) Marchel Stuiver (technical assistant) Peter Crowly University of Galway, Ireland Lila Gierasch University of Massachusetts, Amherst, USA Ann Gershensons University of Massachusetts, Amherst, USA Chemistry, Göttingen Alexander Löwer Max-Delbrück-Center for Molecular Medicine, Berlin and signaling in cells. Nat. Chem. Biol. 12, Kosten J, Binolfi A, Stuiver M, Verzini S, Theillet FX, Bekei B, van Rossum M, Selenko P (2014) Efficient modification of alpha-synuclein Serine 129 by protein kinase CK1 requires phosphorylation of Tyrosine 125 as a priming event. ACS Chem. Neurosci. 5, Europäischer Forschungsrat (8. Forschungsrahmenprogramm), In-cell NMR monitoring of alpha-synuclein aggregation in neuronal cells, ERC-CoG-2014, , Gary Pielak University of North Carolina, Chapel Hill, USA Theillet FX, Binolfi A, Frembgen-Kesner T, Hingorani K, Sarkar M, Kyne C, Li C, Crowley PB, Gierasch L, Pielak GJ, Elcock AH, Gershenson A, Selenko P (2014) Physicochemical properties of cells Adrian Elcock and their effects on intrinsically disordered proteins (IDPs). University of Iowa, Iowa City, USA Chem. Rev. 144, Daron Freedberg Federal Food and Drug Administration, Washington DC, Maryland, USA Freedberg DI, Selenko P (2014) Live cell NMR. Annu. Rev. Biophys. 43, Theillet FX, Rose HM, Liokatis S, Binolfi A, Thongwichian R, Stuiver M, Selenko P (2013) Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts. Nat. Protoc. 8, FMP authors Group members

54 104 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 105 CORE FACILITY A B Fig. 1: Investigation of the decomposition of Phospholysin using a real-time NMR approach. The stability of Phospholysin in aqueous solution was tested using different ph-values. (a) Superposition of a region of two proton spectra of a Phospholysincontaining peptide recorded before and after a period of 20 hours, the intensity changes of the signals corresponding to the phosphorylated and unphosphorylated form can be clearly seen. (b) Decay of the signal of the phosphorylated form extracted from pseudo-2d-nmr-experiment. (Cooperation with J. Bertran-Vicente and C.P.R. Hackenberger) NMR GROUP LEADER HARTMUT OSCHKINAT PETER SCHMIEDER A B SUMMARY The NMR facility was reorganized as a core facility to allow a better organisation of the many requests from inside and outside the FMP, either for the use of the DNP-equipment, the in-cell NMR facilities, or the NMR machines in general. This has eased participation in the European Bio-NMR network as well as the German DFG-funded G-NMR network of facilities. The NMR core facility serves as a platform for the use of NMR spectrometers by non-departmental groups. These include, for example, FMP groups that perform chemical synthesis and thus heavily rely on continuous NMR support as well as groups studying the interaction of proteins with peptides or small molecules that benefit from the strength of NMR to detect weak protein-ligand interactions. In addition, there are requests from groups outside of the FMP for access to the NMR facility, some of which are organized via the European Bio-NMR network. Requests include those for access to the DNP spectrometer and expertise regarding in-cell NMR. In addition, groups from elsewhere in Berlin and from other cities in the north-east of Germany use the equipment of the facility if comparable resources are not available at their own sites. A variety of results have been produced by collaborations in the context of the NMR core facility. One such example of an in-house collaboration is shown in Figure 1. Phosphorylation is a key event in many cellular processes but up until now it is the phosphorylation of Ser, Thr and Tyr that has mainly been investigated. Other amino acids can be phosphorylated as well and the stability of Phospholysine was investigated with the group of Christian Hackenberger using NMR to monitor the reduction in the rate of the phosphorylation in real-time using a pseudo-2d-nmr experiment. A study of a peptideprotein interaction is exemplified in Figure 2. The interaction between the C-terminal peptide of the single-strand DNA binding protein with the C-terminal helicase-binding domain of DnaG primase revealed the binding site of the peptide on the protein. Fig. 2: Investigation of the interaction of the C-terminal helicase-binding domain of DnaG primase with the C-terminal peptide of the single-strand DNA binding protein (SSB-ct). (a) 1 H, 15 N-HSQC titration of the protein with increasing amounts of SSB-ct. (b) The magnitude of the chemical shift changes in the titration experiment is mapped onto the structure of the protein, indicating the binding site. (Cooperation with N. Naue and U. Curth) ZUSAMMENFASSUNG Die NMR-Einrichtung am FMP wurde in eine Core Facility umgewandelt, um die Vielfalt von Anfragen von inner- und außerhalb des FMP besser organisieren zu können. Diese Anfragen betreffen die Nutzung spezieller Geräte wie etwa der Instrumente für Dynamic Nuclear Polarization (DNP), sowie vorhandene NMR-Spektrometer im Allgemeinen. Durch die Etablierung der Core Facility NMR konnte eine Teilnahme am europäischen Bio-NMR Projekt und dem deutschen, DFG-geförderten G-NMR Netzwerk bedeutend erleichtert werden. Die NMR Core Facility dient Forschergruppen als Plattform für die Nutzung der NMR Spektrometer, die nicht dem Bereich Strukturbiologie des FMP angehören. Der Nutzerkreis umfasst beispielsweise chemische Synthesegruppen, für die eine kontinuierliche Unterstützung durch NMR essentiell ist, aber auch Gruppen, die die Wechselwirkung von Proteinen mit Peptiden oder kleinen Molekülen untersuchen, und dabei von NMR-Techniken zur Untersuchung schwacher Protein-Liganden-Wechselwirkungen profitieren. Hinzu kommen Anfragen von Arbeitsgruppen außerhalb des FMP, von denen einige im Bio-NMR-Netzwerk organisiert sind. Hier handelt es sich um Anfragen für die Nutzung von DNP oder von In-Zell-NMR. Zudem ermöglicht die Core Facility Institutionen aus Berlin und anderen Städten im Nordosten Deutschlands Zugang zu NMR-Techniken, die an den eigenen Standorten nicht verfügbar sind. Dies hat zu einer steigenden Zahl an Forschungsergebnissen in Kooperation mit der NMR Core Facility des FMP geführt. SELECTED PUBLICATIONS Bertran-Vicente J, Serwa RA, Schümann M, Schmieder P, Krause E, Hackenberger CPR (2014) Site-specifically phosphorylated lysine peptides. J. Am. Chem. Soc. 136, Niedermeyer TH, Schmieder P, Kurmayer R (2014) Isolation of Microcystins from the Cyanobacterium Planktothrix rubescens Strain No80. Nat. Prod. Bioprospect. 4, Mühlberg M, Siebertz KD, Schlegel B, Schmieder P, Hackenberger CPR (2014) Controlled thioamide vs. amide formation in the thioacidazide reaction under acidic aqueous conditions. Chem. Commun. 50, Schäfer G, Milic J, Eldahshan A, Gotz F, Zühlke K, Schillinger C, Kreuchwig A, Elkins JM, Abdul Azeez KR, Oder A, Moutty MC, Masada, N, Beerbaum M, Schlegel B, Niquet S, Schmieder P, Krause G, von Kries JP, Cooper DM, Knapp S, Rademann J, Rosenthal W, Klussmann E (2013) Highly Functionalized Terpyridines as Competitive Inhibitors of AKAP-PKA Interactions. Angew. Chem. Int. Ed. Engl. 52, Naue N, Beerbaum M, Bogutzki A, Schmieder P, Curth U (2013) The helicase-binding domain of Escherichia coli DnaG primase interacts with the highly conserved C-terminal region of single-stranded DNAbinding protein. Nucleic Acids Res. 41, Hack V, Reuter C, Opitz R, Schmieder P, Beyermann M, Neudörfl J-M, Kühne R, Schmalz H-G (2013). Efficient α-helix Induction in a Linear Peptide Chain by N-Capping with a Bridged-tricyclic Diproline Analogue. Angew. Chem. Int. Ed. Engl. 52, FMP authors Group members

55 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE Screening Unit Group leader Dr. Jens Peter von Kries PAGE 126 Medicinal Chemistry Medizinische Chemie Group leader Dr. Marc Nazaré Mass Spectrometry Massenspektrometrie Group leader Dr. Eberhard Krause PAGE 118 PAGE 122 Chemical Biology II Chemische Biologie II Group leader Prof. Dr. Christian P.R. Hackenberger PAGE 110 CHEMICAL BIOLOGY SECTION BEREICH CHEMISCHE BIOLOGIE Peptide-Lipid Interaction / Peptide Transport Peptid-Lipid-Interaktion / Peptidtransport Group leader Dr. Margitta Dathe PAGE 114 Peptide Synthesis Peptidsynthese Group leader Dr. Rudolf Volkmer PAGE 130

56 108 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 109 Research projects in this section apply innovative synthetic and diagnostic chemical methods to probe the biological function of cellular target molecules and thereby pave the way towards novel approaches in the pharmaceutical and medicinal sciences. Work of the groups is devoted both to the synthesis and identification of novel bioactive molecules of high pharmacological potency and the development of new chemical and analytical tools for the functional study of biologically relevant cellular proteins. During the reporting period, the Chemical Biology section successfully advanced its research activities as well as its service facilities, which were extended in 2011 into a comprehensive Chemical Biology Platform for the development of qualified small molecule tools serving projects from inside and outside the institute. After already several successful recent recruitments, Dorothea Fiedler (Princeton University) has accepted the call for the vacant Chemical Biology chair and director position at FMP and will start in summer With the planned installation of a Chemical Biology junior group, the restructuring process of this section will be completed. The Chemical Biology section is currently headed by Christian Hackenberger, who was appointed Leibniz-Humboldt professor for Chemical Biology in December Research within his department Chemical Biology II is aimed at the synthesis of functional peptides and proteins by combining advanced techniques of organic synthesis with biochemical and biophysical approaches. These chemical tools contributed within the last two years to the engineering of new pharmaceutically active biopolymers and the understanding of posttranslational modifications (PTMs) in protein function. Examples included small PEGylated peptides with intracellular activity, the identification of new targets for medicinal chemistry research in the area of neurodegenerative diseases and new synthetic approaches for the conjugation of ligands for targeted drug delivery. Another recent highlight was performed in close collaboration with the group of Eberhard Krause, who leads the Mass Spectrometry group at the FMP and focuses on high resolution proteomic studies, with a particular focus on the role of protein-protein interactions and PTMs in cell signalling. The two laboratories contributed the first synthesis and MS-analysis of site-specifically phosphorylated lysine peptides. The phosphorylation of lysine is one of the least studied, because labile PTMs with high significance in the regulation of histone function. The Peptide-Lipid Interaction / Peptide Transport group led by MargittaDathe has a long-standing expertise in the development and clinical application of peptide-modified liposomal carriers. In cooperation with the Universität Münster, an enzyme substitution therapy for the rare skin disorder Transglutaminase 1-deficient autosomal recessive Congenital Ichthyosis was developed, which was granted orphan designation by the European Commission in An important aim within the Chemical Biology Platform at the FMP is the chemical optimization and validation of first hits discovered in the SECTION CHEMICAL BIOLOGY BEREICH CHEMISCHE BIOLOGIE small molecule screening of pharmacological targets. The Medicinal Chemistry group led by Marc Nazaré contributed to the development of new chemical tools using strategies like fragment growing, rescaffolding approaches and structure-based design to improve the initial screening hit. These efforts led to new small molecule probes for different kinases, the phosphatase SHP2, tryptophanhydroxylase TPH, and the Poly-ADP-ribosyltransferase tankyrase 1, with some of them now being profiled and progressed to an in vivo evaluation. Moreover, the FMP small molecule screening collection was considerably enhanced to now compounds by incorporation of a new approved drug sub-set and a natural product-derived library. Furthermore, the research activities of the Chemical Biology section are intimately interconnected with most groups in the other sections, particularly the Structural Bioinformatics and Protein Design group led by Gerd Krause and the Drug Design group led by Ronald Kühne with its computational chemistry approaches (both in the Structural Biology section). Significant effort has been invested to provide professional core facilities, in particular the internationally recognized Screening Unit led by Jens von Kries, which is devoted to high-throughput screening of small molecule and RNAi libraries. Scientific highlights of research within the Screening Unit have been the establishment of drug screens with zebrafish embryos and the resulting identification of approved drugs, which rescue heart development. Furthermore, the group has established genome-wide RNA interference in combination with high-content screening for compounds, which are able to rescue defective regulation of cellular water content. The Screening Unit also has served as a central anchoring point for the collaborative activities with the Leibniz research network Drug Research and Biotechnology, the Helmholtz consortium Drug Research Initiative and the neighboring Max-Delbrück-Center for Molecular Medicine. In addition, the Screening Unit provides a focal point of the Chemical Biology Platform, which itself is the central node of the European initiative EU-OPENSCREEN of the ESFRI roadmap, coordinated at the FMP by Ronald Frank. In April 2013, EU-OPEN- SCREEN was officially included into the German Roadmap for Large Research Infrastructures, which also documents the willingness of the Federal Ministry of Education and Research (BMBF) to financially support the future European infrastructure and upgrade of the Berlin site for the operational phase of the network starting in The Chemical Biology Platform and Screening Unit will furthermore support translational projects of the Berlin Institute of Health (BIH). Overall, the Chemical Biology section has contributed important chemical methods and discoveries thus providing privileged access to pharmaceutically active substances through the synthesis, identification / screening, and optimization of new (small) molecules for studies of underexplored protein targets. These assets ideally support the core mission of the FMP to develop new principles at the molecular level for the pharmacological intervention with biological processes that ultimately shall lead to new avenues in the treatment of diseases. Forschungsprojekte aus diesem Bereich dienen der Untersuchung der biologischen Funktion zellulärer Zielmoleküle (Targets) mit innovativen synthetischen und diagnostischen Techniken der Chemie. Sie sollen den Zugang zu neuartigen Ansätzen in Pharmazie und Medizin eröffnen. Die Arbeiten der beteiligten Gruppen widmen sich der Synthese und Identifizierung neuartiger biologisch aktiver Moleküle mit hohem pharmakologischen Potential sowie der Entwicklung neuer chemischer und analytischer Werkzeuge zur funktionellen Analyse biologisch relevanter Proteine. Während des Berichtszeitraums hat der Bereich seine Forschungsaktivitäten sehr erfolgreich weiterentwickelt. Seine Serviceeinheiten wurden 2011 in eine umfassende Plattform für Chemische Biologie ( Chemical Biology Platform ) integriert, die der Entwicklung validierter kleiner Moleküle zum Einsatz als Werkzeug in Projekten innerhalb des Instituts und in Kooperationsprojekten mit anderen Institutionen dient. Im Berichtszeitraum wurde eine Reihe von Stellen erfolgreich und zielführend besetzt. Insbesondere die Berufung von Dorothea Fiedler (Princeton University) als Bereichssprecherin und Direktorin am FMP ist hier zu nennen. Frau Fiedler wird im Sommer 2015 mit ihrer Abteilung die Arbeit am Institut aufnehmen. Mit der geplanten Etablierung einer Nachwuchsgruppe Chemische Biologie wird schließlich die Umstrukturierung des Bereiches abgeschlossen sein. Christian Hackenberger, der im Dezember 2012 als Leibniz-Humboldt- Professor berufen wurde, ist derzeit kommissarischer Sprecher des Bereichs Chemische Biologie. Die Forschung in seiner Abteilung Chemische Biologie II hat ihren Fokus auf der Synthese funktioneller Peptide und Proteine durch Kombination modernster Techniken der organischen Chemie mit biochemischen und biophysikalischen Methoden. Die so zum Einsatz kommenden chemischen Werkzeuge haben in den letzten zwei Jahren entscheidend dazu beigetragen, neue pharmakologisch aktive Biopolymere zu generieren und die Bedeutung posttranslationaler Modifikationen (PTMs) für die Proteinfunktion zu verstehen. Beispiele sind hier kleine PEGylierte Peptide mit intrazellulärer Aktivität, die Identifizierung neuer Targets für die medizinalchemische Forschung im Kontext neurodegenerativer Erkrankungen sowie neue synthetische Methoden zur Konjugation von Liganden für eine zielgerichtete Zuführung von Wirkstoffen zu ihren Targets. Ein weiteres wissenschaftliches Highlight entstand in enger Zusammenarbeit mit Eberhard Krause, der die Arbeitsgruppe Massenspektrometrie am FMP leitet. Diese Gruppe legt ihren Fokus auf hochaufgelöste Proteomstudien und hier insbesondere auf die Rolle von Protein-Protein-Wechselwirkungen und PTMs. Den Gruppen gelang es gemeinsam, erstmalig positionsspezifische Phospholysinpeptide zu synthetisieren und massenspektrometrisch zu analysieren. Die Phosphorylierung von Lysinresten resultiert in einer labilen PTM, die daher eine der am wenigsten untersuchten Modifikationen ist, jedoch hohe Bedeutsamkeit für die Funktion von Histonen besitzt. Die Arbeitsgruppe Peptid-Lipid-Interaktion / Peptidtransport von Margitta Dathe besitzt seit Langem große Expertise in der Entwicklung und klinischen Anwendung von peptidmodifizierten liposomalen Carriern. Die Arbeitsgruppe entwickelte in Kooperation mit der Universität Münster eine Enzymsubstitutionstherapie für die seltene Hautkrankheit Transglutaminase 1-deficient autosomal recessive Congenital Ichthyosis, der 2013 durch die Europäische Kommission eine Orphan Drug -Klassifizierung zuerkannt wurde. Ein sehr wichtiges Ziel der Chemical Biology Platform des FMP ist die chemische Optimierung und die Validierung von biologisch wirksamen kleinen Molekülen (Hits), die initial durch Screening kleiner Moleküle gegen pharmakologische Targets identifiziert werden. Die Arbeitsgruppe Medizinische Chemie von Marc Nazaré ist an der Entwicklung neuer chemischer Werkzeuge beteiligt. Sie nutzt Strategien wie Fragmentwachstum, Neuanordnung von Molekülgerüsten und strukturgeleitetes Design zur Optimierung der Screening-Hits. Die Arbeiten der Gruppe resultierten in neuen kleinen Molekülen, die als Sonden zur Beeinflussung verschiedener Kinasen, der Phosphatase SHP2, der Tryptophanhydroxylase TPH oder der Poly-ADP-Ribosyltransferase Tankyrase 1 wirken. Einige dieser Substanzen werden gegenwärtig physikalisch-chemisch charakterisiert bzw. einer Untersuchung in vivo zugeführt. Zudem wurde die Substanzsammlung des FMP für das Small Molecule Screening durch die Integration neuer Sammlungen zugelassener Medikamente und einer Naturstoff-Sammlung erheblich auf nunmehr Substanzen erweitert. Die Forschungsaktivitäten des Bereiches Chemische Biologie sind eng mit vielen Gruppen der anderen Bereiche verzahnt und verknüpft. Besonders sind hier aus dem Bereich Strukturbiologie die Strukturelle Bioinformatik und Proteindesign -Gruppe von Gerd Krause und die Wirkstoff- Design -Gruppe von Ronald Kühne mit ihren Methoden der Computer-gestützten Chemie zu erwähnen. Erhebliche Anstrengungen wurden unternommen, um hochprofessionelle Core Facilities, insbesondere die international anerkannte Screening Unit unter Leitung von Jens von Kries, aufzubauen. Die Screening Unit ist auf High-Throughput Screening von kleinen Molekülen und von RNAi-Bibliotheken spezialisiert. Wissenschaftlich herausragend ist hier die Durchführung eines Wirkstoff-Screens an Zebrafischembryos, der zur Identifizierung zugelassener Medikamente führte, die Defekte in der Entwicklung des Herzens beheben können. Zudem hat die Arbeitsgruppe genomweit RNA- Interferenz in Kombination mit einem High-Content Screen für solche Substanzen durchgeführt, die einer fehlerhaften Regulation des zellulären Wassergehaltes entgegenwirken. Die Screening Unit dient weiterhin als Dreh- und Angelpunkt für Forschungskooperationen im Leibniz Forschungsverbund Wirkstoffforschung und Biotechnologie, im Helmholtz-Konsortium Wirtstoffforschung und mit dem benachbarten Max-Delbrück-Centrum für Molekulare Medizin (MDC). Gleichzeitig ist die Screening Unit das Zentrum der Chemical Biology Platform des FMP, die ihrerseits eine zentrale Rolle in der von Ronald Frank am FMP koordinierten Initiative zur Einrichtung der europäischen Forschungsinfrastruktur EU-OPENSCREEN der ESFRI-Roadmap spielt. Seit April 2013 ist diese Initiative zudem Teil der deutschen Roadmap für große Forschungsinfrastrukturen, was die Bereitschaft des Bundesministeriums für Bildung und Forschung (BMBF) zeigt, die zukünftige europäische Infrastruktur finanziell zu fördern und die Berliner Einrichtungen von EU-OPENSCREEN für die 2016 beginnende Arbeitsphase von EU-OPENSCREEN zu ertüchtigen und auszubauen. Außerdem werden Screening Unit und die Chemical Biology Platform translationale Projekte des Berliner Instituts für Gesundheitsforschung (BIH) unterstützen. Insgesamt hat der Bereich Chemische Biologie wichtige chemische Methoden entwickelt und Entdeckungen gemacht, die einen einzigartigen Zugang zu pharmakologisch aktiven Substanzen durch Synthese, Identifikation / Screening und die chemische Optimierung neuer kleiner Moleküle für Studien an wenig erforschten zellulären Targets ermöglicht. Durch seine Ergebnisse unterstützt der Bereich den Kernauftrag des FMP in optimaler Weise, neue molekulare Prinzipien der pharmakologischen Beeinflussung biologischer Vorgänge zu erarbeiten, die letztendlich neue Wege in der Behandlung von Krankheiten eröffnen werden.

57 110 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 111 CHEMICAL BIOLOGY II CHEMISCHE BIOLOGIE II GROUP LEADER PROF. DR. CHRISTIAN P.R. HACKENBERGER Fig 1: Conjugation of functional modules to peptides and proteins (Reference: D. Schumacher, C.P.R. Hackenberger, Curr. Opin. Chem. Biol. 2014, 22, 62 69, More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins) bioconjugation BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Undergraduate studies and prediploma in Chemistry (Albert-Ludwigs-Universität Freiburg) Graduate studies and M.Sc. in chemistry with Prof. Samuel H. Gellman (University of Wisconsin / Madison, USA) Ph.D. research with Prof. Carsten Bolm (summa cum laude) at the RWTH-Aachen Postdoctoral scholar (DAAD and DFG) work with Prof. Barbara Imperiali (Massachusetts Institute of Technology, USA) 2004 Research stay with Prof. Sheena E. Radford (University of Leeds, UK) Junior group leader as Liebig-Scholar (FCI) at Freie Universität Berlin Emmy-Noether-Group (DFG) leader at Freie Universität Berlin Habilitation and Associate Professor (W2) for Bioorganic Chemistry at Freie Universität Berlin Since 2008 Speaker of the graduate college Multivalency in Chemistry and Biochemistry within the SFB 765; member of the SFB 765 Since 2011 Speaker DFG priority program SPP 1623 Chemoselective Reactions for the synthesis and application of functional proteins Since 2012 Leibniz-Humboldt Professor (W3) for Chemical Biology funded by the Einstein Foundation Berlin In the densely packed world of a cell, many signaling pathways that support healthy functioning and that are disrupted in disease are controlled by the modification of proteins. The most common of these functionalization events are phosphorylation and glycosylation, but increasingly other so-called post-translational modifications (PTMs), such as acetylation and methylation, are being identified as important toggle switches in health and disease. Chemical biologists increasingly want to selectively control functionalization of proteins in the cell, both to study the biological role of post-translational modifications, and to decorate proteins with fluorescent moieties that permit their visualization or with molecular tags that allow a straightforward purification. The Hackenberger laboratory aims to identify new bioconjugation strategies that allow the functionalization of peptides and proteins, both on isolated biomolecules as well as in living cells and organisms. In this, our main objective is to apply these highly selective organic reactions, together with other established chemoselective or bioorthogonal reactions and biochemical methods, to study the functional consequences of natural protein modifications as well as to generate novel peptide- and protein-conjugates for pharmaceutical and medicinal applications (Figure 1). ZUSAMMENFASSUNG In der dicht gepackten Welt einer Zelle werden viele Signalwege, die deren normales Leben steuern und die bei Krankheit gestört sind, durch Veränderungen an Proteinen reguliert. Am häufigsten sind sind hier Phosphorylierungen und Glycosylierungen zu beobachten; vermehrt werden aber auch andere posttranslationale Modifikationen wie Acetylierung und Methylierung identifiziert, die wie Wechselschalter zwischen Gesundheit und Krankheit wirken können. Chemische Biologen versuchen deshalb zunehmend, die Funktionalisierung von Proteinen in der Zelle selektiv zu kontrollieren, um entweder die biologische Rolle solcher posttranslationalen Modifikationen zu erforschen, oder um Proteine mit fluoreszierenden Gruppen zu versehen, die ihre Visualisierung ermöglichen, oder mit spezifischen molekularen Markierungen ( Tags ), die ihre Aufreinigung erlauben. Das Labor von Christian Hackenberger hat sich zum Ziel gesetzt neue Methoden für die Biokonjugation zu entwickeln, um Peptide und Proteine sowohl in isolierter Form als auch in lebenden Zellen oder Organismen gezielt funktionalisieren zu können. Das Hauptaugenmerk liegt dabei darauf, hochselektive organisch-chemische Reaktionen zur Anwendung zu bringen, die in Kombination mit anderen chemoselektiven und bioorthogonalen Reaktionen und biochemischen Methoden erlauben, die Auswirkungen natürlich vorkommender Modifikationen auf Proteinfunktionen zu untersuchen und neuartige Peptid- und Proteinkonjugate für medizinische und pharmakologische Anwendungen zu entwickeln (Figure 1). A New Chemical Tool for Protein Modifications: Bioorthogonal Staudinger-Phosphite and Phosphonite Reactions In the course of investigating a Lewis acid-catalyzed phosphorimidatephosphoramidate rearrangement, we discovered a quantitative hydrolysis of the phosphorimidate intermediates upon the addition of water. Building on this observation, we used the Staudingerphosphite reaction to convert azides into phosphoramidates, either in solution or on the solid support. In a first biological application of this reaction we developed a chemoselective phosphorylation of proteins which allowed, in combination with unnatural protein translation, a site-specific incorporation of a charged phosphoramidate moiety into a protein that is recognized by a phospho-tyr specific antibody. Recently, we extended this concept to the site-specific phosphorylation of Lys-peptides residues, which represents a very labile PTM and which we were able to analyse in close collaboration with Eberhard Krause (FMP) by ETD-MS (Figure 2). In subsequent studies, we engineered an unsymmetrical version of the Staudinger-phosphite, as well as a Staudinger-phosphonite, reaction for the chemical lipidation, biotinylation and glycosylation of proteins as well as polymeric materials. We demonstrated that the Staudinger-phosphite is an efficient transformation even in a highly crowded bio-environment such as E.coli lysate, and further employed this reaction for an efficient and metal-free PEGylation to deliver a new class of branched oligoethylene glycol scaffolds for the stabilization of peptides in the cytosol. Probing the impact of post-translational modifications on peptide and protein aggregation: Semi-Synthesis of the Alzheimer-relevant Tau Protein In this project we are studying the structural consequences of post-translational modification on model peptide sequences. Building upon recent model studies in which we studied the impact of phosphorylation on aggregating coiled-coils or β-sheets, we also study the effect of phosphorylation and glycosylation on intrinsically unstructured proteins. A prime example is the neuronal Tau protein, which exists in an unstructured soluble form, a microtubule bound state of unknown structure, or a hyperphosphorylated aggregated state found in neurofibrillar tangles in Alzheimer s disease. Currently, we are investigating the complex relationship between phosphorylation and aggregation of Tau by generating otherwise inaccessible homogeneously phosphorylated proteins using protein semi-synthesis. Very recently, we have succeeded in the first semi- synthesis of a homogeneously phosphorylated functional Tau protein. Furthermore, we have extended this study to glycosylated Tau to evaluate whether a perturbed balance between phosphorylation and glycosylation (O-GlcNAc) is associated with Tau aggregation. Other studies (with Guy Lippens, CNRS Lille), in which synthetic phosphorylated peptides were subjected to enzymatic transformations, have already identified two new O-GlcNAc glycosylation sites in Tau and, furthermore, demonstrated the reciprocal relationship between phosphorylation and O-GlcNAcylation. Development of new unnaturally modified glycoproteins In collaboration with Prof. Stephan Hinderlich (Beuth-Hochschule) we recently expanded the repertoire of unnaturally modified glycoproteins by metabolic oligosaccharide engineering. For this purpose C4-substituted ManNAc-derivatives were synthesized and incorporated into novel C7-modified sialic acids in glycoproteins in cells and in living zebrafish. These novel biopolymers will be investigated for the development of new diagnostic and therapeutic glycoproteins and potentially applied to the in vivo modification of living animal models. Site-specific functionalization of proteins for the acquisition of multivalent glycoconjugates In a combined effort with Prof. Budisa (TU Berlin) we employ a combination of classical site-directed mutagenesis, genetic code engineering and bioorthogonal reactions to deliver chemically modified proteins with carbohydrates installed at specific residues. These protein conjugates are employed in multivalent binding studies within the collaborative research center 765, which support the use of proteins as structurally defined scaffolds for the presentation of an exact number of multivalent ligands. Site-specific labelling and cellular delivery of nanobodies In collaboration with Prof. Leonhardt (LMU Munich) and Prof. Cardoso (TU Darmstadt) we develop powerful new methods to analyse and manipulate biochemical processes within living cells using functionalized nanobodies. Nanobodies are small, antigen-binding proteins that remain active within the reductive milieu inside living cells. These properties give them a significant advantage for intracellular applications over conventional antibodies. Our long-term goal within this SPP 1623-funded project is to generate fluorescently labeled nanobodies functionalized with circular cell penetrating peptides (CPPs) for cellular uptake and caged amino acids for photoinducible activation. To install these functional units we use a combinatorial approach of intein expression, amber suppression and bioorthogonal reactions.

58 112 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 113 CHEMOSELECTIVE STAUDINGER- PHOSPHITE REACTION SITE-SPECIFIC SYNTHESIS ETD MS / MS Intensity Nicole Nischan (photo left), Jordi Bertran-Vicente, Anett Hauser m / z Fig 2: Chemoselective synthesis and ETD-MS Analysis of site-specific phosphorylated Lys-peptides (Reference: J. Bertran-Vicente, R.A. Serwa, M. Schümann, P. Schmieder, E. Krause, C.P.R. Hackenberger, J. Am Chem. Soc. 2014, 136(39), , Site-Specifically Phosphorylated Lysine Peptides) Simon Reiske, Divya Agrawal Olaia Nieto Garcia GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Divya Agrawal International National Bertran-Vicente J, Serwa RA, Schümann M, Schmieder P, Krause E, Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623 Dr. Vera Martos Guy Lippens Nedjliko Budisa Hackenberger CPR (2014) Site-Specifically Phosphorylated Lysine Chemoselective reactions for the synthesis and application of functional (co-advised with Dr. Plested) CNRS and Université des Sciences et Technische Universität Berlin Peptides. J. Am. Chem. Soc. 136, proteins, funds for the coordination of the priority programme, Dr. Olaia Nieto Dr. Stefan Reinke Dr. Marcie Jaffee Lukas Artner (doctoral student) Jordi Bertran (doctoral student) Maria Glanz (doctoral student) Andrew Grimes (doctoral student) Marc-André Kasper (doctoral student) Technologies de Lille, France Caroline Smet-Nocca Université des Sciences et Technologies de Lille, France Jim Paulson The Scripps Research Institute, La Jolla, CA, USA Andrew Udit Jens Dernedde Charité Universitätsmedizin Berlin Werner Reutter Charité Universitätsmedizin Berlin Stephan Hinderlich Beuth-Hochschule, Berlin Michael Gerrits RiNA GmbH Mühlberg M, Siebertz KD, Schlegel B, Schmieder P, Hackenberger CPR (2014) Controlled thioamide vs. amide formation in the thioacidazide reaction under acidic aqueous conditions. Chem. Commun. 50, Majkut P, Claußnitzer I, Merk H, Freund C, Hackenberger CPR, Gerrits M (2013) Completion of Proteomic Data Sets by K d Measurement Using Cell-Free Synthesis of Site-Specifically Labeled Proteins. PLoS ONE 8, , Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623, Site-specific functionalization of nanobodies: From labelling to cellular uptake, jointly with H. Leonhardt (LMU München) and C. Cardoso (TU Darmstadt), , Boehringer-Ingelheim Stiftung, Chemoselective Staudinger-reactions for the modification of peptides and proteins, Plus 3 -Programme, Simon Klenk (doctoral student) Occidental College, Los Angeles, CA, USA Volker Haucke e , Michaela Mühlberg (doctoral student) Nicole Nischan (doctoral student) Oliver Reimann (doctoral student) Simon Reiske (doctoral student) Tom Sauer (doctoral student) Dominik Schumacher (doctoral student) Sergej Schwagerus (doctoral student) Roland Brock Radboud University Nijmegen Medical Centre, The Netherlands Kolio Troev Institut of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria Michal Pietrusiewicz Leibniz-Institut für Molekulare Pharmakologie (FMP) and Freie Universität Berlin Eberhard Krause Leibniz-Institut für Molekulare Pharmakologie (FMP) Rainer Haag Freie Universität Berlin Nischan N, Chakrabarti A, Serwa RA, Bovee-Geurts P H M, Brock R, Hackenberger CPR (2013) Stabilization of Peptides for Intracellular Applications by Phosphoramidate-Linked PEG Chains. Angew. Chem. Int. Ed. 52, Vallée MRJ, Artner LM, Dernedde J, Hackenberger CPR (2013) Alkyne phosphonites for sequential azide-azide couplings. Angew. Chem. Int. Deutsche Forschungsgemeinschaft, SFB 765 B05, Synthesis of multivalent ligand binding systems via chemoselective saccharide- and peptideligations (1st funding period ), Site-specific functionalization of proteins for the acquisition of multivalent glycoconjugtes (2nd funding period ), , jointly with N. Budisa (TU Berlin), Kristina Siebertz (doctoral student) Maria Curie-Sklodowska University Lublin, Christian Freund Ed. 52, Deutsche Forschungsgemeinschaft, SFB 765 (1st and 2nd funding Robert Vallée (doctoral student) Poland Freie Universität Berlin period ), Integriertes Graduiertenkolleg des SFB, Dagmar Krause (technical assistant) Eric Strieter Bernd Lepenies , Kristin Kemnitz-Hassanin (technical assistant) University of Wisconsin / Madison, USA MPI Colloid and Interfaces Andreas Herrmann Humboldt Universität zu Berlin Cristina Cardoso Technische Universität Darmstadt Heinrich Leonhardt Ludwig-Maximilians-Universität München FMP authors Group members Deutsche Forschungsgemeinschaft, Emmy Noether Programm, New synthetic methods for naturally modified peptides and proteins, their structural evaluation and biological function, ,

59 114 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 115 PEPTIDE-LIPID INTERACTION / PEPTIDE TRANSPORT PEPTID-LIPID-INTERAKTION / PEPTIDTRANSPORT Fig. 1: Localisation of antimicrobial peptides in E. coli and B. subtilis analysed with fluorescence microscopy. (A) Fluorescein-labelled Buforin II in B. subtilis served as control for cytoplasmic peptide localisation, (B) cw[nbd]w in E. coli and (C) in B. subtilis. While Buforin II was internalised into the cytoplasm, the cyclic hexapepetide bound to the membrane of Gram positive and Gram negative bacteria. Moreover, the peptide was found to accumulate at the septa and polar regions in B. subtilis. GROUP LEADER DR. MARGITTA DATHE A B C BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS 1974 Diploma thesis in physics at the Humboldt-Universität Berlin, GDR 1978 Ph.D. at the Academy of Sciences of the GDR Research Associate at the Institute of Drug Research of the Academy of Sciences of the GDR Team Leader of the Conformational Analysis Group at the FM since 1999 Team Leader of the Peptide Lipid Interaction / Peptide Transport Group at the FMP Our group is interested in the selective interaction of peptides with cellular membranes and their ability to penetrate or permeabilize cellular barriers. We structurally optimize and exploit such peptides as targeting and uptake-mediating tools for lipid-based carrier systems loaded with diagnostic or therapeutic drug molecules. Our activities have been focused on targeting the blood-brain barrier and on the efficient cutaneous delivery of bioactive compounds. Furthermore, we are attempting to elucidate the mode of action of small cyclic membrane-active antimicrobial peptides. Such peptides, rich in particular amino acids such as arginine and tryptophan, appear to utilize a quite novel mechanism of action and thus open new ways to generate antimicrobial peptides for selected applications. ZUSAMMENFASSUNG Unsere Arbeitsgruppe interessiert sich für die selektive Wechselwirkung von Peptiden mit zellulären Membranen und die Fähigkeit mancher dieser Peptide, diese zellulären Barrieren zu überwinden beziehungsweise zu öffnen. Solche Peptide werden in ihrer Struktur optimiert und eingesetzt, um Lipid-basierte Carriersysteme, die mit diagnostisch oder therapeutisch wirksamen Substanzen beladen wurden, zielgerichtet an Zellen heranzuführen und eine zelluläre Aufnahme zu vermitteln. Der Fokus liegt dabei auf der zielgerichteten Zuführung solcher Carrier an die Blut-Hirn-Schranke und auf einer effektiven Aufnahme bioaktiver Substanzen in die Haut. Ein zweiter Schwerpunkt besteht in der Aufklärung des Wirkmechanismus kleiner zyklischer, antimikrobiell wirksamer und membranaktiver Peptide. Diese Peptide besitzen einen hohen Gehalt an den Aminosäuren Arginin und Tryptophan und scheinen einen neuartigen Wirkmechanismus zu nutzen. Das könnte neue Wege zur Erzeugung antimikrobiell wirksamer Peptide für ausgewählte Anwendungen eröffnen. Cationic antimicrobial peptides Optimization of the activity and bacterial selectivity of antimicrobial peptides (AMPs) requires an understanding of their diverse mechanisms of action. Tryptophan (W)- and arginine (R)-rich cyclic hexapeptides of the type cyclo-rrrwfw (cwfw) fulfil the structural prerequisite for membrane insertion due to their charge and amphipathicity. However, they do not exert their killing activity by membrane permeabilization. Our research interest focuses on alternative mechanisms of action such as peptide translocation into the cytoplasm and peptide interaction with components of the phospholipid matrix of the bacterial membrane. Fluorescence microscopy and an HPLC-based strategy to analyse peptide uptake into cells confirmed the cytoplasmic membrane as the major target where cwfw accumulated at distinct sites (Fig 1). Further characterization of peptide-membrane interaction involving live cell imaging showed a distribution of the peptide similar to that of the lipid cardiolipin (CL) within the membrane. Furthermore, isothermal titration calorimetry using model membranes with different bacterial lipid compositions demonstrated a highly preferred affinity for CL-rich phosphatidylethanolamine (PE) matrices. These observations point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cwfw that is not based on membrane permeabilization or translocation into the cytoplasm but rather on pronounced partitioning into lipid layers containing microdomains of both CL and PE. In an ongoing cooperation with the Centre for Bacterial Cell Biology, Newcastle University, UK, the molecular details of how elevated CL levels or phospholipid migration lead to cell death are being investigated and peptide interaction with particular lipid compositions will be further characterized. Influencing lipid organisation directly or indirectly by targeting negative intrinsic curvature lipids could be a new approach for the design of antimicrobial peptides. bioactive compounds have been characterized. P2R12 was found to be a promising vector for selectively delivering the anticancer drug Paclitaxel (PCL), incorporated into PEGylated lipid micelles, into human brain capillary endothelial cells, as well as into glioblastoma cells. Pronounced uptake into glioblastoma cells correlated with high cytotoxicity. Thus, arginine-rich lipopeptides seem to be promising candidates for targeting the BBB as well as brain-located tumours (cooperation with Northeastern University, Boston, USA). Chemical exchange saturation transfer with hyperpolarized xenon nuclei (Hyper-CEST) allows sensitive detection of supramolecular cages such as cryptophane-a (CrA) in non-invasive Magnetic Resonance Imaging (MRI). Studies in cooperation with L. Schröder, FMP, demonstrated efficient delivery of the MRI contrast agent CrA via P2R12-modified liposomes into brain capillary endothelial cells. Liposomal incorporation and cell-selective targeting circumvented chemical modification and the high toxicity of CrA. The results paved the way towards the first in vivo studies with the Xe Hyper-CEST MRI technique. Transglutaminase 1 (TG1)-deficient autosomal recessive congenital ichthyosis (ARCI) is a rare but severe genetic skin disease. P2A2- modified liposomes mediated efficient transmembrane transport and skin penetration of encapsulated recombinant TG1 (rhtg1) and demonstrated in vivo normalization of the ARCI skin (cooperation with University Hospital, Münster) (Fig 2). In 2013, the therapeutic modality received the orphan drug designation from the European Commission (EU 3 / 13 / 1130, OD number EMA / OD / 003 / 13). The combined ongoing activities of the academic partners, together with an influential industrial partner, to advance the preclinical development underline the huge therapeutic and economic potential of this work. Peptide-modified liposomal and micellar carriers Cell-recognising and membrane-translocating peptides provide promising tools for the development of efficient drug delivery systems. Early observations showed that small micelles, formed of an arginine- and lysine-rich lipopeptide (P2A2), selectively internalise into endothelial cells of the blood-brain barrier (BBB) whereas an oligo-arginine lipopeptide (P2R12) mediates efficient uptake of lipid particles independent of carrier size and peptide loading. Based on these results, peptide-modified carrier systems loaded with different

60 116 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 117 A B Kathi Scheinpflug, Heike Nikolenko and Karl Sydow (photo left), Heike Nikolenko Mareike Brehm and Kathi Scheinpflug C D Fig. 2: Phenotype of regenerated TG1-deficient skin grafts in a skin-humanized mouse model before and after application of rhtg1-liposomes. Phenotypic changes in mice treated with 40 ng rhtg1 / cm 2 (n=3) (C) can be observed after the second application (day 5). Grafts treated with 2 ng rhtg1 / cm 2 (n=3) for 14 days show a lower effect suggesting a dose dependency (D). Skin treated with empty liposomes shows no change in phenotype (B) when compared to the untreated grafts (A). (Aufenvenne et al Am J Hum Genet. 93, 620) GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Margitta Dathe (group leader) International National Sydow K, Torchilin VP, Dathe M (2014) Lipopeptide-modified PEG- DFG Peptide-modified micellar nanocarriers to mediate transport at Dr. Oxana Krylova (scientist) Igor Komarov Sonia Waiczies PE-based pharmaceutical nanocarriers for improved cytotoxicity against the blood brain barrier (BBB) DA 324 / 9 1, 06 / / 2014, Kathi Scheinpflug (doctoral student) Shevchenko University, Kiev, Ukraine MDC Berlin, Germany glioma cells. Eur. J. Lip. Sci.Technol. 116, Karl Sydow (doctoral student) Heike Nikolenko (technical assistant) Gabriela Vogelreiter Marina Rautenbach University Stellenbosch, South Africa Vladimir Torchilin Northeastern University, Boston, USA Henrik Strahl Newcastle University, UK Michael Kumke University Potsdam, Germany Alfred Blume MLU Halle, Germany Martin Schulze Institute for Reproduction of Farm Animals Schnurr M, Sydow K, Rose HM, Dathe M, Schröder L (2014) Brain Endothelial Cell Targeting via a Peptide-Functionalized Liposomal Carrier for Xenon Hyper-CEST MRI. Adv. Healthcare Mater. 2014,1 6. Schulze M, Junkes C, Mueller P, Speck S, Ruediger K, Dathe M, Mueller K (2014) Effects of Cationic Antimicrobial Peptides on Liquid- Internationales Büro des Bundesministeriums für Bildung und Forschung (BMBF) Optimierung kleiner Peptide als potentielle Wirkstoffe gegen intrazelluläre menschliche Pathogene, SUA 09 / 048, M. Dathe with M. Beyermann and M. Rautenbach, Stellenbosch University, South Africa, 05 / / 2013, Schoenow, Bernau, Germany Heiko Traupe University Münster, Germany Preserved Boar Spermatozoa. PlosONE 9, e Gehne S, Sydow K, Dathe, M, Kumke M (2013) Characterization of Cell-Penetrating Lipopeptide Micelles by Spectroscopic Methods. J. Phys. Chem. B 117, Alexander v. Humboldt Stiftung Synthesis and study of small antimicrobial peptides containing conformationally constrained arginine analogues, M. Dathe. Cooperation with I. Komarov, Shevchenko University, Kiev 2.3-DEU / , 07 / / 2013, Scheinpflug K, Nikolenko H, Komarove IV, Rautenbach, M, Dathe M (2013) What goes around comes around A comparative study of the influence of chemical modifications on the antimicrobial properties of small cyclic peptides. Pharmaceuticals 6, Aufenvenne K, Larcher F, Hausser I, Duarte B, Oji V, Nikolenko H, Del Rio M, Dathe M, Traupe H (2013) Topical enzyme replacement therapy restores transglutaminase 1-activity and corrects architecture of transglutaminase 1-deficient skin grafts. Am. J. Human Genetics 93, FMP authors Group members

61 118 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 119 MASS SPECTROMETRY Heike Stephanowitz and Annika Manns (photo left), Michael Schümann MASSENSPEKTROMETRIE GROUP LEADER DR. EBERHARD KRAUSE BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS 1975 Diploma degree in physical chemistry at the Humboldt University, Berlin 1982 Dr. rer. nat., Humboldt University, Berlin Research Group Leader Drug Development in the Pharmaceutical Industry Research Associate at the Institute of Drug Research since 1992 Senior Scientist and Head of the Mass Spectrometry Group at the FMP Our group focuses on the development and application of mass spectrometry-based proteomic methods to investigate cellular signaling processes. Our main topics of research have been protein-protein interactions and post-translational modifications and their functional consequences. In this context, and to gain further insight into the role of tyrosine and serine / threonine kinases for T cell function, we are interested in T cell receptor (TCR)- associated protein-protein interactions that are mediated by specific protein phosphorylations which may function as modulators of cellular adhesion and migration processes. Moreover, we have contributed to various proteomic studies and developed improved approaches for the reliable identification of specific protein modifications, as well as quantification of proteins in affinity-purification mass spectrometry experiments. ZUSAMMENFASSUNG Schwerpunkt unserer Forschung ist die Entwicklung und Anwendung massenspektrometrischer Proteomik-Methoden zur Untersuchung zellulärer Signalverarbeitungsprozesse. In der Hauptsache arbeiten wir an Protein-Protein-Wechselwirkungen sowie an posttranslationalen Modifikationen und ihren funktionellen Auswirkungen. In diesem Zusammenhang interessieren wir uns für die Aufklärung der Rolle von Tyrosin- und Serin / Threoninkinasen für die Funktionalität von T-Zellen. Insbesondere geht es hier um T-Zellrezeptor (TCR)- abhängige Protein-Protein-Wechselwirkungen, die durch spezifische Proteinphosphorylierungen vermittelt werden, die möglicherweise Zelladhäsion der Zellmigration modulieren können. Zudem haben wir zu einer Vielzahl von Proteomstudien beigetragen und verbesserte methodische Ansätze für die zuverlässige Identifizierung spezifischer Proteinmodifikationen und für die Quantifizierung von Proteinen in der Affinitäts-Massenspektrometrie entwickelt. Analysis of difficult protein phosphorylation. Reversible phosphorylation is the most widespread post-translational protein modification and is a key regulatory mark in most cellular processes. While the majority of protein phosphorylation research has been focused on hydroxyl amino acids such as serine, threonine, and tyrosine, basic amino acid side-chains of histidine, arginine and lysine residues may also undergo phosphorylation to yield phosphoramidates. Because of the lability of the P-N bond this modification most often remains undetected in conventional phosphoproteomics studies, even though recent studies have indicated the potential importance of phosphohistidine and phosphoarginine in various biological signaling processes. We performed MS studies of phospholysine peptides that were very recently synthesizable via a new synthetic route established by the Hackenberger group at the FMP. Employing the chemoselectivity of the Staudinger-phosphite reaction, the preparation of wellcharacterized, site-specifically phospholysine-containing peptides enables a systematic study of the behavior of such peptides using various mass spectrometric fragmentation techniques. Our data indicate that the stability of the phosphoramidate bond during the ETD process is high, keeping the modified side-chain completely intact during fragmentation (Figure 1). This makes ETD mass spectrometry particularly suitable for proteomic studies of lysine-phosphorylated peptides and proteins as an essential tool for evaluating the biological relevance of lysine phosphorylation (Betran-Vicente et al. JACS 2014). An improved method for affinity purification mass spectrometry experiments. Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein-protein interactions. However, it remains a challenge to identify proteins whose cellular abundance is relatively low. We used the specificity of an enzyme-catalyzed transpeptidation with sortase A to prepare affinity matrices in which the bait remains functionally accessible to protein protein interactions (Figure 2). Peptide and protein substrates (baits) with the C-terminal sortase A recognition motif LPXTG were synthesized via solid-phase peptide synthesis or recombinantly expressed in E. coli. Immobilization was carried out by incubating the LPXTG-tagged baits with the nucleophile pre-loaded beads in the presence of the enzyme. We were able to develop a robust and efficient immobilization protocol and developed a straightforward method for absolute quantification of loaded proteins by isotope dilution analysis. To examine whether this sortase approach can be successfully utilized for interactome analysis, we performed SILAC-based pull-down experiments (Figure 2) using the T-cell adapter protein ADAP and the CD2BP2-GYF domain and thereby demonstrated the suitability of the approach (Kuropka et al. Proteomics 2014). The Cell Surface Proteome of Entamoeba histolytica. The intestinal protozoan Entamoeba histolytica is an important human parasite. After infection, E. histolytica trophozoites are normally present in the intestine and can become a pathogen by penetrating the intestinal mucosa and inducing colitis and abscess formation in the liver. Surface molecules are thought to be of major importance for host-parasite interactions and are predicted to be of prime importance for tissue invasion. We have analyzed the complete surface proteome of E. histolytica. Using cell surface biotinylation and nanolc-tandem mass spectrometry, 693 putative surface-associated proteins were identified. In silico analysis predicted that 26 % of these proteins are membrane-associated, as they contain transmembrane domains, signal sequences, and / or sites of palmitoylation, myristoylation, or prenylation. However, about 50 % of the identified proteins lacked the conventional characteristics associated with membrane localization, suggesting that the plasma membrane is not a static cellular compartment but rather part of a highly interconnected cellular machinery in which intracellular membrane systems are in constant exchange with the plasma membrane (Biller et al. Mol Cell Proteomics 2014).

62 120 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 121 Nonphosphorylated ADAP-peptide or Mutant CD2BP2-GYF domain Phosphorylated ADAP-peptide or Wild-type CD2BP2-GYF domain MS / MS Light Heavy Intensity m / z Ratio L / H (reverse exp.) Elution and combining Trypsin 2-D LC-MS / MS analysis Binding partners Fig. 1: Principle of SILAC-based peptide pull-down experiment. Bait proteins were covalently coupled to agarose beads via sortase-mediated transpeptidation reaction and subsequently incubated with human T cell lysate. After tryptic protein digestion of bound proteins, peptides were separated, identified, and quantified by 2-D LC-MS / MS analysis. Fig. 2: ETD-MS / MS fragment ion spectra of the lysine-phosphorylated peptide Fragment ions resulting from the triply charged precursor ion correspond to peptide sequence and are labeled according to the c- and z-ion nomenclature. In particular, the C4, C5, Z8, and Z9 ions indicate the phosphorylation of the Lys in position 21. Ratio L / H (forward exp.) GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS Annika Manns (doctoral student) Benno Kuropka (doctoral student) Michael Schümann (technical assistant) Heike Stephanowitz (technical assistant) International Remigiusz Serwa Imperial College London, UK George S. Baillie University of Glasgow, Scotland Peter Pohl Johannes Kepler University Linz, Austria Hans G. Börner Humboldt-Universität, Berlin Volker Haucke FMP Christian Hackenberger FMP Christian Freund FU Berlin Kuropka B, Royla N, Freund C, Krause E (2014) Sortase A-mediated site-specific immobilization for identification of protein interactions in affinity purification-mass spectrometry experiments. Proteomics doi: / pmic Bertran-Vicente J, Serwa R, Schümann M, Schmieder P, Krause E, Hackenberger CPR (2014) Site-specifically phosphorylated lysine peptides. J. Am. Chem. Soc. 136, Lang D, Anker S, Kuropka B, Krause E (2013) Combining enzymatic 18O-labeling and 2-D LC-MS / MS for a study of protein interactions in primary T cells. Anal. Methods 5, National Dirk Schwarzer Biller L, Matthiesen J, Kühne V, Lotter H, Handal G, Nozaki T, Saito- Bernd Nürnberg Universität Tübingen Nakano Y, Schümann M, Roeder T, Tannich E, Krause E, Bruchhaus I Universität Tübingen Ralf Schülein (2014) The cell surface proteome of Entamoeba histolytica. Mol. Cell. Iris Bruchhaus FMP Proteomics 13, Bernhard Nocht Institute for Tropical Medicine, Hamburg Norbert Reiling Leibniz Center for Medicine and Biosciences, Borstel Kurt Engeland Universität, Leipzig Ingolf Blasig FMP Michael Veit FU Berlin Michael Schupp Charite, Berlin Pichlo M, Bungert-Plümke S, Weyand I, Seifert R, Bönigk W, Strünker T, Kashikar ND, Goodwin N, Müller A, Körschen HG, Collienne U, Pelzer P, Van Q, Enderlein J, Klemm C, Krause E, Trötschel C, Poetsch A, Kremmer E, Kaupp UB (2014) High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor. J. Cell Biol. 206, U. Benjamin Kaupp Center of Advanced European Studies and Research, Bonn Lang D, Schümann M, Gelato K, Fischle W, Schwarzer D, Krause E (2013) Probing the acetylation code of histone H4. Proteomics 13, FMP authors Group members

63 122 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 123 MEDICINAL CHEMISTRY MEDIZINISCHE CHEMIE Sandra Mischke and Andre Horatschek (photo left), Isabel Fernandez-Bachiller and Hassane Belabed GROUP LEADER DR. MARC NAZARÉ BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Studies in Chemistry, University of Karlsruhe 1995 Diploma in Chemistry, University of Karlsruhe, Prof. H. Waldmann Ph.D. research with Prof. H. Waldmann Medicinal chemist and project leader in drug discovery at Sanofi, Frankfurt am Main Medicinal Chemistry Department, Sanofi, Frankfurt am Main Therapeutic department Thrombosis and Angiogenesis, Sanofi, Frankfurt am Main Therapeutic Strategic Unit Age Related Diseases, Sanofi, Frankfurt am Main Since 2013 Group leader at the Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin Small molecule tools allow one to probe protein functions and elucidate mechanisms and signal transduction pathways by directly interfering with a specific protein of interest. These chemical tools validate hypotheses based on chronic genetic inactivation in knock-down loss of function studies and can serve as progenitors or starting points of new therapeutic approaches and drugs. Our aim is to discover and develop highly active and selective chemical probes for the specific modulation of protein-ligand and protein-protein interactions. Together with our collaboration partners, we are currently interested in the conception and design of specific inhibitors for clathrin-mediated endocytosis, phosphatase Shp2, and tryptophane hydroxylase. To achieve our goals we study the structure-activity relationship (SAR) of the protein ligand interactions by synthesizing distinct small molecule derivatives or small focussed libraries of the initial screening hit structure. Iterative cycles of biological testing make possible the synthesis of new analogues based on the data obtained. A second field of interest is the further expansion and qualitative enhancement of the FMP compound library containing 55,000 commercial compounds and 7,000 small molecules sourced from academic research. Careful extension of the library guarantees an optimal coverage of the screened biological space and high hit rates as well as suitable starting points for the chemical optimization. The Medicinal Chemistry group closely collaborates with the Screening Unit with respect to the FMP library and biological profiling in SAR studies. ZUSAMMENFASSUNG Kleine Moleküle lassen sich als Forschungswerkzeuge einsetzen, mit denen Proteinfunktionen untersucht, molekulare Mechanismen aufgeklärt und Signaltransduktionswege durch direkte Einflussnahme auf spezifische Proteine aufgeschlüsselt werden können. Diese chemischen Werkzeuge können genutzt werden, um Hypothesen aus genetischen Studien, z. B. durch Inaktivierung biologischer Prozesse mittels Knock-Down- / Loss-of-Function-Ansätzen, zu validieren. Die Substanzen können als Vorläufersubstanzen von Pharmaka oder Startpunkte für neuartige Therapien dienen. Ziel unserer Arbeit ist die Entdeckung und Entwicklung hochaktiver, selektiver chemischer Sonden für eine spezifische Modulation von Protein- Liganden- oder Protein-Protein Wechselwirkungen. Mit unseren Kooperationspartnern sind wir derzeit an Konzepten und dem Design von spezifischen Inhibitoren für die Clathrinvermittelte Endozytose, die Phosphatase Shp2 sowie Tryptophanhydroxylase interessiert. Dazu untersuchen wir die Struktur-Wirkungsbeziehung (Structure-Activity Relationship, SAR) von Protein-Liganden-Wechselwirkungen mittels Synthese kleiner Molekülderivate oder kleiner fokussierter Substanzbibliotheken, ausgehend von Strukturen, die sich in initialen Screens als aktiv erwiesen haben (hits). Iterative Zyklen biologischer Tests ermöglichen dann die Synthese neuer, verbesserter Analoga auf Basis der erhobenen Daten. Ein zweiter Fokus unserer Arbeit ist die weitere Entwicklung und die Qualitätserhöhung der FMP Substanzsammlung mit derzeit kommerziellen Verbindungen und kleinen Molekülen aus der akademischen Forschung. Eine behutsame Erweiterung der Sammlung garantiert dabei eine optimale Abdeckung des zu screenenden biologischen Raums, hohe Ausbeuten an Screening-Hits und die Identifizierung geeigneter Startpunkte für chemische Optimierungen solcher Hits. Die Arbeitsgruppe Medizinalchemie arbeitet dazu sowohl bei der Entwicklung der FMP-Sammlung als auch beim biologischen Profiling in SAR-Studien eng mit der Screening Unit des Instituts zusammen. Specific inhibition of clathrin-mediated endocytosis by disruption of clathrin-endocytic protein interactions with Pitstop derivatives Clathrin-mediated endocytosis (CME) regulates many key physiological processes such as the internalization of growth factors and receptors, entry of pathogens (e.g. HIV-1), and synaptic transmission by formation of so-called clathrin coated vesicles. An ELISA-based HTS using 17,000 small molecules from the ChemBioNet library at the Screening Unit of the FMP resulted in the identification of two hit compounds inhibiting the clathrin terminal domain (TD): amphiphysin B / C complex formation (V. Haucke et al. Cell, 2011). Starting from these hits, and in collaboration with the group of Volker Haucke (FU & FMP, Berlin), focused libraries of around 120 compounds were synthesized, providing a structure-activity relationship for Pitstop2. Co-crystallization of Pitstop derivatives with clathrin TD guided the further design and provided insight into key interactions between these Pitstop ligands and clathrin TD. Surprisingly, X-ray structure determination (Haydar Bulut, FU Berlin) of six nearly equipotent inhibitors derived from one core scaffold resulted in four different binding modes. These non-canonical binding modes of the novel Pitstop analogues revealed several new aspects of the structural basis for the disruption of the clathrin TD-endocytic protein interactions. Development of specific inhibitors of the tyrosine phosphatase Shp-2 In stark contrast to their validated significance in signal transduction and disease pathology phosphatases are notoriously difficult to address using small molecules. The protein tyrosine phosphatase Shp-2 plays a critical role in growth factor-mediated processes, primarily by promoting the activation of the RAS / ERK signaling pathway. Aberrant gain-of-function mutations are associated with several metastatic cancers. A rescaffolding approach involving replacing the former framework of a tyrosine phosphatase Shp2 inhibitor (W. Birchmeier et. al, PNAS, 2008), in collaboration with Walter Birchmeier, led to the discovery of novel structural classes and eliminated several unfavorable structural features. These novel compounds are not only active in a sub-micromolar range in the Shp2-enzyme assay, but are also effective in the low micromolar range on HGF-stimulated canine MDCK-C cells, as well as human pancreatic tumor cells for epithelialmesenchymal transition (EMT), a hallmark of cancer cell dissemination. Tryptophan hydroxylase (TPH) inhibitors The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] is causally involved in multiple aspects of mood control in the central nervous system such as regulating sleep, anxiety, drug abuse, and food intake. In peripheral tissues, serotonin regulates vascular tone, gut motility, primary hemostasis, and cell-mediated immune responses, and is associated with diseases like irritable bowel syndrome and carcinoid syndrome. The biosynthesis of serotonin is a highly regulated two-step process, starting with the essential amino acid L-tryptophan (Trp), and tryptophan hydroxylase (TPH) is the initial and ratelimiting enzyme in the biosynthesis of serotonin. In collaboration with the groups of Michael Bader and Udo Heinemann (both MDC) and Jens von Kries (FMP) we have identified and further developed highly active TPH inhibitors that are able to modulate the physiological serotonin levels. The X-ray co-crystal structures obtained with our inhibitors allowed us to elucidate the binding mode and reveal the structural determinants for the remarkably efficient protein-ligand interaction of these inhibitors. Shaping the FMP library with novel scaffolds The optimal design of a chemical library to provide the best possible coverage of chemical space is crucial for the successful outcome of an HTS. The selection of the central scaffold for a drug molecule is a conceptually challenging and decisive task in the design of new small molecule modulators. Whereas the peripheral side-chain decoration of a given hit structure is the first and obvious starting point for variation, the exchange of the scaffold core is inherently more difficult. The availability, i.e. ease of synthetic accessibility, is an important yet often underestimated factor in the successful optimization of a tool compound. Therefore we have investigated and developed new routes for the synthesis of privileged scaffolds like 2H indazoles, pyrazolotriazoles and 1,2-benzodiazepines for the generation of libraries and we have included them in our FMP compound collection.

64 124 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 125 Fig. 1: Overlay of three x-ray co-crystal structures Pitstop2 derivatives / clathrin illustrating the reversed, non-canonical binding modes within the clathrin box. A B C D SCM / impedance / 1h Lioudmila Perepelittchenko and Maria Pascual Lopez Alberca (photo left), Jessica Przygodda, Kevin Mallow and Edgar Specker relative activity (%) concentration Fig. 2: Activity of the novel Shp2 inhibitors in a 2D cellular model for metastasis. Comparison of hepatocyte growth factor (HGF)-induced cell scattering of human pancreas tumor cell line (HPAFII cells). A) cell colonies without HGF stimulation B) after HGF stimulation cells loose contact and migrate C) upon incubation with the Shp2 inhibitor the HGF induced migration is blocked D) concentration dependency of the inhibition of HGF induced migration using impedance measurement. HPAFII / human pancreas tumor cell line IC 50 (SHP2) = 0.83 µm IC 50 (HPAFII) ~ 3.5µM Sandra Mischke, Upendra Anumala and Isabel Fernandez-Bachiller Jens Schöne GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Upendra Anumala Dr. Isabel Fernández Bachiller Dr. Hassen Bel Abed Dr. André Horatscheck Dr. Judith Holz Keven Mallow (technical assistant) Sandra Miksche (technical assistant) Sylvia Oestreich (technical assistant) Dr. María Pascual López-Alberca Dr. Lioudmila Perepelittchenko (technical assistant) Jessica Przygodda (technical assistant) Dipl. Chem. Jens Schöne (doctoral student) International Stefan Krauss University Hospital, Oslo National Bernd Nürnberg Michael Bader MDC, Berlin Walter Birchmeier MDC, Berlin Udo Heinemann MDC, Berlin Lenhard Rudolph FLI, Jena David W. Will EMBL, Heidelberg Halland N, Schmidt F, Weiss, T, Saas J, Li Z, Czech J, Dreyer M, Hofmeister A, Mertsch K, Dietz U, Strübing C, Nazaré M (2015) Discovery of N-[4-(1H- Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors. ACS Med. Chem. Lett. 6, Hu H-Y, Lim N-H, Ding-Pfennigdorff D, Saas J, Wendt KU, Ritzeler O, Nagase H, Plettenburg O, Schultz C, Nazaré M (2015) DOTAM Derivatives as Active Cartilage-targeting Drug Carriers for the Treatment of Osteoarthritis. Bioconj. Chem. 26, Wilde F, Specker E, Neuenschwander M, Nazaré M, Bodtke A, Link A (2014) Tractable synthesis of multipurpose screening compounds with under-represented molecular features for an open access screening platform. Mol. Divers. 18, Nazaré M, Matter H, Will DW, Wagner M, Urmann M, Czech J, Schreuder H, Helmholtz Association Helmholtz Wirkstoffforschung, , SAW DNA damage responses in aging P21 collaboration , The Research Council of Norway Extended biotarget validation studies for the specific inhibitor of Wnt / β-catenin signaling OD270, 2014, VIP Validierung neuartiger Wirkstoffe zur Behandlung von Depressionen durch pharmakologische Aktivierung der Tryptophan-Hydroxylase 2 together with Michael Bader, MDC., , MDC PreGo-Bio Novel compounds for the targeted therapy of prostate cancer together with Heinemann U, MDC., , Dr. Edgar Specker Bauer A, Ritter K, Wehner V (2012) Fragment Deconstruction of Small, Potent Factor Xa Inhibitors: Exploring the Superadditivity Energetics of Fragment Linking in Protein-Ligand Complexes. Angew. Chem. Int. Ed. 124, Zech G, Hessler G, Evers A, Weiss T, Florian P, Just M, Czech J, Czechtizky W, Görlitzer J, Ruf S, Kohlmann M, Nazaré M (2012) Identification of High Affinity P2Y12 Antagonists Based on a Phenylpyrazole Glutamic Acid Piperazine Backbone. J. Med. Chem. 55, FMP authors Group members

65 126 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 127 CORE FACILITY SCREENING UNIT Sabrina Kleißle (MDC) and Katina Lazarow (photo left), Carola Seyffarth and Andreas Oder GROUP LEADER DR. JENS PETER VON KRIES BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS 1987 Diploma in biology at the University Hospital Hamburg-Eppendorf (Prof. Strätling) 1991 Ph.D. at the University Hospital Hamburg-Eppendorf (Prof. Strätling) Research on proteins involved in origin of cancer in the lab of W. Birchmeier Head of Screening Unit of Semaia Pharmaceuticals since 2003 Head of Screening Unit at FMP 2011 Chairman Gemeinsame Fachgruppe Chemische Biologie DECHEMA 2014 Advisory Board SFICAST (University Oslo) The Screening Unit serves as an open access technology platform for automated screening, using either compound libraries such as the ChemBioNet and other collections (60,000 cpds) or genome-wide RNAi libraries (human, mouse, nematodes). Besides supporting assay development, process automation, screening and automated data analysis, the Unit identifies novel screening technologies (such as flow cytometry, impedance measurements, High- Content-Screening, AlphaScreen, capillary electrophoresis, real-time kinetic cell-based assays) and implements these for service. The Unit currently supports compound screening projects in assay development and optimization for High-Throughput Screening (HTS, Silke Radetzki), and in process automation (Martin Neuenschwander), including automated data documentation and analysis. Genome-wide RNAi screening (Katina Lazarow) has been established as a service complementing the identification of cellular targets through similar cellular phenotypes that have been generated either by compounds or RNA-interference. The Unit closely collaborates with the Medicinal Chemistry group for biological profiling of chemical optimization for Structure-Activity Relationship (SAR). ZUSAMMENFASSUNG Die Screening Unit ist eine frei zugängliche Technologieplattform für automatisierte systematische Testungen ( Screenings ); verwendet werden entweder Substanzbibliotheken wie die ChemBioNet- und andere Sammlungen ( chemische Substanzen) oder genomweite RNAi-Bibliotheken (Mensch, Maus, Nematoden). Neben der Unterstützung von Testentwicklung, Prozessautomatisierung, Screening und automatischer Datenanalyse identifiziert die Screening Unit neue Screening-Techniken (wie Durchflusszytometrie, Impedanzmessungen, High-Content-Screening, AlphaScreen, Kapillarelektrophorese, zellbasierte Tests mit Echtzeit-Kinetik) und implementiert diese für den Einsatz. Sie unterstützt derzeit Screeningprojekte mit chemischen Verbidnugen bei der Entwicklung und Optimierung von Testverfahren zum Hochdurchsatz-Screening (HTS, Silke Radetzki), bei der Prozessautomatisierung (Martin Neuenschwander) und bei der automatisierten Datendokumentation und Analyse. Das genomweite RNAi-Screening (Katina Lazarow) wurde als Service-Einheit etabliert, um die Identifizierung von zellulären Zielstrukturen ( Targets ) durch ähnliche zelluläre Phänotypen zu erweitern, die entweder durch Substanzen oder RNA-Interferenz erzeugt wurden. Die Screening Unit arbeitet eng mit der Arbeitsgruppe Medizinalchemie bei der biologischen Profilierung zur chemischen Optimierung im Zusammenhang von Struktur-Wirkungsbeziehungen (SAR) zusammen. Genome-wide RNAi for identification of the Volume-Regulated Anion Channel VRAC The group of Thomas Jentsch and the FMP Screening Unit identified a long-sought channel that helps cells to reduce their volume. For this purpose a human genome-wide RNA-interference library was tested with a cellular reporter system for iodide influx and intracellular quenching of an iodide-sensitive yellow fluorescent protein. The messenger RNA of 21,687 genes was targeted by ~130,000 transfections with interfering RNA. The Volume-Regulated Anion Channel was stimulated by a change of cell culture medium from isotonic to hypotonic conditions. The inhibition of quenching provided a read out using high-speed kinetic imaging (FLIPR Tetra-system, Molecular Devices). Eighty-seven genes were identified as candidates and were taken into a secondary screen with newly designed silencer RNAs for identification of LRRC8 heteromers as essential components of VRAC. Interfering with Influenza infection by inhibition of host cell functions Targeting of host cell functions such as enzymatic activities represents a novel therapy concept against viral infection and was developed by Thomas Meyer. In collaboration with the ANTIFLU consortium (Thomas F. Meyer, Max-Planck Institute for Infection Biology, Berlin) we have started to identify inhibitors of kinases that have been previously validated by genome-wide RNA-interference as playing key roles in viral infection. Validated hits from screens with the kinases CLK1 and ACK1, identified by capillary electrophoresis, have also been validated as interfering with viral growth in host cells. First inhibitors for MALT-1 caspase interfering with NFKB signaling in B-cell lymphoma (ABC-DLBCL) The Screening Unit carried out HTS with the ChemBioNet collection using MALT-1 caspase (Nagel et al. 2012, Cancer Cell) and a FRET assay using a synthetic peptide as the substrate for the protease. The collaboration with the laboratory of Daniel Krappmann (Helmholtz Zentrum München) resulted in identification of inhibitors of MALT-1, which selectively kill diffuse large B-cell lymphoma (ABC-DLBCL) in vitro and in vivo. As these compounds are already widely used as antipsychotic drugs, they will facilitate clinical trials for an off-label use in ABC-DLBCL therapy. plays a key role in the entry of pathogens (HIV, bacteria), but also in the nervous system for synaptic signaling and for recycling of trans-membrane receptors. In collaboration with the research group of Volker Haucke we used an ELISA to identify compounds named pitstops that specifically interfere with clathrin protein interactions and thereby also interfere with HIV infection (von Kleist et al. 2011, Cell). Wnt-signaling inhibitors interfering in vitro and in vivo with growth of tumor cells Canonical Wnt signaling is deregulated in several types of human cancer, where it plays a central role in tumor cell growth and progression. The last step of the signal cascade consists of gene activation by β-catenin in complex with LEF / TCF transcription factors. In collaboration with Stefan Krauss (University of Oslo) we set up a High-Content Screen with automated microscopes. The Screening Unit identified three novel small molecules that specifically inhibit the canonical Wnt pathway at the level of the destruction complex for β-catenin (Cancer Research, 2011). Tankyrase was identified as the target of the inhibitor, which acts through stabilization of Axin2, a negative regulator of Wnt signaling (Cancer Research, 2012). The compounds were also validated in vivo by inhibiting the growth of human tumor cells in mouse xenografts, and in ApcMin mice (multiple intestinal neoplasia, Min), which represent an animal model for the development of human colon cancer. Inhibitors for Met receptor-mediated metastasis of tumor cells The Unit focused its research on the inhibition of Met receptorsinduced scattering of tumor cells in vitro as a cellular model for metastasis. A cellular assay with a human pancreatic tumor cell line was optimized for high content screening using fluorescent staining of DNA, of actin filaments and cytoplasm. Automated object identification of cells growing connected in colonies (unscattered, HGF) and single cells (scattered, +HGF), which left the colonies, was set up (MolDiaPacra, EU-funded and SFMET, EU-funded). Moreover, we established a label-free impedance measurement rocedure for scattering. While imaging allowed us to identify Metinduced scattering in the hours range, the impedance measurements identified significant alterations on a scale of minutes. Specific inhibition of clathrin-mediated endocytosis Cellular transport from the cell s outside border to intracellular compartments, mediated by membrane vesicles and the protein clathrin,

66 128 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 129 Fig. 2 Fig. 1: We set up a 2D cell culture assay for high content screening with automated microscopes, which represents a model for metastasis of tumor cells initiated by addition of Hepatocyte Growth Factor (A, C: no initiation of metastasis, B, D: initiation) and enables one to track the migration of about 2,000 cells by colored lines for 24 hours (C,D). Without initiation, cells build colonies contacting each other and migrating within the colony area (A, C). After initiation, cells lose contact and migrate out of the colony area (B, D). Tumor cells are fluorescently stained red for actin (phalloidin) and green-blue for nuclei (Hoechst 3342, CMFDA). Fig. 2: In the absence of an inhibitor, β-catenin (green fluorescence) mainly localizes in the nuclei of colon cancer cells (left image), while application of the compound blocks nuclear localization and Sabrina Kleißle (MDC) and Silke Radetzki β-catenin localizes at cellular borderlines in adherens junctions (right image). The compound has been shown to inhibit tankyrase enzyme activity and thereby increases axin protein level in cells. Axin stimulates degradation of β-catenin protein, which is Fig. 1 not protected by cadherin protein in the adherens junctions, thus preventing Catenin signaling in the nucleus. GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS EXTERNAL FUNDING Dr. Katina Lazarow (RNAi) Dr. Silke Radetzki (High Content Screening) Dipl. Biologin Jamina Eckhard (RNAi) Dr. Martin Neuenschwander (Process Automation, HTS-Analysis) M.Sc. Marc Wippich (HTS) Dr. Simone Gräber (High Content Screening) Sabrina Kleißle (MDC) Andreas Oder Carola Seyffarth International Stefan Knapp Oxford University Susanne Müller-Knapp Oxford University Len Stephens Babraham Institute, Cambridge, U.K. Stefan Krauss University Oslo National Thomas F. Meyer MPIIB, Berlin Walter Birchmeier MDC, Berlin Claus Scheidereit MDC, Berlin Michael Bader MDC, Berlin Udo Heinemann MDC, Berlin Thomas Gress Uniklinik, Marburg Lenhard Rudolph FLI, Jena Voss FK, Ullrich F, Münch J, Lazarow K, Lutter D, Mah N, Andrade- Navarro MA., von Kries JP, Stauber T, Jentsch TJ (2014) Identification of LRRC8 Heteromers as an Essential Component of the Volume- Regulated Anion Channel VRAC. Science 344, Nagel D, Spranger S,Vincendeau M, Grau M, Raffegerst S, Kloo B, Hlahla D, Neuenschwander M, von Kries JP, Hadian K, Dörken, Lenz P, Lenz G, Schendel DJ, Krappmann D (2012). Pharmacologic Inhibition of MALT1 Protease by Phenothiazines as a Therapeutic Approach for the Treatment of Aggressive ABC-DLBCL. Cancer Cell 22, Waaler J, Machon O, Tumova L, Dinh H, Korinek V, Wilson SR, Paulsen JE, Pedersen NM, Eide TJ, Machonova O, Gradl D, Voronkov A, von Kries JP, Krauss S (2012) A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice. Cancer Cell 22, Helmholtz Wirkstoffforschung BMBF Deutsche Forschungsgemeinschaft, Synthesis, optimisation, and screening of small molecule libraries targeting protein-protein interactions, FOR 806, TP Z1 (RA 895 / 5 1), with J. Rademann, M. Beyermann, , Bundesministerium für Bildung und Forschung, Screening Unit: Assay development screening for lead identification and optimization, 01GU0514 KR, with C. Freund, R. Kühne, , Europäische Kommission, (6. Forschungsrahmenprogramm) PL018771, with J. Rademann, , Ulrich Martin MHH, Hannover Clemens Schmitt Charité, Berlin von Kleist L, Stahlschmidt W, Bulut H, Gromova K, Puchkov D, Robertson MJ, MacGregor KA, Tomlin N, Pechstein A, Chau N, Chircop M, Sakoff J, von Kries JP, Saenger W, Kräusslich H-G, Shupliakov O, Robinson PJ, McCluskey A, Haucke V (2011). Essential Role of the Clathrin Terminal EU-ANTIFLU FP7, Coordinator: Thomas F. Meyer, (MPI-IB, Berlin) Juni 2011 Juni 2015, EU-SFMET FP7-HEALTH-2007-A, HGF / SF and MET in metastasis EU-SFMET, Coordinator: Ermanno Gherardi, (MRC-Cambridge, UK), Domain in Regulating Coated Pit Dynamics Revealed by Small Molecule , Inhibition. Cell 146, MDC, Screening Unit, , Timm T, von Kries JP, Li X, Mandelkow E, and Mandelkow E-V (2011) Microtubule affinity regulating kinase (MARK) activity in living neurons PAKT (FLI-Jena), with W. Rosenthal, , examined by a genetically encoded FRET / FLIM based biosensor: ECRC, with MDC, , Inhibitors with therapeutic potential. J. Biol. Chem. 286, BMBF / MDC, RNAi & REMP, with MDC, J. Rademann, , FMP authors Group members NGFNplus, 5 Projektförderungen á ,

67 130 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 131 CORE FACILITY PEPTIDE SYNTHESIS PEPTIDSYNTHESE GROUP LEADER DR. RUDOLF VOLKMER BIOGRAPHY SUMMARY DESCRIPTION OF PROJECTS Industrial training as a laboratory assistant at Hoechst AG, Frankfurt / Main Chemistry at the University of Frankfurt / Main and FU Berlin Freelancer at the German Trade Union Federation, Frankfurt / Main 1984 Diploma thesis at the FU Berlin (Prof. Rewicki) 1991 Ph.D. at the FU Berlin (Prof. Rewicki) 1991 Teacher at the DRK-nurse s training school Post-Doc at Charité Universitätsmedizin Berlin (Prof. Schneider-Mergener) Since 1999 Group leader Molecular Libraries and Recognition Group at Charité Universitätsmedizin Berlin, Institute of Medical Immunology Since 2013 Guest scientist at the FMP, group leader Peptide Synthesis The group was formed in February 2013 as a service unit for peptide synthesis and is part of the department of Chemical Biology II, headed by Prof. Dr. Christian Hackenberger. Our aim is to provide synthetic peptides to all research groups at the FMP. In general, peptides are prepared according to the solid phase peptide synthesis method. For peptide synthesis, the full repertoire of standard solid phase peptide synthesis is used, resulting in linear peptides with or without side chain and / or termini-modifications. More challenging peptides can also be synthesized following the relevant consultations. For peptide synthesis a small reimbursement for the material costs are requested. Besides the standard peptide synthesis, our group offers access to peptide libraries and peptide arrays on cellulose membranes for screening purposes. These research tools are prepared at the Charité Universitätsmedizin Berlin, Molecular Libraries and Recognition Group. The service unit for peptide synthesis is embedded as a cooperation partner in ongoing research projects at the FMP and at the Charité. ZUSAMMENFASSUNG Unserer Gruppe hat ihre Tätigkeit als Serviceeinheit Peptid Synthese im Februar 2013 aufgenommen. Sie ist Teil der Chemischen Biologie II welche von Prof. Dr. Christian Hackenberger geleitet wird. Die Aufgabe der Serviceeinheit ist es allen Arbeitsgruppen des FMP Zugang zu synthetischen Peptiden anzubieten. In der Regel werden diese über die Technik der Festphasensynthese hergestellt wobei das Standartrepertoire jener Technik eingesetzt wird. Somit können lineare Peptide mit oder ohne Seitengruppen- und / oder Terminus-Modifizierung hergestellt werden. Kompliziertere Peptidstrukturen können nach Rücksprache ebenfalls hergestellt werden. Für die Peptidsynthese wird ein Unkostenbeitrag für die benötigten Materialien erhoben. Neben der Peptidsynthese offerieren wir Zugang zu Peptidbibliotheken und Peptidarrays auf Cellulosemembranen die für Screening Zwecke eingesetzt werden können. Diese werden an der Charité Universitätsmedizin Berlin synthetisiert. Die Serviceeinheit ist als Kooperationspartner in laufende Forschungsprojekte am FMP und an der Charité eingebunden. ProMs: A modular toolkit to inhibit proline-rich motif mediated protein-protein interactions Recently, the service unit for peptide synthesis has been involved in the synthesis and application of Pro-Pro dipeptide mimics (ProMs) with a PPII helix conformation. The project is funded in the context of a VIP BMBF project (ProMiCom, coordinated by Ronald Kühne, FMP). ProMs are chemical tools for constructing cell-permeable, selective inhibitors of heretofore undruggable proline-rich motif mediated protein-protein interactions. The targets are domains specialized in recognizing proline-rich segments (PRS), including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena) / vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. The project allows us, for the first time, to replace PRS using a toolkit of chemical fragments (ProMs) in a modular way. Small, selective, cell-permeable, non-peptidic inhibitors of Ena / VASP EVH1 domains were developed. One such example of a non-peptidic ligand is shown in Figure 1. Highly invasive MDA MB 231 breast cancer cells treated with this ligand showed displacement of VASP from 1 focal adhesions as well as from the front of lamellipodia and yielded a two-thirds reduction in cell invasion. The design strategy appears to be generally applicable to all PRS; however, this requires further substantiation on the basis of ProM-containing ligands designed for PRS-recognizing protein domains. Reducing the pathogenicity of cystic fibrosis: stabilizing the CFTR at the apical membrane using CAL-PDZ inhibitors The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that is mutated in patients with cystic fibrosis (CF), disrupting fluid and ion balance in multiple epithelial tissues. ~90 % of CF patients carry one or two copies of the ΔF508 allele, which encodes a protein that is inefficiently folded, shows limited channel activity, and is rapidly degraded. Compounds have been identified that address the folding and channel defects. Neither provides significant benefits as a monotherapy, but in combination they produce significant improvement in lung function (ΔFEV 1 >10 %) in 25 % of ΔF508 homozygous patients. To reach more patients and increase the functional response, we have proposed the early-stage pharmacological validation of a novel translational strategy to address the remaining defect the breakdown of rescued ΔF508-CFTR. No clinical trials have yet included compounds specifically designed to increase CFTR stability at the apical membrane. Having identified the CFTR-Associated Ligand (CAL) as a key mediator of CFTR degradation, we have localized a critical binding interface, designed peptides (named ical36 and ical42) that block it, and have shown that they act as first-in-class stabilizers of functional ΔF508-CFTR in polarized CF bronchial epithelial cells. Preclinical advancement of our inhibitor-of-cal (ical) approach is currently limited by lead affinity, delivery, and limited data regarding the extent of additional rescue compared to combination therapies currently used in clinical trials. The project focuses on several cell-permeable icals, on demonstrating the intracellular ical-cal interaction, and the stabilizers effect on functional CFTR and ΔF508-CFTR. Cellular models are Caco-2 cells, ΔF-CFBE cells and human biopsies of CF-patients. Our new data confirm substantial additivity for a cell-permeable ical in concert with a known corrector, the small molecule VX-809. Figure 2 depicts the cellular internalization of modified ical peptides into Caco-2 cells, as well as the colocalization of a Tamra-labelled ical36 peptide with CAL in GFP-CAL transfected Caco-2 cells. The project is NIH-funded and our cooperation partners are: Dean Madden, Hanover, USA; Rudolf Volkmer and Nico Derrichs Charité Universitätsmedizin, Berlin; Prisca Boisguerin, Montpellier, France; and Hartmut Oschkinat at the FMP Berlin.

68 132 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 133 A B C MPG-iCAL42, 1μM, 1h Pen-iCAL42, 1μM, 1h Tamra -MPG-iCAL36 GFP-CAL Hoechst Fluorescence Tamra-Pen-iCAL36 Magnification Ac-[2-CI-Phe]-[ProM-2]-[ProM-1]-OEt Transfected Caco-2 cells: GFP-CAL Tamra-MPG-iCAL36: 1μM Nucleus Dye: Hoechst Incubation: 1h Tamra-Myr-iCAL42, 1μM, 1h Tamra-Myr-iCAL42, 1μM, 3h Fig. 1: Structure of a non-peptidic VASP-EVH1 ligand. Starting from a VASP-EVH1 binding peptide (Ac-SFE-FPPPP-TEDEL amid, K D 20µM), a rational design allows transformation to non-peptidic VASP-EVH ligands. One of the shortest, non-peptidic ligands includes the unnatural amino acid 2-chloro-L-phenylalanine (2-Cl-Phe) and two PPII-mimetic modules (ProM-1, ProM-2) and binds VASP-EVH1 with 9 µm affinity. The structure of this non-peptidic VASP-EVH1 ligand Ac-[2-Cl-Phe]-ProM-2-[ProM-1]- OEt is shown. Fig. 2: Cellular internalisation and colocalisation of ical peptides. (A) Confocal laser scanning microscopy of the cellular internalisation of ical42 N-terminally modified with cell-penetrating peptides (MPG, Penetratin) and Myristic acid. Caco-2 cells were used for internalisation. Blue, nucleus stained with Hoechst dye; red, Tamra-Pen-iCAL42, Tamra-MPG-iCAL42, Myr-Lys(Tamra)- ical42. Cell penetrating peptides are able to translocate ical42 within 1h. However, it takes a period of 3 h using myristic acid for translocation. (B) Colocalization of GFP-CAL protein with Tamra-MPG-iCAL36 peptide. Confocal laser scanning microscopy of GFP-CAL transfected and Tamra-MPG-iCAL36 incubated Caco-2 cells. Yellow dots demonstrate the interaction between CAL and Tamra-MPG-iCAL36. (C) Confocal laser scanning microscopy of the Tamra-Pen-iCAL36 internalisation in human rectal biopsies. Blue: nucleus stained with Hoecht dye; red: Tamra-Pen-iCAL36. GROUP MEMBERS COLLABORATIONS SELECTED PUBLICATIONS Ines Kretzschmar (technical assistant) International Dean Madden Geisel School of Medicine at Dartmouth, Hanover, USA Prisca Boisguerin CRBM, Montpellier, France Tim Clausen University of Vienna, Austria Michel Steinmetz Paul Scherer Institut, Switzerland National Enno Klussmann Max-Delbrück-Center for Molecular Medicine, Berlin Karola Rück-Braun Technische Universität Berlin Christian Freund Freie Universität Berlin Jörg Höhfeld University of Bonn Rodriguez Plaza JG, Morales-Nava R, Diener C, Schreiber G, Gonzalez ZD, Lara Ortiz MT, Ortega Blake I, Pantoja O, Volkmer R, Klipp E, Herrmann A, Del Rio G (2014) Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin. J Biol Chem 289, Mastny M, Heuck A, Kurzbauer R, Heiduk A, Boisguerin P, Volkmer R, Ehrmann M, Rodrigues CD, Rudner DZ, Clausen T (2013) CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis. Cell 155, Jaeger IS, Kretzschmar I, Körner J, Weiser AA, Mahrenholz CC, Potty A, Kourentzi K, Willson RC, Volkmer R, Preissner R (2013) Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches. J. Mol. Recognit. 26, Hovestädt M, Kuckelkorn U, Niewienda A, Keller C, Goede A, Ay B, Günther S, Janek K, Volkmer R, Holzhütter HG (2013) Rapid degradation of solid-phase bound peptides by the 20S proteasome. J. Pept. Sci. 19, Marius Sudol Henning Mootz Ulbricht A, Eppler FJ, Tapia VE, van der Ven PF, Hampe N, Hersch N, Institute of Molecular and Cell Biology, University of Münster Vakeel P, Stadel D, Haas A, Saftig P, Behrends C, Fürst DO, Volkmer R, Singapore Michael Ehrmann University of Duisburg-Essen Hoffmann B, Kolanus W, Höhfeld J (2013) Cellular mechanotransduction relies on tension-induced and chaperone-assisted autophagy. Curr. Biol. 23, FMP authors Group members

69 Campus Berlin Buch Campus Berlin Buch All Research Groups Alle Forschungsgruppen PAGE 136 Map Campus Berlin Buch Karte Campus Berlin Buch PAGE 138 APPENDIX ANHANG Administrative and Technical Services Administrative und technische Dienstleistungen PAGE 140 Imprint Impressum

70 136 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 APPENDIX ANHANG 137 ALL RESEARCH GROUPS ALLE FORSCHUNGSGRUPPEN MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE STRUCTURAL BIOLOGY STRUKTURBIOLOGIE CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE DEPARTMENTS ABTEILUNGEN DEPARTMENTS ABTEILUNGEN DEPARTMENTS ABTEILUNGEN Physiology and Pathology of Ion Transport Thomas Jentsch Molecular Pharmacology and Cell Biology Volker Haucke NMR-Supported Structural Biology Hartmut Oschkinat Molecular Biophysics Adam Lange Chemical Biology I Dorothea Fiedler (from 04 / 2015) Chemical Biology II Christian Hackenberger RESEARCH GROUPS FORSCHUNGSGRUPPEN RESEARCH GROUPS FORSCHUNGSGRUPPEN RESEARCH GROUPS FORSCHUNGSGRUPPEN Protein Trafficking Ralf Schülein Molecular Cell Physiology Ingolf E. Blasig Structural Bioinformatics and Protein Design Gerd Krause Solution NMR Peter Schmieder Computational Chemistry / Drug Design Ronald Kühne Mass Spectrometry Eberhard Krause Medicinal Chemistry Marc Nazaré Peptide-Lipid Interaction / Peptide Transport Margitta Dathe JUNIOR RESEARCH GROUPS JUNIOR FORSCHUNGSGRUPPEN JUNIOR RESEARCH GROUPS JUNIOR FORSCHUNGSGRUPPEN Molecular Neuroscience and Biophysics Andrew Plested Membrane Traffic and Cell Motility Tanja Maritzen Proteostasis in Aging and Disease Janine Kirstein LIAISON GROUP NEUROSCIENCE Behavioural Neurodynamics Tatiana Korotkova, Alexey Ponomarenko LIAISON GROUP NEUROSCIENCE Molecular and Theoretical Neuroscience Alexander Walter (from 01 / 2015) In-Cell NMR Philipp Selenko Molecular Imaging Leif Schröder CORE FACILITIES CORE FACILITY CORE FACILITIES Cellular Imaging Burkhard Wiesner / Dmytro Puchkov Animal Facility Natali Wisbrun NMR Hartmut Oschkinat / Peter Schmieder Peptide Synthesis C. Hackenberger / R. Volkmer Screening Unit Jens Peter von Kries

71 138 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 APPENDIX ANHANG 139 CAMPUS BERLIN BUCH CAMPUS BERLIN BUCH RESEARCH Leibniz-Institut für Molekulare Pharmakologie (FMP) C 81 Marthe-Vogt-House C 81.1 NMR I C 81.2 NMR 2 Max Delbrück Center for Molecular Medicine (MDC) C 27 C 31 C 83 C 84 A 10 B 63 Walter-Friedrich-House Max-Delbrück-House Max Delbrück Communications Center Hermann-von-Helmholtz-House Library Research services Shared Facilities by MDC and FMP C 84.1 Research services C 87 Timoféeff-Ressovsky-House CLINICAL RESEARCH COMMON FACILITIES A 8 A 9 A 13 A 14 Gate House with Café Max Reception Life Science Learning Lab; Campus-Info-Center Cafeteria Guesthouses of the MDC B 54 B 61 Hans-Gummel-Guest House Kindergarten; Salvadore-Luria-Guest House COMPANIES

72 140 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 ADMINISTRATIVE AND TECHNICAL SERVICES ADMINISTRATIVE UND TECHNISCHE DIENSTLEISTUNGEN IMPRINT IMPRESSUM DIRECTORATE Prof. Dr. Volker Haucke Director Dr. Henning Otto Dr. Elvira Rohde Scientific Coordinators Silke Oßwald Public Relations Manager Dr. Anne Höner EU-Liaison Officer Dr. Birgit Oppmann Technology Transfer Katrin Wittig PhD-Programme Coordinator Alexandra Chylla Secretary Heidemarie Petschick Secretary OFFICES Andrea Steuer Department of NMR-Supported Structural Biology / Department of Molecular Biophysics Marianne Dreißigacker Department of Chemical Biology Dr. Norma Nitschke Scientific Coordinator Department of Physiology and Pathology of Ion Transport ADMINISTRATION Frank Schilling Head Administration Thomas Ellermann General Administration Marina Spors Personnel Management Christel Otto General Administration Claudia Messing General Administration Mathias Schmidt General Administration (until 05 / 2013) Dennis Bischoff General Administration (since 01 / 2014) Gabriele Schumacher Secretary COMPUTER SERVICES (IT) Ronald Jäckel Chief Information Officer (CIO), System Administration Ingrid Hermann System Administration Ingo Breng Service Engineer Alexander Heyne Service Technician TECHNICAL SERVICES Hans-Jürgen Mevert Marco Mussehl Holger Panzer Michael Uschner Stephanie Wendt Roy Wolschke Pascal Schulz Bernd-Uwe Wagner ANIMAL CARE OFFICER Dr. Nadjeschda Heinrichs WORKPLACE SAFETY Dr. Jens Furkert GENETIC ENGINEERING / BIOLOGICAL SAFETY Dr. Ralf Schülein Leibniz-Institut für Molekulare Pharmakologie (FMP) im Forschungsverbund Berlin e.v. Campus Berlin-Buch Robert-Rössle-Str Berlin Germany Phone Fax osswald@fmp-berlin.de Research Report 2013 / 2014 Editorial Board Christian Hackenberger, Volker Haucke, Thomas Jentsch, Adam Lange, Hartmut Oschkinat Coordination Silke Oßwald, Henning Otto Author Feature Articles and Interview Birgit Herden, Karl-Heinz Karesch Editing Martin McLean, Silke Oßwald, Henning Otto Translations Mick Locke Photography Silke Oßwald Further Photography Maj Brit Jansen (p. 38), Stefan Jentsch (p. 34) 3-D Illustration Barth van Rossum (p. 22) Scientific Figures Sections Michael Lisurek (p. 106), Barth van Rossum (p. 72), Jan Schmoranzer (p. 30) Design and Layout KRAUT & KONFETTI, Berlin Print Druckerei Conrad GmbH, Berlin Berlin, August 2015

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