RESEARCH REPORT 2015 / 2016

Transkript

1 RESEARCH REPORT 2015 / 2016

2 SCIENTIFIC ADVISORY BOARD Prof. Dr. Karl-Heinz Altmann Institut für Pharmazeutische Wissenschaften, ETH Zürich (seit 01 / 2012) Prof. Dr. Nils Brose (Vorsitzender) 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. Stefan Offermanns Max-Planck-Institut für Herz- und Lungenforschung, Bad Nauheim (seit 01 / 2016) Prof. Dr. Petra Schwille Max-Planck-Institut für Biochemie, Martinsried (seit 01 / 2012) Prof. Dr. Rebecca Wade (Stellvertretende Vorsitzende) Heidelberg Institute for Theoretical Studies, HITS ggmbh (seit 01 / 2013) Dr. Matthias Gottwald Bayer Pharma AG, Berlin (seit 01 / 2016) 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) Prof. Dr. Beat Meier Laboratorium für Physikalische Chemie, ETH Zürich (seit 01 / 2013) Stichtag

3 RESEARCH REPORT 2015 / 2016

4 2 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 CONTENTS INHALT PREFACE VORWORT Interview with Director Dorothea Fiedler What's new at the FMP? Was ist neu am FMP? 4 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG The cause of muscle weakness reveals the organisational principle in cells Ursache von vererbter Muskelschwäche aufgeklärt 8 Milestone for Parkinson's research: The amyloid protein α-synuclein is visualised in the cell for the first time Meilenstein für die Parkinson-Forschung: Amyloid-Protein α-synuclein erstmals in Zelle sichtbar gemacht 9 Double mechanism confirmed: How inositol pyrophosphates alter proteins Doppelter Mechanismus bestätigt: Wie Inositol-Pyrophosphate Proteine verändern 10 Interview with Thomas J. Jentsch "We've pushed the door wide open to novel biomedical insights" Wir haben die Tür für neue biomedizinische Erkenntnisse aufgestoßen 11

5 CONTENTS INHALT 3 RESEARCH GROUPS FORSCHUNGSGRUPPEN Section Molecular Physiology and Cell Biology Bereich Molekulare Physiologie und Zellbiologie 14 Physiology and Pathology of Ion Transport Thomas J. Jentsch 20 Molecular Pharmacology and Cell Biology Volker Haucke 24 Protein Trafficking Ralf Schülein 28 Molecular Cell Physiology Ingolf E. Blasig 31 Molecular Neuroscience and Biophysics Andrew J.R. Plested 34 Membrane Traffic and Cell Motility Tanja Maritzen 37 Proteostasis in Aging and Disease Janine Kirstein 40 Behavioural Neurodynamics Tatiana Korotkova / Alexey Ponomarenko 43 Molecular and Theoretical Neuroscience Alexander Matthias Walter 46 Cellular Imaging Burkhard Wiesner / Dmytro Puchkov 49 Animal Facility Natali Wisbrun 52 Section Structural Biology Bereich Strukturbiologie 54 Molecular Biophysics Adam Lange 60 NMR-Supported Structural Biology Hartmut Oschkinat 64 Solution NMR Peter Schmieder 68 Computational Chemistry / Drug Design Ronald Kühne 71 Structural Bioinformatics and Protein Design Gerd Krause 74 In-Cell NMR Philipp Selenko 77 Molecular Imaging Leif Schröder 79 NMR Hartmut Oschkinat / Peter Schmieder 82 Section Chemical Biology Bereich Chemische Biologie 86 Chemical Biology II Christian P. R. Hackenberger 92 Chemical Biology I Dorothea Fiedler 96 Peptide-Lipid Interaction / Peptide Transport Margitta Dathe 100 Mass Spectrometry Eberhard Krause 103 Medicinal Chemistry Marc Nazaré 106 Screening Unit Jens Peter von Kries 109 Peptide Synthesis Rudolf Volkmer 112 APPENDIX ANHANG All Research Groups 116 Map Campus Berlin Buch 118 Administrative and Technical Services 120 Imprint

6 4 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 WAS IST NEU AM FMP? WHAT S NEW AT THE FMP? Prof. Dr. Dorothea Fiedler Director at Leibniz-Forschungsinstitut für Molekulare Pharmakologie Dorothea Fiedler has been director at the FMP since July In an interview, she speaks about her move from the USA to Germany, the focus of her research and about current developments at the FMP. Professor Fiedler, you were a distinguished scientist in the USA, latterly at Princeton University, and have received numerous awards. What motivated your move to Berlin-Buch? There were several reasons. The most important one was undoubtedly that the FMP is an excellent institute, where interdisciplinary research is conducted at the interface of biology and chemistry. To this extent, the science at the institute was a perfect match. In addition, I can collaborate with outstanding people here, both internally and externally. And then the FMP offers exceptional research conditions. All of this was simply too tempting for me to turn down the offer of a W3S professorship linked to the post of director, although I was very happy in the USA. Together with Professor Volker Haucke you form a dual leadership at the FMP. How do you divide up this important task? There was a clear agreement that I would officially take over the management after 18 months. That is now the case as of 1 January, but we continue to complete many tasks together and always consult each other on important decisions. You are responsible for 300 employees. Is there any time left to do your own research in the laboratory? Unfortunately, it has been a while since I ran experiments in the laboratory. But at this point, my co-workers can carry out the work better than me. But naturally I still work closely with my PhD students and postdocs. Research is and will certainly remain the part of my work that I enjoy the most. What does a professor of chemical biology conduct research on? In many areas of cell biology we have now arrived at a point where we are trying to decode cellular processes at the molecular level. For me as a chemist that's really exciting. One focus of our work are the so-called inositol pyrophosphates. These molecules are messenger substances in the cell that, for example, play an important role in disorders of fat metabolism and insulin secretion, but also have an influence on cell migration, specifically metastasis. In effect we are trying to understand the language of chemistry and thus hope to lay the foundations for new therapies. Is it the promise of new therapies that motivates you to conduct basic research? The desire to improve the health of humankind is fundamental to all of us. Admittedly, most of the groups at the FMP are not developing therapies in an actual sense, but it is the basic research conducted at the Institute that paves the way. This can be seen, for example, in the fact that two spin-offs from the Hackenberger and Kühne groups are in preparation. One-third of the research groups at the FMP are junior groups. What's new here? A great deal. In the field of molecular physiology and cell biology, there is a new liaison group, Neurosciences, the Emmy Noether Junior Group led by Alexander Walter. In addition, our junior group leader Andrew Plested has been offered a Heisenberg professorship at the Humboldt University. His group will remain at the FMP as a guest group for an additional five years. In contrast, we will have to say farewell to Philipp Selenko this year. He has successfully led a junior group in structural biology for several years and has now accepted an attractive offer from the Weizmann Institute of Science in Israel. It is all the more pleasing that a new junior group at the interface of NMR and Cryo-EM is to be established in this important field of research, this being within the context of the planned Cryo-EM infrastructure, in which the Berlin Universities and the MDC will also be involved. Our doctoral students are very successful as well, for instance Jean-Philippe Demers from the department of Adam Lange received the Raymond Andrew Prize and the Otto Hahn Medal in 2015, a really remarkable accomplishment.

7 WHAT'S NEW AT THE FMP? WAS IST NEU AM FMP? 5 Matthias Schnurr, Honor Rose, Jabadurai Jayapaul Our junior group leader Leif Schröder established a cooperation with the California Institute of Technology in Pasadena to develop ultra-sensitive magnetic resonance imaging, which allows to detect tumours, for example. At the same time, last year, he secured a Reinhart Koselleck-Project of the Deutsche Forschungsgemeinschaft (DFG) for a similar undertaking. The funding amounts to million euros and was the first ever Koselleck Project for the Leibniz Association. I find all of this quite outstanding. At the beginning of this year, there was then a second Reinhart Koselleck-Project for the FMP and a small sensation for the Institute? That's right, just a few months later, Volker Haucke impressed the DFG with his application for research into neuronal communication. We are very proud of that. Both awards show the high level of research at the FMP. As early as 2015, Philipp Selenko and Andrew Plested were awarded the prestigious Consolidator Grant of the European Research Council (ERC) for their research work. Over the past five years, Thomas J. Jentsch successfully applied for these most prominent and coveted programmes offered by the European Commission, for his research on ion channels he received an Advanced Grant to the amount of 2.5 million euros from the European Research Council (ERC) for the first time in 2012, followed in March this year by a second ERC Advanced Grant. A great success! And what about your specialist field, chemical biology? Here, too, there are plans to establish a junior group, although I'm afraid I can't reveal any details at this stage. However, we are very pleased that Dr. Fan Liu will arrive during the second half of this year to oversee the Institute s Mass Spectrometry Facility. She will be able to build on the excellent infrastructure provided by Eberhard Krause s group, and on top of that establish her own research group with a number of exciting and current projects. We are thrilled about her arrival. The FMP will celebrate it's 25 th birthday this year. Where do you see the Institute in 25 years' time? Despite a consistent mission, the FMP still remains in a certain state of upheaval. In the next couple of years, several colleagues will be retiring, some of whom already worked at the predecessor institute in the Institute of Drug Research, Academy of Sciences of the GDR. It's a shame, but that's the way things go. I think, and I can speak for the past and for the future, that the FMP is an institute that places special value on modern methods and technology. With this strategy, and of course driven by our excellent scientists, we will in the long run secure pioneering results that will be of benefit to the scientific community and ultimately to society. What I would like to see is more international visibility. The FMP is very well connected throughout Germany and also internationally. Nevertheless, the Institute, like the Leibniz Association, is not yet as well known internationally as other German research organisations. We're working on that. Dorothea Fiedler ist seit Juli 2015 Direktorin am FMP. Im Interview spricht sie über ihren Wechsel von den USA nach Deutschland, ihre Forschungsschwerpunkte und über aktuelle Entwicklungen am FMP. Frau Professor Fiedler, Sie waren in den USA, zuletzt an der Universität Princeton, eine angesehene Wissenschaftlerin und haben etliche Auszeichnungen erhalten. Was hat Sie nach Berlin-Buch verschlagen? Es waren mehrere Gründe. Das Wichtigste war sicher, dass das FMP ein exzellentes Institut ist, wo interdisziplinär an der Schnittstelle von Biologie und Chemie geforscht wird. Insofern hat es inhaltlich perfekt gepasst. Außerdem kann ich hier mit hervorragenden Leuten zusammenarbeiten, intern wie extern. Und dann verfügt das FMP über ausgezeichnete Forschungsbedingungen. All das war doch zu reizvoll, um den Ruf auf eine W3S-Professur verbunden mit dem Direktorenposten auszuschlagen, obwohl ich mich in den USA sehr wohlgefühlt habe. Zusammen mit Professor Volker Haucke bilden Sie eine Doppelspitze am FMP. Wie teilen Sie sich diese verantwortungsvolle Aufgabe? Es gab eine klare Absprache, dass ich nach eineinhalb Jahren offiziell die Geschäftsführung übernehmen werde. Seit 1. Januar ist das nun der Fall, dennoch erledigen wir nach wie vor viele Aufgaben gemeinsam und sprechen uns bei wichtigen Entscheidungen immer miteinander ab. Sie sind verantwortlich für 300 Mitarbeiter. Bleibt da noch Zeit, selbst im Labor zu forschen? Im Labor stehe ich leider schon lange nicht mehr. Das können meine Mitarbeiter mittlerweile auch besser. Aber natürlich arbeite

8 6 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 ich weiterhin eng zusammen mit meinen Doktoranden und Postdoktoranden. Die Forschung ist und bleibt sicherlich der Teil meiner Arbeit, der mir am meisten Freude bereitet. Woran forscht eine Professorin für Chemische Biologie? Bei vielen Aspekten der Zellbiologie sind wir jetzt an einem Punkt angekommen, wo man versucht, die Vorgänge in einer Zelle auf molekularer Ebene zu entschlüsseln. Für mich als Chemikerin ist das hoch spannend. Einer meiner Schwerpunkte sind die sogenannten Inositol-Pyrophosphate. Das sind bestimmte Botenstoffe in der Zelle, die zum Beispiel bei Fettstoffwechselkrankheiten und der Insulinsekretion eine wichtige Rolle spielen, aber auch Einfluss auf die Zellmigration, spezifisch die Metastasierung, haben. Wir versuchen sozusagen die chemische Sprache zu verstehen und hoffen, damit die Grundlagen für neue Therapien zu legen. Sind neue Therapien für Sie der Ansporn, Grundlagenforschung zu betreiben? Der Wunsch, die Gesundheit von Menschen zu verbessern, ist das, was uns alle hier antreibt. Wobei ich dazu sagen muss, dass die meisten Gruppen am FMP nicht im eigentlichen Sinne Therapien entwickeln, aber die Grundlagenforschung am Institut stellt dafür die entscheiden Weichen. Das sehen Sie zum Beispiel daran, dass gerade zwei Ausgründungen aus den Gruppen Hackenberger und Kühne in Vorbereitung sind. Ein Drittel der Forschergruppen am FMP sind Juniorgruppen. Was passiert hier Neues? Eine Menge. Im Bereich Molekulare Physiologie und Zellbiologie gibt es eine neue Liaisongruppe Neurowissenschaften, die Emmy-Noether-Juniorgruppe um Alexander Walter. Darüber hinaus erhält unser Juniorgruppenleiter Andrew Plested eine Heisenberg-Professur an der Humboldt-Universität. Seine Gruppe wird für weitere fünf Jahre am FMP als Gastgruppe verbleiben. Abschied werden wir dagegen noch in diesem Jahr von Philipp Selenko nehmen müssen. Er hat seit mehreren Jahren erfolgreich eine Nachwuchsgruppe in der Strukturbiologie geleitet und nun ein attraktives Angebot vom Weizmann Institute of Science in Israel angenommen. Umso erfreulicher ist, dass in diesem wichtigen Forschungsbereich eine neue Nachwuchsgruppe an der Schnittstelle von NMR und Cryo-EM eingerichtet werden soll, und zwar im Rahmen der geplanten Cryo-EM-Infrastruktur, an der auch die Berliner Universitäten und das MDC beteiligt sein werden. Unsere Doktoranden sind übrigens auch sehr erfolgreich, Jean-Philippe Demers aus der Abteilung von Adam Lange hat 2015 den Raymond Andrew Preis und die Otto-Hahn Medalle erhalten! Unser Nachwuchsgruppenleiter Leif Schröder konnte mit dem California Institute of Technology in Pasadena eine Kooperation zur Entwicklung ultra-sensitiver Magnetresonanz-Bildgebung etablieren, mit der etwa Tumoren aufgespürt werden können. Gleichzeitig hat er im vergangenen Jahr für ein ähnliches Vorhaben das Reinhart Koselleck-Projekt der Deutschen Forschungsgemeinschaft (DFG) eingeworben. Die Förderung beläuft sich auf 1,525 Millionen Euro und war das erste Kosseleck-Projekt für die Leibniz-Gemeinschaft überhaupt. Ich finde, das ist alles sehr bemerkenswert. Anfang dieses Jahres gab es dann gleich ein zweites Reinhart Koselleck-Projekt für das FMP und noch eine kleine Sensation für das Institut? Ja, nur wenige Monate später konnte Volker Haucke die DFG mit seinem Projektanatrag zur Erforschung der neuronalen Kommunikation überzeugen. Darauf sind wir sehr stolz. Beide Auszeichnungen zeigen, wie hochranging am FMP geforscht wird. Schon 2015 wurden Robert Puschmann, Sarah Hostachy and Dario Demartin Philipp Selenko und Andrew Plested für ihre Forschungen mit dem hochrangigen Consolidator Grant des Europäischen Forschungsrates (ERC) ausgezeichnet. Mit dieser profiliertesten und begehrtesten Ausschreibung der Europäischen Kommission war Thomas J. Jentsch in den vergangenen fünf Jahren gleich zwei Mal erfolgreich, für seine Forschung an Ionenkanälen erhielt er zum ersten Mal 2012 einen Advanced Grant in Höhe von 2,5 Millionen Euro vom Europäischen Forschungsrat (ERC) und im März dieses Jahres gleich den zweiten ERC Advanced Grant, ein großer Erfolg! Und was ist mit Ihrem Steckenpferd, der Chemischen Biologie? Auch hier ist der Aufbau einer Nachwuchsgruppe in Planung, Details kann ich Ihnen zu diesem Zeitpunkt leider noch nicht verraten. Allerdings sind wir sehr glücklich, dass wir die Gruppe Massenspektrometrie weiterführen und Ende dieses Jahres mit Dr. Fan Liu neu besetzen. Sie wird die exzellente Infrastruktur, die Eberhard Krause über Jahrzehnte etabliert hat, weiterführen und dazu ihre eigene Forschungsgruppe aufbauen mit sehr aktuellen und interessanten Forschungsprojekten. Wir freuen uns sehr darauf, Dr. Liu demnächst bei uns willkommen zu heißen. Das FMP wird in diesem Jahr 25 Jahre alt, wo sehen Sie das Institut in 25 Jahren? Trotz stetiger Mission, befindet sich das FMP immer noch in einem gewissen Umbruch. Schon in den nächsten zwei Jahren werden uns Kollegen und Kolleginnen verlassen und in den Ruhestand treten, die zum Teil bereits im Vorläuferinstitut im Institut für Wirkstofforschung der Akademie der Wissenschaften der DDR gearbeitet haben. Das ist bedauerlich, aber eben der Lauf der Dinge. Ich denke, und da spreche ich für die Vergangenheit und für die Zukunft, dass das FMP ein Institut ist, welches besonderen Wert auf modernste Techniken legt. Mit dieser Strategie und unseren exzellenten Wissenschaftlern sichern wir uns langfristig inhaltlich interessante und zukunftsweisende Ergebnisse, die die wissenschaftliche Gemeinschaft und letztendlich die Gesellschaft bereichert. Was ich mir wünsche ist international mehr Sichtbarkeit. Das FMP ist deutschlandweit aber auch international sehr gut vernetzt. Trotzdem ist das Institut, wie auch die Leibniz- Gemeinschaft, international noch nicht so bekannt, wie andere deutschen Forschungsorganisationen. Daran arbeiten wir.

9 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 7 RESEARCH HIGHLIGHTS

10 8 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 THE CAUSE OF MUSCLE WEAKNESS REVEALS THE ORGANISATIONAL PRINCIPLE IN CELLS URSACHE VON VERERBTER MUSKELSCHWÄCHE AUFGEKLÄRT In hereditary myotubular myopathy, the muscles are severely atrophied and the children rarely survive. In co-operation with French human geneticists, the group led by Volker Haucke has researched what goes wrong on a molecular level in this disease and has stumbled upon a general organisational principle in cells. The disease is caused by a defect in an enzyme that is specialized in transforming certain membrane lipids, the phosphoinositide phosphates (PIPs). As shown by the team using clever experiments and high-resolution imaging from inside the cell, this defect leads to substance transport within the cells coming to a halt. This work has made clear how the dynamic processes in cells are directed by the targeted transformation of PIPs. The compartments and transport vesicles within the cells repeatedly cloak themselves with different PIPs and thus change their identity, says Volker Haucke. This indicates whether a transport container belongs inside the cell or whether it is supposed to discharge its freight outside. In their experiments in cell culture, the FMP researchers were able to restart the transport with a certain active substance. This might be a starting point for the development of drugs for treating this severe and currently incurable hereditary disease. Bei der vererbten Myotubulären Myopathie sind die Muskeln stark verkümmert, die betroffenen Kinder kaum lebensfähig. Die Gruppe um Volker Haucke hat zusammen mit französischen Humangenetikern erforscht, was bei dieser Krankheit auf der molekularen Ebene schiefläuft und ist dabei auf ein allgemeines Organisationsprinzip in Zellen gestoßen. Die Krankheit entsteht durch einen Defekt in einem Enzym, dass darauf spezialisiert ist, bestimmte Membranlipide, die Phosphoinositidphosphaten (PIPs) umzuwandeln. Wie das Team mit trickreichen Experimenten und hochaufgelösten Aufnahmen aus dem Zellinneren zeigen konnte, kommt dadurch der Stofftransport innerhalb von Zellen zum Erliegen. Durch die Arbeit ist klargeworden, wie die dynamischen Abläufe in Zellen durch die gezielte Umwandlung der PIPs dirigiert werden. Die Kompartimente und Transportvesikel innerhalb der Zellen kleiden sich in immer wieder andere PIPs und wechseln dadurch ihre Identität, sagt Volker Haucke. So wird angezeigt, ob ein Transportbehälter ins Zellinnere gehört oder ob er seine Fracht ins Freie entlassen soll. Bei ihren Experimenten in Zellkultur konnten die FMP-Forscher den Transport mit einem bestimmten Wirkstoff wieder in Gang setzten. Dies wäre ein Ansatzpunkt für die Entwicklung von Medikamenten, um die schwerwiegende und derzeit unheilbare Erbkrankheit zu behandeln. (Katharina Ketel) integrin CONTROL CELL integrin LOSS OF MTM1 Accumulation of integrin (red), an important component of muscles, in vesicles (green) from cells without MTM1 (right images, including magnified view) or from control cells (left images, including magnified view). Akkumulation von Integrin (rot), ein wichtiger Baustein von Muskeln, in Vesikeln (grün) aus Zellen ohne MTM1 (rechte Bilder inkl. Vergrößerung) im Vergleich zu Kontrollzellen (linke Bilder inkl. Vergrößerung). vesical vesical Ketel K, Krauss M, Nicot AS, Puchkov D, Wieffer M, Müller R, Subramanian D, Schultz C, Laporte J, Haucke V (2016) A phosphoinositide conversion mechanism for exit from endosomes. Nature 529,

11 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 9 MILESTONE FOR PARKINSON'S RESEARCH: THE AMYLOID PROTEIN α -SYNUCLEIN IS VISUALISED IN THE CELL FOR THE FIRST TIME MEILENSTEIN FÜR DIE PARKINSON-FORSCHUNG: AMYLOID-PROTEIN α -SYNUCLEIN ERSTMALS IN ZELLE SICHTBAR GEMACHT (Philipp Selenko) Protein α-synuclein in healthy cells: The central NAC region (grey) is well protected. The protein ensures that no interaction occurs with the cytoplasm (white) and other cell components. In the case of neurodegenerative changes, the grey areas would grow together and form amyloid structures. Proteins α-synuclein in gesunden Zellen: Die zentrale NAC-Region (grau) ist gut geschützt. Das Protein sorgt dafür, dass es zu keiner Interaktion mit dem Zytoplasma (weiß) und anderen Zell-Komponenten kommt. Bei neurodegenerativen Veränderungen würden die grauen Bereiche zusammenwachsen und Amyloid-Strukturen ausbilden. The amyloid protein α-synuclein plays an important role in Parkinson's disease and other neurodegenerative diseases. It is known that this protein has very concrete structures in the pathological state; however, as isolated, purified protein it does not appear to have any structure at all. What has been missing up until now is an understanding of how α-synuclein is structured in the healthy cell. Scientists from the FMP (Philipp Selenko's research group) have now for the first time visualised the protein in healthy cells with the aid of high-resolution spectroscopic methods. Surprisingly, they found the same structure-less state that the protein has in its purified state. The new findings, which have been published in Nature and Nature Communications, are a milestone for research: We now know that the structure of the protein changes dramatically over the course of the disease. Das Amyloid-Protein α-synuclein spielt bei Parkinson und anderen neurodegenerativen Erkrankungen eine wichtige Rolle. Bekannt ist, dass dieses Protein im krankhaften Zustand über sehr konkrete Strukturen verfügt; als isoliertes, aufgereinigtes Protein scheint es jedoch überhaupt keine Struktur zu besitzen. Was bislang fehlte, war die Erkenntnis, wie α-synuclein in der gesunden Zelle aufgebaut ist. Wissenschaftler vom FMP (Forschungsgruppe Philipp Selenko) konnten das Protein jetzt erstmals mit Hilfe von hochauflösenden spektroskopischen Verfahren in gesunden Zellen sichtbar machen. Überraschendweise fanden sie jenen strukturlosen Zustand vor, den das Protein auch in aufgereinigtem Zustand hat. Die neuen Erkenntnisse, die in Nature und Nature Communications erschienen sind, sind ein Meilenstein für die Forschung: Jetzt weiß man, dass sich die Struktur des Proteins im Krankheitsverlauf dramatisch verändert. Binolfi A, Limatola A, Verzini S, Kosten J, Theillet F X, Rose H M, Bekei B, Stuiver M, van Rossum M, Selenko P (2016) Intracellular repair of oxidation damaged a-synuclein fails to target C-terminal modification sites. Nat Commun. 7, Theillet F X, Binolfi A, Bekei B, Martorana A, Rose H M, Stuiver M, Verzini S, Lorenz D, van Rossum M, Goldfarb D, Selenko P (2016) Structural disorder of monomeric a-synuclein persists in mammalian cells. Nature 530(7588),

12 10 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 DOUBLE MECHANISM CONFIRMED: HOW INOSITOL PYROPHOSPHATES INFLUENCE PROTEINS DOPPELTER MECHANISMUS BESTÄTIGT: WIE INOSITOL-PYROPHOSPHATE PROTEINE BEEINFLUSSEN They are called inositol pyrophosphates and are involved in a wide variety of different processes in the cell, such as insulin secretion and metastasis. With the aid of chemical fishing rods, the team led by Dorothea Fiedler identified over a hundred proteins in yeast cultures to which the messenger substances bind. The bound molecules subsequently altered several of the proteins chemically. Proof that two processes work in tandem here i. e. first the selective binding and then the chemical modification was established when the researchers were able to synthesise stabilised versions of the messenger substances. Thus, the reactive molecules sustained the subsequent tests with the fishing hooks. In this way, the researchers extracted the relevant candidates from more than 6,000 proteins. Some of these proteinmessenger interactions had already been characterized, but in most cases it was not known which of the proteins bound to inositol pyrophosphates. Of course, the investigation will not stop at yeast cultures. In the next step, the researchers want to apply their method to human cells, with even better fishing techniques. Experiments on animal models may then follow. Once we know the exact mechanisms and we have now taken an important first step in this direction it may one day be possible to intervene in the signal functions of these messenger substances therapeutically, says Fiedler. It will be a long journey until then, but in light of the diverse pathological processes in which inositol pyrophosphates are involved, it will be worth it. Sie heißen Inositol-Pyrophosphate und sind an den verschiedensten Prozessen in der Zelle beteiligt, zum Beispiel an der Insulinsekretion oder der Metastasierung. Das Team um Dorothea Fiedler identifizierte mit Hilfe von chemischem Angeln über hundert Proteine in Hefekulturen, an die die Botenstoffe binden. Die nun gebundenen Moleküle veränderten mehrere der Eiweiße anschließend chemisch. Den Nachweis, dass sich hier zwei Prozesse vereinen also erst die selektive Bindung und dann die chemische Modifikation war dadurch gelungen, dass die Forscher stabilisierte Versionen der Botenstoffe synthetisieren konnten. So hielten die reaktiven Moleküle den anschließenden Versuchen mit den Angelhaken stand. Auf diese Weise fischten die Forscher aus über Proteinen die relevanten Kandidaten heraus. Einige dieser Proteine kannte man schon, von den meisten wusste man jedoch nicht, dass sie mit Inositol-Pyrophosphaten interagieren. Bei Hefekulturen wird es natürlich nicht bleiben. Im nächsten Schritt wollen die Forscher ihre Methode an menschlichen Zellen überprüfen, mit noch besseren Angeltechniken. Anschließend könnten Experimente an Tiermodellen folgen. Wenn wir die genauen Mechanismen kennen und hierzu ist uns jetzt ein erster wichtiger Schritt gelungen könnte es eines Tages möglich sein, therapeutisch in die Signalfunktionen dieser Botenstoffe einzugreifen, sagt Fiedler. Bis dahin sei es zwar noch ein langer Weg, aber angesichts der vielfältigen Krankheitsprozesse, an denen Inositol-Pyrophosphate beteiligt sind, ein sehr lohnenswerter. P Control beads S. cerevisiae cell lysate P P P P P P InsP 6 beads Elution / SDS-PAGE Digestion / LC-MS/MS P P P P PCP P 5PCP-InsP 5 beads!" #" $" %" &" '" (" )" *"!+"!!"!#"!$" RNA pol I complex Nucleolus Preribosome Plasma membrane enriched fraction RNA pol III complex Small nucleolar ribonucleoprotein complex RNA polymerase activity Transcription from RNA pol I promoter Regulation of translation rrna processing Glucose metabolic process Analysis Log 10 P Cellular compartment Molecular function Biological process Inositol pyrophosphate affinity reagents identify protein interacting partners, and highlight the unusual ability of these molecules to access two distinct modes of action. Immobilisierte Inositolpyrophosphate können zur Identifizierung ihrer Interaktionspartner genutzt werden, und veranschaulichen, wie diese Moleküle über zwei verschiedene Mechanismen fungieren können. Wu M, Chong L S, Perlman D H, Resnick A C, Fiedler D (2016) The inositol polyphosphates intersect with protein signaling and metabolic networks via two distinct mechanisms. Proc. Nat. Acad. Sci. USA 113, E6757 E6765.

13 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 11 WIR HABEN DIE TÜR FÜR NEUE BIOMEDIZINISCHE ERKENNTNISSE AUFGESTOSSEN WE'VE PUSHED THE DOOR WIDE OPEN TO NOVEL BIOMEDICAL INSIGHTS Prof. Dr. Dr. Thomas J. Jentsch Thomas Jentsch is considered one of the world's leading researchers in the field of ion channels. In this interview, he talks about his latest discoveries and his plans for the coming years. Professor Jentsch, three years ago you discovered the molecular identity of the anion channel VRAC. Since this breakthrough, what else have you been able to find out about this regulator of cell volume? The identification of the proteins constituting VRAC is indeed decisive for understanding this important channel which researchers had been studying for more than 30 years. Only by knowing its molecular composition one can investigate its localisation, details of its molecular working, as well as its diverse physiological functions and role in diseases. We have only recently begun to study these aspects, but we have already discovered that VRAC not only regulates the cell volume, but also transports certain neurotransmitters and anti-cancer drugs. Apart from this, we've learned that VRAC consists of five subunits, which can occur in different combinations. For example, the subunit LRRC8D is essential for transport of the chemotherapeutics cisplatin and carboplatin, which are administered to treat various solid tumours. Are we already involved in clinical research? Let's put it this way: With the identification of VRAC, we have pushed open the door to many new biological, medical and pharmacological insights. At the moment, we are still operating in the field of basic research, and I never tire of stressing the importance of basic research. The identification of VRAC is a further example of how quickly this flows into concrete medical findings. Such as cancer? Yes, for example. Within one year of the identification of VRAC, we were able to show that chemotherapeutics used to treat cancer pass into the cell through this channel. If the VRAC subunit necessary for this transport is missing, we not only observe a lower degree of tumour cell killing in culture, but also chemotherapy resistance in cancer patients. We demonstrated this in co-operation with Dutch scientists. Our colleagues analysed gene expression profiles of ovarian cancer in patients who had been treated with cisplatin or carboplatin. Those who had less of the subunit LRRC8D in their tumours died earlier, or in other words were probably partially resistant to this medication. Had it not been suspected for a long time that VRAC also plays a role in programmed cell death (apoptosis), which chemotherapies are known to activate? This was a hypothesis that we looked into. Impairment of the cell shrinkage that occurs in apoptosis, so the hypothesis went, reduces the induced cell death of cancer cells. Indeed, cells in which we had genetically eliminated VRAC showed significantly lower levels of programmed cell death. VRAC-related drug resistance of tumours thus may involve a dual mechanism, although we currently assume that the reduced intake of anti-cancer drugs is the more important one. Are your findings on the transport of neurotransmitters as similarly far advanced? Here, we have been able to confirm the hypothesis that VRAC transports glutamate and other amino acids. A new finding is that this depends on the subunit composition of VRAC and that this channel

14 12 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Issue of The Journal of Physiology (2015) devoted to the 25th anniversary of the identification of the CLC channels by Thomas Jentsch (picture left) Ausgabe des The Journal of Phyisology (2015) zum 25-jährigen Jubiläum der Identifizierung der CLC Kanäle durch Thomas Jentsch (Abbildung links) Components of the VRAC in the plasma membrane of the cell. (Tobias Stauber) Bestandteile des VRAC in der Plasmamembran in der Zelle. can also transport GABA. We suspect fascinating roles of specific VRACs in physiological signal transmission in the brain but also, for example, in the development of pathologies like stroke. Do you already have your sights on a medical target? In the case of stroke, we know that astrocytes, glial cells found in the central nervous system, swell and release glutamate under hypoxia. This will result in glutamate toxicity that leads to neuronal cell death. If one could prevent VRAC from releasing glutamate, the damaged brain area would probably be smaller. Using genetic mouse models, we are now investigating this hypothesis in co-operation with a group at the Charité. What might a therapeutic intervention look like? It is hoped that drugs may be developed that specifically block those VRACs that allow glutamate to pass, that is channels containing specific combinations of subunits. We are currently searching for substances that modulate the activity of VRAC together with the FMP Screening Unit. But it will no doubt take years to develop new treatment options. At the beginning of the year, you received your second ERC Advanced Grant. What do you intend to do with the 2.5 million euros? Well, a part of the project is dedicated to the characterisation of VRAC and its physiological and pathological roles, an area in which we expect many new and no doubt in part surprising findings. We have already spoken about some of the aspects involved. The second part aims at the molecular identification of two further important ion channels. These two channels may also lead us into completely new terrain, as was the case with VRAC. Was VRAC actually the reason for being awarded a second ERC grant? It wasn't the reason, but an important prerequisite. The identification of VRAC, a central project of my first ERC Advanced Grant, provided not only the basis for the VRAC projects of the second grant, but also demonstrated our ability to carry out such high-risk projects successfully. We will now explore new areas which will hopefully lead to new exciting discoveries. Thomas Jentsch gilt als weltweit führender Forscher auf dem Gebiet der Ionenkanäle. Im Interview spricht er über seine jüngsten Entdeckungen und Pläne für die kommenden Jahre. Herr Professor Jentsch, vor drei Jahren haben Sie die molekulare Identität des Anionenkanals VRAC entdeckt. Was konnten Sie seit diesem Durchbruch Neues über diesen Regulator des Zellvolumens herausfinden? In der Tat war die Identifizierung der Proteine, aus denen VRAC besteht, entscheidend für das Verständnis des Kanals, den man ja schon seit 30 Jahren untersucht hat. Erst dadurch können seine Lokalisation, molekulare Funktionsweise und diverse physiologische Funktionen sowie seine Rolle bei Krankheiten untersucht werden. Wir sind mit diesen Untersuchungen immer noch am Anfang, haben aber schon jetzt herausgefunden, dass der Ionenkanal nicht nur das Zellvolumen reguliert, sondern auch bestimmte Neurotransmitter und Anti- Krebsmedikamente transportiert. Außerdem haben wir gelernt, dass VRAC aus fünf Untereinheiten besteht, die in verschiedenen Kombinationen auftreten können. So ist etwa die Untereinheit LRRC8D für den Transport der Chemotherapeutika Cisplatin und Carboplatin essenziell, die bei verschiedenen soliden Tumoren gegeben werden. Jetzt sind wir schon in der klinischen Forschung? Sagen wir es so: Mit der Identifizierung von VRAC haben wir die Tür zu vielen neuen biologischen, medizinischen und pharmakologischen Erkenntnissen aufgestoßen. Momentan bewegen wir uns noch im Bereich der Grundlagenforschung, und ich werde nicht müde, deren Bedeutung hervorzuheben. Die Identifizierung von VRAC ist ein weiteres Beispiel dafür, wie schnell diese in konkrete medizinische Erkenntnisse mündet. Erkenntnisse über Krebs? Ja, zum Beispiel. Schon im ersten Jahr nach der Identifizierung von VRAC konnten wir zeigen, dass durch diesen Kanal Chemotherapeutika in die Zelle gelangen. Fehlt die für diesen Transport notwendige VRAC Untereinheit, beobachten wir nicht nur ein geringeres Abtöten von Tumorzellen in Kultur, sondern auch eine Chemotherapie-Resistenz bei Tumorpatienten. Dies konnten wir in

15 RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 13 Tobias Münch and Ian Orozco Felizia Voss and Tobias Stauber Zusammenarbeit mit einer holländischen Arbeitsgruppe nachweisen. Die Kollegen hatten Genexpressionsprofile von Eierstocktumoren von Patientinnen analysiert, die mit Cisplatin oder Carboplatin behandelt worden waren. Diejenigen, die weniger von der Untereinheit LRRC8D im Tumor hatten, waren deutlich früher gestorben, also wahrscheinlich relativ resistent gegen das Medikament. Gab es nicht schon lange den Verdacht, dass VRAC auch eine Rolle beim programmierten Zelltod, der Apoptose, spielt, den Chemotherapien ja aktivieren können? Es gab diese Hypothese und wir sind ihr auch nachgegangen. Nach dieser Hypothese ist der induzierte Zelltod von Krebszellen dann verringert, wenn die bei Apoptose typische Zellschrumpfung ausfällt. In der Tat zeigten Zellen, in denen wir VRAC genetisch eliminiert hatten, wesentlich weniger programmierten Zelltod. Bei der Chemo-Resistenz von Tumoren haben wir es also wahrscheinlich mit einem doppelten Mechanismus zu tun, wobei wir derzeit davon ausgehen, dass die reduzierte Medikamenten-Aufnahme der wichtigere ist. Sind Ihre Erkenntnisse zum Transport von Neurotransmittern schon ähnlich konkret? Hier konnten wir die Hypothese bestätigen, dass VRAC Glutamat und andere Aminosäuren transportiert. Neu ist die Erkenntnis, dass dies über verschiedene Kombinationen von Untereinheiten geschieht und unter anderem auch GABA so die Zelle verlassen kann. Wir vermuten faszinierende Rollen bei der physiologischen Signalübertragung im Gehirn, aber zum Beispiel auch beim Schlaganfall. Formen von VRAC medikamentös zu blockieren, die Glutamat durchlassen. Wir suchen bereits mit unserer Screening-Unit nach Substanzen, die die Aktivität von VRAC modulieren. Es wird aber sicher Jahre dauern, bis sich möglicherweise neue Behandlungsmöglichkeiten auftun. Sie haben Anfang des Jahres ihren zweiten ERC Advanced Grant bekommen. Was werden Sie mit den 2,5 Millionen Euro anfangen? Nun, ein Teil des Projektes ist der Charakterisierung von VRAC und seinen physiologischen und pathologischen Rollen gewidmet, wo wir sehr viele neue, zum Teil sicher überraschende, Erkenntnisse erwarten. Über einige Aspekte haben wir eben schon gesprochen. Im zweiten Teil geht es um die molekulare Identifizierung von zwei weiteren Ionenkanälen. Diese beiden Kanäle könnten uns ebenfalls in völlig neues Terrain führen, wie es bei VRAC der Fall war. War VRAC eigentlich der Grund für die abermalige Auszeichnung? Der Grund nicht, aber eine wichtige Voraussetzung. Die Identifizierung von VRAC war ja ein zentrales Projekt meines ersten ERC Advanced Grants und war damit nicht nur die Grundlage für die VRAC-Projekte des zweiten Grants, sondern auch der Beweis, dass wir solche Hochrisikoprojekte erfolgreich durchziehen können. Nun geht es um zwei weitere neue Felder und Entdeckungen, von denen wir heute noch nicht einmal etwas ahnen. Haben Sie schon einen medizinischen Angriffspunkt im Blick? Wir wissen, dass beim Schlaganfall Astrozyten, das sind bestimmte Zellen des zentralen Nervensystems, anschwellen und Glutamat freisetzen. In der Folge kommt es zu der bekannten Glutamattoxizität und Neuronen sterben ab. Würde man nun VRAC an der Glutamatfreisetzung hindern, wäre das geschädigte Hirnareal vermutlich kleiner. Dies können wir jetzt mit Hilfe von genetischen Mausmodellen zusammen mit einer Gruppe an der Charité untersuchen. Wie könnte denn ein therapeutischer Eingriff aussehen? Die Hoffnung ist, gezielt die entsprechend zusammengesetzten Prof. Dr. Dr. Thomas J. Jentsch was named Honorary Doctor by the University Medical Center Hamburg-Eppendorf (UKE) on May 2, Prof. Dr. Dr. Thomas J. Jentsch wurde am 2. Mai 2017 zum Ehrendoktor der Medizinischen Fakultät des Universitätsklinikums Hamburg-Eppendorf (UKE) ernannt.

16 Molecular Cell Physiology Molekulare Zellphysiologie Group leader PD Dr. Ingolf E. Blasig Molecular and Theoretical Neuroscience Molekulare und Theoretische Neurowissenschaften Group leader Dr. Alexander Matthias Walter PAGE 46 PAGE 31 SECTION MOLECULAR PHYSIOLOGY AND CELL BIOLOGY BEREICH MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE Cellular Imaging Zelluläre Bildgebung Group leaders Dr. Burkhard Wiesner (Light Microscopy) Dr. Dmytro Puchkov (Electron Microscopy) PAGE 49 Animal Facility Tierhaltung Group leader Dr. Natali Wisbrun PAGE 52

17 Physiology and Pathology of Ion Transport Physiologie und Pathologie des Ionentransports Group leader Prof. Dr. Dr. Thomas J. Jentsch PAGE 20 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE Molecular Pharmacology and Cell Biology Molekulare Pharmakologie und Zellbiologie Group leader Prof. Dr. Volker Haucke PAGE 24 Proteostasis in Aging and Disease Die Rolle der Proteostase beim Altern und in Krankheit Group leader Dr. Janine Kirstein PAGE 40 Behavioural Neurodynamics Verhaltensneurodynamik Group leaders Dr. Tatiana Korotkova Dr. Alexey Ponomarenko PAGE 43 Molecular Neuroscience and Biophysics Molekulare Neurowissenschaften und Biophysik Group leader Dr. Andrew J.R. Plested PAGE 34 Membrane Traffic and Cell Motility Membrantransport und Zellbeweglichkeit Group leader PD Dr. Tanja Maritzen Protein Trafficking Group leader Prof. Dr. Ralf Schülein PAGE 28 PAGE 37

18 16 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 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 the case of disease, this interplay becomes unbalanced. Research in the Molecular Physiology and Cell Biology section 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 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, to 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 modeling, drug and sirna screening, as well as chemical biology. Two main research 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 exoand endocytosis. Strong links exist between these research 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 Mathias Böhme 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 discovered a mechanism for the local conversion of phosphoinositides, minor phospholipids that couple organelle identity to membrane traffic and signaling, from phosphatidylinositol 3-phosphate to phosphatidylinositol 4-phosphate, to enable exit from the endosomal system. A defect in this phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy in humans (Ketel et al., Nature 2016). The Physiology and Pathology of Ion Transport department, led by Thomas Jentsch, found that volume-regulated LRRC8 (VRAC) channels, only recently identified by the group, transport organic compounds including neurotransmitters and the anti-cancer drug cisplatin depending on the particular subunit composition, proving that LRRC8 proteins form the channel pore. Downregulation of LRRC8D is clinically relevant in tumor drug resistance (Planells- Cases et al., EMBO J 2015). The Molecular Cell Physiology research group, headed by Ingolf Blasig, discovered a novel pathway to overcome pharmacological tissue barriers that can then be targeted to improve drug delivery, while the Protein Trafficking group of Ralf Schülein identified novel inhibitors of the eukaryotic Sec / translocon pathway in cell-based high-throughput screens. The four junior research groups of the section contributed significantly to the scientific output of the section. The Behavioral Neurodynamics junior group, headed by Tatiana Korotkova / Alexey Ponomarenko, used novel optogenetic actuators for opposing control of neuronal activity to show that high-frequency oscillations in the hypothalamus and cerebral cortex enable food-seeking behavior (Carus-Cadavieco et al., Nature 2017). The Molecular Neuroscience and Biophysics group, led by Andrew Plested, produced a glutamate receptor that can report its own activation with a fluorescent signal (Zacchariassen et al., PNAS 2016), the first of its kind and the initial step on the road to a genetically-encoded optical reporter of synaptic transmission. Tanja Maritzen s junior group, Membrane Traffic and Cell Motility, discovered that the thus far uncharacterized endocytic adaptor protein Stonin1 regulates cell motility by mediating the internalization of the glioma-associated proteoglycan NG2 (Feutlinske et al., Nature Commun. 2015), thereby likely also limiting NG2 s oncogenic potential. Janine Kirstein, who leads the junior group Proteostasis in Aging and Disease, identified a novel human chaperone complex that completely suppresses Huntington aggregation and resolubilizes amyloid fibrils formed by this neurodegenerative disease-causing protein. The Liaison Group Neuroscience Molecular and Theroretical Neuroscience, led by Alexander Walter, unraveled the molecular principles of the spatial organization of neurotransmitter release at the synapse and its importance for efficient synaptic transmission, using a combination of super-resolution imaging, physiology, genetics, and mathematical modeling (Böhme et al., Nat. Neurosci. 2016).

19 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 17 Sophie Dithmer Dmytro Puchkov The FMP is closely connected to the Berlin neuroscience community via the groups of Korotkova / Ponomarenko and Alexander Walter, located at the Charité in central Berlin, as well as through the DFG-financed Cluster of Excellence, NeuroCure. 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 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 an SAW grant for the Role of protein homeostasis in cellular aging, as part of a program aimed at creating synergies with other Leibniz Institutes (Leibniz-Institut für Neurobiologie (LIN, Magdeburg) and the Fritz-Lipmann-Institut (FLI, Jena)), and they likewise received intramural grants for collaborative aging-related research. Most recently, an SAW grant was awarded to Volker Haucke, together with Dorothea Fiedler (Chemical Biology Section) and Hartmut Oschkinat, to develop a novel methodology for the quantitative determination of inositol phosphates. Moreover, the section has invested heavily in cellular imaging techniques, a development that was initiated by Volker Haucke and that is supervised by Burkhard Wiesner. 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 carried out in the section is thoroughly interconnected with research being done in the other sections of the FMP, especially 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 deren 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 Modellierunng, 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 entdeckte die Abteilung Molekulare Pharmakologie und Zellbiologie unter der Leitung von Volker Haucke einen Mechanismus der lokalen

20 18 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Claudia Schmid (photo left) and Ljudmila Katchan (photo right) Arthur Gibert (photo left) and Ian Orozco (photo right) Umwandlung von Phosphoinositiden (Phospholipide, die die Identität von Organellen mit Membranverkehr und Signaltransduktion koppeln), der Phosphatidylinositol 3-Phosphat in Phosphatidylinositol 4-Phosphat umwandelt. Eine Störung dieser Reaktion, die für den Transport aus dem endosomalen System wichtig ist, liegt der X-chromosomalen zentronukleären Myopathie beim Menschen zugrunde (Ketel et al., Nature 2016). Die von Thomas Jentsch geleitete Abteilung Physiologie und Pathologie des Ionentransports entdeckte, dass die erst kürzlich von der Gruppe identifizierten volumenregulierenden LRRC8 (VRAC) Kanäle, abhängig von ihrer spezifischen Zusammensetzung aus LRRC8 Untereinheiten, organische Verbindungen wie z. B. Neurotransmitter oder das Krebsmedikament Cisplatin transportieren ein Befund, der beweist, dass LRRC8 Proteine die Pore des Kanals bilden. Eine Herunterregulierung von LRRC8D ist klinisch relevant für die Resistenz bestimmter Krebsformen gegen Zytostatika-Therapie (Planells-Cases et al., EMBO J 2015). Die Arbeitsgruppe Molekulare Zellphysiologie, geleitet von Ingolf Blasig, entdeckte einen neuen Signaltransduktionsweg, der gezielt eingesetzt werden kann, um durch Überwindung von Gewebebarrieren die Wirkstoffeffizienz zu verbessern. Die von Ralf Schülein geleitete Gruppe Protein Trafficking identifizierte in einem zellbasierten Hochdurchsatz-Screen neue Inhibitoren des eukaryotischen Sec / Translocon Weges über die Membran des endoplasmatischen Retikulums. Die vier Nachwuchsgruppen des Bereichs haben ebenfalls signifikant zu dessem wissenschaftlichen Erfolg beigetragen. Die Nachwuchsgruppe Verhaltensneurodynamik von Tatiana Korotkova und Alexey Ponomarenko zeigten mit neuartigen optogenetischen Verfahren zur Manipulation neuronaler Aktivität, dass hochfrequente elektrische Schwingungen im Hypothalamus und in der Hirnrinde das Nahrungssuchverhalten ermöglichen (Carus-Cadavieco et al., Nature 2017). Die Nachwuchsgruppe Molekulare Neurowissenschaft und Biophysik unter Leitung von Andrew Plested stellte einen Glutamatrezeptor her, der seine eigene Aktivierung mit einem Fluoreszenzsignal anzeigt (Zacchariassen et al., PNAS 2016), den ersten seiner Art. Dies ist ein erster Schritt hin zu einem genetisch-kodierten optischen Detektor synaptischer Übertragung. Die Nachwuchsgruppe Membrantransport und Zellbeweglichkeit von Tanja Maritzen entdeckte, dass das bislang noch nicht charakterisierte endozytische Adapterprotein Stonin1 die Zellmotilität durch Vermittlung der Internalisierung des Gliomassoziierten Proteoglycans NG2 reguliert (Feutlinske et al., Nature Commun. 2015), wodurch wahrscheinlich auch das kreberzeugende Potenzial von NG2 eingeschränkt wird. Janine Kirstein, die die Juniorgruppe Rolle der Proteostase beim Altern und in Krankheit leitet, identifizierte einen neuartigen menschlichen Chaperon-Komplex, der die Protein-Aggregation bei der Huntington-Krankheit vollständig unterdrückt und die durch diese neurodegenerative Erkrankung verursachenden Amyloidfibrillen auflöst. Die Liaison Gruppe Neurowissenschaften Molekulare und Theoretische Neurowissenschaften unter der Leitung von Alexander Walter konnte durch eine Kombination von hochauflösender Mikroskopie, Physiologie, Genetik und mathematischer Modellierung molekulare Mechanismen ergründen, die Neurotransmitter-Freisetzung auf der Skala millionstel- Millimeter räumlich präzise an der Synapse organisieren, um eine

21 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 19 Mouhannad Malek and Rashin Roshan Bin (photo above), Annika Scior and Kerstin Steinhagen (photo left), Franziska Bender and Alexey Ponomarenko (photo right) effiziente Signalübertragung zu gewährleisten (Böhme et al., Nat. Neurosci. 2016). Sowohl durch die Gruppen Korotkova / Ponomarenko und Alexander Walter, deren Labore sich an der Charité-Universitätsmedizin im Zentrum Berlins befinden, als auch durch das DFG-finanzierte Exzellenzcluster NeuroCure ist das FMP eng in die neurowissenschaftliche Forschungsszene Berlins 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 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 die gemeinsame Forschung zum Altern erhalten. Jüngst konnte Volker Haucke zusammen mit Dorothea Fiedler (Bereich Chemische Biologie) und Hartmut Oschkinat eine SAW-Förderung zur quantitativen Bestimmung von Inositolphosphaten einwerben. Zudem investiert der Bereich kontinuierlich und substanziell in den Aufbau hochmoderner zellulärer Visualisierungstechniken, eine Entwicklung initiiert von Volker Haucke und geleitet von Burkhard Wiesner. 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, die Screening Unit und die Chemical Biology Platform bereithalten, sowie auf das Proteomik-Portfolio der Arbeitsgruppe Massenspektrometrie im Bereich Chemische Biologie zu. Die Vielfalt der wissenschaftlichen Ansätze und Techniken, zusammen mit unserem hohen Interesse an Zellbiologie und Neurobiologie, bieten eine ausgezeichnete Grundlage um unser Wissen über grundlegende Mechanismen zu vergrößern, die pharmakologischen Eingriffen zugänglich sein könnten.

22 20 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 PHYSIOLOGY AND PATHOLOGY OF ION TRANSPORT PHYSIOLOGIE UND PATHOLOGIE DES IONENTRANSPORTS GROUP LEADER PROF. DR. DR. THOMAS J. JENTSCH BIOGRAPHY Studied Medicine, Studied Physics, Free University of Berlin Staff scientist, Institute of Clinical Physiology (Prof. Wiederholt), Free University of Berlin 1982 Ph.D. in Physics, Fritz-Haber-Institute (Prof. Block), Berlin 1984 M.D., Institute of Clinical Physiology (Prof. Wiederholt), Free University of Berlin Postdoctoral fellow, Whitehead Institute (Harvey F. Lodish, MIT), Cambridge MA Research group leader, Center for Molecular Neurobiology Hamburg (ZMNH), Hamburg University 1991 Habilitation in Cell Biochemistry, Medical School of Hamburg University Full professor (C4) of Molecular Neuropathology, ZMNH, Hamburg University; Director of the Institut für Molekulare Neuropathobiologie & Director of the Center for Molecular Neurobiology Hamburg (ZMNH) Since 2006 Head of department, FMP and MDC, Berlin (joint appointment), Full Professor (W3), Charité University Medicine Berlin Since 2007 Member of NeuroCure Cluster of Excellence Since 2009 Deputy Director, FMP SUMMARY We aim to understand ion transport processes from the molecular 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 knock-out (KO) and knock-in (KI) mice and the analysis of human genetic diseases. We investigate CLC Cl - channels and transporters, KCNQ K + channels, KCC cation-chloride cotransporters, anoctamin Ca 2+ -activated Cl - channels, and the volume-regulated VRAC channel. Key research areas are structure / function analysis, cellular neurobiology, extracellular signaling, volume regulation, and the endosomal-lysosomal system. We study many organs, including the brain, inner ear, olfactory epithelium, skin mechanoreceptors, kidney, and testis. After our breakthrough in identifying the long-sought volume-regulated anion channel VRAC in 2014, we put great emphasis on understanding the structure / function and physiology of this new channel. We are particularly excited by its ability to transport organic signaling molecules and drugs and are generating and analyzing knock-out mouse models for each of its five subunits. These mice have begun to provide novel biological, and medically important, insights. ZUSAMMENFASSUNG Unser Ziel ist es, Ionentransportprozesse von der molekularen über die subzelluläre und zelluläre Ebene bis zur Rolle im gesamten 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 Cl - Kanälen und -Transportern, KCNQ K + Kanälen, KCC-Kation-Chlorid- Kotransportern, Anoctamin Ca 2+ -aktivierten Cl - Kanälen und in letzter Zeit vor Allem Volumen-regulierten VRAC Kanälen. Wir befassen uns mit Struktur / Funktions-Analyse dieser Kanäle, ihrer Rolle im Nervensystem, bei extrazellulärer Signaltransduktion, in der Volumenregulation und in Endosomen und Lysosomen. Dabei untersuchen wir eine Reihe von Organen wie das Gehirn, Innenohr, Mechanorezeptoren der Haut, Riechepithelien, Niere und Hoden. Nachdem uns 2014 der Durchbruch mit der molekularen Identifizierung des schwell-aktivierten Anionenkanals VRAC gelungen ist, entwickeln wir breite Forschungsprogramme zur Aufklärung der Struktur-Funktions-Beziehungen und physiologischen Rollen dieses neuen Kanals. Uns fasziniert insbesondere, dass er auch organische Signalmoleküle und Medikamente transportiert, und wir haben eine Reihe von Mausmodellen für alle seine fünf Untereinheiten erzeugt. Ihre Analyse erlaubt schon jetzt wichtige Einblicke in bisher unbekannte physiologische Prozesse und ergibt medizinisch relevante Einsichten. Since 1992 more than 12 prizes and awards, e. g. Gottfried Wilhelm Leibniz Prize; Prix Louis-Jeantet de médecine; Ernst Jung Preis für Medizin; Feldberg Prize. A detailed list is awailable at: Since 2000 elected member of EMBO and four Academies of Science 2011, 2017 ERC Advanced Grants

23 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 21 DESCRIPTION OF PROJECTS Properties and roles of the long-sought volume-regulated anion channel VRAC Cells must 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 that has been known biophysically for more than 20 years but whose molecular identity had remained obscure. Using a genome-wide sirna screen at the FMP screening facility, we have identified heteromers of LRRC8 proteins, containing four membrane spans and C-terminal leucine-rich repeats, as crucial VRAC components (Voss et al., Science 2014). LRRC8A is required for VRAC activity, but needs at least one other isoform (LRRC8B to -E) to form channels. VRACs are probably hexamers of up to five different LRRC8 isoforms. We have shown that VRAC also conducts various organic compounds, including neurotransmitters and modulators, suggesting it has a role in extracellular signal transduction and diseases such as stroke. We discovered that the subunit composition determines VRAC s permeation properties, with inclusion of LRRC8D enhancing the transport of various compounds, and LRRC8E that of glutamate. Our findings demonstrate that LRRC8 proteins form the pore of VRACs. Excitingly, LRRC8D-containing VRACs also transport the anti-cancer drug cisplatin and down regulation of LRRC8D confers tumor drug resistance. By impairing apoptotic cell volume decrease, disruption of VRAC impairs drug-induced apoptosis. The LRRC8 subunit composition determines the inactivation of VRAC currents and we identified relevant residues for this inactivation. In a major effort to uncover the physiological and pathological roles of VRACs, we generate and analyze multiple mouse models for various LRRC8 subunits. We disrupt Lrrc8a in a cell- and tissue-specific manner as the KO of LRRC8A, which completely abolishes VRAC function, is lethal. 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. 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 - channels. We are now analyzing similar models for ClC-3. 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. We have previously shown that disruption of the plasma membrane Cl - channel ClC-2 leads to leukodystrophy, testicular degeneration, and retinal degeneration. We are now generating and analyzing cell type-specific ClC-2 KOs to better understand these pathologies that are also found in patients with CLCN2 mutations. Anoctamin (TMEM16) Ca 2+ -activated chloride channels We have shown that Ano2 is the Ca 2+ -activated Cl - channel of olfactory sensory neurons, but surprisingly our Ano2 -/- mice showed that Ano2 is dispensable 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 that mutations in KCNQ4 cause a form of deafness. In collaboration with A. Ponomarenko and T. Korotkova we analyzed Kcnq5 dn/dn mice in which channels containing KCNQ5 are inactivated. These studies showed that KCNQ5 is important for controlling synaptic inhibition and network activity. We have recently shown that KCNQ3 is also expressed in extremely sensitive D-hair mechanoreceptors in the skin and modulates their sensitivity, complementing our previous work showing a similar role of KCNQ4 in rapidly adapting skin mechanoreceptors. Potassium-chloride cotransporters We have previously analyzed constitutive KOs of the KCC K + -Cl - - cotransporters KCC1 KCC4 and discovered unexpected roles in various tissues. Neuronal KCC2 lowers cytosolic Cl - concentration, a process needed for the inhibitory response to the neurotransmitters GABA and glycine. Using a mitral cell-specific Kcc2 KO we have now shown that KCC2-dependent synaptic inhibition in the olfactory bulb is crucial for discriminating closely related odors.

24 22 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 1: Dual role of VRAC in anti-cancer drug sensitivity: (1) Uptake of cisplatin / carboplatin and (2) facilitation of apoptosis by mediating apoptotic volume decrease. Channels mediating drug uptake need both the LRRC8A and LRRC8D subunits, whereas volume-regulatory channels require only LRRC8A and any other LRRC8 subunit. For details, see Planells-Cases et al., EMBO J. 34, (2015). Figure adapted from Jentsch, Nature Rev. Mol. Cell Biol. 17, (2016). GROUP MEMBERS Dr. Kathrin Gödde Dr. Maja Hoegg-Beiler Dr. Anna Oliveras Martínez Dr. Ian Orozco Dr. Rosa Planells-Cases Dr. Sonali Saha Dr. Tobias Stauber Dr. Janis Vogt Dr. Felizia Voss Dr. Stefanie Weinert Dr. Joanna Ziomkowska Dr. Pingzheng Zhou 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) Corinna Göppner (doctoral student) Karen López Cayuqueo (doctoral student) Jennifer Lück (doctoral student) Carmen Ludwig (doctoral student) Darius Lutter (doctoral student) Jonas Münch (doctoral student) Karina Oberheide (doctoral student) Maya Polovitskaya (doctoral student) Sebastian Schütze (doctoral student) Till Stuhlmann (doctoral student) Florian Ullrich (doctoral student) Carolin Backhaus (technical assistant) Anyess von Bock (technical assistant) Karolin Fuchs (technical assistant) Petra Göritz (animal care taker) Anika Günther (technical assistant) Johanna Jedamzick (technical assistant) Janet Liebold (technical assistant) Antje Maluck (technical assistant) Ruth Pareja-Alcaraz (technical assistant) Katrin Räbel (technical assistant) Patrick Seidler (technical assistant) Andrea Weidlich (technical assistant) Staff employed within the reporting period COLLABORATIONS International Piet Borst, Netherlands Cancer Institute, Amsterdam, The Netherlands Alan Carleton, Université de Genève, Switzerland Dominique Eladari, Faculté de Médecine, Université Paris-Descartes, France Sven Rottenberg, Netherlands Cancer Institute, Amsterdam, The Netherlands Francisco Sepúlveda, CECS, Valdivia, Chile Guillermo Spitzmaul, INIBIBB, Bahía Blanca, Argentina Chris de Zeeuw, Erasmus MC, Rotterdam and Netherlands Institute for Neuroscience, Amsterdam, The Netherlands National Ulrich Dirnagl, Charité Universitätsmedizin Berlin Maik Gollasch, Charité Universitätsmedizin Berlin Hans-Jürgen Holdt, Universität Potsdam Christian Hübner, Universitätsklinikum Jena Vera Kalscheuer, Max-Planck-Institut für Molekulare Genetik, Berlin Uwe Kornak, Charité Universitätsmedizin Berlin and Max-Planck-Institut für Mole kulare Genetik, Berlin Tatiana Korotkova, Charité Universitätsmedizin Berlin and FMP, Berlin Eberhard Krause, Leibniz-Forschungsinstitut für Mole kulare Pharmakologie (FMP), Berlin Gerd Krause, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin Jens von Kries, Leibniz-Forschungsinstitut für Mole kulare Pharmakologie (FMP), Berlin Trese Leinders Zufall, Universität des Saarlandes, Homburg / Saar Marc Nazaré, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin Alexei Ponomarenko, Charité Universitätsmedizin Berlin and FMP, Berlin Dmytro Puchkov, Leibniz-Forschungsinstitut für Mole kulare Pharmakologie (FMP), Berlin Christian Rosenmund, Charité Universitätsmedizin Berlin Dietmar Schmitz, Charité Universitätsmedizin Berlin Bernd Wollnik, Universität Göttingen Frank Zufall, Universität des Saarlandes, Homburg / Saar Werner Zuschratter, Leibniz-Institut für Neurobiologie (LIN), Magdeburg

25 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 23 a Kcc2reelinDAPI Kcc2 lox/lox MC- Kcc2 b Correct responses (%) Correct responses (%) Ethyl valerate versus ethyl tiglate 90% Criterion Kcc2 lox/lox MC- Kcc2 Chance level Blocks of 20 trials 0.6/0.4% ethyl valerate/ethyl tiglate versus 0.4/0.6% ethyl valerate/ethyl tiglate Fig. 2: Mitral cell-specific disruption of the K + Cl - cotransporter KCC2 in the olfactory bulb (a) entails the inability to distinguish closely related odors in behavioral olfactometry experiments (b). Taken from Gödde et al., Nature Communications 7, (2016). Blocks of 20 trials SELECTED PUBLICATIONS Planells-Cases R, Lutter D, Guyader C, Gerhards N M, Ullrich F, Elger D A, Kucukosmanoglu A, Xu G, Voss F K, Reincke S M, Stauber T, Blomen V A, Vis D J, Wessels L F, Brummelkamp T R, Borst P, Rottenberg S*, Jentsch T J * (2015). VRAC channel composition determines its substrate specificity and cellular resistance to Pt-based anti-cancer drugs. EMBO J. 34, Fidzinski P, Korotkova T, Heidenreich M, Maier N, Schuetze S, Kobler O, Zuschratter W, Schmitz D, Ponomarenko A *, Jentsch T J * (2015). KCNQ5 K+ channels control hippocampal synaptic inhibition and fast network oscillations. Nature Commun. 6, Schütze S, Orozco I J, Jentsch T J * (2016). KCNQ potassium channels modulate sensitivity of skin D-hair mechanoreceptors. J. Biol. Chem. 291, Ullrich F, Reincke S M, Voss F K, Stauber T, Jentsch T J * (2016). Inactivation and anion selectivity of volume-regulated VRAC channels depend on carboxy-terminal residues of the first extracellular loop. J. Biol. Chem. 291, Gödde K, Gschwend O, Puchkov D, Pfeffer C K, Carleton A *, Jentsch T J * (2016). Disruption of Kcc2-dependent inhibition of olfactory bulb output neurons suggests its importance in odour discrimination. Nature Commun. 7, EXTERNAL FUNDING Prix Louis Jeantet, , Deutsche Forschungsgemeinschaft, Exzellenzinitiative an der Humboldt- Universität zu Berlin, Projekt NeuroCure: Towards a better outcome of central nervous system disorders, - Innovation Project 2015: Role of the volume-regulated anion channel VRAC in hearing and deafness, , ; - Innovation Project 2016: The volume-regulated anion channel VRAC and the ventricular system, , 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 A. Zdebik, Continued as: JE 164 / 9-2, , Deutsche Forschungsgemeinschaft, Der CIC-7 / Ostm1 Chloridtransporter in Lysosomen und Osteoklasten, JE 164 / 7-1, Continued as: JE 164 / 7-2, , Deutsche Forschungsgemeinschaft, Funktionelle Charakterisierung ausgewählter Mitglieder der Anoctamin-Kanalfamilie, JE 164 / 10-1, , Deutsche Forschungsgemeinschaft, Volumen-regulierter Anionen- Kanal VRAC und seine Rolle im Gehirn, JE 164 / 12-1, , Deutsche Forschungsgemeinschaft (Aufbau internationaler Kooperationen), Untersuchung der Funktion der Kaliumkanäle KCNQ in den Augen anhand genetischer Mausmodelle, JE 164 / 13-1, , Europäischer Forschungsrat (7. Forschungsrahmenprogramm), Ion homeostasis and volume regulation of cells and organelles (CYTOVO- LION), ERC-2011-ADG_294435, , Bundesministerium für Bildung und Forschung (ERA-NET: E-Rare), CLC chloride channels and Megalencephalic Leucoencephalopathy: molecular mechanisms and therapeutics, 01GM1403, , Leibniz-Gemeinschaft (Leibniz Wettbewerb 2014), Role of proteostasis in cellular aging, SAW-2014-FMP-2, with V. Haucke, H. Oschkinat, J.-P. von Kries, , (pro rata) EMBO Long-Term Fellowship, Sonali Saha, , Alexander von Humboldt Research Fellowship, Pingzheng Zhou, , FMP authors Group members * corresponding authors

26 24 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 MOLECULAR PHARMACOLOGY AND CELL BIOLOGY MOLEKULARE PHARMAKOLOGIE UND ZELLBIOLOGIE GROUP LEADER PROF. DR. VOLKER HAUCKE BIOGRAPHY Studied Biochemistry, Free University of Berlin and Biozentrum, University of Basel PhD (summa cum laude), Department of Biochemistry (Prof. G. Schatz), Biozentrum, University of Basel 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, Free University of Berlin Full Professor and Chair (W3), Department of Membrane Biochemistry, Free University of Berlin since 2007 Member of Neurocure Cluster of Excellence 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, Free University of Berlin 2014 Elected Member of the European Molecular Biology Organization (EMBO) 2016 Reinhart Koselleck-Grant Award of the Deutsche Forschungsgemeinschaft (DFG) SUMMARY 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 cycling of synaptic vesicles at neuronal synapses and its role in brain function and disease and in the physiological functions of inositol lipids. 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 superresolution 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 Zyklus synaptischer Vesikel an neuronalen Synapsen und dessen Rolle bei der Gehirnfunktion und Erkrankungen des Nervensystems sowie der physiologischen Funktion von Inositol-Lipiden. 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 Avanti Award of the American Society for Biochemistry & Molecular Biology (ASBMB)

27 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 25 DESCRIPTION OF PROJECTS Research within the department is conducted within two subgroups (led by V. Haucke and M. Krauss) and covers three major areas: (i) the role of exo-endocytic and endolysosomal membrane dynamics in synapse function and neuronal development; (ii) the regulation of membrane homeostasis and cell signaling by phosphoinositides, and (iii) the function of septins scaffolds in membrane dynamics and organelle contacts. Together with the Core Facility Cellular Imaging, we also develop and use super-resolution light (e. g. multi-color STORM and 3D-gSTED, TIRF-SIM) and electron microscopy approaches for studying these processes. Exo-endocytic and endolysosomal membrane dynamics in the functioning of synapses and in neuronal development 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, regeneration, and axonal transport of SV precursor organelles. A key question in this regard is how exo- and endocytosis are coupled. We have discovered that the endocytic protein AP180 acts as a governess that oversees sorting of the essential vesicular SNARE synaptobrevin 2. Loss of AP180 impairs neurotransmission and leads to excitatory / inhibitory imbalance and fatal epilepsies due to reduced copy numbers of synaptobrevin 2 in SVs. Further studies have shown that endocytic adaptors such as AP180 and its close relative CALM, a protein implicated in Alzheimer's disease, limit the diffusional spread of newly exocytosed SV proteins to prevent their loss into the axon. In our most recent work, we found that at physiological temperature SV endocytosis occurs on several timescales, ranging from less than a second to several seconds, and largely occurs via formin-mediated endocytosis independent of clathrin, whereas clathrin / AP-2 are required for SV reformation from internal structures. Other ongoing studies have revealed unexpected novel endocytosisindependent roles in neuronal development for endocytic proteins such as AP-2 and intersectins, which regulate key signaling processes. Our studies have implications for the understanding and treatment of neurological disorders and for neurodegeneration. Regulation of endocytic and endolysosomal membrane homeostasis and cell signaling by phosphoinositides 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 lipid kinases and phosphatases as well as Bin / Amphiphysin / Rvs homology (BAR) domain proteins, in the spatiotemporal regulation of clathrin-mediated endocytosis. We have addressed the question of how membrane deformation in endocytosis is coupled to dynamin-mediated fission and how dynamin assembly is coupled to endocytic vesicle formation through regulated synthesis and turnover of phosphoinositide lipids (PIs). Other projects aim at unraveling mechanisms of clathrinindependent endocytosis such as macropinocytosis in dendritic cells of the immune system. Genetics, RNA interference, and acute chemical and optogenetic perturbations are used to designate the roles of specific PIs during the progression of endocytosis and to elucidate how PI conversion along the endolysosomal pathway is linked to cell signaling processes, as well as how these processes are regulated. In recent studies we discovered a mechanism for endosomal exocytosis mediated by the PI(3)P phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data show that defective PI conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans. As many PI-metabolizing enzymes are implicated in cancer as well as hereditary disorders such as Charcot-Marie-Tooth disease we also seek to identify novel pharmacological and chemical inhibitors of select PImetabolizing enzymes. Function of septins scaffolds in membrane dynamics and organelle contacts Recent work from our lab has identified key connections between the endosomal system and the microtubule-based, as well as the actin, cytoskeleton. Septin GTPases are another element of the cytoskeleton and play key roles in regulating a variety of cellular functions, ranging from membrane traffic and signaling to cell motility. By employing biochemical, cell biological, and optical techniques we dissect the role of septin-based membrane scaffolds, adaptor proteins and PI-metabolizing enzymes in regulating cytoskeletal dynamics, the formation of organelle contact sites, and protein sorting through the Golgi-endosomal interface during cell signaling and migration, as well as polarized secretion.

28 26 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 integrin vesical CONTROL CELL integrin vesical LOSS OF MTM1 Fig. 1: Accumulation of integrin (red), an important component of muscles, in vesicles (green) from control cells (left image) and from cells without MTM1 (right image). Inset images are magnified views. Taken from Ketel et al. (2016). GROUP MEMBERS Prof. Dr. Michael Krauß (group leader) Dr. Jan Schmoranzer (group leader) Dr. Caroline Bruns Dr. Gaga Kochlamazashvili Dr. Natalia Kononenko Dr. Marijn Kuijpers Dr. Martin Lehmann Dr. Tania Lopez-Hernandez Dr. Marta Maglione (joint postdoc with S. J. Sigrist, FU Berlin) Dr. Mouhannad Malek Dr. Andrea Lynn Marat Dr. Christoph Ott Dr. Tolga Soykan Dr. Domenico Azarnia Tehran Dr. Anna Wawrzyniak Dr. Mirjana Weimershaus Dr. Haibin Wang Gala Claßen (doctoral student) Katrin Diesenberg (doctoral student) Fabian Feutlinske (doctoral student) Niclas Gimber (doctoral student) Claudia Gras (doctoral student) Burkhard Jakob (doctoral student) Maria Jäpel (doctoral student) Natalie Kaempf (doctoral student) Katharina Ketel (doctoral student) André Lampe (doctoral student) Guan-Ting Liu (doctoral student) Wen-Ting Lo (doctoral student) Albert Mackintosh (doctoral student) Giulia Russo (doctoral student) Paula Samsó Ferre (doctoral student) Linda Sawade (doctoral student) Irene Schütz (doctoral student) Kyungyeun Song (doctoral student) Dennis Vollweiter (doctoral student) Alexander Wallroth (doctoral student) Uwe Fink (technical assistant / chemist) Sabine Hahn (technical assistant) Delia Löwe (technical assistant) Maria Mühlbauer (technical assistant) Lena von Oertzen (technical assistant) Silke Zillmann (technical assistant) COLLABORATIONS International Sandra M. Bajjalieh, Univ. of Washington, Seattle, USA Daniel F. Cutler, University College London, London, UK Emilio Hirsch, University of Torino, Italy Jocelyn Laporte, IGBMC, Strasburg, France Adam McCluskey, University of Newcastle, Australia Phillip J. Robinson, Children's Medical Research Institute (CMRI), Sydney, Australia Takeshi Sakaba, Kyoto University, Japan Oleg Shupliakov, Karolinska Institute, Stockholm, Sweden National Oliver Daumke, Max-Delbrück-Center for Molecular Medicine, Berlin Christian Freund, Freie Universität Berlin Gary R. Lewin, Max-Delbrück-Center for Molecular Medicine, Berlin Tobias Moser, Georg-August-Universität, Göttingen Silvio O. Rizzoli, Georg-August-Universität, Göttingen Christian Rosenmund, Charité Universitätsmedizin Berlin Dietmar Schmitz, Charité Universitätsmedizin Berlin Stephan J. Sigrist, Freie Universität Berlin Carsten Schultz, EMBL, Heidelberg Staff employed within the reporting period

29 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 27 Fig. 2: A synaptic governess: The adaptor protein AP180 ensures that Synaptobrevin2 (Syb2) is sorted into fissioning clathrin-coated vesicles. In this way AP180 ensures that the resulting synaptic vesicles contain sufficient Syb2, a prerequisite for efficient neurotransmission. SELECTED PUBLICATIONS Soykan T, Kaempf N, Sakaba T, Vollweiter D, Goerdeler F, Puchkov D, Kononenko N L, Haucke V (2017) Synaptic vesicle endocytosis occurs on multiple timescales and is mediated by formin-dependent actin assembly. Neuron 93, Ketel K, Krauss M, Nicot A S, Puchkov D, Wieffer M, Müller R, Subramanian D, Schultz C, Laporte J, Haucke V (2016) A phosphoinositide conversion mechanism for exit from endosomes. Nature 529, Koo S Y, Kochlamazashvili G, Rost B, Puchkov D, Gimber N, Lehmann M, Tadeus G, Schmoranzer J, Rosenmund C, Haucke V#, Maritzen T# (2015) Vesicular synaptobrevin / VAMP2 levels guarded by AP180 control efficient neurotransmission. Neuron 88, (#co-corresponding authors). Gimber N, Tadeus G, Maritzen T, Schmoranzer J, Haucke V. (2015) Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins. Nature Communications 6, Reubold T, Faelber K, Plattner N, Posor Y, Ketel K, Curth U, Schlegel J, Roopsee A, Manstein D J, Noé F, Haucke V, Daumke O, Eschenburg S. (2015) Crystal structure of the dynamin tetramer. Nature 525, EXTERNAL FUNDING Bundesministerium für Forschung und Technologie (BMBF), Die neurobiologischen Grundlagen Polyamin-induzierter Protektion gegen altersassoziierte Einschränkungen der Gedächtnisfunktionen (SMARTAGE), to V. Haucke, , Deutsche Forschungsgemeinschaft, Reinhart Koselleck-Award (HA 2686 / 13-1), , Deutsche Forschungsgemeinschaft, Functional characterization of the SNARE adaptors AP180 and CALM in synaptic exo- and endocytosis in vivo, HA 2686 / 8-1, with T. Maritzen, , Deutsche Forschungsgemeinschaft, SFB 958, A01, Structural and functional organization of endocytic scaffolds within the periactive zone, with T. Maritzen, , Deutsche Forschungsgemeinschaft, SFB 958, Z02, Super-resolution light microscopy to resolve nanoscale molecular structures, to J. Schmoranzer, Deutsche Forschungsgemeinschaft, SFB 740 / 3, C08, Funktionelle Organisation und Dynamik PI-Kinase-basierter Module für die Proteinsortierung an endosomalen Membranen, , Deutsche Forschungsgemeinschaft, SFB 765, B04, Multivalente Modulation der Clathrin vermittelten Rezeptorendozytose, , Deutsche Forschungsgemeinschaft, TRR 186, A08, Phosphoinositidebased switches in endocytic membrane traffic and signaling, to V. Haucke (w / C. Schultz, EMBL) , Deutsche Forschungsgemeinschaft, TRR 186, A09, Phosphoinositidebased switches in polarized sorting and signaling, to V. Haucke (w / S. Boulant, Heidelberg) , Deutsche Forschungsgemeinschaft, Excellence Initiative, EXC-257 NeuroCure Towards a better outcome of central nervous system disorders, , European Commission, Horizon 2020, ITN, H2020-MSCA-ITN-2015, "Deciphering PI3K biology in health and disease, to V. Haucke, , European Commission, Horizon 2020, H2020-MSCA-IF-2014, The role of autophagy in presynaptic protein turnover (SYNPT), to M. Kuijpers, , 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, , Alexander von Humboldt Foundation, Mirjana Leona Weimershaus, , University of Newcastle, International Collaboration Award (ICA), , Deutsche Forschungsgemeinschaft, SFB 958, A07, Regulation of SH3 domain-containing scaffolds in synaptic vesicle clustering, with C. Freund, , Deutsche Forschungsgemeinschaft, SFB 958, A11, Structural and functional analysis of septin scaffolds mediating endosomal membrane trafficking, to M. Krauss (w/ O. Daumke, MDC Berlin), , FMP authors Group members

30 28 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 PROTEIN TRAFFICKING GROUP LEADER PROF. DR. RALF SCHÜLEIN BIOGRAPHY Biology studies, 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, FMP; work on the trafficking mechanisms of GPCRs 2002 Habilitation in Pharmacology and Toxicology, Charité University Medicine Berlin 2014 Adjunct professorship, Charité University Medicine Berlin SUMMARY G protein-coupled receptors (GPCRs) are arguably the most important of drug targets. These receptors must reach their correct subcellular locations, usually the plasma membrane, in order to function. Their transport is enabled by the secretory pathway and begins with a signal sequence-mediated insertion of the receptors into the membrane of the endoplasmic reticulum (ER) by the translocon complex (Sec translocon pathway). The aim of the Protein Trafficking group is to find novel substances that influence ER insertion of GPCRs and other integral membrane proteins at the level of the Sec translocon pathway. For pharmacological application, two types of inhibitors of this pathway are likely to be important: those that block the translocon in general and thereby inhibit the biosynthesis of all proteins using this pathway (type 1 inhibitors; potential tumor drugs), and those that block specific signal sequences and thereby inhibit the biosynthesis of specific proteins (type 2 inhibitors; potential alternatives to classical antagonists in the case of closely-related proteins). ZUSAMMENFASSUNG G-Protein-gekoppelte Rezeptoren (GPCR) sind die wichtigsten Zielproteine für Arzneimittel. 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) über den Translokon-Komplex (Sec-Translokon-Mechanismus). Ziel der Arbeitsgruppe Protein Trafficking ist es, neue Substanzen zu finden, die die Integration von GPCR und anderen Membranproteinen in die ER-Membran auf der Ebene des Sec-Translokon-Wegs beeinflussen. Für eine pharmakologische Anwendung wären zwei Typen von Inhibitoren interessant: 1. Substanzen, die das Translokon generell blockieren und damit die Synthese aller Proteine hemmen, die diesen Weg nützen (Typ 1-Inhibitoren, mögliche neue Medikamente für die Behandlung von Tumoren). 2. Substanzen, die spezifische Signalsequenzen und damit nur die Synthese von einzelnen Proteinen unterbinden (Typ-2-Inhibitoren, mögliche Alternativen zu klassischen Antagonisten für sehr ähnliche Proteine).

31 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 29 Fig. 1: Sec translocon pathway for an integral membrane protein with an extracellular tail. The scheme summarizes the individual steps from the formation of the ribosome / nascent chain / SRP complex to the final integration of the protein into the ER membrane. See the text for details. DESCRIPTION OF PROJECTS Functional significance of the signal peptides of CRF receptors. Selectivity of cotransin, an inhibitor of the Sec61 complex The basic mechanism of the Sec translocon pathway for an integral membrane protein is outlined in Figure 1. In the beginning, the protein is synthesized at cytosolic ribosomes. The signal sequence is bound by the signal recognition particle (SRP) and translation of the nascent chain is arrested. The resulting complex is targeted to the SRP receptor of the translocon machinery at the ER membrane, which consists mainly of the heterotrimeric Sec61 complex (Sec61α, Sec61β, Sec61γ; Sec61α represents the protein-conducting channel). The ribosome binds to Sec61 and the signal sequence destabilizes the closed conformation of the protein-conducting channel. Translation then resumes and the transmembrane domains are released into the ER membrane. Extracellular domains are translocated into the ER lumen while cytosolic domains remain at the opposite site. The cyclodepsipeptide cotransin was described as a Secc61α inhibitor that acts in a signal sequence-discriminatory manner (mixed type 1 / type 2 inhibitor). Originally, cotransin was shown to inhibit the biosynthesis of only a small subset of proteins. However, cotransin selectivity was unknown, nor was it known which properties of a signal sequence were responsible for its cotransin sensitivity. To address these questions, we performed a proteomic study using cotransin-treated cells and the stable isotope labeling by amino acids in cell culture (SILAC) technique, in combination with quantitative mass spectrometry (in cooperation with the mass spectrometry group of the FMP). Using a saturating concentration of cotransin, we found that the biosynthesis of almost all secreted proteins was cotransin-sensitive. In contrast, the biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Moreover, we were able to identify the first conformational consensus motif in signal anchor sequences mediating cotransin sensitivity. Although cotransin has a very interesting mechanism of action, its pharmacological application seems to be precluded due to its mixed type 1 / type 2 inhibitor properties. We therefore decided to perform a high-throughput screen to identify pure type 1 and type 2 inhibitors (in cooperation with the Screening Unit of the FMP). Identification of novel type 1 and type 2 inhibitors of the Sec translocon pathway by high-throughput screening. Setting up a high-throughput screening assay for small molecule inhibitors of the Sec translocon pathway, in particular for Sec61α, is notoriously difficult. Sec61α has no enzymatic activity, is expressed only in the ER membrane, and its isolation and functional reconstitution is problematic in large quantities. Our novel whole cell screening assay included two steps, the first of which was a primary screen using the GFP-tagged corticotropin-releasing factor receptor type 1 (CRF 1 R.GFP) as a target, a GPCR which uses the Sec translocon pathway. Following pre-treatment of the cells with the library of compounds, CRF 1 R.GFP expression was induced and receptor expression quantified by measuring the GFP fluorescence signals. Hit compounds of the primary screen reduce CRF 1 R. GFP expression which means that they could inhibit trancription, translation or the Sec translocon pathway. To specifically identify inhibitors of the Sec translocon pathway, a secondary screen was performed using the unfused cytosolic GFP protein, a target which does not use this pathway. Compounds behaving as inhibitors in the primary screen, but not in the secondary screen, were considered as real hits. These remaining substances were rated according to their IC50 value (cut off = 10 µm) and tested for biosynthesis inhibition of various target proteins in a cellular selectivity assay. We also used a cell-free assay to assess for inhibition of the reconstituted Sec translocon pathway (in cooperation with Kurt Vermeire, University of Leuven, Belgium). Four compounds were found to inhibit the reconstituted Sec translocon pathway. One of these, , seems to be specific for the screening target CR- F 1 R.GFP and may represent a novel type 2 inhibitor for the signal sequence of CRF 1 R (Figure 2). Another of these compounds, , seems to block the Sec translocon pathway in general (Figure 2). Protease protection experiments showed that does indeed block co-translational translocation at the level of the Sec61 complex (data not shown in Fig. 2).

32 30 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / Fig. 2: Compounds and behave as type 1 and type 2 inhibitors of the Sec translocon pathway, respectively. Left panels: cellular biosynthesis assay. To analyze activity and selectivity of the compounds, HEK 293 cells were transiently transfected with the original screening target CRF 1 R.GFP (red column) and various GFP-tagged integral membrane proteins. Cells were treated with the compounds (concentration = 5 IC 50 ) or DMSO for 19 hours and the total GFP fluorescence of the cells was analyzed using flow cytometry as a measure of biosynthesis. Compound affects the biosynthesis of all target proteins and behaves as a type 1 inhibitor, whereas compound impairs only biosynthesis of CRF 1 R.GFP and behaves as a type 2 inhibitor. Right panels: cell-free assay (reconstituted Sec translocon pathway). The signal peptide of the original screening target CRF 1 R.GFP was fused to a preprolactin sequence. A truncated PCR fragment without stop codon (78mer) was transcribed and translated using a reticulocyte lysate and [35S]Met labeling (nascent chain = NC). When rough microsomal membranes (RMs) were added, the nascent chain engages the translocon but remains attached to the trna and cannot be processed and released from Sec61α because the stop codon is missing (NC-tRNA). Release and signal peptide cleavage is induced by puromycin (Pu) treatment. The read out was done by SDS PAGE and autoradiography. Note that the mature, processed construct without signal sequence (red arrow) was strongly reduced upon treatment with compounds and , indicating that they inhibit the Sec translocon pathway (although obviously by different mechanisms; see above). Protein abbreviations: AQP2, aquaporin2; CRF 1 R, corticotropin-releasing factor receptor type 1; CRF 2(a) R, corticotropin-releasing factor receptor type 2a, ET B R, endothelin B receptor; LHR, luteinizing hormone receptor; PAR1, proteaseactivated receptor 1; TSHR, thyrotropin receptor; V 2 R, vasopressin 2 receptor. GROUP MEMBERS COLLABORATIONS Dr. Jens Furkert Dr. Claudia Rutz Arthur Gibert (doctoral student) Wolfgang Klein (doctoral student) Bettina Kahlich (technical assistant) Staff employed within the reporting period International Ulrike Steckelings, University of Southern Denmark, Odense, Denmark Giovanna Valenti, University of Bari, Italy Kurt Vermeire, University of Leuven, Belgium National Heike Biebermann, Charité University Medicine Berlin Duska Dragun, Charité University Medicine Berlin Mathias Dreger, Caprotec Bioanalytics GmbH, Berlin Gunnar Kleinau, Charité University Medicine Berlin SELECTED PUBLICATIONS Gibert A, Wiesner B, Schülein R (2017) The monomer / homodimer equilibrium of G protein-coupled receptors: formation in the secretory pathway and functional significance. Curr Mol Pharmacol. (Epub ahead of print) doi, / Vezzoli V, Duminuco P, Vottero A, Kleinau G, Schülein R, Minari R, Bassi I, Bernasconi S, Persani L, Bonomi M. (2015) A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects receptor biogenesis causing leydig cell hypoplasia. Hum Mol Genet 24, Hinz K M, Meyer K, Kinne A, Schülein R, Köhrle J, Krause G (2015) Structural insights into thyroid hormone transport mechanisms of the L-type amino acid transporter 2. Mol Endocrinol 29: EXTERNAL FUNDING Deutsche Forschungsgemeinschaft, Ableitung von Struktur- / Funktionsbeziehungen spezifischer Hemmstoffe der Biosynthese G-Proteingekoppelter Rezeptoren, SCHU 1116 / 2-1, Rutz C, Klein W, Schülein R (2015) N-Terminal Signal Peptides of G Protein-Coupled Receptors: Significance for Receptor Biosynthesis, Trafficking, and Signal Transduction. Prog Mol Biol Transl Sci 132, Klein W, Westendorf C, Schmidt A, Conill-Cortés M, Rutz C, Blohs M, Beyermann M, Protze J, Krause G, Krause E, Schülein R (2015) Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study. PLoS One 10, e FMP authors Group members

33 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 31 MOLECULAR CELL PHYSIOLOGY MOLEKULARE ZELLPHYSIOLOGIE GROUP LEADER PD DR. INGOLF E. BLASIG BIOGRAPHY Studied biology and biochemistry in Leipzig, diploma thesis on cancer research, Robert- Rössle-Hospital in Berlin-Buch 1984 Dissertation on the pharmacology of myocardial infarction, 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, FMP, and teaching of pharmacology, functional biochemistry, neurochemistry, universities in Potsdam and Berlin Awarded project leader, NIH, Washington DC, USA SUMMARY The group focuses on the structure, function, and modulation of cell-cell contacts to explore tight junctions (TJs) in tissue barriers with the aim of revealing pathological mechanisms and improving therapies. One intended outcome of this work is to propose new strategies for manipulating neurological and other barriers in order to improve drug delivery or to enhance drug efficacy, and to prevent barrier dysfunction. 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 exploring the TJ proteome and interactome, the detailed processes that underlie the oligomerization of key proteins in tissue barriers, and mechanisms to open and / or reconstitute TJs after injury. We organize the annual international symposium Signal Transduction at the Blood- Brain Barriers to disseminate news of progress in the field, with the aim of stimulating collaborations in order to generate publications and new joint projects. ZUSAMMENFASSUNG Wir untersuchen die Struktur, Funktion und Modulation von Zellkontakten und erforschen darin die sogenannten Tight Junctions (TJs, permeationsdichte Abschnitte zwischen Zellen) in Gewebeschranken. 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 von Behandlungsansätzen zu verbessern (Drugenhancer) 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 TJs noch nicht vollständig aufgeklärt ist, ist auch unklar, wie Struktur, Regulationsmechanismen und Wechselwirkungen dieser Proteine die Barrierefunktion steuern. Wir konzentrieren uns bei der Aufklärung des Gesamtkomplexes der TJs, auf die Prozesse, die der Oligomerisierung von Schlüsselproteinen der Gewebsbarrieren zugrunde liegen, sowie auf Mechanismen, die 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.

34 32 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 DESCRIPTION OF PROJECTS Elucidation of tightening mechanism at tight junctions: The interaction potential between the main TJ proteins (Fig. 1) was analyzed in detail. The majority of highly homologous claudins are able to associate homo- and heterophilically. In particular, extracellular loops are involved in the associations (Dabrowski et al., 2015), while to a lesser extent the TAMPs interact homo- and heterophilically with TAMPs and claudins apart from occludin and tricellulin, which do not interact with each other. Direct binding of TAMPs with classic claudins has also been demonstrated (Cording et al., 2014). These 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 with other constituents of TJs. At the blood-brain barrier (BBB), interactions found between TJ proteins have provided deeper insights into the principles of TJ assembly (Milatz et al., 2015; Gehne et al., 2015). As claudin-5 is essential for the tightness of the BBB, and claudin-1 for the perineurium that ensheathes peripheral nerves, both claudins were established as new pharmacological and diagnostic targets (Dabrowski et al., 2014; Staat et al., 2015). New regulatory role of tight junction proteins: The molecular function of TAMPs is still unknown. A new concept was developed and proven for how occludin and tricellulin are involved in redox-dependent signal-transduction mechanisms and how redox-sensitive domains of TAMPs are crucial in the redox regulation of protein interactions at TJs. In addition, it was found that occludin acts as an oxidase under reducing conditions, which is a new regulatory mechanism. These data are highly relevant for diseases related to oxidative stress and for possible pharmacological interventions (Castro et al., 2016; Marko et al., 2016; collaboration with the University of Miami and the MDC/Charité Berlin, respectively). New modulators of tight junctions: Modulation of TJs is a key topic of our studies. Different synthetic peptides of non-claudin origin were generated and tested at a three cell-model of the rat BBB consisting of endothelial cells, astrocytes, and pericytes, and at human colon epithelial cells. Five of these peptides transiently opened the cell barriers (Bocsik et al., 2016; in collaboration with the Hungarian Academy of Sciences, Szeged). Claudin-5, and in particular its first and second extracellular loops, was found to contribute to the interaction between TJ proteins. Specific peptidomimetics of the first loop were generated with ß-sheet structural properties that transiently increased the paracellular permeability for ions, high and low molecular weight compounds, as well as antitumor agents through cellular BBB models (see Figure 2). Intravenous injection in mice facilitated the uptake of small molecules into the brain (Dithmer et al., 2017 under revision). In addition, a new peptidomimetic based on the second extracellular loop of tricellulin, named trictide, was designed that is able to open epithelial barriers for molecules up to 10 kda and that affects not only tricellulin, but also occludin (Arzlan et al., 2017 under revision). In conclusion, novel tools were developed to improve the delivery of pharmaceutical agents through neurological barriers. Investigations for a better understanding of neuropathologies The identification of biomarkers is a field of growing importance for the diagnosis and treatment of neurological diseases. In collaboration with the Pasteur Institute (Paris, France), proteomic investigations aimed at discovering potential biomarkers of lysosomal storage diseases were carried out in dog models of Hurler and Sanfilippo syndromes. A variety of biomarker candidates were identified in the cerebrospinal fluid (CSF) for both pathologies. In connection with the studies directed at the identification of potential biomarkers, methodological experiments were performed to assess the transferability of data obtained from human and dog CSF (Günther et al., 2015). 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 TJ proteins. (A) Scheme of protein composition in TJs at the blood-brain barrier with special consideration of members of the claudin protein family and the TJ-associated marvel proteins (TAMPs) occludin and tricellulin (tetraspanning). Single membrane-spanning JAMs (junctional adhesion molecules), as well as membrane-associated zonula occludens proteins (ZO), or multi-pdz domain protein 1 (MUPP-1), are not directly involved in paraendothelial tightening. 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 microscopy of two adhering CECs visualizing TJ elements sealing the intermembrane gap, arrows indicating TJ. (C) Freeze-fracture transmission electron microscopy of TJ strands in mouse BBB endothelial cells. Note that particles are associated nearly equally at the E- and P-faces. Arrows indicate TJ particles. EF / PF, exoplasmic and protoplasmic face of intramembranous TJ strands. intracellular C1C2 extracellular C5C2 cell membrane intracellular mclaudin-5 mclaudin-1 drug no paracellular paracellular drug delivery drug delivery pre-incubation with C5C2 C1C2 Figure 2. Scheme of how a peptidomimetic agent based on an extracellular loop of a tightening claudin can cause transient opening of the paracellular cleft in tissue barriers to improve drug delivery. Example: Peptide C5C2 from murine claudin-5.

35 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 33 GROUP MEMBERS Dr. Christian Bellmann Dr. Rosel Blasig Dr. Reiner Haseloff Dr. Lars Winkler Basak Arslan (doctoral student) Philipp Berndt (doctoral student) Olga Breitkreutz-Korff (doctoral student) Jimmi Cording (doctoral student, postdoc) Sophie Dithmer (doctoral student) Nora Gehne (doctoral student) Sebastian Pfeil (Dabrowski) (doctoral student) Christian Staat (doctoral student, postdoc) Ramona Günther (technical assistent) Heike Meyer (technical assistent) Katrin Schildele (technical assistent) Staff employed within the reporting period COLLABORATIONS International European consortium Brains4brain Anuska Andjelkovic, University of Michigan Medical School, Ann Arbor, USA Maria Deli, Biological Research Centre, Szeged, Hungary Jean-Michel Heard, Institut Pasteur, Paris, France Zhihai Qin, Chinese Acad. Sci., Beijing, PR China Britta Engelhardt, Universität Bern, Schweiz Birger Brodin, University Copenhagen, Denmark Michal Toborek, University of Miami, USA National Jörg Piontek, Charite Berlin Heike Rittner, Universitätsklinikum Würzburg Kai Schmidt-Ott, MDC / Charite Berlin Hartwig Wolburg, Eberhard-Karls-Universität Tübingen SELECTED PUBLICATIONS Markó L, Vigolo E, Hinze C, Park J-K, Roël G, Balogh A, Choi M, Wübken A, Cording J, Blasig I E, Luft F C, Scheidereit C, Schmidt-Ott K M, Schmidt-Ullrich R, Müller D N (2016) Tubular epithelial NF-κB activity regulates acute ischemic kidney injury. J. Am. Soc. Nephrol. 27, EXTERNAL FUNDING VIP Projekt, BMBF, EASYPERM, Modulatoren der Blut-Hirnschranke als Drugenhancer für ZNS-Pharmaka, , ,00 Dabrowski S, Staat C, Zwanziger D, Sauer R S, Bellmann C, Günther R, Haseloff R F, Rittner H, Blasig I E (2015) Structure and function of the first extracellular loop of the cell-cell contact protein claudin-1 lessons from peptide to animal. Antioxid. Redox Signal. 22, Günther R, Krause E, Schümann M, Blasig I E, Haseloff R F (2015) Depletion of highly abundant proteins from human cerebrospinal fluid: a cautionary note. Mol. Neurodegener. 10, 53. Cording J, Günther R, Vigolo E, Tscheik C, Winkler L, Schlattner I, Lorenz D, Haseloff R H, Schmidt-Ott K M, Wolburg H, Blasig I E (2014) Redox regulation of cell contacts by tricellulin and occludin: Redoxsensitive cysteine sites in tricellulin regulate both tri- and bicellular junctions in tissue barriers as shown in hypoxia and ischemia. Antioxid. Redox Signal. 23, Staat C, Coisne C, Dabrowski S, Stamatovic S M, Andjelkovic A V, Wolburg H, Engelhardt B, Blasig I E (2015) Mode of action of claudin peptidomimetics in the transient opening of cellular tight junction barriers. Biomaterials 54, FMP authors Group members

36 34 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 MOLECULAR NEUROSCIENCE AND BIOPHYSICS MOLEKULARE NEUROWISSEN- SCHAFTEN UND BIOPHYSIK GROUP LEADER DR. ANDREW J.R. PLESTED BIOGRAPHY 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 Pharma cology (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 2015 ERC Consolidator Grant SUMMARY Our principal research interests are glutamate receptors and excitatory synapses. These receptors are essential for brain function, being necessary components of excitatory synaptic transmission. Synapses themselves are implicated in cognition and numerous complex brain diseases. We aim to understand the molecular basis of fast excitatory transmission, and to develop methods to observe and alter synapse activity. 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 and biochemistry. Combining these approaches with fluorescence microscopy has enabled us to visualize receptor activity directly. 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 enzymes and other 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 neuster 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.

37 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 35 Sites that funnel neurotransmitter into the binding site of a glutamate receptor Mutation of a site identified as a hotspot for funneling glutamate into the binding cleft (A) slows AMPA receptor activation (B) and deactivation (C), consistent with removal of a preferential binding pathway. DESCRIPTION OF PROJECTS Neurotransmitter funneling focuses glutamate receptor activation Bilobed ligand-binding domains (LBDs), such as those found in ionotropic glutamate receptors (iglurs), are common architectural elements found in hundreds of small-molecule receptor proteins. Despite the ubiquity of these domains, processes essential to receptor activation, such as pathways along which a ligand is guided into its binding site, metastable protein-ligand interactions, and the coupling of ligand binding to protein conformational changes, are poorly understood. In our work, we took advantage of long molecular simulations performed on custom hardware that show the process of glutamate binding to the AMPA-type iglur LBD for the first time. Charged sidechains on the surface of the LBD are found to bind glutamate transiently and funnel it into its recessed binding pocket. Electrophysiological recordings show that eliminating these transient binding sites reveals a selective and unique kinetic signature of slowed activation and deactivation. These results suggest that preferential binding pathways have evolved to optimize rapid responses of glutamate receptors at central nervous system synapses. Structural dynamics of glutamate receptor activation A recent major achievement has been understanding how certain molecules weakly activate the glutamate receptor. When we consider the maximum activation of the receptor i. e. when it is continuously in the on state we expect the receptor to adopt a certain geometry (or set of geometries). However some molecules, called partial agonists, bind to the receptor in a way that causes non-maximal activation. These partial agonists can be synthetic, but some also occur in nature. We believe that the reasons for non-maximal activation occurring in the first place probably relate to how the receptor fundamentally works. One explanation for this might be that the receptor spends some of its time activated and some of its time resting. In this interpretation, the receptor cycles between active and resting states and the agonist is unable to prevent this cycling from happening, like a lazy runner who stops a lot. An alternative interpretation is that the partial agonists very effectively coax the receptor into a shape that is not especially active, which would be analogous to walking in a running race. In order to assess these competing theories, we measured the number of different geometries that the AMPA receptor can adopt for activators of different strengths. We observed that partial agonists that are bad at activating the receptor allow many different conformations to form. Partial agonists that cause about 50 % activity are more selective. Therefore, and perhaps unsurprisingly, the answer is that both theories are true. The different activators drive the receptor into overlapping sets of geometries, which they occupy for different lengths of time on average. What is surprising is that most of the arrangements are inactive. We suggest that this plethora of inactive states allows the receptor activation to be rapid and selective. This idea will be the subject of further tests during the project GluActive. This work was a combined study of structural biology and functional experiments and is in press in Nature Communications (Salazar et al. 2017). Gating the transmembrane domain of glutamate receptors We have continued our investigation of AMPA receptor gating with unnatural amino acids that are sensitive to UV light. We performed an unbiased screen of 23 sites in the transmembrane region with two different unnatural amino acids, azido-phenylalanine and benzoylphenylalanine. This work has revealed details of the gating mechanism, and has produced mutants that offer very potent control of receptor activity with light. In particular, we see that helical contacts at the periphery of the ion channel are critical for controlling gating and desensitization. We now aim to exploit these mutants to control receptors in neurons. Fluorescence studies of AMPA receptor complexes We have developed the first FRET pair between the AMPA receptor and an auxiliary protein, stargazin. This tool employs fluorescent proteins fused to the subunits of interest and enables us to monitor conformational changes in the complex during receptor activation by glutamate. It also allows us to detect optically if the complex dissociates, which is a controversial topic in glutamate receptor biology. With this tool, we are able to see that fast conformational changes occur during gating, but we do not detect any dissociation of the receptor from the auxiliary protein on the timescale of normal gating (seconds).

38 36 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fluorescence resonance energy transfer (FRET) between AMPA receptor and stargazin Insertion of the green fluorescent protein into GluA2 at two sites (383 and 391) produces a construct that efficiently FRETs to a RFP fused to stargazin. Spectra from live cells indicate the red emission (above 560 nm) from 488 nm excitation in a cotransfection. GROUP MEMBERS Dr. Jelena Baranovic Dr. Anna Carbone Dr. Clarissa Eibl Dr. Valentina Ghisi Dr. Ljudmila Katchan Dr. Hector Salazar Sonja Minniberger (PhD Student) Yuchen Hao (PhD Student) Sebastian Opfermann (PhD Student) Irene Riva (PhD Student) Anahita Poshtiban (PhD Student) Antje Maluck (Technician) Ronny Schaefer (Technician) Marcus Wietstruk (Technician) COLLABORATIONS International Daniel Choquet, Interdisciplinary Institute for Neuroscience, Bordeaux, France Teresa Giraldez, University of La Laguna, Spain Eric Gouaux, Howard Hughes Medical Institute & Oregon Health Sciences University, USA Albert Lau, Johns Hopkins University, Baltimore, MD, USA National Oliver Daumke, Max-Delbrück-Center for Molecular Medicine, Berlin Peter Hegemann, HU-Berlin Jana Kusch, Jena Philip Selenko, FMP-Berlin Staff employed within the reporting period SELECTED PUBLICATIONS Salazar H, Eibl CE, Chebli M & Plested AJR (2017) Mechanism of Partial Agonism in AMPA-type glutamate receptors. Nature Communications 8, These authors contributed jointly. Zachariassen LG, Katchan L, Jensen AG, Pickering DS, Plested AJR* & Kristensen AS* (2016) Structural rearrangement of the intracellular domains during AMPA Receptor activation. PNAS 113, E These authors contributed jointly. * Corresponding authors. Baranovic J, Chebli M, Salazar H, Carbone AL, Fälber K, Lau AY, Daumke O, Plested AJR (2016) Dynamics of the ligand binding domain layer during AMPA receptor activation. Biophysical Journal 110, These authors contributed jointly. Carbone AL and Plested AJR (2016) Superactivation of glutamate receptors by auxiliary proteins. Nature Communications 7, EXTERNAL FUNDING DFG SFB / TRR 186 A07 Optical Control of Calcium Switches that Orchestrate Fast Signaling in the Brain (with Peter Hegemann, HU Berlin) DFG PL619 / 2 Optical control of glutamate receptors using genetically encoded unnatural amino acids ERC CoG GluActive FMP authors Group members

39 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 37 MEMBRANE TRAFFIC AND CELL MOTILITY MEMBRANTRANSPORT UND ZELLBEWEGLICHKEIT GROUP LEADER PD DR. TANJA MARITZEN BIOGRAPHY Biochemistry and Molecular Biology studies, University of Hamburg Ph.D. student, Center for Molecular Neurobiology Hamburg (Prof. T.J. Jentsch), Dr. rer. nat. (summa cum laude), University of Hamburg 2006 Hans-Dietrich Herrmann- Promotionspreis for Molecular Medicine Postdoctoral Research Associate, Center for Molecular Neurobiology 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 since 2016 Privatdozent following habilitation at Freie Universität Berlin SUMMARY In order to interact with the extracellular environment cells rely to a large extent on cell surface-localized proteins. The binding of extracellular ligands to these cell surface receptors triggers diverse intracellular signaling cascades that determine, for instance, whether a cell starts to proliferate, to differentiate, to migrate, or to trigger an immune response. Accordingly, the number of these signal receptors at the cell surface has to be tightly regulated in each situation to achieve the right level of responsiveness to a specific extracellular cue. Endocytosis, in conjunction with intracellular membrane transport, constitutes a highly effective way in which to regulate the levels of diverse proteins at the cell surface. Endocytosis also confines the localization of these proteins to specific sites on the plasma membrane, which is essential for processes such as 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, immune functions, and neurotransmission. ZUSAMMENFASSUNG Zellen interagieren mit ihrer Umgebung im Wesentlichen über Proteine, die an der Zell oberfläche 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, zusammen mit intrazellulären Membrantransportprozessen, ermöglicht es Zellen, den Anteil 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 inter agieren und wie ihre Internalisierung in die Zelle gesteuert wird. Mit einer Kombination aus zellbiologischen, optischen und genetischen Methoden untersucht unsere Gruppe die Rolle von endozytotischen und endosomalen Gerüst- und Adaptorproteinen für die Regulation der Lokalisation von Zelloberflächenproteinen. Besonders interessiert uns dabei die Bedeutung von Endozytose und Membrantransport für die zelluläre Beweglichkeit, Immunfunktionen und Neurotransmission.

40 38 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Furthermore, membrane traffic has to be coordinated with actin cytoskeleton dynamics in order for cellular movement to occur. Proteins that connect these processes are likely to be 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 regulates endosomal vesicle dynamics, and also functions as an inhibitor of the actinnucleating ARP2 / 3 complex which plays a crucial role in cell migration. Therefore Gadkin seems a likely candidate for coordinating membrane transport and cytoskeletal dynamics during cell migration. Having previously established that loss of Gadkin promotes melanoma cell migration, we recently demonstrated that Gadkin also affects the motility of dendritic cells, which need to efficiently migrate in order to fulfill their function as sentinels against pathogens. Fig. 1: The endocytic adaptor protein Stonin1 (green) localizes specifically to endocytic structures directly behind focal adhesions (red) at the leading edge of a mouse embryonic fibroblast (arrow indicates direction of migration; cell nucleus is stained in blue). Diverse proteoglycans modulate cell motility by interacting with extracellular ligands and triggering intracellular signaling cascades that modulate actin dynamics. However, their endocytosis-based regulation is poorly understood. We identified the proteoglycan NG2 / CSPG4 as cargo of the so-far orphan endocytic adaptor Stonin1 and showed that its adaptor-dependent internalization is integral to normal cell migration. As NG2 is a known oncogene that promotes glioma growth, we are currently investigating the potential relevance of the Stonin1 NG2 / CSPG4 interaction for tumorigenesis. Role of membrane transport for immune cell functions Membrane transport is not only relevant to immune cell migration, but also affects many facets of immune cell life. The main function of dendritic cells is the sampling of antigens for presentation to T-cells in order to trigger immune responses against pathogens. To this end, dendritic cells phagocytose pathogens and process their proteins into small peptides that are loaded onto MHCII molecules that are then presented to T-cells on the dendritic cell membrane. 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 which might therefore be relevant to immune defense. Fig. 2: Migration parameters such as speed and directionality of movement are measured by tracking cells as they move along a chemotactic gradient. DESCRIPTION OF PROJECTS Membrane transport in cell motility Cell motility is not only crucial during human development, but also thereafter for immunity in the healthy adult. Its dysregulation is involved in autoimmune disorders and 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 the proteins that mediate cell adhesion and migratory signaling such as integrins and proteoglycans. Role of endocytic adaptors and scaffold proteins for brain function Neurons communicate by releasing neurotransmitters from synaptic vesicles (SVs). When these vesicles fuse with the presynaptic membrane, the SV proteins become stranded in this membrane. They have to be retrieved to regenerate SVs for sustained neurotransmission. Thus the internalization of cell surface-localized proteins constitutes an essential step in SV recycling. In fact, loss-of-function mutants of the main components of the endocytic machinery lead to premature death. In collaboration with the group of Volker Haucke, and as part of the SFB958 in a DFG-funded project, we investigate the importance of endocytic adaptor proteins for the high-fidelity retrieval of crucial SV proteins. We have shown that the adaptor protein Stonin2 mediates the sorting of the SV Ca 2+ sensor synaptotagmin1, while AP180 is crucial for internalizing the SV protein synaptobrevin2. Currently, we are dissecting the neuronal role of the AP180-related adaptor protein CALM, a protein that has been implicated in Alzheimer s disease. We also analyze the mechanisms by which the endocytic scaffold intersectin affects brain function. To elucidate the physiological relevance of these proteins in an organismic context we employ mouse genetics in conjunction with live cell imaging approaches and behavioural studies.

41 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 39 GROUP MEMBERS PD Dr. Tanja Maritzen (group leader) Dr. Domenico Azarnia Tehran Dr. Hannah Schachtner Marietta Browarski (doctoral student) Fabian Feutlinske (doctoral student) Marine Gil (doctoral student) Fabian Lukas (doctoral student) Dennis Vollweiter (doctoral student) Lennart Hoffmann (student) Claudia Schmidt (technical assistant) Staff employed within the reporting period COLLABORATIONS International Daniel Legler BITg at the University of Konstanz, Kreuzlingen, Switzerland Laura M. Machesky Beatson Institute for Cancer Research, Glasgow, UK William B. Stallcup Sanford-Burnham Medical Research Institute, CA, USA Nicolas Chevrier FAS Center for Systems Biology, Harvard University, MA, USA National Volker Haucke, Eberhard Krause FMP, Germany MinChi Ku, Sonia Waizcies, Thoralf Niendorf MDC, Berlin, Germany Uta Hoepken MDC, Berlin, Germany Matthias Selbach MDC, Berlin, Germany Christian Rosenmund Charité Universitätsmedizin Berlin, Germany Annette Schürmann DIFE, Potsdam, Germany Tobias Moser Georg-August-Universität Göttingen, Göttingen, Germany Rainer Glass Ludwig-Maximilians-Universität München, München, Germany Frank Schmitz Universität des Saarlandes, Homburg / Saar, Germany SELECTED PUBLICATIONS Feutlinske F, Browarski W, Ku MC, Trnka P, Waiczies S, Niendorf T, Stallcup W B, Glass R, Krause E, Maritzen T (2015) Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility. Nat Commun. 6, Koo S J, Kochlamazashvili G, Rost B, Puchkov D, Gimber N, Lehmann M, Tadeus G, Schmoranzer J, Rosenmund C, Haucke V *, Maritzen T * (2015) Vesicular Synaptobrevin / VAMP2 levels guarded by AP180 control efficient neurotransmission. Neuron. 88(2), Schachtner H, Weimershaus M, Stache V, Plewa N, Legler D F, Höpken U E, Maritzen T (2015) Loss of Gadkin affects dendritic cell migration in vitro. PLoS One. 10(12), e Jung S *, Maritzen T *, Wichmann C *, Jing Z, Neef A, Revelo N H, Al-Moyed H, Meese S, Wojcik S M, Panou I, Bulut H, Schu P, Ficner R, Reisinger E, Rizzoli S O, Neef J, Strenzke N, Haucke V, Moser T. (2015) Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing. EMBO J. 34(21), EXTERNAL FUNDING Deutsche Forschungsgemeinschaft, Untersuchung der Bedeutung von Stonin1 für die Dynamik fokaler Adhäsionen und die Unterdrückung von Tumoren, MA 4735 / 2-1, , 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 zone, with V.Haucke, , Deutsche Forschungsgemeinschaft, Funktionelle Charakterisierung der SNARE Adaptoren AP180 und CALM bei der synaptischen Exo- und Endozytose in vivo, MA 4735 / 1-1, with V. Haucke, , Maritzen T *, Schachtner H, Legler D F * (2015) On the move: Endocytic trafficking in cell migration. Cell Mol Life Sci. 72(11), FMP authors Group members * contributed equally

42 40 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 PROTEOSTASIS IN AGING AND DISEASE PROTEINHOMÖOSTASE IM ALTERN UND IN KRANKHEITEN GROUP LEADER DR. JANINE KIRSTEIN BIOGRAPHY 2003 Diploma / MSc., University of Greifswald 2007 Ph.D., Free University Berlin (summa cum laude) Postdoc, 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, Northwestern University, IL, USA Since 2013 Group Leader in the Molecular Physiology and Cell Biology Section of the FMP Since 2013 Member of NeuroCure Cluster of Excellence 2015 / 2016 DFG NeuroCure Innovation Award SUMMARY Our research goal is to advance our understanding of the protein-folding problem. We want to gain mechanistic insight into the complexity, function, and dynamics of molecular chaperones. Molecular chaperones assist the folding of nascent polypeptide chains upon their synthesis at the ribosome, they facilitate folding of unfolded or denatured protein stretches, they prevent misfolding and aggregation, and they also disaggregate aggregated proteins. Our group studies the contribution of molecular chaperones in the context of aging and neurodegenerative diseases that are characterized by the accumulation of amyloid fibrils formed by aggregation-prone peptides and proteins. We mimic these neurodegenerative diseases by expressing the disease proteins in cells and the nematode C. elegans. We have also established a number of in vitro assays that allows us to monitor the activity of chaperones and chaperone complexes in amyloid fibrilization and disaggregation of disease-associated proteins. ZUSAMMENFASSUNG Das Ziel unserer Forschung ist es, ein besseres Verständis zur Protein-Faltungs Problematik zu gewinnen. Wir wollen mechanistische Erkenntnisse zur Komplexität, Funktion und Dynamik molekularer Chaperone erlangen. Molekulare Chaperone helfen bei der Faltung von neusynthetisierten Proteinen am Ribosom, ermöglichen die Faltung von ungefalteten und denaturierten Proteinen und können auch Proteinaggregate resolubilisieren. Unsere Gruppe erforscht die Funktion der Chaperone während des Alterns und in neurodegenerativen Krankheiten, die durch eine Anhäufung von amyloiden Proteinfibrillen gekennzeichnet sind. Wir stellen diese Szenarien in Zellkultur und im Nematoden C. elegans durch gezielte Synthese dieser krankheitserzeugenden Peptide und Proteine künstlich nach. Zusätzlich haben wir eine Reihe von in vitro Assays etabliert, die es uns erlauben, die Aktivität einzelner Chaperone oder Chaperonkomplexe auf Fibrilisierung der amyloiden Proteine und deren Disaggregation zu untersuchen.

43 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 41 Autophagy is induced in response to the expression of amyloid proteins. The image depicts C. elegans expressing the autophagy marker LGG-1:GFP. On the left: control; on the right: an animal co-expressing Aβ DESCRIPTION OF PROJECTS The trimeric chaperone complex Hsp70 / Hsp110 / J-protein suppresses Htt amyloid formation and resolubilizes preformed Htt fibrils Huntington s disease (HD) is a neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats within the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. The first observations of chaperones being able, to a certain extent, to decrease the aggregation propensity of HttQn in in vitro assays were obtained using bacterial or yeast Hsp70 and Hsp40 (J-protein) chaperones. In vivo, overexpression of Hsp70, J-protein, Hsp110, or TRiC reduces the aggregation toxicity of Htt in cultured cells, flies, and mice HD models. It has also been observed that expression of two chaperones (Hsp70 / J-protein or Hsp110 / J-protein) synergistically suppress Htt aggregation. These findings are in agreement with previous reports that these chaperones form functional complexes, and suggest that they cooperate in vivo to prevent or reverse polyq aggregation. Despite these efforts many unanswered questions remain. Do chaperones interfere with nucleation events of beta-sheet formation and subsequent elongation into amyloids or seeding activities? Can chaperones resolubilize Htt once it is assembled into amyloid fibrils? Are there specific chaperones or chaperone complexes that recognize distinct moieties of misfolded and aggregated Htt? The diversity within the chaperone families has increased over the course of evolution. A pronounced expansion in the number of distinct chaperones occurred, e. g. within the J-protein family, suggesting an increased functional specialization of chaperones. By using a novel FRET-based in vitro assay to monitor the amyloid formation of Htt proteins and the effect of molecular chaperones on the fibrilization kinetics, we have demonstrated that a trimeric chaperone complex composed of a member each of the Hsp70, Hsp110, and type II J-protein families can completely suppress the formation of amyloid fibrils by HttQ n. We also demonstrated for the first time a disaggregation of HttExon1Q 48 fibrils by this trimeric chaperone complex. The composition of the chaperone complex is very dynamic. The combination of different HSP-70 and J-protein chaperones, together with HSP-110, forms distinct chaperone complexes that exhibit different suppression and disaggregation activities. Depletion of these chaperones in HD patient-derived neuronal progenitor cells (NPCs) lead to a pronounced increase in aggregation of the endogenous Htt protein (Q 44 ). We have confirmed the importance of these chaperones for maintaining the solubility of HttQ n and related polyq proteins on an organismal level in C. elegans. To do so, we employed nematode lines that express HttQ n and SCA3Q n proteins and depleted the chaperone expression using RNAi. Knockdown of the chaperone genes lead to a substantial increase in aggregation of the polyq proteins. To confirm the vital role of molecular chaperones for the maintenance of the solubility of polyq proteins we overexpressed a specific chaperone (the J-protein DNAJB1) in HEK cells expressing robustly aggregating HttExon1Q 97. This J-protein is the rate-limiting chaperone in the in vitro chaperone assays and in vivo it is the least abundant protein of the three chaperones. Importantly, overexpression of the J-protein results in a pronounced decrease of HttExon1Q 97 aggregation. Currently we are investigating the functional spectrum of this trimeric chaperone complex. We have already demonstrated that the same chaperone complex disaggregates fibrils formed by α-synuclein, suggesting that this chaperone complex has a broad substrate spectrum. Interplay between disaggregation and proteolytic clearance pathways The cell has three strategies to cope with protein aggregates: (i) deposition of aggregates into cellular assemblies such as INQ, CytoQ and IPOD, (ii) clearance via disaggregation by molecular chaperones, and (iii) proteolytic clearance by the ubiquitin proteasome system (UPS) or autophagy. Little is known regarding if and how the clearance pathways communicate with each other to maintain a balanced proteostasis with the progression of aging or in disease. We set out to address this question by analyzing the disaggregation, UPS, and autophagy capacity under control conditions and upon impairment of one of the aggregate clearance pathways. We developed tools to monitor all three activities in vitro and in living animals. Interestingly, depletion of chaperones involved in disaggregation lead to an induction of autophagy. Autophagy appears to compensate for the lack of disaggregation activity as a means to cope with the accumulation of misfolded and aggregated proteins. It is noteworthy that the UPS activity and the abundance of 20S subunits decreased upon knockdown of chaperones that mediate disaggregation. We observed the same response upon expression of aggregation-prone disease-causing peptides and proteins such as Aβ 1-42 and polyq that lead to an induction of autophagy and a reduction of UPS capacity. We are now aiming to elucidate the mechanism of the interplay between these clearance pathways in the context of aging and neurodegeneration.

44 42 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 EM picture of α-synuclein fibrils with DNJ-13. The image depicts an immunostain of DNJ-13 with α-synuclein fibrils. The J-protein binds to α-synucleinfibrils independently of other chaperones and probably recruits the cooperating chaperones HSP-70 and HSP-110 to initiate the disaggregation process. GROUP MEMBERS Dr. Katrin Jünemann Dr. Annika Scior Kristin Arnsburg (doctoral student) Diogo Feleciano (doctoral student) Christian Gallrein (doctoral student) Manuel Iburg (doctoral student) Lucia Pigazzini (doctoral student) Marc Bohlmann (MSc student) Linda Bergemann (MSc student) Kalina Tosheva (MSc student) Christina Hildebrandt (BSc student) Wolfram Thielicke (BSc student) Kerstin Steinhagen (technical assistant) Staff employed within the reporting period COLLABORATIONS International Harm Kampinga, UMCG, Groningen, Netherlands Funda Sar, Koc University, Istanbul, Turkey Ansgar Siemer, University of Southern California, USA National Erich Wanker, MDC, Berlin Alessandro Prigione, MDC Josef Priller, Charite, Berlin Britta Eickholt, Charite, Berlin Bernd Bukau, ZMBH-DKFZ Alliance, Heidelberg Baris Tursun, MDC, Berlin Ralf Schülein, FMP, Berlin Eberhard Krause, FMP, Berlin Hartmut Oschkinat, FMP, Berlin Phil Selenko, FMP, Berlin Dmytro Puchkov, FMP, Berlin Christian Hackenberger FMP, Berlin SELECTED PUBLICATIONS Lechler M C, Crawford E D, Groh N, Widmaier K, Jung R, Kirstein J, Trinidad J C, Burlingame A L, David D C (2017) Reduced Insulin / IGF-1 Signaling Restores the Dynamic Properties of Key Stress Granule Proteins during Aging. Cell Reports 18, Scior A, Juenemann K, Kirstein J (2016) Cellular strategies to cope with protein aggregation. Essays in Biochemistry 60, Feleciano D R, Arnsburg K, Kirstein J (2016) Interplay between redox and protein homeostasis. Worm 5. Nillegoda N B, Kirstein J, Szlachcic A, Berynskyy M, Stank A, Stengel F, Arnsburg K, Gao X, Scior A, Aebersold R, Guilbride D L, Wade R C, Morimoto R I, Mayer M P, Bukau B (2015) Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature 524, Kirstein J *, Morito D, Kakihana T, Sugihara M, Minnen A, Hipp M S, Nussbaum-Krammer C, Hartl F U, Nagata K, Morimoto R I (2015) Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments. EMBO J 34, EXTERNAL FUNDING DFG EXC 257 NeuroCure ( ) DFG EXC 257 NeuroCure Innovation Award ( ) DFG SFB740 B8 (01/ / 2018) DFG SPP1623 (01/ / 2018) DFG KI1988 / 3-1 (01/ / 2018) Daimler und Benz Stiftung (Annika Scior; 02 / / 2017): AXA Research Fund (Katrin Jünemann; 10 / / 2018): EU-COST STSM Fellowship (Diogo Feleciano; / 2016): FMP authors Group members * co-corresponding author

45 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 43 BEHAVIOURAL NEURODYNAMICS VERHALTENSNEURODYNAMIK GROUP LEADERS DR. TATIANA KOROTKOVA DR. ALEXEY PONOMARENKO BIOGRAPHY Tatiana Korotkova Studied biology and physiology, 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) Post-Doc, FMP, NeuroCure Fellow since 2012 Junior group leader, FMP / NeuroCure Alexey Ponomarenko Studied biology and physiology, 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, Rutgers University (Prof. G. Buzsaki), Newark, USA Post-Doc, University Clinic for Neurology, Heidelberg (Prof. H. Monyer) SUMMARY 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. Using genetic mouse models, we are focusing on the role of voltage-gated channels and GABAA receptors in the operation of hippocampal networks. 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, particularly in 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. since 2009 Junior group leader, FMP / Neuro Cure

46 44 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Simultaneous recordings of multiple neurons in behaving mice, isolation of action potential trains emitted by individual neuron. Botton, right: optogenetic, cell-type specific, stimulation of neurons in lateral hypothalamus in vivo. DESCRIPTION OF PROJECTS Control of hippocampal excitability in vivo by KCNQ3 and 5 channels In collaboration with Prof. T. J. Jentsch, we have investigated the role of KCNQ channels in the control of neuronal excitability in vivo. This project focuses 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 had been unknown. We found that the two channels play distinct and important roles in controlling neuronal excitability in the hippocampus of mice. Their functions include shaping the discharge mode of hippocampal neurons, determining the efficacy of fast network synchronization and the precision of neural representations of space, and 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 because 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. The peak of the power spectral density of optogenetically-paced theta oscillations matched the 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., Nature Communications, 2015). 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 the 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. Activation or inhibition of GABAergic neurons in the LH also affected feeding behavior. Furthermore, neuronal activity of LH neurons was behavior- and state-dependent (Herrera et al., Nature Neuroscience, 2016; Carus-Cadavieco et al., Nature, 2017).

47 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 45 GROUP MEMBERS Dr. Alexey Ponomarenko (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) Suzanne van der Veldt (graduate student) Emmanouela Volitaki (graduate student) Staff employed within the reporting period COLLABORATIONS International Antoine Adamantidis, McGill University, Montreal, Canada Christoph Börgers, Tufts University, Boston, USA Denis Burdakov, National Institute for Medical Research, London, UK Karl Deisseroth, Stanford University, USANancy Kopell, Boston University, Boston, USA Genela Morris, University of Haifa, Israel National Thomas J. Jentsch, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin Lisa Marshall, University of Lübeck Dietmar Schmitz, Charité Universitätsmedizin Berlin Achim Schweikard, University of Lübeck SELECTED PUBLICATIONS Carus-Cadavieco M *, Gorbati M *, Ye L, Bender F, van der Veldt S, Kosse C, Börgers C, Lee S Y, Ramakrishnan C, Hu Y, Denisova N, Ramm F, Volitaki E, Burdakov D, Deisseroth K, Ponomarenko A *, Korotkova T * (2017) Gamma oscillations organize top-down signaling to hypothalamus and enable food seeking. Nature, 542(7640), Herrera C G, Carus-Cadavieco M, Jego S, Ponomarenko A, Korotkova T, Adamantidis A (2016) Hypothalamic feed-forward inhibition of thalamocortical network controls arousal and consciousness. Nature Neuroscience, 19(2), Stempel A V, Stumpf A, Zhang H-Y, Özdogan T, Pannasch U, Theis A-K, Otte D M, Wojtalla A, Rácz I, Ponomarenko A, Xi Z-X, Zimmer A and Schmitz D (2016) Cannabinoid Type 2 Receptors Mediate a Cell Type-Specific Plasticity in the Hippocampus. Neuron, 90(4), Bender F *, Gorbati M *, Carus-Cadavieco M, Denisova N, Gao X, Holman C, Korotkova T *, Ponomarenko A * (2015) Theta oscillations regulate speed of locomotion via hippocampus to lateral septum pathway. Nature Communications, 6, Fidzinski P *, Korotkova T *, Heidenreich M *, Maier N, Schuetze S, Kobler O, Zuschratter W, Schmitz D, Ponomarenko A, Jentsch T J (2015) KCNQ5 K+ channels control hippocampal synaptic inhibition and fast network oscillations. Nature Communications, 6, EXTERNAL FUNDING The Human Frontier Science Program, Neural basis of behavioural multitasking and coordination by specific hypothalamic circuits, ; $ Die Deutsche Forschungsgemeinschaft, Priority Program 1665 (Schwerpunktprogramm), Resolving and manipulating neuronal networks in the mammalian brain from correlative to causal analysis ; ; Die Deutsch-Israelische Stiftung für wissenschaftliche Forschung und Entwicklung (GIF). Midbrain dopamine and GABA inputs onto hippocampus and medial prefrontal cortex: function in motivation, spatial learning and memory ; Die Deutsche Forschungsgemeinschaft, Cluster of Excellence NeuroCure: Towards a better outcome of central nervous system disorders, PI position A. Ponomarenko, ; Deutsche Forschungsgemeinschaft, Cluster of Excellence NeuroCure: Towards a better outcome of central nervous system disorders, Habilitationsgrant für Nachwuchswissen-schaftlerinnen (including PI position), T. Korotkova, , Korotkova T, Ponomarenko A (2016) Optogenetic control of neuronal network oscillations: combination of electrophysiological recordings and optogenetics in behaving mice. In Vivo Neuropharmacology and Neurophysiology, Series Neuromethods, Springer Science. FMP authors Group members * equal first author corresponding author

48 46 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 MOLECULAR AND THEORETICAL NEUROSCIENCE MOLEKULARE UND THEORETISCHE NEUROWISSENSCHAFTEN GROUP LEADER DR. ALEXANDER MATTHIAS WALTER BIOGRAPHY University Course in Chemistry, University of Göttingen, Germany Master s in Neuroscience, Max Planck Research School Göttingen, Germany & Karolinska Institute, Stockholm, Sweden Dr. rer. nat., Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry (Prof. Erwin Neher, Prof. Jakob Sørensen, Prof. Reinhard Jahn) Post-Doc, Center for Neurogenomics and Cognitive Research, Free University Amsterdam, The Netherlands (Prof. Matthijs Verhage) Post-Doc, Institute for Neuro science and Pharmacology, Copenhagen University, Denmark (Prof. Jakob Sørensen) Post-Doc, Department of Molecular Pharmacology and Cell Biology, FMP Berlin, Germany (Prof. Volker Haucke) Since 2015 Group Leader, FMP Berlin Emmy Noether fellow of the DFG SUMMARY We aim to understand the molecular mechanisms of synaptic transmission. This communication between the neurons of the brain forms the basis of survival, cognition, and behaviour. Synaptic dysfunction is linked to many neurological diseases. We are particularly interested in understanding how synaptic molecules function together to produce the complex features of neurotransmission, which is not only optimized for speed but which can also plastically adapt to evade interference or to store information. To do so we combine experimental and theoretical approaches. Our experimental system is the neuromuscular junction of the fruit fly Drosophila melanogaster, a powerful genetic model system. Despite its simplicity, human and Drosophila synaptic genes are remarkably similar and the majority of genes implicated in neurological disorders are conserved from flies to humans. We measure synaptic transmission by electrophysiology and live cell imaging. In addition, super-resolution imaging enables us to uniquely define the topology of the molecular machinery driving transmission on the nanometre scale. To arrive at a conceptual framework of how synaptic molecules enable, control, and adapt synaptic transmission, we generate mathematical models based on parameters derived from our experiments. These are used to generate hypotheses which are then tested experimentally. Iterating between experiments and theoretical work allows us to constantly refine our models and to increase their predictive value, furthering our understanding of molecular synapse function. ZUSAMMENFASSUNG Wir möchten die molekularen Mechanismen synaptischer Signalübertragung aufklären. Diese Kommunikation zwischen Nervenzellen ist Grundlage von Überleben, Kognition und Verhalten. Fehlerhafte Synapsenfunktionen sind der Grund vieler neurologischer Erkrankungen. Insbesondere versuchen wir zu verstehen, wie einzelne synaptische Moleküle gemeinsam komplexe Eigenschaften neuronaler Kommunikation bedingen. Diese Kooperation ist nicht nur auf unglaubliche Geschwindigkeiten ausgelegt, sondern passt sich auch plastisch an, um störenden Einflüssen entgegenzuwirken, aber auch um Informationen zu speichern. Wir untersuchen diese Eigenschaften mithilfe experimenteller und theoretischer Methoden an der neuromuskulären Endplatte der Fruchtfliege Drosophila melanogaster. Trotz seiner Einfachheit ist das Drosophila Genom hinsichtlich synaptischer Gene dem menschlichen sehr ähnlich. Um synaptische Signalübertragung messen zu können, führen wir elektrophysiologische Experimente und lebend-mikroskopie durch. Zudem nutzen wir hochauflösende Mikroskopie, um die molekulare Nanomaschinerie der synaptischen Kommunikation zu visualisieren. Um herauszufinden, wie die einzelnen Komponenten die Transmission kontrollieren und anpassen, konstruieren wir zudem mathematische Modelle auf Grundlage experimenteller Daten. Mit diesen stellen wir neue Hypothesen auf, die im Experiment getestet werden. Dieser Wechsel zwischen experimenteller und theoretischer Arbeit führt zu einer fortwährenden Verbesserung der Aussagekraft unserer Modelle, und treibt unser Verständnis molekularer Funktionen der Synapse voran.

49 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 47 female male Fig.1: Model system and methods used in the Walter lab We use the Drosophila melanogaster larval neuromuscular junction to investigate molecular mechanisms of synaptic transmission. Here the Drosophila life cycle is shown from mating adults to embryos, and on to three consecutive larval stages. Before the late third instar, when larvae crawl out of the food and pupate, they are collected for experiments using several existing techniques employed in the lab, ranging from optical methods such as confocal, super-resolution and functional imaging of synaptic transmission, to electrophysiological methods. Our experimentally obtained data are then used for computational assays to model and simulate synaptic mechanisms. (Modified from and adapted from Kononenko et al., 2014). pupa prepupa super-resolution Drosophila life cycle 3rd instar larva embryo 1st instar larva 2nd instar larva confocal analysis electrophysiology mutant ctrl synaptic transmission activity tracking local synapse activity DESCRIPTION OF PROJECTS Single synapse sub-architecture and its plastic modulation Neurotransmitters are released from a specialized sub-synaptic region known as the active zone (AZ), where evolutionarily conserved proteins orchestrate synaptic vesicle (SV) fusion. Ca 2+ channels open in response to action potential (AP)-induced depolarisation and trigger rapid, locally confined Ca 2+ elevations that activate the SV Ca 2+ sensor synaptotagmin to induce fusion. Owing to the steep Ca 2+ concentration gradient from the Ca 2+ source, its distance to the SV release site is a major determinant of synaptic efficacy. We are investigating which molecules generate release sites and which protein-protein interactions are required for their correct placement. Using live imaging and superresolution STED microscopy we have found that residence times and nanoscale distribution of AZ-proteins differ vastly. While some proteins specifically and very stably (on the scale of hours) localize to sub-az regions where SVs typically fuse, others show broad distribution and high fluctuation (on the scale of minutes). Strikingly, these characteristics are not static, but change with the functional status of the synapse. For instance, induction of synaptic plasticity that increases SV release greatly increases the localisation specificity and lifetime of proteins which are unstable and promiscuously localised when at rest. Identifying the molecular principles of this modulation and how they influence SV coupling distances to Ca 2+ channels is a subject of ongoing investigation. Characterising lipids as regulators of synaptic transmission While we possess considerable knowledge about how AZ proteins contribute to SV release, much less is known about membrane lipids. However, we do know that the key step in transmission concerns SV fusion, where SV and plasma membrane lipids merge. The phosphoinositide PI(4,5)P 2 is enriched at synapses, known to influence secretion, and directly interacts with proteins of the release machinery, such as synaptotagmin. This makes a direct function of PI(4,5)P 2 in SV fusion likely. Classical methods of altering PI(4,5)P 2 levels rely on long-lasting changes which may affect upstream reactions or change the levels of PI(4,5)P 2 metabolites. To investigate direct PI(4,5)P 2 function on the timescale relevant for synaptic transmission, we use caged PI(4,5)P 2 variants, generated by our collaborator Carsten Schultz from the EMBL in Heidelberg, which are loaded into the presynapse. An intense burst of UV light uncages PI(4,5)P 2 in the plasma membrane within milliseconds. Electrophysiological characterisation has revealed that increasing PI(4,5)P 2 rapidly augments synaptic transmission by increasing the likelihood of SV fusion, implying a direct action of PI(4,5)P 2. The exocytosis Syt-pHluorin FV fused vesicle m cooperativity mathematical modeling endocytosis exo- k CME k CIE molecular requirements on PI(4,5)P 2 binding proteins are currently being identified in the laboratory using Drosophila mutants. Mathematical Modeling of synaptic transmission Using theoretical methods, we seek to develop a realistic model of SV release at the synapse. As reactions at the synapse are typically governed by few SVs and Ca 2+ channels, deterministic approaches may fail to viably reproduce these processes. Our computational approach employs stochastic Ca 2+ channel gating and vesicle positioning based on experimentally determined distributions. We have found that current models fail to predict synaptic physiology when taking the actual spatial distribution of SVs into account as they predict rapid fatigue of transmission upon repetitive activation. We are currently exploring which molecular mechanisms ensure performance during continued activity. Molecular basis of synaptic heterogeneity Although core components driving SV fusion are conserved, AZ activity, even among AZs of the same neuron, is largely heterogeneous. This heterogeneity is the neural basis of information processing. We are currently investigating the molecular principles involved using live cell imaging, where synaptic transmission is visualized by postsynaptic expression of Ca 2+ -sensitive fluorescence reporters. As postsynaptic glutamate receptors of the fly neuromuscular junction (NMJ) are permeable to Ca 2+, this allows for the tracking of AZ activity with high temporal and spatial resolution. As a developmental system, the larval NMJ contains a mixture of AZs in various states of maturation. By comparing the activity of single AZs with their molecular composition we identified proteins that locally set the functional status. Identifying local regulators of synaptic transmission will allow us to understand how developmental trajectories generate functional AZs and optimize transmission. F/F FV FV m 0 clathrin k CME clathrin dependent rate k CIE clathrin independent rate endocytosis experiment simulation Time [s]

50 48 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 2: Sketch of neurotransmitter release promoted by Unc13A and -B Bruchpilot (BRP; green) and RBP (red) localize Unc13A at a distance of 70 nm from the Ca 2+ source (Cac; blue). Unc13B is localized at a larger distance of 120 nm. The color transition from dark to light blue indicates the different Ca 2+ concentrations the vesicles sense. This precise vesicle positioning is essential to control synaptic transmission. GROUP MEMBERS Dr. Mathias Böhme Andreas Till Grasskamp (doctoral student) Anthony McCarthy (doctoral student) Meida Jusyte (student) Sabine Hahn (technical assistant) Gabriela Pimenta Dos Reis (research intern) Staff employed within the reporting period COLLABORATIONS Carsten Schulz, EMBL Heidelberg, Heidelberg, Germany Synthesis and analysis of signalling lipids André Nadler, MPI for Mol. Cell Biology and Genetics, Dresden, Germany / Investigating the role of signalling lipids in exocytosis Stephan Sigrist, Free University Berlin, Berlin, Germany / Neurogenetics and ultrastructural analysis of the synapse Stefan Hell, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany / Superresolution (STED) microscopy Susanne Ditlevsen, University of Copen hagen, Copenhagen, Denmark Mathematical modeling of synaptic transmission Jakob Sørensen, University of Copenhagen, Copenhagen, Denmark / Investigating the molecular mechanisms of neurosecretion Matthijs Verhage, Free University of Amsterdam, Amsterdam, The Netherlands / Mathematical modeling of synaptic transmission Nils Brose, Max Planck Institute for Experimental Medicine, Göttingen, Germany / Mathematical modeling of neurosecretion SELECTED PUBLICATIONS Bohme M A, Beis C, Reddy-Alla S, Reynolds E, Mampell M M, Grasskamp A T, Lutzkendorf J, Bergeron D D, Driller J H, Babikir H, Gottfert F, Robinson I M, O'Kane C J, Hell S W, Wahl M C, Stelzl U, Loll B, Walter A M *, Sigrist S J * (2016) Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling. Nat Neurosci. 19, *: equally contributing senior author Muhammad K, Reddy-Alla S, Driller J H, Schreiner D, Rey U, Bohme M A, Hollmann C, Ramesh N, Depner H, Lutzkendorf J, Matkovic T, Gotz T, Bergeron D D, Schmoranzer J, Goettfert F, Holt M, Wahl M C, Hell S W, Scheiffele P, Walter A M, Loll B, Sigrist S J (2015) Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function. Nat Commun. 6, Schotten S *, Meijer A *, Walter A M *, Huson V, Mamer L, Kalogreades L, ter Veer M, Ruiter M, Brose N, Rosenmund C, Sorensen J B, Verhage M, Cornelisse L N (2015) Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate. elife 4, e *: equally contributing first author Walter A M, Kurps J, de Wit H, Schoning S, Toft-Bertelsen T L, Lauks J, Ziomkiewicz I, Weiss A N, Schulz A, Fischer von Mollard G, et al. (2014). The SNARE protein vti1a functions in dense-core vesicle biogenesis. The EMBO Journal 33, Walter A M *, Pinheiro P S, Verhage M, Sorensen J B (2013). A sequential vesicle pool model with a single release sensor and a ca(2+)-dependent priming catalyst effectively explains ca(2+)-dependent properties of neurosecretion. PLoS Comp Biol 9, e *: corresponding author EXTERNAL FUNDING Deutsche Forschungsgemeinschaft, TRR 186 Molecular switches in the spatio-temporal regulation of cellular signal transduction, , Deutsche Forschungsgemeinschaft, Emmy Noether funding, Investigating how the active zone cytomatrix orchestrates neuronal exo- and endocytosis, , FMP authors Group members

51 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 49 CORE FACILITY CELLULAR IMAGING ZELLULÄRE BILDGEBUNG GROUP LEADERS DR. BURKHARD WIESNER LIGHT MICROSCOPY DR. DMYTRO PUCHKOV ELECTRON MICROSCOPY SUMMARY Since 2006 our group has functioned as a core facility. To provide optimal support, our facility is split into two sub-units that focus on light 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 fluorescence imaging technology, including labeling and analysis expertise for studying biological samples including living and fixed cells, small organisms, tissue, and solutions. Our central role is to establish single cell techniques and apply diverse microscopic methods to describe intracellular transport and signal transduction processes. Established microscopic methods include widefield, confocal, and super-resolution imaging. Techniques such as FRET (Fluorescence Resonance Energy Transfer), FRAP (Fluorescence Recovery After Photobleaching), FLIM (Fluorescence Lifetime Imaging Microscopy), TIRFM (Total Internal Reflection Fluorescence Microscopy), FCS (Fluorescence Correlation Spectroscopy), as well as Ca 2+ measurements and caged compounds, are well established in our group. Furthermore, we assist with image analysis and develop novel algorithms for biophysical data analysis. 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. Immunogold labeling, correlative light and electron microscopy (CLEM), and tomographical 3D reconstruction can then be applied in projects showing promising phenotypes in preliminary screening experiments. In addition to our main focus on cell biology, we assist with the visualization of in vitro structures such as proteins, fibril structures, and liposomes, either as cryosamples or by using negative staining. ZUSAMMENFASSUNG Seit 2006 fungiert unsere Gruppe als Technologieplattform des fmp und ist zur Bereitstellung einer besseren technischen Unterstützung in die beiden Service-Gruppen Licht-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 Publikationen mit den Forschungsgruppen führen. Lichtmikroskopie Die Lichtmikroskopie-Gruppe unterstützt alle Forschungsgruppen des FMP mit ihren bildgebenden Fluoreszenz-Technologien, ihrer Kompetenz bei der optischen Markierung sowie Datenanalyse beim Studium biologischer Proben, einschließlich lebender und fixierter Zellen, kleinen Organismen, Geweben und Lösungen. Unsere zentrale Rolle dabei ist es, Einzelzelltechniken zu etablieren und verschiedene mikroskopische Methoden anzuwenden um intrazelluläre Transportprozesse sowie die zelluläre Signalübertragung untersuchen

52 50 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Three-color dstorm image of COS-7 cells immunolabeled for microtubules (CF568), f-actin (phalloidin CF647) and clathrin (CF680), scale bar 3 µm. und beschreiben zu können. Zu den etablierten bildgebenden Methoden gehören die Weitfeld-, konfokale und hochauflösende Mikroskopie. FRET (Fluoreszenz Resonanz Energie Übertragung), FRAP (Fluoreszenz-Rückgewinnung nach Photobleichung), FLIM (Fluoreszenz-Lebensdauer Mikroskopie), TIRFM (Totale Interne Reflexion Fluoreszenz-Mikroskopie), FCS (Fluoreszenz- Korrelation-Spektroskopie) sowie Ca 2+ -Messungen und das Arbeiten mit caged Coumpounds (Verbindungen, die durch das Einbringen einer sogenannten Schutzgruppe, Käfig, biologisch inaktiv sind) sind ebenfalls gut etablierte Methoden in unserer Gruppe. Darüber hinaus geben wir Unterstützung bei der Bildanalyse und entwickeln neuartige Algorithmen für die biophysikalische Datenanalyse. 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. Immunogold-Markierungen, Korrelative Licht- und Elektronenmikroskopie (KLEM), tomographische 3D-Rekonstruktionen werden bei Projekten mit vielversprechenden Phenotypen angewendet. 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. DESCRIPTION OF PROJECTS The light microscopy core facility In neuronal synapses and in clathrin-mediated endocytosis (CME), membranes and proteins are organized on the nanometer scale, which is below the diffraction limit of light (< 250 nm). Recently developed super-resolution microscopy techniques like STED and STORM overcome this diffraction barrier and achieve molecular resolutions down to 20 nm. In collaboration with Jan Schmoranzer (AG Haucke) we screened more than 30 organic fluorophors and optimized imaging conditions to establish robust multicolor STORM and STED imaging. We used multicolor STORM to visualize the nanoscale organisation of CME proteins, the actin cytoskeleton, intracellular organelles, and synaptic proteins in mouse brain slices. In collaboration with Volker Haucke and Frank Noe (FU Berlin) we combined multicolor STORM imaging with mathematical modeling, electron microscopy, and quantitative live cell imaging to investigate the role of the BAR protein SNX9 in the late stages of CME. A variety of collaborators make use of our multicolor STED setup to investigate the molecular organisation of tight junctions (AG Blasig), Golgi transport (AG Krauss), neuronal synapses (AG Haucke), and active zone proteins in the fly neuromuscular junction (AG Sigrist, FU Berlin). With Dmytro Puchkov we are currently establishing correlative light and electron microscopy for cells and tissue samples. The heptahelical G protein-coupled receptors (GPCRs) are important drug targets. Following activation by their ligands, they 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. It is known that for some GPCRs 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 fluorescence resonance energy transfer (FRET), fluorescence-lifetime imaging microscopy (FLIM) and fluorescence cross-correlation spectroscopy (FCCS). The electron microscopy core facility The major interest of the group is the regulation of intracellular trafficking that underlies or governs the majority of physiological functions in eukaryotic cells. In cooperation with the groups of Volker Haucke (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, and Stonin 2, scaffolding and accessory proteins such as clathrin and intersectin, and different phosphatidylinositol kinases and phosphatases, to endocytosis, endosomal sorting and organelle maturation. We conduct these investigations in cells and, in particular, in neuronal synapses. In cooperation with Thomas Jentsch (Physiology and Pathology of Ion Transport, FMP) we investigate 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 Martin Lehmann (light microscopy) we develop correlative light and electron microscopy approaches for understanding organelle dynamic identity. In addition to our work on cell biological projects, we provide support with negative staining for groups studying protein structures.

53 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 51 Epoxy resin section of Mitral cell and perisomatic reciprocal synapses from olfactory bulbs of KCC2 knockout mice (AG Jentsch). GROUP MEMBERS Light Microscopy Dr. Martin Lehmann Jenny Eichhorst (technical assistant) Electron Microscopy Svea Hohensee (research assistant) Martina Ringling (technical assistant) Staff employed within the reporting period COLLABORATIONS International Thomas Walther, Hull University, Hull, UK Pavel Nedvetsky, Catholic University Leuven, Leuven, Belgium National Dorothea Eisenhardt, Freie Universität Berlin Stephan Sigrist, Frei Universität Berlin Karin Müller, Leibniz Institute for Zoo and Wildlife Research, Berlin Alexander Wenig, Charité Universitätsmedizin Berlin Maria Maares, Technische Universität Berlin Max-Delbrück Center for Molecular Medicine, Berlin: Enno Klussmann, Dominique Müller Matthew Poy, Bettina Purfürst, Anje Sporbert, Alessandro Prigione, Thomas Wiglenda Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP): Ingolf Blasig, Margitta Dathe, Volker Haucke, Thomas Jentsch, Gerd Krause, Michael Krauß, Ronald Kühne, Tanja Maritzen, Jan Schmoranzer, Ralf Schülein SELECTED PUBLICATIONS Opitz R, Müller M, Reuter C, Barone M, Soicke A, Roske Y, Piotukh K, Huy P, Beerbaum M, Wiesner B, Beyermann M, Schmieder P, Freund C, Volkmer R, Oschkinat H, Schmalz HG, Kühne R (2015) A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions. Proc Natl Acad Sci U S A Ketel K, Krauss M, Nicot A S, Puchkov D, Wieffer M, Müller R, Subramanian D, Schultz C, Laporte J, Haucke V (2016) A phosphoinositide conversion mechanism for exit from endosomes. Nature 529, Herrada I, Samson C, Velours C, Renault L, Östlund C, Chervy P, Puchkov D, Worman H J, Buendia B, Zinn-Justin S (2015) Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Selfassembly Properties. ACS Chem Biol. 10, Lehmann M, Lichtner G, Klenz H, Schmoranzer J (2016) Novel organic dyes for multicolor localization-based super-resolution microscopy. J Biophotonics. 9, Schröter F, Jakop U, Teichmann A, Haralampiev I, Tannert A, Wiesner B, Müller P, Müller K (2016) Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques. Eur Biophys J. 45, FMP authors Group members

54 52 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 CORE FACILITY ANIMAL FACILITY TIERHALTUNG GROUP LEADER DR. NATALI KRISTINA WISBRUN BIOGRAPHY Until 1999 Studied Veterinary Medicine, University Zagreb, Croatia and Free University of Berlin 1999 Veterinary degree, Faculty of Veterinary Medicine, Free University of Berlin 2006 Dissertation, Cloning and Expression of Enzymes involved in the salvage pathway of L-fucose 2009 Graduate in Veterinary Medicine, specializing in laboratory animal science PROFESSIONAL BACKGROUND Since 2013 Group leader and animal welfare officer, FMP Berlin Head of animal experimental facilities, Philipp University of Marburg, Marburg SUMMARY The animal facility manages the housing and breeding of laboratory animals for use in scientific projects. Animal welfare legislation and the highest scientific standards are enforced to guarantee reliable, ethically obtained scientific results. We support and give advice to scientists on all matters regarding planning and performing experiments involving animals. We also provide practical support to FMP scientists, for instance by taking samples and in keeping proper documentation on their behalf. Furthermore, we organize the export and import of laboratory animals and 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 Head of an animal experimental facility and Animal Welfare Officer, Max Delbrück Center for Molecular Medicine, Berlin Buch Research Fellow and animal welfare officer, Central Animal Laboratory of the University Clinic Essen, Essen Research Fellow, Institute for Biochemistry and Molecular biology, Charité-Universitätsmedizin Berlin GROUP MEMBERS Dr. Nadja Heinrich (Animal Welfare Officer) Nadja Daberkow-Nitsche (veterinarian) Eva Lojek (animal keeper) Sina Scholz (animal keeper) Jannette Unnasch (animal keeper) Elisabeth Lettau (studentische Hilfskraft / student of molecular bioscience) Marco Walther (studentische Hilfskraft / student of biology) Staff employed within the reporting period COLLABORATIONS National Animal Facility Max-Delbrück-Center for Molecular Medicine, Berlin Animal Facilities Charité, Berlin AG Blasig AG Haucke AG Jentsch AG Maritzen AG Plested AG Schröder

55 MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 53 DESCRIPTION OF PROJECTS We provide FMP scientists with animal welfare officers who ensure that experiments using animals are in compliance with the Animal Welfare Act. The facility supplies animal housing and manages all genetically modified mice, frogs, and rats, including overseeing performance standards, veterinary care, qualified animal care staff, and the use of standard operating procedures and their related documentation. Our service also includes the education and training of personnel responsible for animal care and of personnel carrying out experimental procedures (research technicians), along with all scientists involved in experimental trials using animals. Our aim is to establish a carefully designed, well-constructed, and properly maintained laboratory animal facility that manages its responsibilities for animal care in compliance with all applicable laws and regulations. INTERESTS AND FOCUS The Animal Facility oversees all research at the FMP that involves animals, including: Managing the breeding of a variety of genetically modified mice, frogs, and rats in compliance with animal welfare regulations. Organizing veterinary care and health monitoring in accordance with FELASA guidelines. Implementing European guidelines on animal welfare in high-quality research. Organizing imports and exports of animals, embryos, stem cells, and related materials. Consulting for scientists who are designing experiments that use animals. Supporting scientists in implementing experiments using animals. SELECTED PUBLICATIONS Pohlmann A, Karczewski P, Ku M C, Dieringe B, Waiczies H, Wisbrun N, Kox S, Palatnik I, Reimann H M, 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, NMR Biomed. 27, FMP authors Group members

56 Molecular Biophysics Molekulare Biophysik Group leader Prof. Dr. Adam Lange PAGE 60 NMR-Supported Structural Biology NMR-Unterstützte Strukturbiologie Group leader Prof. Dr. Hartmut Oschkinat PAGE 64 Structural Bioinformatics and Protein Design Struktur-Basierte Bioinformatik und Proteindesign Group leader Dr. Gerd Krause PAGE 74 Computational Chemistry / Drug Design Wirkstoff-Design Group leader Dr. Ronald Kühne PAGE 71 Solution NMR Lösungs-NMR Group leader Dr. Peter Schmieder PAGE 68 Molecular Imaging Molekulare Bildgebung Group leader Dr. Leif Schröder PAGE 79

57 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE SECTION STRUCTURAL BIOLOGY BEREICH STRUKTURBIOLOGIE In-Cell NMR NMR in Zellen Group leader Dr. Philipp Selenko PAGE 77 NMR Group leaders Prof. Dr. Hartmut Oschkinat Dr. Peter Schmieder PAGE 82

58 56 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 SECTION STRUCTURAL BIOLOGY BEREICH STRUKTURBIOLOGIE Molecular pharmacology requires three-dimensional representations of supramolecular 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 offers a strong incentive to employ Nuclear Magnetic Resonance (NMR) spectroscopy. With this in mind, the Structural Biology section develops and applies solution and solid-state NMR techniques to investigate pharmacologically relevant proteins in their native environments, or even in intact biological systems such as cells or functional modules. Beyond this, the insights gained into molecular interactions are used to develop NMR reporters for diagnostic imaging purposes where fluorescence detection fails. For example, the departments of Adam Lange and Hartmut Oschkinat apply solid-state NMR to membrane proteins in native lipid bilayers, Philipp Selenko detects proteins and their modifications in live cells by solution NMR, and Leif Schröder images cells or organisms by means of molecule-specific contrast agents. In concert with molecular modeling and structure-function studies provided by the groups of Gerd Krause and Ronald Kühne, atomic-resolution structural data are derived that are indispensable to potential pharmacological interference, a process 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 this section. The department of Adam Lange uses solid-state NMR methods in combination with complementary techniques, such as scanning transmission electron microscopy (STEM) and cryo-em, to investigate protein structure and dynamics. One focus of the group is on bacterial supramolecular assemblies involved in infection processes. Recent examples include the Shigella type-iii secretion needle, the type-i pilus of uropathogenic E. coli, and cytoskeletal bactofilin filaments. Furthermore, the group is interested in solid-state NMR methods development. In the department of Hartmut Oschkinat, the application of microwave-based hyperpolarisation (DNP) has recently offered a picture of the nascent polypeptide chain growing inside the ribosome. Further applications of the technique yielded well-resolved spectra of proteins at temperatures around 200 K. The Solution NMR group of Peter Schmieder continued to investigate protein dynamics in MHC complexes to obtain information that will help to explain the interactions between T-cell receptors and MHC complexes. In addition, several projects seeking to clarify the constitution of small molecules (natural products or products of chemical synthesis) were initiated. The In-cell NMR junior group headed by Philipp Selenko explores novel NMR-based methodologies that allow monitoring of proteins inside cells. Their projects include profiling kinase activities via peptide-based Kinase Activity Reporters (KARs), which allow dynamic changes of cellular kinase activities to be measured under a variety of physiological and pathophysiological conditions. Investigation of the structure of α-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 magnetic resonance imaging (MRI) as a major technique for exploring the possibility of a biosensor-based NMR imaging approach, employing hyperpolarisation to achieve unprecedented sensitivity. In the course of this work, Leif Schröder and his colleagues devised a highly efficient combination of hyperpolarization and controlled depolarization of xenon, which is then used to detect membrane-embedded and receptorattached probes with hyperpolarised nuclei. In this way, enhanced MRI can detect specific cell markers, raising the possibility of tissue- or molecule-specific contrasting that could be used for non-invasive detection of various diseases at an early stage. 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 the impressive task of developing a set of smallmolecule 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 tested the compounds developed in their biological laboratories, thereby closing the gap between structural studies and computational modeling, as well as biochemical and cell physiological analyses. With the arrival of the group of Dorothea Fiedler and the new department of Chemical Biology I, the demand for solution-state NMR support has grown considerably and the services provided by the NMR spectroscopy core facility have been extended. This has also lead to an increase in the collaborations between the group of Peter Schmieder and the Chemical Biology section. In der molekularen Pharmakologie ist die Kenntnis der drei-dimensionalen Struktur supramolekularer 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

59 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 57 Veniamin Chevelkov Jonas Protze, Katrin M. Hinz (in front) and students 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. Darüber hinaus werden die Erkenntnisse über molekulare Wechselwirkungen eingesetzt, um daraus NMR-Reporter für solche diagnostische Bildgebungszwecke zu entwickeln, bei denen Fluoreszenz-Bildgebung nicht anwendbar ist. Die Abteilungen von Hartmut Oschkinat und von Adam Lange nutzen beispielsweise Festkörper-NMR für Membranproteine in nativen Lipidmembranen, und Philipp Selenko detektiert Proteine und Proteinmodifikationen in lebenden Zellen. Leif Schröder arbeitet an der Bildgebung von 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 pharmakologischen Einflussnahme auf Zielproteine zu finden. Diese Untersuchungen werden gewöhnlich von der Lösungs-NMR - Gruppe unter der Leitung von Peter Schmieder unterstützt. Hinsichtlich des Designs von biologisch aktiven Wirkstoffen ist insbesondere die Entwicklung von Inhibitoren von Protein-Wechselwirkungen ein Leitthema des Bereichs. Die Abteilung von Adam Lange verwendet Festkörper-NMR Methoden in Kombination mit komplementären Techniken wie der Scanning Transmission Electron Microscopy (STEM) und der Cryo-EM um die Struktur und Dynamik von Proteinen zu untersuchen. Ein Schwerpunkt der Forschung der Arbeitsgruppe liegt auf bakteriellen supramolekularen Strukturen, z. B. Typ-3 Sekretionsnadeln, dem Typ I-Pilus und Bactofilinfilamenten, einem Bestandteil des bakteriellen Zytoskeletts. Gleichzeitig beschäftigt die Gruppe sich mit der Weiterentwicklung von Festkörper-NMR-Methoden. Durch die Abteilung von Hartmut Oschkinat wurde kürzlich die Struktur der naszierenden Polypeptidkette innerhalb des Ribosoms aufgeklärt. Dies geschah mittels Mikrowellen-gestützter Hyperpolarisation (DNP). Weitere Anwendungen dieser Technik erbrachten gutaufgelöste Spektren von Proteinen bei Temperaturen um 200 K. Die Gruppe Lösungs-NMR von Peter Schmieder hat ihre Arbeiten zur Untersuchung der Dynamik in MHC-Komplexen fortgesetzt, um Informationen zu erhalten, die helfen werden die Wechselwirkung zwischen T-Zell-Rezeptoren und MHC-Komplexen besser zu verstehen. Daneben wurden mehrere Projekte begonnen, die die Konstitutionsbestimmung kleiner Moleküle (Naturstoff oder Syntheseprodukte) zum Ziel haben. Die Nachwuchsgruppe NMR in Zellen von Philipp 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 Kinaseaktivitäts-

60 58 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Reportern (KARs). Diese ermöglichen es, dynamische Änderungen zellulärer Kinaseaktivitäten unter verschiedenen physiologischen und pathophysiologischen Bedingungen zu messen. Ein weiteres Projekt, die Untersuchung der Struktur von α-synuklein, zielt auf ein molekulares Verständnis der Parkinson-Krankheit. Die Nachwuchsgruppe Molekulare Bildgebung von Leif Schröder nutzt die Magnetresonanztomographie (MRT) zur Bildgebung auf der physiologischen Ebene. Die Arbeitsgruppe entwickelt einen neuartigen, auf Biosensoren beruhenden Ansatz zur Bildgebung, der Hyperpolarisation nutzt um eine bislang unerreichte Sensitivität zu erzielen. Es gelang den Wissenschaftlern, eine sehr effiziente Kombination aus Hyperpolarisation und gezielter Depolarisation von Xenon zu entwickeln, die dann benutzt wird, um membranständige oder Rezeptor-gebundene Sondenmoleküle mit hyperpolarisierten Atomkernen zu detektieren. So gelang es durch signalverstärkte MRT, spezifische Zellmarker darzustellen. Damit steht der Weg für eine Weiterentwicklung in Richtung gewebe- oder molekülspezifischer Kontrastmittel offen, die zum Beispiel für eine nicht-invasive Früherkennung von verschiedenen Krankheiten eingesetzt werden könnten. Die Cheminformatik- / Bioinformatik-Gruppen nutzen Strukturdaten um Inhibitoren für Proteinwechselwirkungen abzuleiten und um Proteinfunktionen, insbesondere von G-Proteingekoppelten 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 Prolin-reichen 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. Durch die Etablierung der Gruppe von Dorothea Fiedler als Abteilung Chemische Biologie I hat die Nachfrage nach Lösungs-NMR-Unterstützung stark zugenommen und der von der Core Facility NMR-Spektroskopie angebotene NMR-Service wurde ausgedehnt. Das hat auch zu einer verstärkten Zusammenarbeit der Gruppe von Peter Schmieder mit dem Bereich Chemische Biologie geführt. Matthias Schnurr, Honor Rose and Jabadurai Jayapaul (photo left), Marleen van Rossum and Stamatios Liokatis (photo right) Chaowei Shi and Songhwan Hwang

61 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 59 Hafiza Nayab and Michael Lisurek (photo above), Peter Schmieder and Monika Beerbaum (photo left), Martin Ballaschk and Anne Diehl (photo right)

62 60 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 MOLECULAR BIOPHYSICS MOLEKULARE BIOPHYSIK GROUP LEADER PROF. DR. ADAM LANGE BIOGRAPHY 10 / / 2002 Studied Physics, Georg-August-University, Göttingen 11 / / 2006 Ph.D. / Postdoctoral studies, Max Planck Institute for Biophysical Chemistry, Göttingen 10 / / 2004 Research visit, National Institutes of Health, Bethesda, USA 2006 Ernst Award of the German Chemical Society 2006 Otto-Hahn Medal of the Max Planck Society 10 / / 2008 Postdoctoral fellow, Laboratory of Physical Chemistry, ETH Zürich, Switzerland, EMBO long-term fellowship 09 / / 2014 Independent Group Leader, Max Planck Institute for Biophysical Chemistry, Göttingen, Emmy Noether fellowship 2013 ERC Starting Grant 3D structures of bacterial supramolecular assemblies by solid-state NMR" Since 04 / 2014 Department Head, FMP, Berlin and W3-S Professor, Humboldt University of Berlin 2016 ICMRBS Founders Medal SUMMARY We study protein structure and dynamics using nuclear magnetic resonance in the solid state (solid-state NMR) and a variety of other biophysical methods. 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 stronger than the earth s magnetic field) and spun rapidly (up to 100,000 rotations per second). Spinning around an axis that is inclined to the magnetic field by a magic angle of 54.7 emulates the conditions of fast and freely tumbling molecules in solution. By means of magic-angle spinning, NMR spectra with high resolution and sensitivity can be achieved for solid-state protein samples. A focus of our group is bacterial supramolecular protein assemblies including T3SS needles, the type I pilus, and cytoskeletal bactofilin filaments. Furthermore, we are interested in solid-state NMR methods development. ZUSAMMENFASSUNG Wir untersuchen mittels Festkörper-Kernspinresonanz (Festkörper-NMR) und anderen biophysikalischen Methoden die Struktur und Dynamik von Proteinen. In den vergangenen zehn Jahren hat sich die Festkörper-NMR zu einer wichtigen Technik in der Strukturbiologie entwickelt, die Zugang zu Strukturinformationen an solchen Systemen ermöglicht, die unlöslich oder nicht zu kristallisieren sind. Membranproteine in ihrer Lipidbilayer- Umgebung beispielsweise oder supramolekulare Strukturen wie die Nadel des Typ-3- Sekretionssystems (T3SS), die aus vielen Kopien eines einzelnen kleinen Proteins aufgebaut ist, 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 (bis zu Umdrehungen pro Sekunde), was die Situation von sich frei und schnell bewegenden Molekülen in Lösung simuliert. Die Rotationsachse der Probe ist gegenüber dem Magnetfeld um den magischen Winkel von 54,7 gekippt. Dieses sogenannte magic-angle spinning führt zu hoher Auflösung und Sensitivität der NMR-Spektren von Proteinen im Festkörper. Ein Schwerpunkt der Forschung unserer Arbeitsgruppe liegt auf bakteriellen supramolekularen Strukturen, z. B. T3SS-Nadeln, dem Typ I-Pilus und Bactofilinfilamenten, einem Bestandteil des bakteriellen Zytoskeletts. Gleichzeitig beschäftigen wir uns mit der Weiterentwicklung von Festkörper-NMR-Methoden.

63 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 61 DESCRIPTION OF PROJECTS 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. Once injected, 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 it is the 80-residue protein PrgI. We have previously shown 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 modeling. 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 through the needle in an unfolded state and then 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 have 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 subunits per two turns, similar to the arrangement observed in the related flagellar filament. The structure of bactofilin, an element of the bacterial cytoskeleton For the study of the cytoskeletal bactofilin protein BacA from Caulobacter crescentus, a uniformly 13 C- and 15 N-labeled sample was prepared and investigated by solid-state NMR spectroscopy. From the resulting NMR spectra we were able to determine essentially complete resonance assignments. Additional information was provided by STEM measurements that yielded a restraint for the mass-per-length of the assembly. In combination with homology modeling, a β-helical fold with six windings per subunit was proposed [Vasa et al., PNAS, 2015]. A β-helical fold such as this has never been observed for any other cytoskeletal filament. Subsequently, we determined a high-resolution solid-state NMR structure of BacA [Shi et al., Science Advances, 2015]. In this work, which was based on numerous distance restraints from sparsely 13 C-labeled samples and 4D HN-HN data from a deuterated sample, we were able to confirm and refine our initial model and determine unambiguously that the β-helix is right-handed. The structure of the type I pilus of uropathogenic E. coli Using solid-state NMR we also studied uniformly and sparsely labeled samples of polymerized FimA, the main subunit of the type I pilus of uropathogenic E. coli. As with the bactofilin study, we obtained complementary STEM data. Based on the combination of solid-state NMR data, STEM data, and a solution NMR structure of a soluble FimA construct (called FimAa) we determined a hybrid model of the FimA assembly [Habenstein et al., Angewandte Chemie International Edition, 2015]. Solid-state NMR method development The focus of our solid-state NMR method development work 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 them to deuterated T3SS needles [Chevelkov et al., J Magn Reson, 2014]. By means of band-selective homonuclear cross polarization (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].

64 62 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Some of the methods that we developed and applied for the determination of the BacA structure [Shi et al., Science Advances, 2015] are relatively difficult to perform. In order to facilitate the implementation of these methods, namely a set of experiments for protein resonance assignment based on proton-detected solid-state NMR on deuterated samples, we developed a user-friendly protocol. This protocol was recently accepted for publication [Fricke et al., Nature Protocols, in press]. Menschl. Zelle Bakterielle Proteine T3SS NADEL Basis 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 microscopy and computer modeling [Loquet et al., Acc Chem Res, 2013]. Bakt. Zelle Bakterielle Proteine GROUP MEMBERS Adam Lange (PI) Dr. Veniamin Chevelkov Dr. Matthias Herrera Glomm Dr. Oxana Krylova Dr. Sascha Lange Dr. Stamatios Liokatis Dr. Chaowei Shi Dr. Yong-hui Zhang Claudia Bohg (Student) Jean-Philippe Demers (Ph.D. Student) Pascal Fricke (Ph.D. Student) Songhwan Hwang (Ph.D. Student) Kitty Hendriks (Ph.D. Student) Eve Ousby (Ph.D. Student) Maximilian Zinke (Ph.D. Student) Dagmar Michl (Technical Assistant) Susanne Wojtke (Technical Assistant) Stefanie Schneider (Secretary) COLLABORATIONS International David Baker, University of Washington, Seattle, USA Nikolaos Sgourakis, UC Santa Cruz, USA Yusuke Nishiyama, RIKEN, Kobe, Japan Sophie Zinn-Justin, CEA, Gif-Sur-Yvette, France Changlin Tian, University of Science and Technology of China, Hefei, China National Martin Thanbichler, Philipps-Universität Marburg Stefan Becker, Max Planck Institute for Biophysical Chemistry, Göttingen Michael Kolbe, Helmholtz Centre for Infection Research, Hamburg Michael Habeck, Max Planck Institute for Biophysical Chemistry, Göttingen Staff employed within the reporting period

65 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 63 A C B D E 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]. F SELECTED PUBLICATIONS Vasa S, Lin L, Shi C, Habenstein B, Riedel D, Kühn J, Thanbichler M, and Lange A (2015) β-helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR. Proceedings of the National Academy of Sciences, 112, E127 E136. Habenstein B, Loquet A, Hwang S, Giller K, Vasa SK, Becker S, Habeck M, and Lange A (2015) Hybrid structure of the type I pilus of uropathogenic Escherichia coli. Angewandte Chemie-International Edition, 54, Shi C, Fricke P, Lin L, Chevelkov V, Wegstroth M, Giller K, Becker S, Thanbichler M, and Lange A (2015) Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR. Science Advances 1, e Fricke P, Mance D, Chevelkov V, Giller K, Becker S, Baldus M, and Lange A (2016) High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles. Journal of Biomolecular NMR 65, Fricke P, Chevelkov V, Zinke M, Giller K, Becker S, and Lange A, Backbone assignment of perdeuterated proteins by solid-state NMR using proton-detection and ultrafast magic-angle spinning. Nature Protocols, in press. EXTERNAL FUNDING Emmy Noether Grant (DFG) Solid-state NMR characterization of tau in paired helical filaments and bound to microtubules as well as of toxic and non-toxic oligomers of a-synuclein and tau. (DFG GZ.: LA 2705 / 1-1); Duration: 10 / / 2015; 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: DFG Eigene Stelle (Dr. Stamatios Liokatis) Post-translationale Protein Modifikationen, Querregulierung und Auswirkungen auf Histone H3 Tail Struktur und Dynamik: Eine strukturelle und mechanistische Studie an intakten Nukleosomen mittels hochauflösender Kernresonanzspektroskopie (DFG GZ.: LI 2402 / 2-2); Duration: 04 / / 2019; Total amount: Kekulé Ph.D. Scholarship to Pascal Fricke Alexander-von-Humboldt Fellowship to Dr. Yong-hui Zhang (former Postdoc) FMP authors Group members

66 64 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 NMR-SUPPORTED STRUCTURAL BIOLOGY NMR-UNTERSTÜTZTE STRUKTURBIOLOGIE GROUP LEADER PROF. DR. HARTMUT OSCHKINAT BIOGRAPHY Chemistry degree, University of Frankfurt Chemistry Diploma, University of Frankfurt Visit to the laboratory of Prof. Dr. Ray Freeman, Oxford, England Completion of dissertation in Prof. Kessler's Laboratory, 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, University of Lausanne, Switzerland NMR-spectroscopist, Max-Planck-Institute for Biochemistry, Martinsried, Germany, first in the Clore / Gronenborn group and later independently in the department of Prof. Huber 1992 Habilitation in Biophysical Chemistry, Technical University of Munich Group leader, EMBL, Heidelberg Since 1998 Head of the department NMR-supported Structural Biology, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Professor of Structural Chemistry, Free University of Berlin 1998 Elected member of the European Molecular Biology Organization (EMBO) 2013 Elected Member of National Magnetic Resonance Society of India 2014 Günther Laukien Prize SUMMARY Magic-angle-spinning (MAS) solid state NMR provides high-resolution structural information on complex samples, independent of their molecular weight and without the need for crystallization. It is an attractive method for structural investigations of difficult systems such as proteins embedded in lipid bilayers or attached to the cytoskeleton. In the long run, we aim to carry out structural investigations within the real space of a cell, capitalizing on a fold increase in the signal-to-noise ratio afforded by the use of dynamic nuclear polarization (DNP). For this purpose, we have been improving and testing DNP methods on biological samples and have used this methodology to investigate the nascent chain emerging from the ribosome as well as we refinal esomerisation in channelrhodopsin. In addition, we study membrane proteins in native (or native-like) lipid environments, with a focus on bacterial membrane proteins such as OmpG and YadA. A new research area of ours involves high-frequency magicangle spinning for studying biological samples. At high MAS frequencies, high-resolution proton spectra can be obtained using a minimal amount of sample. We have been systematically optimizing conditions and performing pilot studies on various proteins (SH3 domain, ABC transporter, bacterial biofilms). Finally, we investigate protein systems involved in protein homeostasis, including small heat shock proteins. ZUSAMMENFASSUNG Die Magic-Angle-Spinning (MAS)-Festkörper-NMR-Spektroskopie ermöglicht die Bestimmung der dreidimensionalen Struktur von Proteinen in komplexen Proben mit atomarer Auflösung, unabhängig vom Molekulargewicht der Biomoleküle und ohne vorherige Kristallisation. Sie stellt damit eine attraktive Methode zur Bearbeitung schwierig zu untersuchender Systeme dar. Dazu gehören beispielsweise Proteine, die in Lipid-Doppelschichten eingebaut oder mit dem Zytoskelett verbunden sind. Unser langfristiges Ziel ist es, Strukturuntersuchungen innerhalb von Zellen oder Organellen durchzuführen, was insbesondere durch die Anwendung der dynamischen Kernpolarisation (dynamic nuclear polarization, DNP) und der damit verbundenen fachen Verbesserung des Signal-Rausch-Verhältnisses ermöglicht wird. Zu diesem Zweck haben wir DNP-Methoden an biologischen Proben getestet, optimiert und diese Methodik zur Untersuchung von am Ribosom wachsenden Proteinketten sowie des Protonentransports in Kanalrhodopsin eingesetzt. Darüber hinaus wenden wir besonders schnelle Rotation, um den magischen Winkel (> 60 khz) zur Strukturbestimmung von Membranproteinen in möglichst nativer Lipidumgebung an, wobei unser Schwerpunkt auf bakteriellen Membranproteinen wie OmpG und YadA liegt. Als neues Forschungsgebiet hinzugekommen ist die Hochfrequenz-MAS zur Erforschung biologischer Proben. Bei hohen MAS-Frequenzen können mit minimalen Probenmengen Protonenspektren mit hoher Auflösung erzielt werden. Wir haben die Bedingungen systematisch optimiert und bereits an verschiedenen Proteinen (SH3-Domäne, ABC-Transporter, OmpG, bakterielle Biofilme) Pilotstudien durchgeführt. Schließlich bearbeiten wir Proteinsysteme, die an der Proteinhomöostase beteiligt sind, u. a. kleine Hitzeschockproteine.

67 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 65 DESCRIPTION OF PROJECTS Dynamic Nuclear Polarization Methods that enable structural studies on membrane-integrated receptor systems without the necessity of protein purification or on proteins bound to the cytoskeleton are attractive prospects for structural biology. Dynamic nuclear polarization magic angle spinning NMR allows the investigation of such systems by delivering the required sensitivity. In this method, the very strong polarization of electron spins is transferred to nuclear spins, which can then be detected at a much higher signal-to-noise ratio. With DNP, we have studied 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 ribosomes. Initial results from these studies include the first chemical shift assignments of residues in the nascent protein chain emanating from the ribosome and analysis of its secondary structure. Special emphasis has been placed on making DNP 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 measured at 200 K. We developed a methodology that enabled us to record sufficiently resolved spectra of protein samples while retaining an appreciable signal enhancement of around 20 to 40 in the temperature range of 180 to 200 K. Future applications of DNP comprise the interaction of ribosome-bound signal peptides with interaction partners as well as studies of the chromophore states of channelrhodopsin, during the photocicle. Structures of membrane proteins in native lipids by solid-state NMR We have determined the structure of OmpG reconstituted in native lipids by a new solid-state NMR structure determination methodology. Using a combination of 13 C-detected experiments on fully protonated samples with different 13 C labeling schemes and 1 H-detected experiments on deuterated samples for which the exchangeable sites were protonated, we were able to assign most atoms in the membrane spanning β-sheets. A combination of distance and torsion angle restraints were used in the structure calculation. Our structure shows a β-barrel with strands of different lengths whose minimal height is consistent with the thickness of a natural lipid-bilayer. Most of the extracellular loops appear as very flexible beyond the membrane boundary. However, we found that two loops (3 and 4) appear well ordered over much of their length, and we speculate that these represent a binding site. We have used solution and solid state NMR to investigate proton transport in channelrhodopsin, an important protein used in neurobiology to probe the light-induced activation of nerve cells. In these studies the use of DNP was indispensible for showing that in the fully dark-adapted state, the retinal chromophore resides exclusively in the all-trans configuration. 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 little explored drug targets. Following structural work on αb-crystallin we are now investigating its mechanism of activation and interaction with substrates such as β- and γ-crystallins. Our 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 interactions, using PDZ (PSD-95, Dlg, ZO-1) domains as an example. They play important roles in cellular signaling pathways and are structurally characterized by a hydrophobic pocket surrounded 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, 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 PDZligand complexes provide an excellent basis for rational design. We identified inhibitors with low to medium affinity for several PDZ domains and collected members of the respective substance classes in a PDZ library. 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.

68 66 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 1: Solid-state NMR structure of OmpG in lipid bilayers. Regular secondary structure is shown in blue, loop regions in red. The structures to the right are turned by 90. Extracellular GROUP MEMBERS Dr. Umit Akbey Dr. Ying Wing Chow Dr. Anne Diehl Dr. Trent Franks Dr. Matthias Hiller Dr. Madhu Nagaraj Dr. Andrew Nieuwkoop Dr. Shakeel Ahmad Shahid Dr. Barth van Rossum Michel Andreas Geiger (doctoral student) Johanna Münkemer (doctoral student) Joren Retel (doctoral student) Mahsheed Sohrabi (doctoral student) Daniel Stöppler (doctoral student) Anja Voreck (doctoral student) Arndt Wallmann (doctoral student) Natalja Erdmann (technical assistant) Liselotte Handel (technical assistant) Martina Leidert (technical assistant) Kristina Rehbein (technical assistant) Nils Cremer (technical assistant) Staff employed within the reporting period COLLABORATIONS 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 Angela Gronenborn, Elena MateiUniversity of Pittsburgh, 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 (inspin), Interdisciplinary Nanoscience Center (inano) and Aarhus University, Denmark Michael Nilges, Institut Pasteur, Paris, France Melanie Rosay, Bruker BioSpin, Billerica, USA Paul Tordo, Aix-Marseille Université, France Shimon Vega, The Weizmann Institute of Science, Rehovot, Israel 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

69 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 67 Fig. 2: Representation of the stalled nascent chain interacting with ribosomal proteins along the exit tunnel. The DsbA signal sequence is shown in a stretched conformation (left panel) and in a partial alpha-helical conformation (right panel). Two of the three proteins which form the constriction point (L4 and L22) as well as the trna are shown in blue. SELECTED PUBLICATIONS Nieuwkoop AJ, Franks WT, Rehbein K, Diehl A, Akbey Ü, Engelke F, Emsley L, Pintacuda G, Oschkinat H (2015) Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 khz magic-angle-spinning. J Biomol NMR 61(2), Bruun S, Stoeppler D, Keidel A, Kuhlmann U, Luck M, Diehl A, Geiger MA, Woodmansee D, Trauner D, Hegemann P, Oschkinat H, Hilde brandt P, Stehfest K (2015) Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion Between the all-trans and 13-cis Retinal Isomers. Biochem 54, Nagaraj M, Franks TW, Saeidpour S, Schubeis T, Oschkinat H, Ritter C, van Rossum BJ (2016) Surface binding of TOTAPOL assists structural investigations of amyloid fibrils by dynamic nuclear polarization NMR spectroscopy. ChemBioChem 17(14), Lange S, Franks WT, Rajagopalan N, Döring K, Geiger MA, Linden A, van Rossum BJ, Kramer G, Bukau B, Oschkinat H (2016) Structure analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR. Sci Adv 2(8), e Geiger MA, Orwick-Rydmark M, Märker K, Franks WT, Akhmetzyanov D, Stöppler D, Zinke M, Specker E, Nazaré M, Diehl A, van Rossum BJ, Aussenac F, Prisner T, Akbey Ü, Oschkinat H (2016) Temperature dependence of cross-effect dynamic nuclear polarization in rotating solid: advantages of elevated temperatures. PCCP, 18(44), FMP authors Group members EXTERNAL FUNDING Deutsche Forschungsgemeinschaft, SFB 765 / C4, Multivalente Protein-Protein-Interaktionen zwischen WW-Domänen und Prolinreichen Segmenten, with Christian Freund, , Deutsche Forschungsgemeinschaft, SFB 740 / B07-2, Untersuchungen an Komplexen kleiner Hitzeschockproteine mit Substraten mittels Festkörper-NMR-Spektroskopie und dynamischer Kernpolarisation, , Deutsche Forschungsgemeinschaft, SFB 1078 / B01, Strukturelle Dynamik von Kanalrhodopsinen, , ; , Deutsche Forschungsgemeinschaft, DIP Dynamic Nuclear Polarization: Integrating fundamentals and new applications, OS 106 / 12-1, , Deutsche Forschungsgemeinschaft, Bacterial adhesin CsgA: A structural basis of nucleator-mediated amyloid formation and host protein interactions studied by solid-state NMR, RO 3496 / , Alexander von Humboldt-Stiftung, Forschungskostenzuschuss als Gastgeber von Dr. Andrew Nieuwkoop, , ; , inext, Infrastructure for NMR, EM and X-rays for Translational Research, GA , , ; , (Access Costs) UCB PHARMA S.A, Protofibril-drug interaction investigated by solid-state NMR / Research Services Agreement, ,

70 68 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 SOLUTION NMR LÖSUNGS-NMR GROUP LEADER DR. PETER SCHMIEDER BIOGRAPHY Studied Chemistry, Johann Wolfgang Goethe University, Frankfurt / Main 1988 Diploma thesis (Prof. Kessler), Johann Wolfgang Goethe University, Frankfurt / Main Ph.D.(Prof. Kessler), Technical University of Munich 1990 Hans-Fischer-Preis der TU München Post-Doc (Prof. Wagner), Harvard Medical School, Boston MA since 1995 Group leader Solution NMR spectroscopy, FMP SUMMARY The group focuses on applying 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 labeling schemes and other biophysical techniques. Recently we have been interested in a quantitative determination of the role of dynamics in biomolecules. We have been exploring the role of mobility in the presentation of peptides by major histocompatibility complex (MHC) class I molecules and their recognition by T-cell receptors, exploiting the ability of NMR spectroscopy to provide such information at 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 such as vaccine design. Furthermore, we have pursued a variety of projects in collaboration 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 a collaboration with the group of Ronald Kühne. Here, NMR was used to analyze the constitution of ProM -molecules developed by his group, potential side products of their synthesis, and their interaction with EVH1 protein domains. ZUSAMMENFASSUNG Der Fokus unserer Gruppe liegt auf der Anwendung Lösungs-NMR-spektroskopischer Techniken zur Untersuchung der Struktur und Dynamik von Biomolekülen mit atomarer Auflösung. In Kombination mit geeigneten Markierungsmethoden und anderen biophysikalischen Techniken wird das gesamte Spektrum an mehrdimensionalen NMR-Techniken eingesetzt. In den letzten Jahren haben wir uns für die quantitative Bestimmung der Beweglichkeit in Biomolekülen interessiert. Dabei wurde die Rolle der Dynamik bei der Präsentation von Peptiden durch MHC-Moleküle der Klasse 1 und deren Erkennung durch T-Zell-Rezeptoren (TCR) untersucht, unter Nutzung der Fähigkeit der NMR-Spektroskopie diese Information mit atomarer Auflösung zu erhalten. Da die Interaktion von MHC-Molekülen und TCRs nicht auf der Basis von statischen Strukturen alleine verstanden werden kann, können diese Arbeiten zum Verständnis immunologischer Prozesse und zu Anwendungen wie dem Entwurf von Impfstoffen beitragen. Außerdem haben wir eine Vielzahl von Projekten in Zusammenarbeit mit anderen Gruppen verfolgt und dabei entweder die Interaktion von Biomolekülen und Bindungspartnern oder die Konstitution von kleinen Molekülen oder biologisch relevanten Naturstoffen untersucht. Ein Beispiel ist die Zusammenarbeit mit der Gruppe von Ronald Kühne am FMP bei der NMR dazu genutzt wurde, die Konstitution von in dieser Gruppe entwickelten ProM - Molekülen zu bestätigen, gegebenenfalls Nebenprodukte ihrer Synthese zu charakterisieren und ihre Interaktion mit EVH1-Protein-Domänen zu untersuchen.

71 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 69 Fig. 1: Investigation of the dynamics of MHC class I complexes in a peptide- and subtype-dependent manner. (a) X-ray structure of HLA-B27*09 in complex with the peptide pvipr, β 2 m is shown in grey, the heavy chain in green, and the peptide in a stick representation. Residue 116 (His in the 09-subtype, Asp in the 05-subtype), as well as the two Trp residues of β 2 m, are highlighted. The region of β 2 m that is flexible in the complex is coloured in orange. (b-e) Order parameters of the α1 / α2 domain resulting from a Lipari-Szabo-type analysis obtained for four complexes. Warmer colours indicate more flexibility. While it was to be expected that the loops are more flexible than elements of secondary structure, the two helices also show flexibility in a peptide- and subtype-dependent manner. The four complexes are: B*27:05 / pvipr (b), B*27:05 / TIS (c), B*27:09 / pvipr (d), B*27:09 / TIS (e). DESCRIPTION OF PROJECTS NMR-spectroscopic investigation of micropolymorphismdependent 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 cannot 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 have determined the differences between the dynamics of the two subtypes when in complex with different peptides. While we focused initially on β 2 m, recently we have performed a full Lipari-Szabo analysis of the relaxation times of the heavy chain. Four different complexes with the two types of heavy chain (B27*05 and B27*09), and two different peptides (pvipr and TIS), have been investigated. While the α3 domain exhibits no large differences, variations can be found in the α1 / α2 domain in a subtype- and peptide-dependent manner. Dynamics occur on different timescales, as can be seen from peak doubling and disappearance (msec timescale) and the order factors of a Lipari-Szabo analysis (nsec timescale). Figure 1b-e shows the results of the latter, where the magnitude of the order parameters is indicated by different colors. One can see that not only are the loop regions of the proteins (which might be expected to be rather mobile) dynamic, but so too are the two helices that form the binding groove for the peptide, exhibiting a differential mobility not only between different complexes, but also within the same complex. NMR-spectroscopic analysis of ProM constitution and EVH1-interaction Protein-protein interactions are of major importance in many processes within cells and the ability to interfere with these interactions in a controlled manner would consequently be of pharmaceutical interest. The group of Ronald Kühne has succeeded in developing small molecule inhibitors of the interaction between poly-pro sequences and EVH1 domains using a rational approach based on modeling and X-ray crystallography. The compounds were synthesized by the group of Prof. Schmalz in Köln. The result was a library of proline mimetics (ProMs) with promising activity and the ability to interfere with the binding of the FPPPP motif to the EVH1 domain. An example for one of the compounds (Ac-Cl-Phe-ProM2-ProM1-NH 2 ) is shown in Figure 2a. NMR spectroscopy was important within this project in two respects: Several ProM-based molecules were tested for binding to different EVH1 domains using a 1 H, 15 N-HSQC-based assay. As a prerequisite for this assay the proteins had to be assigned using standard triple resonance experiments. An example for a titration of an-evh1 domain with a ProM-based molecule is shown in Figure 2b. We were able to not only measure the strength of binding but could also determine the binding site based on the available NMR assignments. In addition, since the ProM-based molecules are the result of an elaborate synthesis, proof of their constitution after the complete assembly of the molecules was necessary. Moreover, purification usually yielded side products that needed to be identified. This was done using NMR spectroscopy with the molecules dissolved in methanol, where the molecule exhibits two sets of signals that are most likely based on a cis / trans-isomerization of the Phe-ProM2-peptide bond. The two sets of signals are quite similar, rendering the analysis more difficult. A full assignment was nevertheless possible and the correct constitution of the molecules was confirmed. Smaller amounts of side products could also be characterized. Figure 2c shows a part of the 1 H, 13 C correlation of the ProM-based molecule shown in Figure 2a.

72 70 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 2: Investigating the constitution and protein-interaction of ProM-based molecules. (a) Molecular formula of Ac-ClPhe-ProM2-ProM1-NH 2. (b) NMR titration of the Homer1-EVH1 with the ProM-based molecule shown in (a), the shifts in several resonances indicate an interaction and reveal the site of interaction. (c) 1 H, 13 C correlation of the ProM-based molecule shown in (a), a full assignment was obtained for both conformations of this molecule in methanol to verify the compound s constitution. GROUP MEMBERS Dr. Peter Schmieder (group leader) Martin Ballaschk (doctoral student) Monika Beerbaum (technical assistant) Brigitte Schlegel (technical assistant) Nils Trieloff (technical assistant) Staff employed within the reporting period COLLABORATIONS National A. Ziegler und B. Uchanska-Ziegler Charité Universitätsmedizin Berlin T. Niedermeyer U Tübingen T. Müller U Würzburg C. Rademacher MPI für Kolloid und Grenzflächenforschung A. Weng FU Berlin FMP-intern Anne Diehl Ronald Kühne Michael Krauß Volker Haucke Dorothea Fiedler Christian Hackenberger Marc Nazare SELECTED PUBLICATIONS Boschert V, Frisch C, Back J W, van Pee K, Weidauer S E, Muth E M, Schmieder P, Beerbaum M, Knappik A, Timmerman P, Mueller T D (2016) The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6. Open Biol, 6(8). D'Andrea E D, Diehl A, Schmieder P, Oschkinat H, Pires J R (2016) Chemical shift assignments and secondary structure prediction for Q4DY78, a conserved kinetoplastid-specific protein from Trypanosoma cruzi. Biomol NMR Assign 10(2), Hanske J, Aleksic S, Ballaschk M, Jurk M, Shanina E, Beerbaum M, Schmieder P, Keller B G, Rademacher C (2016) Intradomain Allosteric Network Modulates Calcium Affinity of the C-Type Lectin Receptor Langerin. J Am Chem Soc 138(37), Opitz R, Muller M, Reuter C, Barone M, Soicke A, Roske Y, Piotukh K, Huy P, Beerbaum M, Wiesner B, Beyermann M, Schmieder P, Freund C, Volkmer R, Oschkinat H, Schmalz H G, Kühne R (2015) A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions. Proc Natl Acad Sci U S A 112(16), Preisitsch M, Heiden S E, Beerbaum M, Niedermeyer T H, Schneefeld M, Herrmann J, Kumpfmuller J, Thurmer A, Neidhardt I, Wiesner C, Daniel R, Muller R, Bange F C, Schmieder P, Schweder T, & Mundt S (2016) Effects of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. CAVN2. Marine Drugs 14(1), 21. EXTERNAL FUNDING Deutsche Forschungsgemeinschaft NMR spectroscopic investigation of micropolymorphism-dependent dynamics of human major histocompatibility antigens, SCHM 880 / 9-1, Peter Schmieder, , (davon 1xE13 67 %) FMP authors Group members

73 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 71 COMPUTATIONAL CHEMISTRY / DRUG DESIGN WIRKSTOFF-DESIGN GROUP LEADER DR. RONALD KÜHNE BIOGRAPHY Studied Biochemistry, Martin-Luther-Universität Halle Wittenberg 1973 Diploma in Biochemistry Research associate, Zentralins titut für Molekularbiologie und Medizin der Akademie der Wissenschaften Research associate, Institut für Wirkstofforschung der Akademie der Wissenschaften 1980 Doctorate degree 1993 Group leader, Leibniz-Forschungsinstitut für Molekulare Pharmakologie SUMMARY 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. In recent years we have focused on targeting protein-protein interactions mediated by protein domains specifically recognizing proline-rich motifs. These domains are involved in many diseaserelevant 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. Fig. 1. (a) Schematic representation of proposed flux-coupled gating mechanism. (b) Ion occupancy in potassium channel selectivity filters: MD simulations performed on TRAAK and Kv1.2 reveal relative ion occupancies for the binding sites S1-S4. (c) TRAAK upon mutation of a threonine in the SF (T103C) leads to a loss of binding at S1 and S4. (d) TRAAK with Rb + and Cs + show differences of ion occupancies at the S1-S4 sites.

74 72 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 DESCRIPTION OF PROJECTS Molecular dynamics simulations of K2P channels Two-pore domain (K2P) K + channels are major regulators of excitability that endow cells with an outwardly rectifying background leak conductance. In some K2P channels, strong voltage-dependent activation has been observed despite the channels not containing a canonical voltage-gating domain. In our study, we showed that voltage-dependent gating is common to most of the K2P channels. Experimental mutagenesis, as well as molecular dynamics simulation of permeation, revealed that the voltage-dependent gate is located within the selectivity filter. Based on the experimental and theoretically calculated gating charge that is coupled to pore opening, we propose that the voltage sensitivity originates in the movement of three to four K + ions into the high electric field of an inactive filter (see Figure 1). Overall, this ion-flux gating mechanism generates a one-way check valve within the filter because outward movement of K + induces filter opening, whereas in inward permeation it promotes inactivation. 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). At this point more than 15 new chemical entities have been synthesized using an innovative modular synthesis concept developed at the Universität zu Köln (Inst. Organische Synthese, Prof. H.G. Schmalz). As a proof of concept we were able to develop, for the first time, low molecular weight inhibitors of Ena / VASP EVH1 domains. The optimization of EVH1 inhibitors was supported by detailed insights into their binding modes (Fig. 2). Based on X-ray structures (resolutions in the range of 1.0 Å to 2.5 Å) of different ligand-enah- / EVL-EVH1 complexes we identified an additional binding epitope on the EVH1 surface. The combination of experimental structural biology data and computer simulation yielded further optimization of low molecular weight EVH1 inhibitors. Functional studies using the new EVH1 inhibitors The Ena / VASP protein family is involved in modulating the actin cytoskeleton, a complex and highly regulated process that is the driving force of directed cell migration and which plays important roles in disease-relevant processes like tumor metastasis. We found that inhibition of Ena / VASP EVH1 domains disrupts co-localisation Fig. 2. A Inhibitor 1 Ac-[2-Cl-F][ProM-2] [ProM-1]-OH with ProM-2 in red and ProM-1 in green (right) and the corresponding peptid Ac-FPPPP- OH (left). B Overlay of Mena-EVH1 with Ac-FPPPPT-OH in green (PDB: 1EVH) and EnaH-EVH1 with Ligand 1 in blue (PDB: 4MY6). C Binding constants to VASP-, EnaH- and EVL-EVH1 domains.

75 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 73 of Ena / VASP-mediated protein-protein interactions at the focal adhesions, the leading edge, and the tips of filopodia. Consequently, our novel EVH1 inhibitors block cell migration and motility of invasive tumor cells. We have shown that our compounds were able to inhibit tumor metastasis in a zebrafish metastasis model. Cheminformatics, Library design, and Molecular Modelling Cheminformatics, bioinformatics, and molecular modeling are disciplines important for supporting the rational design of chemical probes. In combination with experimental chemical biology, these 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. Within the Helmholtzinitiative Wirkstoffforschung we developed a biannually updated, Web-based database of commercially available compounds (DACS) containing more than 80 million compounds. The combination of our toolbox and DACS allows fast design of hit- or target-focused 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. GROUP MEMBERS Dr. Michael Lisurek Dr. Matthias Müller Dr. Robert Opitz Dr. Kiril Piotoukh Dr. Bernd Rupp Dr. Han Sun Matthias Barone (PhD student) Hafiza Nayab (PhD student) Raed Al-Yamori (Techn. Informatics) Kathrin Motzny (Techn. Ass.) Staff employed within the reporting period COLLABORATIONS FMP Dr. P.Schmieder Dr. G. Krause Dr. B. Wiesner Dr. E. Krause Dr. M. Dathe International Prof. P. van Dijke, Univ. Leiden, Netherlands National Prof. H.-G. Schmalz, Inst. Organische Chemie, Universität zu Köln Prof. Chr. Freund, FU Berlin Prof. R. Hiesinger, FU Berlin Prof. U. Stein, MDC Berlin Prof. U. Heinemann, MDC Berlin SELECTED PUBLICATIONS Schewe M, Nematian-Ardestani E, Sun H, Musinszki M, Cordeiro S, Bucci G, de Groot BL, Tucker SJ, Rapedius M, Baukrowitz (2016 ) A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels. Cell 164, Opitz R, Müller M, Reuter C, Barone M, Soicke A, Roske Y, Piotukh K, Huy P, Beerbaum M, Wiesner B, Beyermann M, Schmieder P, Freund C, Volkmer R, Oschkinat H, Schmalz HG, Kühne R (2015 ) A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions. Proc Natl Acad Sci U S A. 112, EXTERNAL FUNDING Innovative Inhibitoren Polyprolin-Motiv-erkennender Protein- Protein-Interaktionsdomänen als Ausgangspunkt zur Validierung neuer pharmakologisch relevanter Zielproteine und zur Entwicklung neuartiger Ansätze in der Krebstherapie, BMBF 03V0475, 07 / / 2016, Förderumfang: Reuter C, Opitz R, Soicke A, Dohmen S, Barone M, Chiha S, Klein MT, Neudörfl JM, Kühne R, Schmalz HG (2015) Design and Stereoselective Synthesis of ProM-2: A Spirocyclic Diproline Mimetic with Polyproline Type II (PPII) Helix Conformation. Chemistry 21, Khatri Y, Ringle M, Lisurek M, von Kries J-P, Zapp J, Bernhardt R (2016) Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1α-Hydroxylated Products from C-19 Steroids. Chembiochem. 17, Chenge JT, Duyet LV, Swami S, McLean KJ, Kavanagh ME, Coyne AG, Rigby SE, Cheesman MR, Girvan HM, Levy CW, Rupp B, von Kries J-P, Abell C, Leys D, Munro AW (2017) Structural Characterization and Ligand / Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1. J Biol Chem. 292, FMP authors Group members

76 74 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 STRUCTURAL BIOINFORMATICS AND PROTEIN DESIGN STRUKTUR-ORIENTIERTE BIOIN- FORMATIK UND PROTEINDESIGN GROUP LEADER DR. GERD KRAUSE BIOGRAPHY Studied chemistry, University of Leipzig 1982 Ph.D. in biochemistry, Martin Luther University, Halle Researcher at the Institute of Drug Design, Berlin on behalf of pharmaceutical industry Research Position, Institute of Drug Design, Berlin Visiting Scientist, Washington University (Prof. Marshall), St. Louis, MO, USA Project leader at research institute of molecular pharmacology, FMP, Berlin since 1998 Group Leader of Structural Bioinformatics and Protein Design, FMP, Berlin SUMMARY The group focuses on analyzing the relationship between the sequences and structures of membrane proteins using structural bioinformatics, combined with experimental studies of the functions of altered sequences. Our aim is to reveal the structure-function relationships of proteins and their potential interaction partners. A major activity of ours is developing bioinformatic tools for investigating such structure-function relationships; another is to apply these to particular molecular biological projects of protein-ligand, protein-substrate, or protein-protein interactions. To verify our structure-function hypotheses we use our models to guide site-directed mutagenesis of specific residues and analyze available mutation data. Bioinformatic tools / database development and molecular biology applications mutually support one another. The main aims of the group are to achieve: (1) A detailed understanding of the 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) prediction of small molecules or modifications of biosimilar molecules for potential pharmacological interventions. ZUSAMMENFASSUNG Die Arbeitsgruppe 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 Modulation 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 oder biologisch ähnlichen Molekülen mit dem Potenzial für eine pharmakologische Intervention.

77 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 75 Fig. 1: The wild type C-terminal domain of Clostridium perfringens enterotoxin (ccpe) does not interact with claudin-5 an important constituent of the tight junctions (TJ) in the blood-brain barrier (BBB). Molecular model-guided modifications of ccpe (surface, grey) result in ccpe mutants that are able to interact with the membrane-associated claudin-5 (cartoon, red). The two model-derived substitutions, Y306W and S313H (highlighted in cyan), are able to bind to the extracellular loop 2 of Claudin 5 (inset). This opens the BBB temporarily and might allow the usage of drugs for pharmacological interventions in the brain (cooperation with J. Piontek). DESCRIPTION OF PROJECTS Pharmacological intervention of tight junctions by modified Clostridium perfringens enterotoxin (Piontek, Protze) Claudins (Cld) the main constituents of tight junctions (TJ) regulate paracellular permeability across epithelial and endothelial monolayers and maintain their tightness. In our project we utilize the ability of Clostridium perfringens enterotoxin (CPE) to bind to certain members of the Cld family. We have shown that modifications of the non-cytotoxic C-terminal domain of CPE (ccpe) allow a targeted interaction with distinct Cld-subtypes for potential pharmacological alterations of TJ. We have also shown that ccpe-wt modulates the epidermal barrier in vivo via interaction with an ortholog of Cld4 in Zebrafish. (Zang et al., Exp. Dermatol 2015). Using structure-guided mutagenesis we created ccpe-mutants that bind to Clds differently than ccpe-wt; e. g. ccpe-y306w / S313H (Fig.1) binds to Cld5 (Protze et al., CMLS 2015), which is strongly expressed in the blood-brain-barrier (BBB). ccpe-y306w / S313H opens the BBB transiently and reversibly in vivo and in vitro (Liao et al., Neuroscience 2016). Several Cld structures were solved recently and confirmed our predicted structural determinants that are important for CPE-Cld interactions (Krause et al., Sem Cell Dev. Biol. 2015). Modulation of G-protein-coupled receptors (GPCRs) (Marcinkowski, Hoyer, Kreuchwig) Glycoprotein hormone receptors (GPHRs) are a type of GPCRs that play an important role in pharmacology. Both the lutropin (LH) / choriogonadotropin (CG) receptor (LHCGR) and the thyroidstimulating hormone receptor (TSHR) are GPHRs. LHCGR is activated by two closely-related gonadotrophic glycoprotein hormones. The binding event of both hormones to the receptor's extracellular leucine-rich-repeat domain (LRRD) is similar, however a detailed study combining homology modeling with successive experimental characterization (in cooperation with B. Wiesner and R. Schülein) revealed that the functional differences in signal activation between hlh and hcg are mediated by structural changes in the extracellular hinge region (Grzesik et al., Front Endocrin. 2015). Pathogenic activation of human TSHR by autoantibodies results in dysregulation of the thyroid hormone status. Allosteric small molecule antagonists identified by HTS in the FMP screening unit (in cooperation with J von Kries) that were synthetically modified (by E. Specker) are able to block pathogenic TSHR activation (Krause G, Hoyer I, Marcinkowski P, Specker E, Kries J von et al. 2016, EU Patent, EP ). Structure-function studies of other GPCRs revealed the ligand-binding site of the peptidic urotensin-ii receptor agonist (Bandholtz et al. J Med Chem 2016). Structural bioinformatics webserver as GPHR research resource and GPCR modeling platform (Kreuchwig, Sargent) Of central importance for several GPHR-related projects studying structural-functional properties of the wild-type receptor is the fact that amino acid side-chain substitutions often modify phenotypes. Therefore, our web-based research resource ( de) compiles a huge amount of functional data gathered from both naturally occurring and designed mutations (> 1500) of the GPHRs, along with structural information for detailed structure-function analyses. Despite recent advances in crystallizing GPCRs, it is still difficult to obtain their crystal structures. For this reason, a web server for fragment-based homology modeling of class A GPCRs has been developed ( This server uses a finger print correlation scoring strategy to select the best template per transmembrane helix based on the presence or absence of fingerprint features. Currently, over 1000 models of human, mouse, and rat GPCRs have been pre-calculated and the results made available to users. Structural determinants of transmembrane transporter MCT8 and LAT2 for thyroid hormone import and export (Kinne, Hinz, Protze) Thyroid hormones (TH) are transported via specific transmembrane (TM) transporters into and out of the cell. We utilize homology models for two TH-transporters, the monocarboxylate transporter 8 (MCT8) and the L-type amino acid transporter 2 (LAT2), and combined them with directed mutagenesis to delineate molecular mechanisms of TH transport. Both transporters show a secondary structure with 12 TM helices but differ in their overall fold and substrate specificity. Structural and molecular determinants common for import and export of T 3 and T 4 have been elucidated in MCT8 (Fig. 2) (Protze et al., CMLS 2017). We have shown (in cooperation with R. Schülein) that LAT2 imports amino acids and specific THs, e. g. 3,3 -T 2 and T 3, but not rt 3 and T 4 (Kinne et al., Eur. Thyroid J. 2015), and provided initial structural insights into TH transport mechanisms of LAT2 (Hinz et al., Mol. Endocrinol. 2015). Based on the LAT2 models, we selected several mutants for structure-function analysis in Xenopus leavis oocytes. Just one pocket-widening mutation enabled T 4, and increased T 3, import, but did not alter their export. In addition, we have shown that LAT2 allows only the unidirectional import of T 2 into the cells. In fact LAT2 exports amino acids, but not any TH, out of the cell (Hinz et al., Cell Mol. Endocrinol. 2017). Momentan ist kein Platz für Fig. 2!

78 76 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 2: The monocarboxylate transporter 8 (MCT8) mediates thyroid hormone (TH) transport across the plasma membrane of the cells. Our MCT8 molecular models of extracellular opened, extracellular partly occluded, and intracellular opened conformations. suggest that T3 (green) adheres to transport-sensitive residues during tilting (red arrows) of the N-terminal (cyan) and C- terminal (sand) 6-TMH bundles around the central cavity. T3 is thus lifted through the central traversing cavity and released towards the intracellular side and vice versa. GROUP MEMBERS Dr. Inna Hoyer Dr. Anita Kinne Dr. Annika Kreuchwig Dr. Anna Piontek (Veshnyakova) Dr. Jonas Protze Dr. Catherine Sargent Miriam Eichner (doctoral student) Katrin M. Hinz (doctoral student) Patrick Marcinkowski (doctoral student) Dominik Neef (student) Carolin Scheffner (student) Staff employed within the reporting period COLLABORATIONS International T. Visser Uni Rotterdam, The Netherlands National S. Kaufmann MPI für Infektionsbiologie, Berlin J. Piontek, M. Fromm, H. Biebermann, G. Kleinau, 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, Furkert J, Rutz C, A. Diehl, H. Oschkinat, J.vKries, T. Jentsch FMP SELECTED PUBLICATIONS Hinz K M, Neef D, Rutz C, Furkert J, Koehrle J, Schuelein R, Krause G (2017) Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids Mol Cell Endocrinology Mar 5, 443, Hinz K M, Meyer K, Kinne A, Schülein R, Köhrle J, Krause G (2015) Structural insights into thyroid hormone transport mechanisms of Lat2 Mol Endocrinology Jun 29(6), Protze J, Eichner M, Piontek A, Dinter S, Rossa J, Blecharz K G, Vajcoczy P, Piontek J, Krause G (2015), Directed structural modifications of Clostridium perfringens enterotoxin to enhance binding to claudin-5, Cell Mol Life Sci. Apr 72:7, Grzesik P, Kreuchwig A, Rutz C, Furkert J, Wiesner B, Schülein R, Kleinau G, Gromoll J, Krause G (2015), Differences in signal activation mediated by L H and hcg are triggered by the LH / CG receptor`s extracellular hinge region, Frontiers in Endocrinology Sep 22, 6, 140. 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 H J, 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 Aug 28, 512(7515), EXTERNAL FUNDING Deutsche Forschungsgemeinschaft (DFG), SPP 1629, Thyroid Trans Act- Role of L-type amino acid transporter Lat2 in transport of thyroid hormones KR1273 / 5-1, KI1751 / 1-1, 11 / / Mundi Pharma Research GmbH: Proteininteraktionen 9 / / DFG, SPP1629, -Molekulare Determinanten unterschiedlicher Mechanismen des Schilddrüsenhormon-Imports / -Exports der L-type Aminosäuretransporter KR1273 / 5-2; 6 / / 2019, DFG, Modulatoren für den Thyrotropin Rezeptor: Molekulare Mechanismen allosterischer Bindung und Wirkungsweise kleiner Moleküle, KR1273 / 4-2,1 0 / / ,650 DFG: Molekulare Architektur Claudin-basierter Tight Junction Stränge und parazelluläre Ionenkanäle, KR1273 / 8-1, 4 / / Wilhelm Sander Stiftung: , Targeting Claudin überexprimierender Lungen und korektal Karzinome mittels modifiziertem Clostridium Perfringens Enterotoxin, 2 / / DFG, Searching for transport proteins for TRIAC or DITPA acting as T3 /TH substitutes, KR1273/9-1; PR1616/2 1(eigene Stelle J. Protze) 4/2017 4/2020, FMP authors Group members

79 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 77 IN-CELL NMR NMR IN ZELLEN GROUP LEADER DR. PHILIPP SELENKO BIOGRAPHY 2002 Ph.D., European Molecular Biology Laboratory (EMBL), Heidelberg Post-Doc, 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, FMP, Emmy Noether fellowship by the Deutsche Forschungsgemeinschaft (DFG) SUMMARY 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 ERC Consolidator Grant DESCRIPTION OF PROJECTS One question that we address with in-cell NMR spectroscopy is how intrinsically disordered proteins (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. Many IDPs are key factors in prevalent human neurodegenerative disorders including Alzheimer s, Parkinson s, and Huntington s disease. In all of these disorders, individual IDPs convert into highly ordered, β-sheet structures termed amyloids, which deposit in different areas of the brain. Interestingly, these amyloid structures are found in specific cell types, suggesting that their protein components have different aggregation tendencies in different cellular environments. Our goal is to understand why certain IDPs change their structures in one cell type but not another. Or, in other words, how different intracellular environments trigger structural conversions that lead to amyloid formation. To this end, we use in-cell NMR spectroscopy to characterize the structural properties of neurodegenerative disease IDPs in different types of neuronal and non-neuronal cells. We determined a full-scale atomic-resolution description of the structures of the human amyloid protein alpha-synuclein, the main culprit in Parkinson s disease, in five different mammalian cell types under healthy physiological conditions. Surprisingly, we found that alpha-synuclein remains disordered in all the tested cell types and adopts structures in which its most amyloidogenic NAC region is shielded from exposure to the cytoplasm (Figure 1, NAC region in black). We believe that this prevents the protein from spontaneous amyloid formation under healthy cell conditions. At the same time, it implies that large structural rearrangements are necessary for alpha-synuclein to develop amyloid structures in diseased cells. We are currently inducing such disease conditions to directly monitor the amyloid formation process by in-cell NMR spectroscopy. Insights into this process will enable the design of novel drugs to treat Parkinson s disease.

80 78 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 1: Intracellular structures of human alpha-synuclein in healthy cells. Cartoon representations of alpha-synuclein structures in the crowded environment of human cells. Cytoplasmic components are represented by white spheres. GROUP MEMBERS Dr. Andres Binolfi Dr. Cedric Eichmann Dr. Reeba Jacob Dr. Antonio Limatola Dr. Francois-Xavier Theillet Jonas Kosten (PhD Student) Marchel Stuiver (technical assistant) Marleen van Rossum (technical assistant) COLLABORATIONS International Daniella Goldfarb Weizmann Institute of Science, Rehovot, Israel Richard Treisman The Crick Research Institute, London, UK National Wolfgang Fischle Max Planck Institute of Biophysical Chemistry, Göttingen Staff employed within the reporting period SELECTED PUBLICATIONS Thongwichian R, Kosten J, Benary U, Rose H M, Stuiver M, Theillet F X, Dose A, Koch B, Yokoyama H, Schwarzer D, Wolf J, Selenko P (2015) A multiplexed NMR-reporter approach to measure cellular kinase and phosphatase activities in real-time. J Am Chem Soc. 137(20), EXTERNAL FUNDING ERC Consolidator Grant, NeuroInCellNMR Danielsson J, Mu X, Lang L, Wang H, Binolfi A, Theillet FX, Bekei B, Logan D T, Selenko P, Wennerström H, Oliveberg M (2015) Thermodynamics of protein destabilization in live cells. Proc Natl Acad Sci USA. 112(40), Stützer A, Liokatis S, Kiesel A, Schwarzer D, Sprangers R, Söding J, Selenko P, Fischle W (2016) Modulations of DNA contacts by linker histones and post-translational modifications determine the mobility and modifiability of nucleosomal H3 tails. Mol Cell. 61(2), Müntener T, Häussinger D, Selenko P, Theillet F X (2016) In-cell protein structures from 2D NMR experiments. J Phys Chem Lett. 7(14), Mylona A, Theillet F X, Foster C, Cheng T M, Miralles F, Bates P A, Selenko P, Treisman R (2016) Opposing effects of Elk-1 phosphorylation shape its response to ERK activatio. Science 354(6309), FMP authors Group members

81 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 79 MOLECULAR IMAGING MOLEKULARE BILDGEBUNG GROUP LEADER DR. LEIF SCHRÖDER BIOGRAPHY Studies in Physics and Chemistry, Georg-August Universität Göttingen Studies in 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 2005 Philips Research Prize for Medical Physics, awarded by the German Society for Medical Physics (DGMP) Research Fellow, Lawrence Berkeley National Laboratory 2007 Gorter Award of the International Society for Magnetic Resonance in Medicine (German Chapter) 2008 Dr. Emil-Salzer-Award for Cancer Research of the Federal State of Baden-Württemberg, awarded by the German Cancer Research Center 2009 Emmy Noether Fellow of the DFG, Group Leader at FMP ERC Starting Grantee at FMP (first grant of its kind in the Leibniz Association) 2009 Young Scientist Prize for Medical Physics of the International Union of Pure and Applied Physics (IUPAP) since 2014 Independent Group Leader, FMP 2015 Reinhart Koselleck Grant Award of the DFG (first grant of its kind in the Leibniz Association) SUMMARY Targeted imaging reporters have revolutionized our understanding of many biological processes. Fluorescence imaging is the prime example for translating biochemical knowledge into tools for studying processes in live cells, yet it still has one fundamental limitation: it fails as a non-invasive modality in larger whole organisms. Nuclear Magnetic Resonance (NMR) and its imaging modality, MRI, have the potential to address this need, but it requires a fundamentally new approach to overcome the intrinsic sensitivity limitations that accompany conventional NMR / MRI protocols. By optimizing the steps of preparation, manipulation, and encoding of spin magnetization, our group strives to design ultra-sensitive NMR reporters. These are essential for imaging the spatial distribution of disease markers for the concept of personalized medicine, the tracking of drug carriers, and the detection of particular molecular interactions. Xenon biosensors have an outstanding potential to improve early disease detection and for the visualization of drug delivery response, which is critical for individualized therapy. To explore this potential, our group engages in programs supporting highly innovative research, including the Koselleck Program of the DFG and the European Research Council. We develop methodologies to obtain MRI contrast from molecular targets at extremely low concentrations and sense molecular interactions within a few minutes, where conventional MRI protocols would theoretically require thousands of years. ZUSAMMENFASSUNG Zielgerichtete Kontrastmittel haben unser Verständnis vieler biologischer Prozesse revolutioniert. Die Fluoreszenz-Bildgebung ist das beste Beispiel für die Umsetzung biochemischen Wissens in Methoden zur Untersuchung von Prozessen in lebenden Zellen, aber sie hat nichtsdestotrotz eine fundamentale Einschränkung: für größere, komplette Organismen ist sie als nicht-invasive Modalität nicht einsetzbar. Die Kernspinresonanz (NMR) mit ihrer Bildgebungs-Modalität (MRT) hat das Potenzial, diesen Bedarf zu decken. Dabei erfordert diese Herausforderung einen grundsätzlich neuen Ansatz, um die geringe intrinsische Empfindlichkeit, die mit herkömmlichen NMR / MRI-Protokollen einhergeht, zu überwinden. Durch die Optimierung der Schritte zur Vorbereitung, Manipulation und Kodierung der Spin-Magnetisierung zielt unsere Gruppe auf die Entwicklung ultra-sensitiver NMR-Reporter ab. Diese sind essentiell für die Darstellung der räumlichen Verteilung von Krankheitsmarkern in der personalisierten Medizin, sowie für die Verfolgung von Wirkstoffträgern und den Nachweis bestimmter molekularer Wechselwirkungen. Xenon-Biosensoren haben ein herausragendes Potenzial zur frühzeitige Diagnose und Visualisierung der Therapieantwort, was entscheidend für individuelle Therapien ist. Um dieses Potenzial zu erforschen, engagiert sich unsere Gruppe in Programmen für hochinnovative Forschung wie dem Koselleck-Programm der DFG und dem Europäischen Forschungsrat. Wir entwickeln Methoden, um MRT-Kontrast von molekularen Markern in extrem geringer Konzentration zu erzielen und molekulare Wechselwirkungen innerhalb weniger Minuten zu detektieren, wo herkömmliche MRT-Protokolle andernfalls theoretisch tausende von Jahren erfordern würden.

82 80 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 DESCRIPTION OF PROJECTS Cell Surface Glycan Imaging with Ultra-Sensitive MRI Reporters We demonstrated the first case of NMR mapping of a diagnostic target that is inaccessible with existing MRI reporters. Cell surface glycans are an excellent example of this. Their modification through metabolic oligosaccharide engineering has not been able to be combined with MRI in previous studies. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry (see Fig. 1). While conventional relaxivity agents require micromolar target concentrations, this xenon biosensor could be detected at nanomolar concentrations. This first demonstration of live cell glycan-targeted MRI presents an excellent test bed for moving forward with xenon biosensors as these reporters continue to progress in vivo to investigate unsolved diagnostic questions, including glycome analysis. Optimized Xenon Host-Guest Complexes The expanding use of the Hyper-CEST technique (chemical exchange saturation transfer with hyperpolarized nuclei) for MR applications requires quantitative characterization of the exchange dynamics and optimizing the physico-chemical behaviour of xenon hosts. We could achieve the first quantitative comparison of various hosts, including cryptophane cages, cucurbit[n]urils, and genetically engineered bacterial gas vesicles. By introducing the gas turnover rate as a novel quantitative parameter, our work has demonstrated, for instance, that the fast exchanging host cucurbit[6]uril is approximately 100-fold more potent as an MRI reporter than cryptophane-a. CEST Image Contrast Modeling Xenon biosensors represent an emerging class of contrast agents with detection limits down to the nano- and picomolar regimes. However, build-up of the measured Hyper-CEST effect is complex and relies on the specific Xe-host system properties and the applied saturation pulse strength and duration. In this project, we introduced optimal saturation pulse parameters for a maximum, but still spectrally narrow, Hyper-CEST effect. The parallel development of sensors and their respective detection technique will accelerate the transition to the first in vivo studies, where knowledge about efficient signal build-up is critical. MRI Visualization of Enzymatic Activity Based on the promising potential of cucurbit[n]urils (CB) for reversible binding of Xe and their established use in switchable fluorescence detection, we devised an enzyme activity reporter for MRI purposes. Our design relies on hyperpolarized Xe NMR spectroscopy, in which we 1) use CB hosts as contrast agents, 2) rationally exploit the molecular recognition properties of these hosts, and 3) for the first time apply an optimized magnetization transfer (MT) experiment for the Xe MRI of enzyme activity. This extends the use of biosensors by introducing a signal switch capability in which Xe is gradually displaced from its host as an enzymatic reaction progresses (see Fig. 2). Critically, this approach circumvents the shortcomings in previous attempts by others where the enzyme activity was detected as a minor shift in resonance frequency of caged Xe. A cellular target metabolic labeling biochemical selectivity xenon host cells + Ac 4 ManNAz glycan N 3 scaffold cells + Man azide label N 3 modified sugar N 3 N 3 N 3 N N N chemoselective in situ coupling glycobiosynthesis N 3 B laser-induced magnetization Rb Rb Xe Rb Rb polarized photons Xe magnetization build-up RF off controlled depolarization RF on Xe signal contrast Xe Xe Xe Xe Xe Xe Xe Xe MRI detection cells + Ac 4 ManNAz + sensor cells + sensor Hyper-CEST effect [%] transfer to sample spatial encoding Fig. 1: Implementation of metabolic oligosaccharide engineering (MOE) for ultra-sensitive MRI detection of cell surface glycans. (A) Target cells characterized by certain glycan structures can be metabolically labeled with a modified sugar. This incorporates pre-labeling of the cells through a chemoselective group prior to MRI acquisition. (B) Hyperpolarized Xe is prepared outside the MRI scanner. The noble gas is transferred into the sample where it participates in reversible binding with the glycan-coupled host. We can then selectively image areas that contain metabolically labeled cells in an opaque test volume.

83 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 81 A substrate product product Xe Fig. 2: MRI visualization of enzymatic activity based on a displacement assay. (A) Lysine decarboxylase (LDC) produces cadaverine with a high affinity for LDC cucurbit[7]uril (CB7) and displaces Xe from this molecular container. (B) The absence of enzymatic activity maintains free exchange access for Xe into enzymatic conversion Xe displacement CB7 and yields an unchanged, high magnetization transfer (MT) signal. The production of cadaverine (Cad) suppresses the Xe exchange; hence, the MT response of reversibly bound Xe is lost and this appears in the MRI data as a B Lys Xe Xe@CB7 Xenon Magnetization Transfer MR Imaging Cad@CB7 Xe change in switchable signal contrast. enzyme 2 mm + enzyme GROUP MEMBERS COLLABORATIONS Dr. Jabadurai Jayapaul Dr. Jan Oliver Jost Dr. Martin Kunth Dr. Honor Rose Dr. Christopher Witte Jörg Döpfert (doctoral student) Stefan Klippel (doctoral student) Ursula Pfeiffer (doctoral student) Federica Rossella (doctoral student) Matthias Schnurr (doctoral student) Nils Bogdanoff (HiWi) International Mikhail Shapiro, Caltech, USA Alexander Pines, UC Berkeley, USA David Wemmer, UC Berkeley, USA Matthew Francis, UC Berkeley, USA Ville-Veikko Telkki, University of Oulu, Finland Yoram Cohen, Tel Aviv University, Israel Xin Zhou, WIPM, China Paul Beer, University of Oxford, UK National Andreas Hennig, Jacobs University Bremen Gil Westmeyer, TU München Jörg Piontek, Charité, Berlin Lorenz Mitschang, PTB, Berlin Salim Seyfried, MDC, Berlin Hartmut Kühn, Charité, Berlin Daniel Messroghli, Deutsches Herzzentrum, Berlin Jörg Matysik, Universität Leipzig Staff employed within the reporting period SELECTED PUBLICATIONS Korchak S, Kilian W, Schröder L, Mitschang L (2016) Design and Comparison of Exchange Spectroscopy Approaches to Cryptophane- Xenon Host-Guest Kinetics. J. Magn. Reson. 265, Schnurr M, Sloniec-Myszk J, Döpfert J, Schröder L, Hennig A (2015) Supramolecular Assays for Mapping Enzyme Activity by Displacement- Triggered Change in Hyperpolarized 129 Xe Magnetization Transfer NMR. Angew. Chem. Int. Ed. 54, Kunth M, Witte C, Hennig A, Schröder L (2015) Identification, Classification, and Signal Amplification Capabilities of High-Turnover Gas Binding Hosts in Ultra-Sensitive NMR. Chem. Sci. 6, (highlighted as cover article). Kunth M, Witte C, Schröder L (2015) Continuous-wave Saturation Considerations for Efficient Xenon Depolarization. NMR Biomed. 28, Witte C, Martos V, Rose H M, Reinke S, Klippel S, Schröder L, Hackenberger C P R (2015) Live-cell MRI of Xenon Hyper-CEST Biosensors Targeted to Metabolically-labeled Cell-surface Glycans. Angew. Chem. Int. Ed. 54, (highlighted as VIP and inside back cover article; recommended by F1000Prime). FMP authors Group members EXTERNAL FUNDING International Human Frontiers Science Program Organization, Cell Profiling with Xenon Biosensors, Long-Term Postdoctoral Fellowship for Christopher Witte, ; International Human Frontiers Science Program Organization, Imaging Cellular Function Non-invasively with genetically Engineered Reporters for Hyperpolarized MRI, Program Grant with Mikhail Shapiro, Chemical Engineering, California Institute of Technology ; $, FMP share: $ Deutsche Forschungsgemeinschaft, Multivalent Hosts for Hyperpolarized Xenon Enabling in vivo MRI Visualization of Tumor Cell Surface Glycans, Reinhard Koselleck-Förderung (first of its kind in the Leibniz Association); GZ SCHR 995 / 5 1; ; Michael J. Fox Foundation for Parkinson s Research, Developing a Molecular Imaging Tool That Binds to Alpha-Synuclein and Inhibits Its Formation, Porgram for Improved Biomarkers & Clinical Outcome Measures; Grant ID: 12549; ; $ Deutsche Forschungsgemeinschaft, Internationale Kooperationsanbahnung mit Prof. X. Zhou, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics, The Chinese Academy of Sciences; Live Animal MRI of Hyperpolarized Xenon for Hyper-CEST Applications ; GZ SCHR 995 / 6 1; ; Deutsche Forschungsgemeinschaft, Research Training Group BIOQIC Biophysical Quantitative Imaging Towards Clinical Diagnosis ; administered through Prof. I. Sack, Department of Radiology, Charité Universitätsmedizin Berlin; GRK 2260 / 1; ; , FMP share:

84 82 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 CORE FACILITY NMR GROUP LEADERS HARTMUT OSCHKINAT PETER SCHMIEDER SUMMARY The NMR facility responds to the many requests from inside and outside the FMP for the use of the DNP-equipment, the in-cell NMR facilities, or the NMR instruments in general. It also participates in the German DFG-funded G-NMR network of facilities, the inext initiative, and worldwide NMR studies such as the Fab-NMR-study by NIST. With the arrival of the group of Dorothea Fiedler the number of researchers performing chemical synthesis has increased, and so too has the demand for NMR support. Since the dedicated 300 MHz-spectrometer has been unable to meet the demands of all groups from the chemical biology department, one of the 600 MHz spectrometers has been converted into a second open-access spectrometer. In addition, a cryoprobe that is especially suited for 31 P-NMR helps to address more specific questions from within the chemical biology department, where several research projects are examining phosphorylation. Groups at the FMP that study the interaction of proteins with peptides or small molecules frequently rely on the capability of NMR spectroscopy for detecting weak protein-ligand interactions. Furthermore, the facility receives requests for access to standard NMR instruments from groups outside of the FMP, as well as from commercial companies situated near to the institute. These requests are fulfilled in the form of collaborations or as a NMR service provided by the facility. One example of an in-house collaboration is shown in Figure 1. As part of a study of the application of reductive caging of fluorescent dyes in super-resolution microscopy, the question of the mechanism of reduction arose and was clarified by the application of multidimensional NMR (Figure 1). This work was done in collaboration with the groups of Volker Haucke, Christian Hackenberger, and the Core Facility Cellular Imaging. A study of a peptide-protein interaction is exemplified in Figure 2. The interaction between a SEPT9-derived peptide and the SH3 domain of Cin85 revealed the binding site of the peptide on the protein and, together with other biophysical and biochemical methods, helped to show the downregulation of EGFR by SEPT9. This was done in collaboration with the group of Michael Krauß in the Department of Molecular Pharmacology and Cell Biology. ZUSAMMENFASSUNG Die NMR-Core-Facility bearbeitet die Vielfalt von Anfragen von inner- und außerhalb des FMP die Nutzung spezieller Geräte wie etwa der Instrumente für Dynamic Nuclear Polarization (DNP) oder der vorhandene NMR-Spektrometer im allgemeinen betreffen. Sie nimmt außerdem am deutschen DFG-geförderten G-NMR Netzwerk, an der inext-initiative sowie an weltweiten NMR-Studien wie der Untersuchung von Fab- Fragmenten durch das NIST mittels der NMR teil. Durch die Gründung der Gruppe von Dorothea Fiedler ist die Zahl der Wissenschaftler die chemische Synthese durchführen weiter angestiegen und damit auch die Nachfrage nach NMR-Unterstützung. Da das nur für die Synthese-Chemie genutzte 300 MHz-Spektrometer

85 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 83 Fig. 1: 1 H- and 13 C-NMR-spectra of atto488 (a) and reduced atto488 (b) were used to determine the constitution of reduced atto488, one of several dyes that can be used for reductive caging in supraresolution spectroscopy. The site of attachment of the proton transferred by NaBH4 was clarified using multidimensional NMR spectra.

86 84 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 2: Investigation of the interaction of a SEPT9-derived peptide with the SH3 domain of Cin85. (a) Overlay of 1 H, 15 N HSQC spectra of 15 N-labeled CIN85 SH3 containing increasing amounts of the SEPT9-derived peptide PxxxPR (no peptide, 0.56, 16, 26, 106 molar equivalents). (b) Ribbon model of CIN85 SH3A based on the X-ray structure B2Z8. Residues for which peptide-induced chemical shift changes were observed are highlighted in red and orange nicht in der Lage war, den Messzeit-Bedarf aller Gruppen des Bereichs Chemische Biologie zu decken wurde eines der 600 MHz-Spektrometer in ein weiteres Service-Spektrometer mit open access -Betrieb umgewandelt. Außerdem wurde ein Cryo-Probenkopf, der besonders für Messungen von 31P geeignet ist, beschafft um bei den zahlreichen Projekten in der Chemischen Biologie, die sich mit Phosphorylierung befassen, mitzuwirken. Gruppen am FMP die sich mit der Interaction von Proteinen mit Peptiden oder kleinen Molekülen befassen nutzen immer wieder die Fähigkeit der NMR-Spektroskopie schwache Protein- Ligand-Wechselwirkungen zu detektieren. Außerdem beantwortet die Facility die Anfragen zur Nutzung der Standard -NMR- Spektrometer entweder durch Gruppen außerhalb des FMP oder durch Firmen auf dem Campus Berlin-Buch. Diese Anfragen werden entweder in Form von Kooperationen oder als Dienstleistungen bearbeitet.

87 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 85 SELECTED PUBLICATIONS Bertran-Vicente J, Penkert M, Nieto-Garcia O, Jeckelmann J M, Schmieder P, Krause E, Hackenberger C P (2016) Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides. Nat Commun 7, Lehmann M, Gottschalk B, Puchkov D, Schmieder P, Schwagerus S, Hackenberger C P, Haucke V, Schmoranzer J (2015) Multicolor Caged dstorm Resolves the Ultrastructure of Synaptic Vesicles in the Brain. Angew Chem Int Ed Engl 54(45), Bertran-Vicente J, Schumann M, Schmieder P, Krause E, Hackenberger C P (2015) Direct access to site-specifically phosphorylated-lysine peptides from a solid-support. Org Biomol Chem 13(24), Diesenberg K, Beerbaum M, Fink U, Schmieder P, & Krauss M (2015) SEPT9 negatively regulates ubiquitin-dependent downregulation of EGFR. Journal of Cell Science 128(2), FMP authors Group members

88 Medicinal Chemistry Medizinische Chemie Group leader Dr. Marc Nazaré PAGE 106 Chemical Biology II Chemische Biologie II Group leader Prof. Dr. Christian P.R. Hackenberger PAGE 92 Chemical Biology I Chemische Biologie I Group leader Prof. Dr. Dorothea Fiedler PAGE 96 Peptide-Lipid Interaction / Peptide Transport Peptid-Lipid-Interaktion / Peptidtransport Group leader Dr. Margitta Dathe PAGE 100

89 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE Screening Unit Group leader Dr. Jens Peter von Kries PAGE 109 Mass Spectrometry Massenspektrometrie Group leader Dr. Eberhard Krause PAGE 103 SECTION CHEMICAL BIOLOGY BEREICH CHEMISCHE BIOLOGIE Peptide Synthesis Peptidsynthese Group leader Dr. Rudolf Volkmer PAGE 112

90 88 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 SECTION CHEMICAL BIOLOGY BEREICH CHEMISCHE BIOLOGIE Research projects in this section apply innovative synthetic and diagnostic chemical methods to probe the biological functions of cellular target molecules and thereby pave the way towards novel approaches in the pharmaceutical and medicinal sciences. Work carried out by the research groups in this department is devoted both to the synthesis and identification of novel bioactive molecules of high pharmacological potency and to the development of new chemical and analytical tools for the functional study of biologically relevant proteins and pathways. The Chemical Biology section is headed by Christian Hackenberger, who is now a Leibniz-Humboldt professor for Chemical Biology. 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. In the last two years these chemical tools have contributed to the engineering of new pharmaceutically active biopolymers and the understanding of post-translational modifications (PTMs) in protein function. Examples include the intracellular delivery of functional proteins and the identification of new targets for medicinal chemistry research in the area of viral infection and neurodegenerative diseases. Furthermore, site-specific protein conjugation methods, developed in the Hackenberger group, are currently being applied to pharmacologically relevant antibodies for targeted drug delivery and will be the basis for a planned start-up company. Another recent research highlight took place in close collaboration with the group of Eberhard Krause, who leads the Mass Spectrometry group at the FMP that focuses on high-resolution proteomic studies, with a particular focus on the role of protein-protein interactions and PTMs in cell signaling. The two laboratories contributed the first synthesis and MS-analysis of site-specifically phosphorylated lysine, as well as cysteine peptides. The Mass Spectrometry group also collaborates closely with Dorothea Fiedler s group in an effort to detect a labile PTM termed pyrophosphorylation. Dorothea Fiedler joined the FMP in 2015 and heads the Chemical Biology I department. She holds an appointment at the Humboldt University of Berlin, and is one of the Directors at the FMP. Research in the Fiedler group centers on using chemical approaches to elucidate signaling and metabolic networks, and their deregulation in disease. One particular set of molecules focused on by Fiedler s program is the so-called inositol pyrophosphates, a group of messengers involved in the regulation of body weight, life span, and fertility. To decipher their specific signaling functions, the group employs synthetic chemistry, host-guest chemistry, and chemical genetics, in combination with genome editing techniques and proteomics. For example, the group has identified the protein binding partners of the inositol pyrophosphate messengers from baker s yeast, using immobilized non-hydrolyzable analogs. In addition to bona fide protein binding partners, this analysis revealed the isolation of targets of protein pyrophosphorylation, an unusual modification mediated by the inositol pyrophosphates. To annotate endogenously pyrophosphorylated proteins, the lab has developed reagents for the enrichment of peptides containing pyrophosphoryl groups and teamed up with Eberhard Krause s group to implement a robust strategy for detecting this modification using mass spectrometry. By analyzing the signaling properties of the inositol pyrophosphates in healthy and diseased states, the group plans to explore how inositol pyrophosphate metabolism can be harnessed for therapeutic purposes. Robert Puschmann

91 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 89 The Peptide-Lipid Interaction group, led by Margitta Dathe, has a long-standing expertise in the development of peptide-modified liposomal drug carriers. In cooperation with the University of Münster they developed an enzyme substitution therapy for the rare skin disorder transglutaminase 1-deficient ichthyosis, granted orphan designation by the European Commission in Current efforts are focused on the development of a pathogenesis-based therapy for the peeling skin disease. An important aim within the Chemical Biology Platform at the FMP is the chemical optimization and validation of first hits discovered in the small molecule screening of pharmacological targets. The Medicinal Chemistry group led by Marc Nazaré is developing new chemical tools using strategies like fragment growing, re-scaffolding approaches, and structure-based design to improve the initial screening hit rate. These efforts have led to new small molecule probes for kinase PI3KC2α, the phosphatase SHP2, tryptophanhydroxylase TPH1, and the Poly-ADP-ribosyltransferase tankyrase, with some of these now being profiled in pharmacological models in vivo. Moreover, the FMP small molecule screening collection was considerably enhanced (it now stands at 66,000 compounds) through academic donations, a newly approved drug sub-set, and its own synthetic libraries. The platform s research activities are deeply 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. Significant effort has been invested to provide professional core facilities, in particular the internationally recognized Screening Unit, led by Jens von Kries, that supports high-throughput screening of small molecule and RNAi libraries. Scientific highlights of research within the Screening Unit include the identification of approved drugs to rescue heart development in zebrafish embryos and work led by Thomas Jentsch using genome-wide RNA interference that led to the identification of the gene-encoding VRAC that is essential for regulation of cellular water content in cells. The Screening Unit has also served as a central anchoring point for projects within the Leibniz research network Drug Research and Biotechnology, the Helmholtz consortium Drug Research Initiative, the Berlin Institute of Health (BIH), and the neighboring Max-Delbrück-Center for Molecular Medicine. In addition, the Screening Unit provides a focal point of the Chemical Biology Platform, as a key partner of the EU- OPENSCREEN initiative. Overall, the Chemical Biology section has contributed important chemical methods and discoveries to research at the FMP, 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 activities support the core mission of the FMP to develop new principles at the molecular level for pharmacological intervention in biological processes that ultimately shall lead to new possibilities in the treatment of diseases. Michael Schümann Kristina Siebertz and Oliver Reimann

92 90 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014 Liudmila Perepelittchenko and María Pascual López-Alberca 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 in diesem Bereich 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. Christian Hackenberger, der als Leibniz-Humboldt-Professor berufen ist, fungiert als 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 der Transport funktionaler Proteine in lebende Zellen und die Identifizierung neuer Targets für die medizinisch-chemische Forschung im Kontext viraler Infektion und neurodegenerativer Erkrankungen. Des Weiteren werden neu entwickelte ortspezifische Konjugationsmethoden in der Herstellung pharmakologisch relevanter Antikörper-Konjugate für einen gezielten Wirkstofftransport ( Targeted Drug Delivery ) verwendet. Diese Methoden dienen als Grundlage für eine Firmengründung, die in naher Zukunft vollzogen wird. Ein weiteres neues Highlight wurde in enger Zusammenarbeit mit der Gruppe von Eberhard Krause durchgeführt, der die Massenspektrometrie-Gruppe am FMP leitet und sich auf hochauflösende Proteomstudien konzentriert, wobei der Schwerpunkt auf der Rolle von Protein- Protein-Wechselwirkungen und PTMs in der Signalweiterleitung in der Zelle liegt. Die beiden Labore trugen zur ersten Synthese und MS-Analyse von ortsspezifisch phosphoryliertem Lysin sowie Cystein-Peptiden bei. Die Gruppe Massenspektrometrie arbeitet auch eng mit der Arbeitsgruppe von Dorothea Fiedler zusammen, mit dem Ziel, eine labile PTM, die Pyrophosphorylierung, nachweisen zu können. Dorothea Fiedler kam im Jahr 2015 zum FMP und leitet dort die Abteilung Chemische Biologie I. Sie wurde an die Humboldt- Universität zu Berlin berufen und ist eine der Direktoren am FMP. Die Forschung in der Fiedler-Gruppe konzentriert sich auf die Verwendung von chemischen Ansätzen zur Erkennung von Signal- und Stoffwechselnetzen und deren Deregulierung bei Krankheiten. Im Fokus ihres Forschungsprogramms stehen dabei sogenannte Inositol-Pyrophosphate, eine Gruppe von Botenstoffen, die bei der Regulierung des Körpergewichts, der Lebenserwartung und der Fruchtbarkeit eine Rolle spielen. Um deren spezifische Signalfunktionen zu entschlüsseln, setzt die Gruppe synthetische Chemie, Wirt-Gast-Chemie und chemische Genetik in Kombination mit Geneditierungstechniken und Proteomik ein. So ist es der Gruppe z.b gelungen, mit immobilisierten, nichthydrolysierbaren Analoga Protein-Bindungspartner der Inositol- Pyrophosphat-Botenstoffe aus Bäckerhefe zu identifizieren. Neben bona fide Protein-Bindungspartnern führte diese Analyse zur Isolierung von Substraten der Protein-Pyrophosphorylierung, einer ungewöhnlichen durch Inositol-Pyrophosphate vermittelten Modifikation. Um die endogen pyrophosphorylierten Proteine näher zu charakterisieren, wurden in ihrem Labor Reagenzien zur Anreicherung von Peptiden, die Pyrophosphatgruppen enthalten, entwickelt. Und gemeinsam mit der Arbeitsgruppe von Eberhard Krause soll mithilfe der Massenspektrometrie eine robuste Strategie zum Nachweis dieser Modifikation implementiert werden. Durch Analyse der Signalübertragungseigenschaften der Inositol- Pyrophosphate in gesundem und erkranktem Zustand möchte die

93 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 91 Edgar Specker, Martin Neuenschwander and Silke Radetzki Gruppe herausfinden, wie der Inositolpyrophosphat-Stoffwechsel für therapeutische Zwecke genutzt werden kann. Die Arbeitsgruppe Peptid-Lipid-Interaktion von Margitta Dathe besitzt seit Langem große Expertise in der Entwicklung von peptidmodifizierten liposomalen Carriern. Die Arbeitsgruppe entwickelte in Kooperation mit der Universität Münster eine Enzymsubstitutionstherapie für die seltene Hautkrankheit Transglutaminase 1-deficient Ichthyosis, der 2013 durch die Europäische Kommission der Orphan Drug -Status zuerkannt wurde. Gegenwärtige gemeinsame Aktivitäten richten sich auf die Entwicklung einer Therapie zur Behandlung der Peeling Skin Disease. 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 entwickelt neue chemische Werkzeuge und nutzt dabei zur Optimierung der Screening-Hits Strategien wie Fragmentwachstum, Neuanordnung von Molekülgerüsten und strukturgeleitetes Design. Die Arbeiten der Gruppe resultierten in neuen kleinen Molekülen, die als Sonden zur Beeinflussung der PI3KC2α Kinase, der Phosphatase SHP2, der Tryptophanhydroxylase TPH1 oder der Poly-ADP-Ribosyltransferase Tankyrase wirken. Einige dieser Substanzen werden gegenwärtig in pharmakologischen Tiermodellen in vivo evaluiert. Zudem wurde die Substanzsammlung des FMP für das Small Molecule Screening durch die Integration von akademischen Substanz-Spenden, zugelassenen Medikamenten und eigenen synthetischen Verbindungen auf nunmehr Substanzen erheblich erweitert. Die Forschungsaktivitäten des Bereiches Chemische Biologie sind eng mit vielen Gruppen der anderen Bereiche verzahnt. Besonders sind hier aus dem Bereich Strukturbiologie die Strukturelle Bioinformatik und Proteindesign -Gruppe von Gerd Krause und die Wirkstoff-Design -Gruppe von Ronald Kühne 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 unterstützt Hochdurchsatz-Screening von kleinen Molekülen und RNAi- Bibliotheken. Wissenschaftlich herausragend ist hierbei die Identifizierung von zugelassenen Arzneimittel-Wirkstoffen, die Defekte in der Entwicklung des Herzens von Zebrafischembryos beheben können und somit für entsprechende humane Krankheiten eingesetzt werden könnten. Darüber hinaus wurde in der Unit durch RNA-Interferenz in Zusammenarbeit mit Thomas Jentsch das Gen für den Ionenkanal identifiziert, der in jeder Zelle den Wasserhaushalt reguliert. Die Screening Unit dient weiterhin als Dreh- und Angelpunkt für Forschungskooperationen im Leibniz Forschungsverbund Wirkstoffforschung und Biotechnologie, im Helmholtz-Konsortium Wirtstoffforschung, mit dem Berliner Instituts für Gesundheitsforschung (BIH) und mit dem benachbarten Max-Delbrück-Centrum für Molekulare Medizin (MDC). Gleichzeitig ist die Screening Unit mit der Chemical Biology Platform des FMP ein zentraler Partner der EU-OPENSCREEN- Initiative. 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.

94 92 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 CHEMICAL BIOLOGY II CHEMISCHE BIOLOGIE II GROUP LEADER PROF. DR. CHRISTIAN P.R. HACKENBERGER BIOGRAPHY 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), 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), Free University of Berlin Emmy-Noether-Group (DFG) leader, Free University of Berlin Habilitation and Associate Professor (W2) for Bioorganic Chemistry, Free University of Berlin 2013 Margaret and Harlan Goering Visiting Professor, University of Wisconsin / Madison, USA 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 SUMMARY In the densely packed world of a cell, protein modifications control many signaling pathways that support healthy functioning and that are disrupted in disease. Common examples include protein phosphorylation and glycosylation, but increasingly other post-translational modifications (PTMs) such as acetylation, ubiquitylation, and methylation, are being identified as important toggle switches in health and disease. Chemical biologists want to control these protein modifications in the cell, both to study the biological role of PTMs and to decorate proteins with fluorescent moieties that permit their visualization. The Hackenberger laboratory aims to identify new chemical reactions that allow the modification 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 bioconjugation reactions to study the functional consequences of natural protein modifications, as well as to generate novel peptide- and protein-conjugates, in particular antibody-drug conjugates (ADCs), for pharmaceutical and medicinal applications (Figure 1). ZUSAMMENFASSUNG In der dicht gepackten Welt einer Zelle werden viele Signalwege, die normales Leben steuern und bei Krankheit gestört sind, durch Veränderungen an Proteinen reguliert. Am häufigsten sind hierbei Phosphorylierungen und Glycosylierungen zu beobachten; vermehrt werden aber auch andere solcher posttranslationaler Modifikationen wie Acetylierung, Ubiquitylierung und Methylierung identifiziert, die wie Wechselschalter zwischen Gesundheit und Krankheit wirken können. Wissenschaftler / innen in der Chemischen Biologie versuchen deshalb zunehmend die Modifizierung von Proteinen in der Zelle zu kontrollieren, um entweder die biologische Rolle solcher posttranslationalen Modifikationen zu erforschen oder um Proteine mit fluoreszierenden Gruppen zu versehen, die ihre Visualisierung ermöglichen. Das Labor von Christian Hackenberger hat sich zum Ziel gesetzt neue chemische Reaktionen 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 auf der Entwicklung hochselektiver organisch-chemischer Methoden für die Biokonjugation, welche die Auswirkungen natürlich vorkommender Modifikationen auf die Funktion von Proteinen untersuchen und neue medizinische und pharmakologische Anwendungen, beispielsweise durch Antikörper-Wirkstoff-Konjugate (antibody-drug-conjugates, ADCs), ermöglichen (Figure 1). Since 2012 Leibniz-Humboldt Professor (W3) for Chemical Biology funded by the Einstein Foundation Berlin

95 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 93 DESCRIPTION OF PROJECTS Bioorthogonal Staudinger Reactions: chemical phosphorylation, PEGylation, and more Over the years, we have introduced the Staudinger-phosphite reaction as a bioorthogonal reaction for the modification of azide-containing biomolecules. In a first biological application of this reaction we engineered a chemoselective phosphorylation of proteins, which allowed the site-specific incorporation of a phospho-tyr mimetic into full-length proteins. Recently, we extended this concept to the site-specific phosphorylation of Lys-peptides, which represents a very labile PTM and which we were able to analyse using electron transfer dissociation tandem mass spectrometry (ETD / MS / MS) in collaboration with Eberhard Krause (FMP Berlin). Furthermore, the use of nucleophilic phosphites also allowed us to obtain site-specifically phosphorylated Cys-peptides, which were again analysed by ETD / MS / MS (Figure 2). With this protocol in hand we detected the phosphorylation of a Cys-residue in the glycolysis pathway. In subsequent studies, we used 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 Staudinger-reactions are efficient transformations even in lysates, and further employed these reactions for an efficient and metal-free peptide or protein PEGylation to deliver a new class of branched oligoethylene glycol scaffolds for the stabilization against degradation in plasma or the cytosol. Probing the impact of post-translational modifications on peptide and protein aggregation: semi-synthesis of the Alzheimer s disease-relevant Tau protein In this project we are studying the structural consequences of post-translational modifications on pharmacologically relevant proteins, in particular 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, and 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 with the recent 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 Caroline Smet-Nocca, 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. Chemical approaches to modulate sialylation in vivo In collaboration with Prof. Stephan Hinderlich (Beuth- Hochschule) we recently expanded the repertoire of unnaturally modified sialylated 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 are of interest for the development of new diagnostic and therapeutic glycoproteins and potentially can be applied to the in vivo modification of living animal models. Furthermore, in collaboration with the late Werner Reutter (Charité Berlin), we have identified mannosamine-based diselenides as highly potent inhibitors of the ManNAc-kinase, a key enzyme in the biosynthesis of sialic acid, and showed that addition of these inhibitors to a cell model system reduced sialylation. 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. Chemoenzymatic labeling and cellular delivery of proteins and antibodies In collaboration with Prof. Leonhardt (LMU Munich) and Prof. Cardoso (TU Darmstadt) we aim to develop powerful new methods for the chemoenzymatic modification of proteins as well as antibodies. Our long-term goal within this project is to generate new antibody-drug conjugates (ADCs) as well as cell-permeable antigen-recognizing proteins. To reach this aim we have recently

96 94 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 engineered so-called Tub-tag labeling, which allows the modular C-terminal modification of proteins by the enzyme tubulin tyrosine ligase and which will be the basis of a new start-up company in the near future (Tubulis Technologies). Additionally, in a proof of concept study we were able to transport a functional full length protein to the cytosol and the nucleus of living cells by conjugation of the protein with cyclic cell-penetrating peptides (ccpps). Next we will combine these approaches for the fluorescent labeling of nanobodies, small antigen-binding proteins that remain active within the reductive milieu inside living cells, functionalized with ccpps to directly detect intracellular targets after cellular uptake. 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 GROUP MEMBERS Dr. Debasish Bhowmick Dr. Wenyi Li Dr. Olaia Nieto Lukas Artner (doctoral student) Lorenzo Assennato (doctoral student) Alice Baumann (doctoral student) Jordi Bertran (doctoral student) Maria Glanz (doctoral student) Marc-André Kasper (doctoral student) Simon Klenk (doctoral student) Michaela Mühlberg (doctoral student) Nicole Nischan (doctoral student) Martin Penkert (co-supervised with Dr. Eberhard Krause) Oliver Reimann (doctoral student) Simon Reiske (doctoral student) Tom Sauer (doctoral student) Dominik Schumacher (doctoral student) Anselm Schneider (doctoral student) Sergej Schwagerus (doctoral student) Kristina Siebertz (doctoral student) Kristin Kemnitz-Hassanin (technical assistant) Dagmar Krause (technical assistant) Inez Kretzschmar (technical assistant, Peptide service facility) Staff employed within the reporting period COLLABORATIONS International Caroline Smet-Nocca, Université des Sciences et Technologies de Lille, France Roland Brock, Radboud University Nijmegen Medical Centre, The Netherlands Eric Strieter, University of Massachusetts- Amherst, MA, USA Ron Raines, Massachusetts Institute of Technology (MIT), MA, USA National Nedjliko Budisa, Technische Universität Berlin Jens Dernedde, Charité Universitäts medizin Berlin Werner Reutter, Charité Universitätsmedizin Berlin Stephan Hinderlich, Beuth-Hochschule, Berlin Eberhard Krause, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Jens-Peter von Kries, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Janine Kirstein, Leibniz-Forschungsinstitut für Mole kulare Pharmakologie (FMP) Rainer Haag, Freie Universität Berlin Bettina Keller, Freie Universität Berlin Roland Netz, Freie Universität Berlin Bernd Lepenies, Leibniz-Universität Hannover Andreas Herrmann, Humboldt Universität zu Berlin Cristina Cardoso, Technische Universität Darmstadt Heinrich Leonhardt, Ludwig-Maximilians-Universität München

97 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 95 Fig 2: Chemoselective synthesis and ETD-MS / MS analysis of site-specifically phosphorylated Cys-peptides (Reference: J. Bertran-Vicente, M. Penkert, O. Nieto-Garcia, J.-M. Jeckelmann, P. Schmieder, E. Krause, C.P.R. Hackenberger, Nature Comm. 2016, 7, Article number: 12703, Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides) electrophilic disulfide Ellman's reagent CHEMICAL PHOSPHORYLATION ETD MS / MS SELECTED PUBLICATIONS Bertran-Vicente J, Penkert M, Nieto-Garcia O, Jeckelmann J-M, Schmieder P, Krause E, Hackenberger C P R * (2016) Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides. Nature Comm. 7, Article number, Nieto-Garcia O, Wratil P R, Nguyen L D, Böhrsch V, Hinderlich S, Reutter W*, Hackenberger C P R * (2016) Inhibition of key enzyme of sialic acid biosynthesis by C6-Se modified N-acetylmannosamine analogs. Chem. Sci. 7, Schumacher D, Helma J, Mann F A, Pichler G, Natale F, Krause E, Cardoso M C, Hackenberger C P R *, Leonhardt H* (2015) Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase. Angew. Chem. Int. Ed. 54, 46: Vielseitige, effiziente und ortsspezifische Proteinfunktionalisierung durch das Enzym Tubulin Tyrosin Ligase. Angew. Chem. 127, 46, Nischan, N., Herce, H.D., Natale, F., Bohlke, N., Budisa, N., Cardoso, M.C.,* Hackenberger C P R * (2015) Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability. Angew. Chem. Int. Ed. 54, 6: Kovalente Verknüpfung cyclischer TAT-Peptide mit GFP resultiert in der direkten Aufnahme in lebende Zellen mit sofortiger biologischer Verfügbarkeit. Angew. Chem. 127, 6, Reimann O, Smet-Nocca C, Hackenberger C P R * (2015) Traceless Purification and Desulfurization of Tau Protein Ligation Products. Angew. Chem. Int. Ed. 54, 1: Spurlose Aufreinigung und Desulfurierung von Ligationsprodukten des Tau-Proteins. Angew. Chem. 127, 1, EXTERNAL FUNDING Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623 Chemoselective reactions for the synthesis and application of functional proteins, funds for the coordination of the priority programme, , 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), , Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623, Chemoselective Staudinger-induced Michael-additions to antibodies to analyze protein homeostasis in C.elegans, jointly with J. Kirstein (FMP Berlin), , Boehringer-Ingelheim Stiftung, Chemoselective Staudinger-reactions for the modification of peptides and proteins, Plus 3 - Programme, , Deutsche Forschungsgemeinschaft, SFB 765 B05, Synthesis of multivalent ligand binding systems via chemoselective saccharide- and peptide-ligations (1st funding period ), Site-specific functionalization of proteins for the acquisition of multivalent glycoconjugtes (2nd and 3rd funding period ), , jointly with N. Budisa (TU Berlin), Deutsche Forschungsgemeinschaft, SFB 765 (1st and 2nd funding period ), Integriertes Graduiertenkolleg des SFB, , FMP authors Group members

98 96 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 CHEMICAL BIOLOGY I CHEMISCHE BIOLOGIE I GROUP LEADER PROF. DR. DOROTHEA FIEDLER BIOGRAPHY Undergraduate studies and Diploma in Chemistry, Ludwigs-Maximilians-Universität Würzburg Diploma thesis with Prof. John Arnold, University of California at Berkeley, USA Graduate studies in Inorganic and Supramolecular Chemistry with Prof. Kenneth N. Raymond and Prof. Robert G. Bergman, University of California at Berkeley, USA Postdoctoral research in Molecular Biology / Chemical Biology with Prof. Kevan M. Shokat, University of California at San Francisco, USA 2009 NIH Pathway to Independence Award Assistant Professor of Chemistry, Princeton University, USA 2013 NIH Director s New Innovator Award 2013 Rita Allen Scholar Award Since 2015 Full Professor (W3-S) of Chemical Biology, Humboldt University of Berlin, and Director at the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin SUMMARY Our group seeks to develop a better understanding of the multiple ways in which nature utilizes phosphate in both protein signaling cascades and metabolic networks. Signaling and metabolic pathways are very complex, but the individual steps within these cascades depend on simple chemical reactions and often involve the reversible addition of phosphoryl groups. As deregulation of cellular information transfer is associated with a wide range of diseases, a detailed annotation of signaling events in healthy and diseased states can help to highlight new avenues for therapeutic intervention. One group of densely phosphorylated messengers of particular interest to our group are the inositol pyrophosphates (PP-InsPs). These molecules have emerged as central regulators of cell homeostasis, and genetic studies in mice and humans implicate PP-InsPs in a host of processes including weight gain, height regulation, fertility, and longevity. The rapid cellular turnover of PP-InsPs has led to the hypothesis of an inositol pyrophosphate code, in which the temporally and spatially controlled production of a particular PP-InsP dictates specific downstream signaling events. However, how these molecules exert their effects at the molecular level is not well understood. Using a multi-disciplinary approach that employs techniques from inorganic and organic chemistry, chemical genetics and genetics, molecular biology, and proteomics, it is our goal to decipher the concrete signaling functions of PP- InsPs and ultimately to guide the development of new therapeutic strategies against cancer, diabetes, and obesity. ZUSAMMENFASSUNG Unsere Gruppe versucht, ein besseres Verständnis zu entwickeln, wie die Natur Phosphat in Protein-Signalkaskaden und metabolischen Netzwerken verwendet. Signal- und Stoffwechselwege sind sehr komplex, aber die einzelnen Schritte innerhalb dieser Kaskaden hängen von einfachen chemischen Reaktionen ab und beinhalten oft die reversible Addition von Phosphorylgruppen. Da die Deregulierung des zellulären Informationstransfers mit einer Vielzahl von Krankheiten einhergeht, kann eine ausführliche Erläuterung von Signalereignissen in gesunden und kranken Zuständen dazu beitragen, neue Wege für therapeutische Interventionen zu finden. Von besonderem Interesse ist eine Gruppe von phosphorylierten Botenstoffen, die Inositolpyrophosphate (PP-InsPs). Diese Moleküle sind als zentrale Regulatoren der Zellhomöostase bekannt, und genetische Untersuchungen an Mäusen und Menschen implizieren PP-InsPs in einer Vielzahl von Prozessen, einschließlich Gewichtszunahme, Wachstum, Fruchtbarkeit und Langlebigkeit. Der schnelle zelluläre Umsatz von PP-InsPs hat zur Hypothese eines Inositolpyrophosphat-Codes geführt, bei dem die zeitlich und räumlich kontrollierte Produktion eines bestimmten PP-InsPs spezifische nachgeschaltete Signalereignisse vorschreibt. Doch wie diese Moleküle ihre Wirkung auf molekularer Ebene ausüben, ist noch nicht ausreichend untersucht. Mit einem multidisziplinären Ansatz, der Techniken aus der anorganischer und organischer Chemie, der chemischen Genetik und der Genetik, der Molekularbiologie und der Proteomik einsetzt, ist es unser Ziel, die konkreten Signalisierungsfunktionen von PP-InsPs zu entschlüsseln und letztlich die Entwicklung neuer therapeutischer Strategien gegen Krebs, Diabetes und Fettleibigkeit zu begleiten.

99 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 97 DESCRIPTION OF PROJECTS New analytical approaches for the detection of cellular inositol poly- and pyrophosphate species Despite their essential functions in cell physiology, the analysis of inositol polyphosphates (InsPs) and PP-InsPs remains a formidable challenge. As a result, information on the production, local concentrations, biosynthetic pathways, and exact chemical nature of InsP messengers is sparse and sometimes controversial. In collaboration with H. Oschkinat (FMP) and V. Haucke (FMP) we are seeking to adress these questions by using 13 C-labeled inositol, in combination with NMR spectroscopy, to enable the detection and quantification of InsPs and PP-InsPs in complex samples. Through chemical synthesis of 13 C-labeled InsPs and PP-InsPs, we have established the relevant reference spectra, and metabolic labeling of cells with 13 C-inositol proceeds smoothly without the side-effects of using radioactive materials (which is the standard procedure). In a parallel effort, we have also developed a derivatization strategy for the highly charged InsPs / PP-InsPs to enable their detection using mass spectrometry with high sensitivity. In the long term, these convenient bioanalytical approaches will find widespread applications in the fields of membrane traffic and inositol signaling, and which help elucidate the multifaceted effects of these central molecules in a wide range of biological contexts. Characterization of inositol pyrophosphate binding proteins It is the general consensus that inositol phosphates regulate protein function by binding to their targets, but the literature on PP- InsP interacting proteins is sparse. Therefore, our group set out to develop affinity reagents to identify PP-InsP binding partners on a proteome-wide scale. To achieve this goal we have designed, synthesized, and characterized PP-InsP analogs that incorporate a bisphosphonate moiety in place of the pyrophosphate group. We have shown that the bisphosphonate group adequately mimics the pyrophosphate group with regards to both chemical and biochemical properties, while providing improved synthetic accessibility and increased stability. metabolic networks that maintain cellular energy homeostasis. Notably, we also isolated several known and novel substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by PP-InsPs. Our findings not only demonstrate that PP- InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action. Chemical tools for annotating the pyrophosphoproteome In addition to the traditional binding mechanism, a covalent protein modification termed protein pyrophosphorylation has been proposed for PP-InsPs. Efforts to characterize protein pyrophosphorylation have exclusively relied on in vitro labeling strategies; consequently, many questions about the regulation of pyrophosphorylation in vivo still linger. To address these shortcomings, our group has implemented a set of complementary methods with the goal of comprehensively annotating in vivo pyrophosphorylated proteins. We have developed a convenient synthetic approach to obtain pyrophosphorylated peptides. These peptides are currently being used to investigate the feasibility of mass spectrometry-based detection in complex mixtures (in collaboration with E. Krause, FMP). In parallel, we devised a method for the incorporation of a stabilized pyrophosphoserine moiety into peptide sequences. With the goal of generating antibodies against the pyrophosphoserine group. To complement these antibodies, which may display sequence sensitivity, an affinity reagent for the enrichment of pyrophosphoserine-containing proteins from cell lysates was developed. This reagent contains a cavity that allows for selective recognition of pyrophosphate esters. A combination of these newly developed techniques is now on the verge of providing the first comprehensive analysis of endogenous pyrophosphoproteins. The analogs were converted to affinity reagents via attachment to a solid phase resin and applied to cell lysates from S. cerevisiae. Over 150 putative PP-InsP interacting proteins were identified, among them a large proportion of proteins involved in phosphate metabolism, glucose metabolism, and ribosome biogenesis. Expanding this approach to other organisms is expected to provide the molecular details of how PP-InsPs can intersect with protein signaling and

100 98 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 PP-InsP binding proteins Pyrophosphorylation substrates P P P P PCP P P P P P PCP P 5PP-InsP 5 beads P P P P PCP P Nucleotide metabolism Polyphosphate metabolism Protein phosphorylation Carbohydrate metabolism Ribosome biogenesis Fig 1: Inositol pyrophosphates are ubiquitous eukaryotic messengers and are involved in numerous cellular processes. By applying chemically synthesized affinity reagents, inositol polyphosphate binding proteins were comprehensively annotated in the model organism Saccharomyces cerevisiae. The protein targets are diverse in function and highlight the complex regulation of cellular signaling and metabolic networks by inositol pyrophosphates. GROUP MEMBERS Dr. Barbara Dul Dr. Anastasia Hager Dr. Sarah Hostachy Dr. Javier Moreno Dr. Florence Williams John Conway (Ph.D. student) Jeffrey Bratz (Ph.D. student) Nathaniel Brown (Ph.D. student) Robert Harmel (Ph.D. student) Alan Marmelstein (Ph.D. student) Cesar Perez Ramirez (Ph.D. student) Robert Puschmann (Ph.D. student) Mingxuan Wu (Ph.D. student) Lisa Yates (Ph.D. student) Natascha Heinsohn (Master student) Lucy Chong (Visiting scholar) Katy Franke (technical assistant) Lena von Oertzen (technical assistant) Staff employed within the reporting period COLLABORATIONS International Adam Resnick, University of Pennsylvania, USA Solomon Snyder, Johns Hopkins University, USA Rashna Bhandari, CDFD, India Adolfo Saiardi, University College London, Great Britain Roberto Docampo, University of Georgia, USA Stephen Shears, NIH, Triangle Park, North Carolina, USA Christopher Barker, Karolinska Institutet, Sweden Andreas Mayer, University of Lausanne, Switzerland Michael Hothorn, University of Geneva, Switzerland Sebastian Hiller, University of Basel, Switzerland Robbie Loewith, University of Geneva, Switzerland Julianne Djordjevic, University of Sydney, Australia Nicolas Veiga, University of Montevideo, Uruguay National Henning Jessen, Universität Freiburg Thomas Schrader, Universität Duisburg-Essen Eberhard Krause, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Volker Haucke, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Hartmut Oschkinat, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)

101 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 99 Fig 2: Protein pyrophosphorylation is a new post-translational modification. a) Protein pyrophosphorylation is mediated by inositol pyrophosphate messengers. b) Synthetic peptide standards have guided the development of an enrichment procedure and enabled the detection of these species by mass spectrometry. SELECTED PUBLICATIONS Wu M, Chong L S, Perlman D H, Resnick A C, Fiedler D (2016) The inositol polyphosphates intersect with protein signaling and metabolic networks via two distinct mechanisms. Proc. Nat. Acad. Sci. USA 113, E6757 E6765. Brown N W, Marmelstein A M, Fiedler D (2016) Chemical tools for interrogating inositol pyrophosphate structure and function. Chem. Soc. Rev. 45, Hager A, Wu M, Wang H, Brown Jr. N W, Shears S B, Veiga N, Fiedler D (2016) Cellular cations control conformational switching of inositol pyrophosphate analogs. Chem. Eur. J. 22, Williams F J, Fiedler D (2015) A fluorescent sensor and gel stain for detection of pyrophosphorylated proteins. ACS Chem. Biol. 10, Conway J H, Fiedler D (2015) An affinity reagent for recognition of pyrophosphorylated peptides. Angew. Chem. Int. Ed. 54, EXTERNAL FUNDING National Institute of Health, NIH Director s New Innovator Award Program. Understanding phosphate metabolism in cancer and metastasis , US $ Sidney Kimmel Foundation for Cancer Research, Kimmel Scholar Program. Does inorganic phosphate promote metastasis to bone? , US $ Rita Allen Foundation, Rita Allen Scholars Program. Understanding phosphate metabolism in cancer and metastasis , US $ Swiss National Science Foundation, Sinergia. Joint with A. Mayer, S. Hiller, M. Hothorn. Discovery and mechanistic dissection of novel signaling pathways controlling phosphate homeostasis in eukaryotes , CHF Leibniz Gemeinschaft, Leibniz Wettbewerb. Joint with H. Oschkinat, V. Haucke. Systems level analysis of inositol messengers in nutrient signaling , FMP authors Group members

102 100 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 PEPTIDE-LIPID INTERACTION / PEPTIDE TRANSPORT PEPTID-LIPID-INTERAKTION / PEPTIDTRANSPORT GROUP LEADER DR. MARGITTA DATHE BIOGRAPHY 1974 Diploma thesis in Physics, Humboldt-University Berlin, GDR 1978 Ph.D, Academy of Sciences of the GDR Research Associate, Institute of Drug Research of the Academy of Sciences of the GDR Team Leader of the Conformational Analysis Group, FMP since 1999 Team Leader of the Peptide Lipid Interaction / Peptide Transport Group, FMP 2015 Leibniz Drug of the Year Award of the Leibniz Research Alliance Bioactive Compounds and Biotechnology SUMMARY Our group is interested in the interaction of cell-penetrating and cell-permeabilizing peptides with membranes of human and bacterial cells. We exploit membrane-translocating peptides as targeting and uptake-mediating tools for lipid-based carrier systems loaded with diagnostic or therapeutic drug molecules. Currently, our efforts are focused on targeting the blood-brain barrier and delivering bioactive compounds via the skin. Furthermore, we are attempting to elucidate the mode of action of small cyclic arginine and tryptophan-rich antimicrobial peptides. Unlike the common membrane-permeabilizing mechanism of antimicrobial peptides, these compounds activate a novel mode of action against prokaryotic cells and are able to penetrate the membrane of eukaryotic cells. In this way they might offer new ways to generate antimicrobial compounds for selected applications against intracellular pathogens. ZUSAMMENFASSUNG Uns interessiert die Zell-penetrierende und Membran-permeabilisierende Wirkung von Peptiden auf menschlichen Zellen und Bakterien. Zell-penetrierende Peptide werden genutzt, um die Aufnahme Lipid-basierter Carriersysteme, die mit diagnostisch oder therapeutisch wirksamen Substanzen beladen wurden, in Zellen zu vermitteln. Gegenwärtig ist unsere Forschung auf die Überwindung der Blut-Hirn-Schranke und auf eine effektive Aufnahmevermittlung bioaktiver Substanzen in die Haut gerichtet. Ein weiterer Forschungsschwerpunkt ist das antimikrobielle Wirkungsprinzip zyklischer Peptide mit einem hohen Anteil an den Aminosäuren Arginin und Tryptophan. Sie wirken nicht durch Permeabilisierung der Bakterienmembran sondern aktivieren einen neuartigen Wirkmechanismus und sind außerdem in der Lage, die Membran von Säugerzellen zu überwinden. Damit eröffnen sie neue Wege zur Entwicklung antimikrobieller Peptide zur Bekämpfung intrazellulärer Pathogene.

103 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 101 DESCRIPTION OF PROJECTS Small cyclic antimicrobial peptides Optimizing the activity and bacterial selectivity of antimicrobial peptides (AMPs) requires an understanding of their mechanism of action. With its amphipathic structure and high content of arginine residues, the synthetic cyclic hexapeptide cwfw (cyclo (RRRWFW)) is highly membrane-active. However, in contrast to most antimicrobial peptides, cwfw neither permeabilizes the membrane nor translocates into the cytoplasm of bacteria. In a collaboration with the Centre for Bacterial Cell Biology, Newcastle University, UK, we have shown that cwfw instead triggers a rapid reduction of membrane fluidity, both in live Bacillus subtilis cells and in bacterial model membranes. This immediate activity is accompanied by the formation of distinct membrane domains which differ in local membrane fluidity and which severely disrupt membrane protein organization by segregating peripheral and integral proteins into domains of different rigidity (Fig. 1). We consider these major membrane disturbances as key events that cause specific inhibition of bacterial cell wall synthesis, and trigger autolysis. Additionally, the peptide was found to be non-toxic against eukaryotic cells (HELA) and to translocate into their cytoplasm using an endocytotic uptake route. The uptake is concentration- and time-dependent, and modified by efflux pumps. Peptide-bearing lysosomes seem to accumulate in the endoplasmic reticulum of the cells. Fig. 1: cwfw-triggered formation of lipid domains and segregation of membrane proteins (a) Phase contrast, GFP-protein fluorescence, Nile red staining of lipid domains and fluorescent color overlays for cells expressing different integral membrane proteins (upper panels) and different peripheral membrane proteins (lower panels) in the presence of cwfw (20 min incubation with 12 μm). Whereas there is uniform membrane fluorescence of GFP-proteins and Nile red in non-treated cells (not shown), the formation of domains of different fluidity is observed after incubation with cwfw. Integral proteins accumulate in Nile red-free regions whereas peripheral proteins co-localize with Nile red, the regions in which cwfw also accumulates. Strains used: (a) B. 816 subtilis BS23 (AtpA-GFP), B. subtilis HS41 (YhaP-GFP), B. subtilis HS64 (WALP23-GFP), 817 (b) B. subtilis KR318 (SpoVM-GFP), B. subtilis HS65 (GFP-MinDMTS) and B. subtilis HS (SepFMTS-GFP), Reference: Scheinpflug K. et al. (2017) Antimicrobial peptide cwfw kills by combining lipid phase separation with autolysis. Scheinpflug et al. (2017) Scientific Reports 7: The novel antibacterial mode of action carries a low risk of inducing bacterial resistance and with its membrane-translocating ability the peptide provides a valuable basis for the design of new synthetic antimicrobial compounds for the treatment of intracellular pathogens. Peptide-modified micellar and liposomal 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). 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). We generated a lipopeptide, PCrAA2, with a covalently attached CrA. Studies in cooperation with L. Schröder (FMP) confirmed cell selectivity of PCrAA2 micelles and allowed us to distinguish BBB endothelial cells from control aortic endothelial cells based on high local cage concentration and reliable quantification of the signal molecule (Fig. 2). Thus, the micelles combine a high selectivity for human brain capillary endothelial cells with the great sensitivity of Xe Hyper-CEST MRI and might be a potential tool in MRI-based brain diagnostics. Ongoing activities with industrial partners to advance the preclinical development of a P2A2 peptide-modified liposomal preparation of recombinant Transglutaminase 1 (in cooperation with University Hospital, Münster) to treat autosomal recessive congenital ichthyosis (ARCI) underline the huge therapeutic and economic potential of this formulation. Further studies using liposomes decorated with an oligo-arginine lipopeptide showed that they are highly suitable for transporting Corneodesmosin to its site of action, the membrane of skin keratinocytes, and provide a promising basis for the development of a pathogenesis-based therapy for Peeping Skin Disease (in cooperation with University Hospital, Münster).

104 102 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 Fig. 2: Hyper-CEST MRI of human brain capillary endothelial cells (HBMEC, inner compartment) and aortic endothelial cells (HAoEC, outer compartment) incubated with PCrAA2 micelles (5 µm CrA; cells): a) axial Hyper-CEST MIR; b) pixel histogram illustrating the distribution of the Hyper-CEST effect. The figure illustrates the pronounced Hyper-CEST effect in the HBMEC compartment. GROUP MEMBERS Dr. Margitta Dathe (group leader) Dr. Oxana Krylova Kathi Scheinpflug (doctoral student, postdoc) Heike Nikolenko (technical assistant) Sven Richter (master student) Ines Volk (master student) Staff employed within the reporting period COLLABORATIONS International Henrik Strahl, Newcastle University, UK Marina Rautenbach, University Stellenbosch, South Africa National Heiko Traupe, University Münster, Germany Vincence Oji, University Münster, Germany Sonia Waiczies, MDC Berlin, Germany Alfred Blume, MLU Halle, Germany Martin Schulze, Institute for Reproduction of Farm Animals Schoenow, Bernau, Germany SELECTED PUBLICATIONS Scheinpflug K, Wenzel M, Krylova O, Bandow J E, Dathe M, Strahl H (2017) Antimicrobial peptide cwfw kills by combining lipid phase separation with autolysis. Scientific Report 7, Schnurr M, Sydow K, Rose HM, Dathe M, Schröder L (2015). Brain Endothelial Cell Targeting via a Peptide-functionalized Liposomal Carrier for Xenon Hyper-CEST MRI. Adv Healthcare Mat 4, Sydow K, Nikolenko H, Lorenz D, Müller RH, Dathe M (2016) Lipopeptide-based micellar and liposomal carriers: Influence of surface charge and particle size on cellular uptake into blood brain barrier cells. Euro J Pharm Biopharm 109, Rautenbach M, Troskie A M, Vosloo J A, Dathe M (2016) Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi. Biochimie 130, Scheinpflug K, Krylova O, Nikolenko H, Thurm C, Dathe M (2015) Evidence for a novel mechanism of antimicrobial action of a cyclic R-, W-rich hexapeptide. PlosONE 10(4) e FMP authors Group members

105 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 103 MASS SPECTROMETRY MASSENSPEKTROMETRIE GROUP LEADER DR. EBERHARD KRAUSE BIOGRAPHY 1975 Diploma degree in Physical Chemistry, Humboldt University, Berlin 1982 Dr. rer. nat., Humboldt University Research Group Leader Drug Development in the Pharmaceutical Industry Research Associate, Institute of Drug Research, Berlin since 1992 Senior Scientist and Head of Mass Spectrometry Group, FMP SUMMARY Our group focuses on the development and application of 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, 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, developed new mass spectrometry-based methods for the discovery of uncommon protein modifications, and improved approaches for the identification and quantification of proteins in affinity-purification mass spectrometry experiments. ZUSAMMENFASSUNG Schwerpunkt unserer Forschung ist die Entwicklung und Anwendung von 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 T-Zellrezeptor (TCR)-abhängige Protein-Protein-Wechselwirkungen, die durch spezifische Proteinphosphorylierungen vermittelt werden. Zudem haben wir zu einer Vielzahl von Proteomstudien beigetragen und neue massenspektrometrische Methoden für die Identifizierung ungewöhnlicher, bisher kaum untersuchter Proteinmodifikationen entwickelt.

106 104 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 DESCRIPTION OF PROJECTS Serine phosphorylation-dependent protein-protein interactions of the T cell adaptor protein ADAP. Reversible protein phosphorylation is an important feature of T cell signaling, regulating many of the signal transduction pathways required for proper T cell functioning. The human adhesion and degranulation promoting adaptor protein (ADAP) plays a central role in T cell signaling. Upon T cell receptor stimulation ADAP is strongly tyrosinephosphorylated and serves as a hub for SH2 domain-containing effector proteins. In addition, phosphoproteomics revealed several serine and threonine phosphorylation sites within the N-terminal domain of ADAP. However, the role of these phosphorylation events in T cell signaling events has not been investigated. Using quantitative SILAC-based peptide and protein pull-down approaches, we demonstrate for the first time that two distinct phosphorylated serine residues in the N-terminal region of ADAP bind specifically to isomers (Figure 1). Since proteins are known for playing an important role in T cell signaling, this new phosphorylation-dependent interaction between ADAP and proteins may yield new insights into T cell signaling pathways. Analysis of uncommon protein phosphorylation. Reversible phosphorylation is the most widespread posttranslational 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, amino acid side-chains of histidine, arginine, cysteine and lysine residues may also undergo phosphorylation. However, because of the instability of the P-N and P-S bonds, these modifications most often remain undetected in conventional phosphoproteomics studies by mass spectrometry (MS), even though recent studies have indicated their potential importance in various biological signaling processes. We performed MS studies of phosphocysteine and pyrophospholysine peptides that very recently became synthesizable via new synthetic routes established by the Hackenberger group at the FMP. The preparation of well-characterized, site-specifically modified peptides enables a systematic study of the behavior of such peptides using various mass spectrometric fragmentation techniques. Our data indicate that the stability of both modifications during electron- A B Fig. 1: Results of SILAC-based pull-down experiments with site-specifically serinephosphorylated ADAP sequences. Scatter plots of heavy / light and light / heavy ratios of identified proteins from pull-down experiments using (A) ADAP-pSer155 and (B) ADAP-pSer235 show that seven isomers can be considered as phosphorylation-dependent interaction partners. GROUP MEMBERS Annika Manns (doctoral student) Martin Penkert (doctoral student) Michael Schümann (technical assistant) Heike Stephanowitz (technical assistant) Staff employed within the reporting period COLLABORATIONS International Remigiusz Serwa, Imperial College London, UK Edward W. Tate, Imperial College London, UK Dorothea Fiedler, Princeton University, USA National Ria Baumgrass, DRFZ, Berlin Beate Braun, IZW, Berlin Kurt Engeland, Universität, Leipzig U. Benjamin Kaupp, Center of Advanced European Studies and Research, Bonn Hans G. Börner, Humboldt-Universität, Berlin Jörg Rademann, FU Berlin Volker Haucke, FMP Christian Hackenberger, FMP Dorothea Fiedler, FMP Christian Freund, FU Berlin Dirk Schwarzer, Universität Tübingen Ralf Schülein, FMP Michael Veit, FU Berlin

107 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 105 transfer dissociation (ETD) mass spectrometry is high, keeping the modified side-chain completely intact during fragmentation and making ETD particularly suitable for proteomic studies as an essential tool for evaluating the biological relevance of these uncommon protein modifications. To demonstrate the use of ETD MS-based proteomics in resolving biological questions, we identified an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in carbohydrate uptake in the bacterial phosphotransferase system (Betran-Vicente et al. Nature Commun. 2016). Another labile phospho-modification that has eluded detection by conventional MS methods is protein pyrophosphorylation. Located close to polyacidic amino acid stretches, this modification occurs on phosphorylated serine residues and is mediated by inositol pyrophosphate messengers. While perturbation of inositol pyrophosphate biosynthesis has revealed a wide range of functions for these messengers, including insulin signaling and central energy metabolism, the role of protein pyrophosphorylation in regulating these processes has not been elucidated and remains a controversial topic of debate. MS analysis of a set of synthesized, site-specifically modified peptides which were very recently accessible via a new synthetic approach developed by the Fiedler group at Princeton University (now at the FMP) have shown that using conventional, collision-based MS / MS methods such as CID and HCD, pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178 and 196 Da, preventing the identification of the modification site. In contrast, ETD combined with higher energy collision dissociation (EThcD) provides useful tandem MS spectra for direct and unambiguous assignment of the site of pyrophosphorylation. Based on the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation, we developed a data-dependent neutral-loss-triggered EThcD acquisition method (Figure 2) that allows for the reliable characterization of protein pyrophosphorylations (Penkert et al. Anal. Chem. 2017) and, in combination with a selective enrichment procedure, uncovered the first pyrophosphorylation sites in vivo. A B Fig. 2: Identification of protein pyrophosphorylation sites. (A) Proteins were reduced, alkylated and digested with trypsin. Tryptic peptides were passed over an affinity reagent to enrich the concentration of pyrophosphorylated peptides followed by nano- LC-MS / MS analysis applying the data-dependent neutral-loss-triggered (DDNL) EThcD method. (B) Principle of DDNL-EThcD analysis. After a highresolution survey scan measured in the orbitrap, low collision energy CID is performed in the ion trap. CID MS / MS spectra of protonated peptides have to exhibit neutral losses of 98 and 178 Da above a relative intensity and have to belong to the five most intense peaks to trigger an EThcD for sequence analysis. The chosen trigger requirements are based on the results of the low collision energy CID studies. SELECTED PUBLICATIONS Penkert M, Yates L M, Schümann M, Perlman D H, Fiedler D, Krause E (2017) Unambiguous identification of serine and threonine pyrophosphorylation using neutral-loss-triggered EThcD mass spectrometry. Anal. Chem. 89, Bertran-Vicente J, Penkert M, Nieto-Garcia O, Jeckelmann JM, Schmieder P, Krause E, Hackenberger C P R (2016) Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides. Nature Commun. 7, Kuropka B, Witte A, Sticht J, Waldt N, Majkut P, Hackenberger C P R, Schraven B, Krause E, Kliche S, Freund C (2015) Analysis of phosphorylation-dependent protein interactions of ADAP reveals novel interaction partners required for chemokine-directed T cell migration. Mol. Cell. Proteomics 14, Bertran-Vicente J, Schümann M, Hackenberger C P, Krause E (2015) Gas-phase rearrangement in lysine phosphorylated peptides during electron-transfer dissociation tandem mass spectrometry. Anal. Chem. 87, Kuropka B, Royla N, Freund C, Krause E (2015) Sortase A-mediated site-specific immobilization for identification of protein interactions in affinity purification-mass spectrometry experiments. Proteomics 1, FMP authors Group members

108 106 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 MEDICINAL CHEMISTRY MEDIZINISCHE CHEMIE GROUP LEADER DR. MARC NAZARÉ BIOGRAPHY Studies in Chemistry, University of Karlsruhe 1995 Diploma in Chemistry with Prof. H. Waldmann, University of Karlsruhe Ph.D. research with Prof. H. Waldmann, University of Karlsruhe Medicinal chemist and project leader in drug discovery, 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, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin SUMMARY Small molecule tools allow one to probe protein functions and elucidate mechanisms and signal transduction pathways by directly interfering with specific proteins. These chemical tools validate hypotheses based on chronic genetic inactivation in knock-down loss of function studies and can serve as starting points for 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 tryptophan hydroxylase, among other targets. To achieve our goals we study the structure-activity relationship (SAR) of protein-ligand interactions by synthesizing distinct, small molecule derivatives or small focused libraries of the initial screening hit structure. Iterative cycles of biological testing, design, and synthesis of new analogues based on the data obtained yield optimized chemical tools for proof-of-concept studies. A second field of interest is the further expansion and qualitative enhancement of the FMP compound library that currently contains 60,000 commercial compounds and 7,000 small molecules sourced from academic research. Careful expansion of this library should guarantee optimal coverage of the biological space being screened, along with high hit rates as well as suitable starting points for chemical optimization. The Medicinal Chemistry group closely collaborates with the Screening Unit and the Computational Chemistry group on work for 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 entschlüsselt 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. Diese Substanzen können als Vorläufer von Medikamenten oder als 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 unter anderem derzeit an Konzepten und dem Design von spezifischen Inhibitoren für die Clathrin-vermittelte 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 auf Basis der erhobenen Daten das Design und die Synthese neuer, verbesserter Analoga für proof-of-concept Studien. Ein zweiter Fokus unserer Arbeit ist die weitere Entwicklung und die Qualitätserhöhung der FMP Substanzsammlung mit derzeit kommerziellen Verbindungen und 7,000 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 Medizinische Chemie arbeitet dazu sowohl bei der Entwicklung der FMP Sammlung als auch beim biologischen Profiling in SAR-Studien eng mit der Screening Unit und der Arbeitsgruppe Computerchemie des Instituts zusammen.

109 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 107 Fig. 1: Overlay of three X-ray co-crystal structures for Pitstop2 derivatives / clathrin illustrating the reversed, non-canonical binding modes within the clathrin box. DESCRIPTION OF PROJECTS 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 high-throughput screen (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 that inhibit complex formation between the clathrin terminal domain (TD) and amphiphysin B / C (V. Haucke et al. Cell, 2011). Starting from these hits, and in collaboration with the group of Volker Haucke (FU & FMP, Berlin), we synthesized focused libraries of around 150 compounds, providing a structure-activity relationship for Pitstop2. Co-crystallization of Pitstop derivatives with the clathrin TD guided the further design and provided insights into the key interactions between these Pitstop ligands and the clathrin TD. Surprisingly, X-ray structure determination (Haydar Bulut, FU Berlin) of six nearly equipotent inhibitors derived from one core scaffold showed 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 clathrin TDendocytic protein interactions. For cellular studies we are developing a new photocleavable protecting group (photocage) that allows for the spatio-temporally controlled intracellular release of the non-cell permeable Pitstop1. 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 inhibit 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. In collaboration with Walter Birchmeier, a re-scaffolding approach that involves replacing the former framework of a tyrosine phosphatase Shp2 inhibitor (W. Birchmeier et al., PNAS, 2008), led to the discovery of novel structural classes and eliminated several chemical liabilities, i. e. unfavorable structural features. These novel compounds are not only active in a submicromolar range in the Shp2-enzyme assay, but are also effective in the low micromolar range on hepatocyte growth factor (HGF)-stimulated canine MDCK-C cells, as well as human pancreatic tumor cells for epithelial-mesenchymal 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), while tryptophan hydroxylase (TPH) is the initial and rate-limiting 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 physiological serotonin levels. The X-ray co-crystal structures obtained with our inhibitors allowed us to elucidate the binding mode and to reveal the structural determinants for the remarkably efficient protein-ligand interaction of these inhibitors. Several inhibitors are currently undergoing in vivo efficacy studies in mice. 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 a 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. 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 and efficient routes for the synthesis of privileged scaffolds like 2H indazoles and pyrazolo-triazoles for generating libraries, and we have included them in our FMP compound collection.

110 108 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 A B C Fig. 2: Upper panel: Structure of original hit (right) and optimized novel Shp2 inhibitor (left). Colors indicate the structural modifications. Lower panel: 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 lose 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. GROUP MEMBERS Dr. Upendra Anumala Dr. Isabel Fernández Bachiller Dr. Hassen Bel Abed Dr. André Horatscheck Dr. María Pascual López-Alberca Dr. Vera Martos Dr. Lioudmila Perepelittchenko Dr. Edgar Specker Benjamin Jakob Brennecke (doctoral student) Thais Gazzi (doctoral student) Jens Schöne (doctoral student) Keven Mallow (technical assistant) Sandra Miksche (technical assistant) Jessica Przygodda (technical assistant) Staff employed within the reporting period COLLABORATIONS International Uwe Grether, F.Hoffmann-La Roche, Basel, Switzerland Haiyu Hu, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College Stefan Krauss, University Hospital, Oslo, Norway National Michael Bader, MDC, Berlin Walter Birchmeier, MDC, Berlin Udo Heinemann, MDC, Berlin Hans-Jürgen Holdt, University of Potsdam, Potsdam Christoph Rademacher, MPI, Potsdam K. Lenhard Rudolph, FLI, Jena Claus Scheidereit, MDC, Berlin David W. Will, EMBL, Heidelberg SELECTED PUBLICATIONS Bel Abed H, Schoene J, Christmann M, Nazare M (2016) Organophosphorus-mediated N-N bond formation: facile access to 3-amino-2H-indazoles. Org. Biomol. Chem. 14, Kozian D H, von Haeften E, Joho S, Czechtizky W, Anumala U R, Roux P, Dudda A, Evers A, Nazare M (2016) Modulation of Hexadecyl-LPA-Mediated Activation of Mast Cells and Microglia by a Chemical Probe for LPA5. ChemBioChem 17, Aretz J, Kondoh Y, Honda K, Anumala U R, Nazaré M, Watanabe N, Osada H, Rademacher C (2016) Chemical fragment arrays for rapid druggability assessment. Chem. Commun. 52, 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, Juretschke H-P, Ding-Pfennigdorff D, Florian P, Kohlmann M, Kandira A, von Kries J, Saas J, Rudolphi K A, Wendt K U, Nagase H, Plettenburg O, Nazare M, Schultz C (2015) In vivo visualization of osteoarthritic hypertrophic lesions. Chem.Sci. 6, EXTERNAL FUNDING Helmholtz Wirkstoffforschung Berlin Institute of Health (BIH) SAW DNA damage responses in aging P21 collaboration with FLI Jena 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 Udo Heinemann, MDC DFG, NA 1274 / 1-1 Development and characterization of specific small molecule inhibitors of class II phosphatidylinositol 3-kinase C2alpha function together with Volker Haucke, FMP Sino-German Center for Research Promotion, GZ 1271, Tumor- targeting SMART Imaging Agents together with Haiyu-Hu, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China FMP authors Group members

111 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 109 CORE FACILITY SCREENING UNIT GROUP LEADER DR. JENS PETER VON KRIES BIOGRAPHY Diploma & Ph.D. in biology, University Hospital Hamburg-Eppendorf (Prof. Strätling) Research on proteins involved in origin of cancer in the lab of W. Birchmeier Head of Screening Unit, Semaia Pharmaceuticals since 2003 Head of Screening Unit, FMP; set up and technology development 2005 Core facility for Helmholtz-Initiative für Wirkstoffforschung 2015 Core facility for Chemical Biology, Berlin Institute of Health 2017 Core facility for EU-OPENSCREEN Chairman of Gemeinsame Fachgruppe Chemische Biologie, DECHEMA 2014 Advisory Board SFICAST, University Oslo SUMMARY 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 compounds) or genome-wide RNAi libraries (human, mouse, nematodes). The platform is typically a part of scientific collaborations and its primary aim is to make possible the use of drugs in academic research for analysis of molecular mechanisms in disease and development. Besides supporting assay development, process automation, screening and automated data analysis, the Unit engages in the identification of novel screening technologies that may prove useful in its services. The Unit currently supports compound screening projects in assay development and optimization of 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 is building a central core facility for drug screening on the Campus Berlin-Buch for the Helmholtz-Initiative für Wirkstoffforschung, the Berlin Institute of Health (BIH), and EU-OPENSCREEN. 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 und implementiert diese für den Einsatz. Sie unterstützt derzeit Screeningprojekte mit chemischen Verbindungen 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.

112 110 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 DESCRIPTION OF PROJECTS Therapeutic targeting of Hodgkin lymphoma Classical Hodgkin lymphoma (chl) reflects a clinical challenge when presenting as primary refractory or relapsed disease. Interestingly, chl is an archetypical example of malignant plasticity. chl is characterized by a virtual lack of gene products whose expression constitutes the B-cell phenotype. Restoring the B-cell phenotype may render chl susceptible to clinically established antibody therapies, targeting B-cell surface receptors, or small compounds interfering with B-cell receptor signaling. We supported a high-throughput pharmacological screening based on more than 28,000 compounds in chl cell lines carrying a CD19 reporter to identify drugs that promote re-expression of the B-cell phenotype. Three compounds were identified that robustly enhanced CD19 transcription. Subsequent chromatin immune precipitation-based analyses indicated that the action of two of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the anti-leukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of chl cell lines. Furthermore, restoration of the B-cell phenotype also rendered chl cells susceptible to the B-cell non-hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we identified a conceptually novel, redifferentiation-based treatment strategy for chl. (Figure 1, Blood. 2017;129(1):71-81). STOML3 inhibitors affecting tactile-driven chronic pain. The skin is equipped with specialized mechanoreceptors that allow perception of the slightest pressure. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. We identified small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. Under pathophysiological conditions following nerve injury or diabetic neuropathy, even the slightest touch can cause pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. In this way, small molecules applied locally to the skin may be used to modulate touch and could serve as peripherally-acting drugs to treat tactile-driven pain following neuropathy. Wnt inhibitors for suppression of self-renewal of cancer stem cells and tumorigenesis Wnt / b-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon, head, and neck. With Walter Birchmeier (MDC) we performed a high-throughput screen using purified proteins in AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between β-catenin and the transcription factor TCF4. We found a 4-thioureidobenzenesulfonamide derivative that robustly inhibits this interaction in colon cancer cells. Remarkably, the self-renewal capacity of cancer stem cells was also blocked by the inhibitor, as demonstrated by sphere formation of colon, head, and neck cancer stem cells under nonadherent conditions. (Cancer Res; 76(4); AACR). 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 (VRAC) 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 used in a secondary screen with newly designed silencer RNAs for identification of LRRC8 heteromers as essential components of VRAC. Fig. 1: Expression of the B-cell-specific surface marker CD20 is observed in B-NHL samples (follicular lymphoma [FL], left; diffuse large B-cell lymphoma [DLBCL], right), but not in chl samples (with CD30 as a typical chl marker). Standard hematoxylin and eosin (HE) staining was used to visualize morphology. Note that CD20 - HRS (arrowheads) are surrounded by a few infiltrating CD20 + non-malignant B cells in the chl sections. (Clemens Schmitt, Charité)

113 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 111 GROUP MEMBERS Dr. Katina Lazarow (RNAi) Dipl. Ing. Romy Leu (HCT) Dr. Martin Neuenschwander (Process Automation, HTS-Analysis) Dr. Silke Radetzki (High Content Screening, HCT) Sabrina Kleißle (MDC) (RNAi, cellular test systems) Andreas Oder (protein-ligand interactions surface plasmon resonance, FRET, AlphaScreen) Carola Seyffarth (capillary electrophoresis, enzyme screens) M.Sc. Marc Wippich (HTS) Staff employed within the reporting period COLLABORATIONS International Andrew W. Munro, Manchester University Stefan Krauss, University Oslo National Salim Seyfried, Universität Potsdam & MHH Clemens Schmitt, Charité Berlin Karoline Krause, Charité Berlin Björn Schuhmacher, Universität Köln Stefan Kubick, IZI Fraunhofer Potsdam-Golm Thomas F. Meyer, MPIIB, Berlin Walter Birchmeier, MDC, Berlin Claus Scheidereit, MDC, Berlin Michael Bader, MDC, Berlin Udo Heinemann, MDC, Berlin Lenhard Rudolph, FLI, Jena Ulrich Martin, MHH, Hannover SELECTED PUBLICATIONS Du J, Neuenschwander M, Yu Y, Däbritz JHM, Neuendorff N-R, Schleich K, Bittner A, Milanovic M, Beuster G, Radetzki S, Specker E, Reimann M, Rosenbauer F, Mathas S, Lohneis P, Hummel M, Dörken B, von Kries JP, Lee S, Schmitt CA (2017) Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma. Blood 129, Wetzel C, Pifferi S, Picci C, Gök C, Hoffmann D, Bali KK, Lampe A, Lapatsina L, Fleischer R, Smith ESJ, Bégay V, Moroni M, Estebanez L, Kühnemund J, Walcher J, Specker E, Neuenschwander M, von Kries JP, Haucke V, Kuner R, Poulet JFA, Schmoranzer J, Poole K, Lewin GR (2017) Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity. Nat. Neurosci 20, Chenge JT, Le DV, Swami S, McLean KJ, Kavanagh ME, Coyne AG, Rigby SEJ, Cheesman MR, Girvan HM, Levy CW, Rupp B, von Kries JP, Abell C, Leys D, Munro AW (2016). Structural Characterization and Ligand / Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1. J. Biol. Chem. 292, Fang L, Zhu Q, Neuenschwander M, Specker E, Wulf-Goldenberg A, Weis WI, Kries von JP, Birchmeier W (2016) A Small-Molecule Antagonist of the β-catenin / TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis. Cancer Res. 76, Khatri Y, Ringle M, Lisurek M, Kries von JP, Zapp J, Bernhardt R (2016) Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1α-Hydroxylated Products from C-19 Steroids. Chembiochem 17, EXTERNAL FUNDING EU-OPENSCREEN, BMBF Berlin Institute of Health, BMBF CCMCURE, E-RARE 2014; BMBF; , Coordinator Prof. Salim Seyfried; ,80 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, , 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), , MDC, Screening Unit, , PAKT (FLI-Jena), with W. Rosenthal, , ECRC, with MDC, , BMBF / MDC, RNAi & REMP, with MDC, J. Rademann, , NGFNplus, 5 Projektförderungen á , FMP authors Group members

114 112 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 CORE FACILITY PEPTIDE SYNTHESIS PEPTIDSYNTHESE GROUP LEADER DR. RUDOLF VOLKMER BIOGRAPHY Industrial training as a laboratory assistant, Hoechst AG, Frankfurt / Main Studied Chemistry, University of Frankfurt / Main and FU Berlin Freelancer, German Trade Union Federation, Frankfurt / Main 1984 Diploma thesis, FU Berlin (Prof. Rewicki) 1991 Ph.D., FU Berlin (Prof. Rewicki) 1991 Teacher at the DRK-nurse s training school Post-Doc, Charité University Medicine Berlin (Prof. Schneider-Mergener) Since 1999 Group leader Molecular Libraries and Recognition Group, Charité University Medicine Berlin, Institute of Medical Immunology Since 2013 Guest scientist and group leader Peptide Synthesis, FMP SUMMARY The Peptide Synthesis 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 using solid-phase peptide synthesis. The full repertoire of standard solid phase peptide synthesis methods are 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. Since October 2016 SPOT synthesis technologies have been available directly from the peptide service group at the FMP. This technology can be applied to the mapping and screening of protein-protein binding sites. The service unit for peptide synthesis is a collaborative and service 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. Seit Oktober 2016 bietet die Servicegruppe zusätzlich die Möglichkeit der SPOT Synthese an. Diese Technologie eignet sich hervorragend um Protein-Protein Kontaktstellen aufzuspüren und zu kartieren. Die Serviceeinheit ist als Kooperations- und / oder Servicepartner in laufende Forschungsprojekte am FMP und an der Charité eingebunden.

115 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 113 A B MPG-iCAL42, 1μM, 1h Pen-iCAL42, 1μM, 1h Tamra -MPG-iCAL36 GFP-CAL Hoechst 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 C Figure 1. 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 one hour. However, it takes a period of three hours 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. Fluorescence Magnification Tamra-Pen-iCAL36 DESCRIPTION OF PROJECTS Peptide synthesis service at the FMP The peptide synthesis service unit at the FMP has been operating since At this time three FMP labs, namely those of Blasig, Haucke and Kühne, were our first customers for peptide synthesis. Four years later, we are periodically receiving peptide synthesis orders from 12 FMP groups. These orders include not only simple linear peptides (on a milligram to gram scale), but also dye-labeled peptides, head-to-tail cyclic peptides with and without dye-labeling, peptides without gene-encoded amino acids, building blocks or proline-mimetics, D-amino acids or polyethylene glycol molecules, and phosphorylated peptides. In its early years, the peptide service unit synthesized approximately 140 peptides per year. In 2016, Ines Kretzschmar synthesized 255 peptides, an increase of almost 75 %. In total, 771 peptides have been synthesized since March In addition to its services, the peptide synthesis unit is embedded in several FMP research projects. In addition to standard peptide synthesis, the SPOT synthesis technology for preparing peptide arrays has also been offered since October 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. Most 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 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

116 114 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 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 on 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 data confirm substantial additivity for a cell-permeable ical in concert with a known corrector, the small molecule VX-809. Figure 1 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 is a collaboration with Dean Madden, Hanover, USA; Rudolf Volkmer and Nico Derrichs, Charité Universitätsmedizin, Berlin; Prisca Boisguerin, Montpellier, France; and Hartmut Oschkinat at the FMP Berlin. GROUP MEMBERS Ines Kretzschmar (technical assistant) Staff employed within the reporting period COLLABORATIONS International Dean Madden, Geisel School of Medicine at Dartmouth, Hanover, USA Prisca Boisguerin, CRBM, Montpellier, France Michel Steinmetz, Paul Scherer Institut, Switzerland Marius Sudol, Institute of Molecular and Cell Biology, Singapore 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 Henning Mootz, University of Münster Michael Ehrmann, University of Duisburg-Essen SELECTED PUBLICATIONS Kathage B, Gehlert S, Ulbricht A, Lüdecke L, Tapia V E, Orfanos Z, Wenzel D, Bloch W, Volkmer R, Fleischmann B K, Fürst D O, Höhfeld J (2017) The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mtorc1.biochim Biophys Acta 1864, Eccles R L, Czajkowski M T, Barth C, Müller P M, McShane E, Grunwald S, Beaudette P, Mecklenburg N, Volkmer R, Zühlke K, Dittmar G, Selbach M, Hammes A, Daumke O, Klussmann E, Urbé S, Rocks O (2016) Bimodal antagonism of PKA signalling by ARHGAP36. Nat Commun 7, Manatschal C, Farcas A M, Degen M S, Bayer M, Kumar A, Landgraf C, Volkmer R, Barral Y, Steinmetz M O (2016) Molecular basis of Kar9-Bim1 complex function during mating and spindle positioning. Mol Biol Cell 27, Tapia Mancilla V E, Volkmer R (2016) Peptide Arrays on Planar Supports. Methods Mol Biol 1352, Opitz R, Müller M, Reuter C, Barone M, Soicke A, Roske Y, Piotukh K, Huy P, Beerbaum M, Wiesner B, Beyermann M, Schmieder P, Freund C, Volkmer R, Oschkinat H, Schmalz H G, Kühne R (2015) A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions. Proc Natl Acad Sci U S A. 112, FMP authors Group members

117 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 115 Campus Berlin Buch Campus Berlin Buch APPENDIX ANHANG All Research Groups Alle Forschungsgruppen PAGE 116 Map Campus Berlin Buch Karte Campus Berlin Buch PAGE 118 Administrative and Technical Services Administrative und technische Dienstleistungen PAGE 120 Imprint Impressum

118 116 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 ALL RESEARCH GROUPS ALLE FORSCHUNGSGRUPPEN MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE THOMAS J. JENTSCH STRUCTURAL BIOLOGY STRUKTURBIOLOGIE ADAM LANGE DEPARTMENTS ABTEILUNGEN DEPARTMENTS ABTEILUNGEN Physiology and Pathology of Ion Transport Thomas J. Jentsch Molecular Pharmacology and Cell Biology Volker Haucke Molecular Biophysics Adam Lange RESEARCH GROUPS FORSCHUNGSGRUPPEN Protein Trafficking Ralf Schülein Molecular Cell Physiology Ingolf E. Blasig RESEARCH GROUPS FORSCHUNGSGRUPPEN Solution NMR Peter Schmieder Computational Chemistry / Drug Design Ronald Kühne 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 JUNIOR RESEARCH GROUPS JUNIOR FORSCHUNGSGRUPPEN In Cell-NMR Philipp Selenko Molecular Imaging Leif Schröder CORE FACILITIES Cellular Imaging Burkhard Wiesner / Dmytro Puchkov Animal Facility Natali Wisbrun CORE FACILITY NMR Hartmut Oschkinat / Peter Schmieder

119 APPENDIX ANHANG 117 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE CHRISTIAN HACKENBERGER NMR-Supported Structural Biology Hartmut Oschkinat DEPARTMENTS ABTEILUNGEN Chemical Biology II (Leibniz-Humboldt) Christian Hackenberger Chemical Biology I Dorothea Fiedler Structural Bioinformatics and Protein Design Gerd Krause RESEARCH GROUPS FORSCHUNGSGRUPPEN Peptide-Lipid-Interaction / Peptide Transport Margitta Dathe Mass Spectrometry Eberhard Krause Medicinal Chemistry Marc Nazaré CORE FACILITIES Peptide Synthesis Christian Hackenberger / Rudolf Volkmer Screening Unit Jens Peter von Kries

120 118 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 CAMPUS BERLIN BUCH CAMPUS BERLIN BUCH ROBERT-RÖSSLE-STR BERLIN RESEARCH Leibniz-Forschungsinstitut 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

121 APPENDIX ANHANG 119

122 120 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016 ADMINISTRATIVE AND TECHNICAL SERVICES ADMINISTRATIVE UND TECHNISCHE DIENSTLEISTUNGEN DIRECTORATE Prof. Dr. Dorothea Fiedler Director, Managing Director since 01 / 2017 Prof. Dr. Volker Haucke Director, Managing Director until 12 / 2016 Dr. Henning Otto Dr. Elvira Rohde Scientific Coordinators Silke Oßwald Public Relations Dr. Anne Höner EU-Liaison Officer (until 09 / 2016) Dr. Franziska Ringleb EU-Liaison Officer (since 10 / 2016) 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 Marianne Dreißigacker Department of Chemical Biology Dr. Norma Nitschke Scientific Coordinator Department of Physiology and Pathology of Ion Transport Stefanie Schneider Department of Molecular Biophysics ADMINISTRATION Frank Schilling Head Administration Thomas Ellermann General Administration Marina Spors Personnel Management Christel Otto General Administration Claudia Messing General Administration Dennis Bischoff General Administration 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

123 IMPRINT IMPRESSUM Leibniz-Forschungsinstitut 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 2015 / 2016 Editorial Board Dorothea Fiedler, Christian Hackenberger, Volker Haucke, Thomas Jentsch, Adam Lange, Hartmut Oschkinat Coordination Silke Oßwald Author Feature Articles and Interview Beatrice Hamberger, Birgit Herden (p. 8) Editing Martin McLean Translations Mick Locke, Claudia Hecker Photography Silke Oßwald Further Photography David Ausserhofer (p. 11), Meida Jusyte (p. 16), Stefan Jentsch (p. 20), Maj Brit Jansen (p. 31) 3-D Illustration Barth van Rossum Scientific Figures Sections Jan Schmoranzer (p. 14), Michael Lisurek (p. 54), Barth van Rossum (p. 86) Design and Layout KRAUT & KONFETTI, Berlin Print Druckerei Conrad GmbH, Berlin Berlin, Mai 2017

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