RESEARCH REPORT 2015 / 2016 SCIENTIFIC ADVISORY BOARD

Prof. Dr. Karl-Heinz Altmann Prof. Dr. Stefan Offermanns Institut für Pharmazeutische Max-Planck-Institut für Herz- und Wissenschaften, ETH Zürich Lungenforschung, Bad Nauheim (seit 01 / 2012) (seit 01 / 2016)

Prof. Dr. Nils Brose Prof. Dr. Petra Schwille (Vorsitzender) Max-Planck-Institut für Biochemie, Max-Planck-Institut für Experimentelle Martinsried Medizin, Göttingen (seit 01 / 2012) (seit 01 / 2012) Prof. Dr. Rebecca Wade Prof. Dr. Ulrike Eggert (Stellvertretende Vorsitzende) Randall Division of Cell and Molecular Heidelberg Institute for Theoretical Biophysics, King´s College London Studies, HITS gGmbH (seit 01 / 2013) (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 31.12.2016 RESEARCH REPORT 2015 / 2016 2 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

CONTENTS INHALT

PREFACE Interview with Director Dorothea Fiedler VORWORT What's new at the FMP? Was ist neu am FMP? 4

RESEARCH HIGHLIGHTS The cause of muscle weakness reveals the organisational principle in cells AKTUELLES AUS Ursache von vererbter Muskelschwäche aufgeklärt 8 DER FORSCHUNG 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 CONTENTS INHALT 3

RESEARCH GROUPS Section Molecular Physiology and Cell Biology FORSCHUNGSGRUPPEN 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 All Research Groups 116 ANHANG Map Campus Berlin Buch 118 Administrative and Technical Services 120 Imprint 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 are trying to decode cellular processes at the molecular level. For me as 2015. In an interview, she speaks about her move from the a chemist that's really exciting. One focus of our work are the so-called USA to Germany, the focus of her research and about current inositol pyrophosphates. These molecules are messenger substances in developments at the FMP. the cell that, for example, play an important role in disorders of fat meta- bolism and insulin secretion, but also have an influence on cell migration, Professor Fiedler, you were a distinguished scientist in the specifically metastasis. In effect we are trying to understand the language USA, latterly at Princeton University, and have received of chemistry and thus hope to lay the foundations for new therapies. numerous awards. What motivated your move to Berlin-Buch? There were several reasons. The most important one was undoubtedly Is it the promise of new therapies that motivates you to conduct that the FMP is an excellent institute, where interdisciplinary research basic research? is conducted at the interface of biology and chemistry. To this extent, The desire to improve the health of humankind is fundamental to all of us. the science at the institute was a perfect match. In addition, I can Admittedly, most of the groups at the FMP are not developing therapies collaborate with outstanding people here, both internally and exter- in an actual sense, but it is the basic research conducted at the Institute nally. And then the FMP offers exceptional research conditions. All of that paves the way. This can be seen, for example, in the fact that two this was simply too tempting for me to turn down the offer of a W3S spin-offs from the Hackenberger and Kühne groups are in preparation. professorship linked to the post of director, although I was very happy in the USA. One-third of the research groups at the FMP are junior groups. What's new here? Together with Professor Volker Haucke you form a dual lead- A great deal. In the field of molecular physiology and cell biology, ership at the FMP. How do you divide up this important task? there is a new liaison group, “Neurosciences”, the Emmy Noether There was a clear agreement that I would officially take over the Junior Group led by Alexander Walter. In addition, our junior group management after 18 months. That is now the case as of 1 January, leader Andrew Plested has been offered a Heisenberg professorship but we continue to complete many tasks together and always consult at the Humboldt University. His group will remain at the FMP as each other on important decisions. 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 You are responsible for 300 employees. Is there any time left a junior group in structural biology for several years and has now to do your own research in the laboratory? accepted an attractive offer from the Weizmann Institute of Science Unfortunately, it has been a while since I ran experiments in the in Israel. It is all the more pleasing that a new junior group at the laboratory. But at this point, my co-workers can carry out the work interface of NMR and Cryo-EM is to be established in this better than me. But naturally I still work closely with my PhD important field of research, this being within the context of the students and postdocs. Research is and will certainly remain the planned “Cryo-EM infrastructure”, in which the Berlin Universities part of my work that I enjoy the most. and the MDC will also be involved. Our doctoral students are very successful as well, for instance Jean-Philippe Demers from the What does a professor of chemical biology conduct research on? department of Adam Lange received the Raymond Andrew Prize and In many areas of cell biology we have now arrived at a point where we the Otto Hahn Medal in 2015, a really remarkable accomplishment. 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 co- I think, and I can speak for the past and for the future, that the operation with the California Institute of Technology in Pasadena FMP is an institute that places special value on modern methods to develop ultra-sensitive magnetic resonance imaging, which and technology. With this strategy, and of course driven by our allows to detect tumours, for example. At the same time, last excellent scientists, we will in the long run secure pioneering results year, he secured a Reinhart Koselleck-Project of the Deutsche that will be of benefit to the scientific community and ultimately Forschungsgemeinschaft (DFG) for a similar undertaking. The to society. What I would like to see is more international visibility. funding amounts to 1.525 million euros and was the first ever The FMP is very well connected throughout Germany and also Koselleck Project for the Leibniz Association. I find all of this quite internationally. Nevertheless, the Institute, like the Leibniz Association, outstanding. is not yet as well known internationally as other German research organisations. We're working on that. At the beginning of this year, there was then a second Reinhart Koselleck-Project for the FMP and a small sensation for the Dorothea Fiedler ist seit Juli 2015 Direktorin am FMP. Im Institute? Interview spricht sie über ihren Wechsel von den USA nach That's right, just a few months later, Volker Haucke impressed the Deutschland, ihre Forschungsschwerpunkte und über aktuelle DFG with his application for research into neuronal communication. Entwicklungen am FMP. 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 Frau Professor Fiedler, Sie waren in den USA, zuletzt an der were awarded the prestigious “Consolidator Grant” of the European Universität Princeton, eine angesehene Wissenschaftlerin Research Council (ERC) for their research work. Over the past five und haben etliche Auszeichnungen erhalten. Was hat Sie years, Thomas J. Jentsch successfully applied for these most prominent nach Berlin-Buch verschlagen? and coveted programmes offered by the European Commission, for his Es waren mehrere Gründe. Das Wichtigste war sicher, dass research on ion channels he received an Advanced Grant to the amount das FMP ein exzellentes Institut ist, wo interdisziplinär an der of 2.5 million euros from the European Research Council (ERC) for Schnittstelle von Biologie und Chemie geforscht wird. Insofern hat the first time in 2012, followed in March this year by a second ERC es inhaltlich perfekt gepasst. Außerdem kann ich hier mit hervor- Advanced Grant. A great success! ragenden Leuten zusammenarbeiten, intern wie extern. Und dann verfügt das FMP über ausgezeichnete Forschungsbedingungen. And what about your specialist field, chemical biology? All das war doch zu reizvoll, um den Ruf auf eine W3S-Professur Here, too, there are plans to establish a junior group, although verbunden mit dem Direktorenposten auszuschlagen, obwohl ich I'm afraid I can't reveal any details at this stage. However, we are mich in den USA sehr wohlgefühlt habe. very pleased that Dr. Fan Liu will arrive during the second half of this year to oversee the Institute’s Mass Spectrometry Facility. Zusammen mit Professor Volker Haucke bilden Sie eine Doppel- She will be able to build on the excellent infrastructure provided spitze am FMP. Wie teilen Sie sich diese verantwortungsvolle by Eberhard Krause’s group, and on top of that establish her own Aufgabe? research group with a number of exciting and current projects. We Es gab eine klare Absprache, dass ich nach eineinhalb Jahren offiziell are thrilled about her arrival. die Geschäftsführung übernehmen werde. Seit 1. Januar ist das nun der Fall, dennoch erledigen wir nach wie vor viele Aufgaben The FMP will celebrate it's 25th birthday this year. Where do gemeinsam und sprechen uns bei wichtigen Entscheidungen immer you see the Institute in 25 years' time? miteinander ab. Despite a consistent mission, the FMP still remains in a certain state of upheaval. In the next couple of years, several colleagues will be Sie sind verantwortlich für 300 Mitarbeiter. Bleibt da noch retiring, some of whom already worked at the predecessor institute in Zeit, selbst im Labor zu forschen? the “Institute of Drug Research, Academy of Sciences of the GDR”. Im Labor stehe ich leider schon lange nicht mehr. Das können It's a shame, but that's the way things go. meine Mitarbeiter mittlerweile auch besser. Aber natürlich arbeite 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, Grundlagenfor- schung 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 ent- Robert Puschmann, Sarah Hostachy and Dario Demartin wickeln, 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. Philipp Selenko und Andrew Plested für ihre Forschungen mit dem Ein Drittel der Forschergruppen am FMP sind Juniorgruppen. hochrangigen „Consolidator Grant“ des Europäischen Forschungsrates Was passiert hier Neues? (ERC) ausgezeichnet. Mit dieser profiliertesten und begehrtesten Eine Menge. Im Bereich Molekulare Physiologie und Zellbiologie Ausschreibung der Europäischen Kommission war Thomas J. gibt es eine neue Liaisongruppe „Neurowissenschaften“, die Jentsch in den vergangenen fünf Jahren gleich zwei Mal erfolgreich, Emmy-Noether-Juniorgruppe um Alexander Walter. Darüber für seine Forschung an Ionenkanälen erhielt er zum ersten Mal hinaus erhält unser Juniorgruppenleiter Andrew Plested eine 2012 einen „Advanced Grant“ in Höhe von 2,5 Millionen Euro vom Heisenberg-Professur an der Humboldt-Universität. Seine Gruppe Europäischen Forschungsrat (ERC) und im März dieses Jahres gleich wird für weitere fünf Jahre am FMP als Gastgruppe verbleiben. den zweiten ERC Advanced Grant, ein großer Erfolg! Abschied werden wir dagegen noch in diesem Jahr von Philipp Selenko nehmen müssen. Er hat seit mehreren Jahren erfolgreich Und was ist mit Ihrem Steckenpferd, der Chemischen Biologie? eine Nachwuchsgruppe in der Strukturbiologie geleitet und nun Auch hier ist der Aufbau einer Nachwuchsgruppe in Planung, ein attraktives Angebot vom Weizmann Institute of Science in Details kann ich Ihnen zu diesem Zeitpunkt leider noch nicht Israel angenommen. Umso erfreulicher ist, dass in diesem wichtigen verraten. Allerdings sind wir sehr glücklich, dass wir die Gruppe Forschungsbereich eine neue Nachwuchsgruppe an der Schnittstelle Massenspektrometrie weiterführen und Ende dieses Jahres mit von NMR und Cryo-EM eingerichtet werden soll, und zwar im Dr. Fan Liu neu besetzen. Sie wird die exzellente Infrastruktur, die Rahmen der geplanten „Cryo-EM-Infrastruktur“, an der auch die Eberhard Krause über Jahrzehnte etabliert hat, weiterführen und Berliner Universitäten und das MDC beteiligt sein werden. Unsere dazu ihre eigene Forschungsgruppe aufbauen mit sehr aktuellen Doktoranden sind übrigens auch sehr erfolgreich, Jean-Philippe und interessanten Forschungsprojekten. Wir freuen uns sehr darauf, Demers aus der Abteilung von Adam Lange hat 2015 den Raymond Dr. Liu demnächst bei uns willkommen zu heißen. Andrew Preis und die Otto-Hahn Medalle erhalten! Das FMP wird in diesem Jahr 25 Jahre alt, wo sehen Sie das Unser Nachwuchsgruppenleiter Leif Schröder konnte mit dem Institut in 25 Jahren? California Institute of Technology in Pasadena eine Kooperation Trotz stetiger Mission, befindet sich das FMP immer noch in zur Entwicklung ultra-sensitiver Magnetresonanz-Bildgebung einem gewissen Umbruch. Schon in den nächsten zwei Jahren etablieren, mit der etwa Tumoren aufgespürt werden können. werden uns Kollegen und Kolleginnen verlassen und in den Gleichzeitig hat er im vergangenen Jahr für ein ähnliches Vorhaben Ruhestand treten, die zum Teil bereits im Vorläuferinstitut im das Reinhart Koselleck-Projekt der Deutschen Forschungs- „Institut für Wirkstofforschung der Akademie der Wissenschaften gemeinschaft (DFG) eingeworben. Die Förderung beläuft sich auf der DDR“ gearbeitet haben. Das ist bedauerlich, aber eben der Lauf 1,525 Millionen Euro und war das erste Kosseleck-Projekt für die der Dinge. Ich denke, und da spreche ich für die Vergangenheit und Leibniz-Gemeinschaft überhaupt. Ich finde, das ist alles sehr für die Zukunft, dass das FMP ein Institut ist, welches besonderen bemerkenswert. Wert auf modernste Techniken legt. Mit dieser Strategie und unseren exzellenten Wissenschaftlern sichern wir uns langfristig Anfang dieses Jahres gab es dann gleich ein zweites Reinhart inhaltlich interessante und zukunftsweisende Ergebnisse, die die Koselleck-Projekt für das FMP und noch eine kleine Sensation wissenschaftliche Gemeinschaft und letztendlich die Gesellschaft für das Institut? bereichert. Was ich mir wünsche ist international mehr Sichtbarkeit. Ja, nur wenige Monate später konnte Volker Haucke die DFG mit Das FMP ist deutschlandweit aber auch international sehr seinem Projektanatrag zur Erforschung der neuronalen Kommuni- gut vernetzt. Trotzdem ist das Institut, wie auch die Leibniz- kation überzeugen. Darauf sind wir sehr stolz. Beide Auszeichnungen Gemeinschaft, international noch nicht so bekannt, wie andere zeigen, wie hochranging am FMP geforscht wird. Schon 2015 wurden deutschen Forschungsorganisationen. Daran arbeiten wir. RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 7

RESEARCH HIGHLIGHTS 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 Bei der vererbten Myotubulären Myopathie sind die Muskeln stark atrophied and the children rarely survive. In co-operation with verkümmert, die betroffenen Kinder kaum lebensfähig. Die Gruppe French human geneticists, the group led by Volker Haucke has um Volker Haucke hat zusammen mit französischen Humangenetik- researched what goes wrong on a molecular level in this disease and ern erforscht, was bei dieser Krankheit auf der molekularen Ebene has stumbled upon a general organisational principle in cells. schiefläuft und ist dabei auf ein allgemeines Organisationsprinzip in Zellen gestoßen. The disease is caused by a defect in an enzyme that is specialized in transforming certain membrane lipids, the phosphoinositide Die Krankheit entsteht durch einen Defekt in einem Enzym, dass phosphates (PIPs). As shown by the team using clever experiments darauf spezialisiert ist, bestimmte Membranlipide, die Phosphoinos- and high-resolution imaging from inside the cell, this defect leads itidphosphaten (PIPs) umzuwandeln. Wie das Team mit trickreichen to substance transport within the cells coming to a halt. This work Experimenten und hochaufgelösten Aufnahmen aus dem Zellinneren has made clear how the dynamic processes in cells are directed zeigen konnte, kommt dadurch der Stofftransport innerhalb von by the targeted transformation of PIPs. “The compartments and Zellen zum Erliegen. Durch die Arbeit ist klargeworden, wie die transport vesicles within the cells repeatedly cloak themselves with dynamischen Abläufe in Zellen durch die gezielte Umwandlung der different PIPs and thus change their identity,” says Volker Haucke. PIPs dirigiert werden. „Die Kompartimente und Transportvesikel “This indicates whether a transport container belongs inside the innerhalb der Zellen kleiden sich in immer wieder andere PIPs und cell or whether it is supposed to discharge its freight outside.” wechseln dadurch ihre Identität“, sagt Volker Haucke. „So wird angezeigt, ob ein Transportbehälter ins Zellinnere gehört oder ob er In their experiments in cell culture, the FMP researchers were able seine Fracht ins Freie entlassen soll.“ to restart the transport with a certain active substance. This might be a starting point for the development of drugs for treating this Bei ihren Experimenten in Zellkultur konnten die FMP-Forscher severe and currently incurable hereditary disease. 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.

CONTROL CELL 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,

(Katharina Ketel) integrin integrin including magnified view).

Akkumulation von Integrin (rot), ein wichtiger vesical vesical Baustein von Muskeln, in Vesikeln (grün) aus Zellen ohne MTM1 (rechte Bilder inkl. Vergrößerung) im Vergleich zu Kontrollzellen (linke Bilder inkl. Vergrößerung).

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, 408 – 412. 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 war die Erkenntnis, wie α-synuclein in der gesunden Zelle aufgebaut Parkinson's disease and other neurodegenerative diseases. It is known ist. Wissenschaftler vom FMP (Forschungsgruppe Philipp Selenko) that this protein has very concrete structures in the pathological konnten das Protein jetzt erstmals mit Hilfe von hochauflösenden state; however, as isolated, purified protein it does not appear to spektroskopischen Verfahren in gesunden Zellen sichtbar machen. have any structure at all. What has been missing up until now is an Überraschendweise fanden sie jenen strukturlosen Zustand vor, den understanding of how α-synuclein is structured in the healthy cell. das Protein auch in aufgereinigtem Zustand hat. Die neuen Erkennt- Scientists from the FMP (Philipp Selenko's research group) have now nisse, die in „Nature“ und „Nature Communications“ erschienen for the first time visualised the protein in healthy cells with the aid of sind, sind ein Meilenstein für die Forschung: Jetzt weiß man, dass sich high-resolution spectroscopic methods. Surprisingly, they found the die Struktur des Proteins im Krankheitsverlauf dramatisch verändert. 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 Binolfi A, Limatola A, Verzini S, Kosten J, Theillet F X, Rose H M, Bekei the structure of the protein changes dramatically over the course of B, Stuiver M, van Rossum M, Selenko P (2016) Intracellular repair of the disease. oxidation damaged a-synuclein fails to target C-terminal modification sites. Nat Commun. 7,10251. Das Amyloid-Protein α-synuclein spielt bei Parkinson und anderen Theillet F X, Binolfi A, Bekei B, Martorana A, Rose H M, Stuiver M, neurodegenerativen Erkrankungen eine wichtige Rolle. Bekannt Verzini S, Lorenz D, van Rossum M, Goldfarb D, Selenko P (2016) ist, dass dieses Protein im krankhaften Zustand über sehr konkrete Structural disorder of monomeric a-synuclein persists in mammalian cells. Strukturen verfügt; als isoliertes, aufgereinigtes Protein scheint es Nature 530(7588), 45 – 50. jedoch überhaupt keine Struktur zu besitzen. Was bislang fehlte, 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 Sie heißen Inositol-Pyrophosphate und sind an den verschiedensten variety of different processes in the cell, such as insulin secretion Prozessen in der Zelle beteiligt, zum Beispiel an der Insulinsekretion and metastasis. With the aid of chemical fishing rods, the team led oder der Metastasierung. Das Team um Dorothea Fiedler identifizierte by Dorothea Fiedler identified over a hundred proteins in yeast mit Hilfe von chemischem Angeln über hundert Proteine in Hefe- cultures to which the messenger substances bind. The bound molecules kulturen, an die die Botenstoffe binden. Die nun gebundenen subsequently altered several of the proteins chemically. Proof that two Moleküle veränderten mehrere der Eiweiße anschließend chemisch. processes work in tandem here – i. e. first the selective binding and Den Nachweis, dass sich hier zwei Prozesse vereinen – also erst die then the chemical modification – was established when the researchers selektive Bindung und dann die chemische Modifikation – war were able to synthesise stabilised versions of the messenger substances. dadurch gelungen, dass die Forscher stabilisierte Versionen der Thus, the reactive molecules sustained the subsequent tests with Botenstoffe synthetisieren konnten. So hielten die reaktiven Moleküle the fishing hooks. In this way, the researchers extracted the relevant den anschließenden Versuchen mit den Angelhaken stand. Auf diese candidates from more than 6,000 proteins. Some of these protein- Weise fischten die Forscher aus über 6.000 Proteinen die relevanten messenger interactions had already been characterized, but in most Kandidaten heraus. Einige dieser Proteine kannte man schon, von den cases it was not known which of the proteins bound to inositol pyro- meisten wusste man jedoch nicht, dass sie mit Inositol-Pyrophosphaten phosphates. Of course, the investigation will not stop at yeast cultures. interagieren. Bei Hefekulturen wird es natürlich nicht bleiben.

In the next step, the researchers want to apply their method to human Im nächsten Schritt wollen die Forscher ihre Methode an menschlichen cells, with even better fishing techniques. Experiments on animal Zellen überprüfen, mit noch besseren Angeltechniken. Anschließend models may then follow. “Once we know the exact mechanisms – and könnten Experimente an Tiermodellen folgen. „Wenn wir die genau- we have now taken an important first step in this direction – it may one en Mechanismen kennen – und hierzu ist uns jetzt ein erster wichtiger day be possible to intervene in the signal functions of these messenger Schritt gelungen – könnte es eines Tages möglich sein, therapeutisch substances therapeutically,” says Fiedler. It will be a long journey until in die Signalfunktionen dieser Botenstoffe einzugreifen“, sagt Fiedler. then, but in light of the diverse pathological processes in which inositol Bis dahin sei es zwar noch ein langer Weg, aber angesichts der vielfäl- pyrophosphates are involved, it will be worth it. tigen Krankheitsprozesse, an denen Inositol-Pyrophosphate beteiligt sind, ein sehr lohnenswerter.

S. cerevisiae cell lysate !" RNA pol I complex #" Nucleolus $" Preribosome P P P P P %" Plasma membrane enriched fraction P P P P P PCP &" RNA pol III complex P P '" Small nucleolar ribonucleoprotein complex Control beads InsP6 beads 5PCP-InsP5 beads (" )" RNA polymerase activity *" Elution / SDS-PAGE !+" Transcription from RNA pol I promoter !!" Regulation of translation !#" rRNA processing Digestion / LC-MS/MS !$" Glucose metabolic process 0 2 4 6 8 10 12 14 -Log10P Analysis 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. Wu M, Chong L S, Perlman D H, Resnick A C, Fiedler D (2016) The inositol Immobilisierte Inositolpyrophosphate können zur Identifizierung ihrer polyphosphates intersect with protein signaling and metabolic networks via Interaktionspartner genutzt werden, und veranschaulichen, wie diese two distinct mechanisms. Proc. Nat. Acad. Sci. USA 113, E6757 – E6765. Moleküle über zwei verschiedene Mechanismen fungieren können. RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 11

„WIR HABEN DIE TÜR FÜR NEUE BIOMEDIZINISCHE ERKENNTNISSE AUFGESTOSSEN“ “ WE'VE PUSHED THE

DOOR WIDE Prof. Dr. Dr. Thomas J. Jentsch OPEN TO NOVEL BIOMEDICAL INSIGHTS”

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

Bestandteile des VRAC in der Plasmamembran in der Zelle. (Tobias Stauber) (Tobias

can also transport GABA. We suspect fascinating roles of specific Thomas Jentsch gilt als weltweit führender Forscher auf dem VRACs in physiological signal transmission in the brain but also, for Gebiet der Ionenkanäle. Im Interview spricht er über seine example, in the development of pathologies like stroke. jüngsten Entdeckungen und Pläne für die kommenden Jahre.

Do you already have your sights on a medical target? Herr Professor Jentsch, vor drei Jahren haben Sie die moleku- In the case of stroke, we know that astrocytes, glial cells found lare Identität des Anionenkanals VRAC entdeckt. Was konnten in the central nervous system, swell and release glutamate under Sie seit diesem Durchbruch Neues über diesen Regulator des hypoxia. This will result in glutamate toxicity that leads to neuronal Zellvolumens herausfinden? cell death. If one could prevent VRAC from releasing glutamate, the In der Tat war die Identifizierung der Proteine, aus denen VRAC damaged brain area would probably be smaller. Using genetic mouse besteht, entscheidend für das Verständnis des Kanals, den man ja schon models, we are now investigating this hypothesis in co-operation seit 30 Jahren untersucht hat. Erst dadurch können seine Lokalisation, with a group at the Charité. molekulare Funktionsweise und diverse physiologische Funktionen sowie seine Rolle bei Krankheiten untersucht werden. Wir sind mit What might a therapeutic intervention look like? diesen Untersuchungen immer noch am Anfang, haben aber schon It is hoped that drugs may be developed that specifically block those jetzt herausgefunden, dass der Ionenkanal nicht nur das Zellvolumen VRACs that allow glutamate to pass, that is channels containing reguliert, sondern auch bestimmte Neurotransmitter und Anti- specific combinations of subunits. We are currently searching for Krebsmedikamente transportiert. Außerdem haben wir gelernt, substances that modulate the activity of VRAC together with the dass VRAC aus fünf Untereinheiten besteht, die in verschiedenen FMP Screening Unit. But it will no doubt take years to develop new Kombinationen auftreten können. So ist etwa die Untereinheit treatment options. LRRC8D für den Transport der Chemotherapeutika Cisplatin und Carboplatin essenziell, die bei verschiedenen soliden Tumoren At the beginning of the year, you received your second gegeben werden. ERC Advanced Grant. What do you intend to do with the 2.5 million euros? Jetzt sind wir schon in der klinischen Forschung? Well, a part of the project is dedicated to the characterisation of Sagen wir es so: Mit der Identifizierung von VRAC haben wir die Tür VRAC and its physiological and pathological roles, an area in which zu vielen neuen biologischen, medizinischen und pharmakologischen we expect many new and no doubt in part surprising findings. We Erkenntnissen aufgestoßen. Momentan bewegen wir uns noch im have already spoken about some of the aspects involved. The second Bereich der Grundlagenforschung, und ich werde nicht müde, deren part aims at the molecular identification of two further important Bedeutung hervorzuheben. Die Identifizierung von VRAC ist ein ion channels. These two channels may also lead us into completely weiteres Beispiel dafür, wie schnell diese in konkrete medizinische new terrain, as was the case with VRAC. Erkenntnisse mündet.

Was VRAC actually the reason for being awarded a second Erkenntnisse über Krebs? ERC grant? Ja, zum Beispiel. Schon im ersten Jahr nach der Identifizierung von It wasn't the reason, but an important prerequisite. The identification of VRAC konnten wir zeigen, dass durch diesen Kanal Chemothera- VRAC, a central project of my first ERC Advanced Grant, provided not only peutika in die Zelle gelangen. Fehlt die für diesen Transport the basis for the VRAC projects of the second grant, but also demonstrated notwendige VRAC Untereinheit, beobachten wir nicht nur ein our ability to carry out such high-risk projects successfully. We will now geringeres Abtöten von Tumorzellen in Kultur, sondern auch eine explore new areas which will hopefully lead to new exciting discoveries. Chemotherapie-Resistenz bei Tumorpatienten. Dies konnten wir in RESEARCH HIGHLIGHTS AKTUELLES AUS DER FORSCHUNG 13

Tobias Münch and Ian Orozco Felizia Voss and Tobias Stauber

Zusammenarbeit mit einer holländischen Arbeitsgruppe nachweisen. Formen von VRAC medikamentös zu blockieren, die Glutamat Die Kollegen hatten Genexpressionsprofile von Eierstocktumoren durchlassen. Wir suchen bereits mit unserer Screening-Unit nach von Patientinnen analysiert, die mit Cisplatin oder Carboplatin be- Substanzen, die die Aktivität von VRAC modulieren. Es wird aber handelt worden waren. Diejenigen, die weniger von der Untereinheit sicher Jahre dauern, bis sich möglicherweise neue Behandlungsmög- LRRC8D im Tumor hatten, waren deutlich früher gestorben, also lichkeiten auftun. wahrscheinlich relativ resistent gegen das Medikament. Sie haben Anfang des Jahres ihren zweiten ERC Advanced Gab es nicht schon lange den Verdacht, dass VRAC auch eine Grant bekommen. Was werden Sie mit den 2,5 Millionen Euro Rolle beim programmierten Zelltod, der Apoptose, spielt, den anfangen? Chemotherapien ja aktivieren können? Nun, ein Teil des Projektes ist der Charakterisierung von VRAC Es gab diese Hypothese und wir sind ihr auch nachgegangen. Nach und seinen physiologischen und pathologischen Rollen gewidmet, dieser Hypothese ist der induzierte Zelltod von Krebszellen dann ver- wo wir sehr viele neue, zum Teil sicher überraschende, Erkenntnisse ringert, wenn die bei Apoptose typische Zellschrumpfung ausfällt. In erwarten. Über einige Aspekte haben wir eben schon gesprochen. der Tat zeigten Zellen, in denen wir VRAC genetisch eliminiert hatten, Im zweiten Teil geht es um die molekulare Identifizierung von zwei wesentlich weniger programmierten Zelltod. Bei der Chemo-Resistenz weiteren Ionenkanälen. Diese beiden Kanäle könnten uns ebenfalls in von Tumoren haben wir es also wahrscheinlich mit einem doppelten völlig neues Terrain führen, wie es bei VRAC der Fall war. Mechanismus zu tun, wobei wir derzeit davon ausgehen, dass die redu- zierte Medikamenten-Aufnahme der wichtigere ist. War VRAC eigentlich der Grund für die abermalige Auszeichnung? Sind Ihre Erkenntnisse zum Transport von Neurotransmittern Der Grund nicht, aber eine wichtige Voraussetzung. Die Identifi- schon ähnlich konkret? zierung von VRAC war ja ein zentrales Projekt meines ersten ERC Hier konnten wir die Hypothese bestätigen, dass VRAC Glutamat Advanced Grants und war damit nicht nur die Grundlage für die und andere Aminosäuren transportiert. Neu ist die Erkenntnis, dass VRAC-Projekte des zweiten Grants, sondern auch der Beweis, dass dies über verschiedene Kombinationen von Untereinheiten geschieht wir solche Hochrisikoprojekte erfolgreich durchziehen können. Nun und unter anderem auch GABA so die Zelle verlassen kann. Wir geht es um zwei weitere neue Felder und Entdeckungen, von denen vermuten faszinierende Rollen bei der physiologischen Signalüber- wir heute noch nicht einmal etwas ahnen. tragung im Gehirn, aber zum Beispiel auch beim Schlaganfall.

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 Glutamat- freisetzung hindern, wäre das geschädigte Hirnareal vermutlich Prof. Dr. Dr. Thomas J. Jentsch was named Honorary Doctor by the kleiner. Dies können wir jetzt mit Hilfe von genetischen Mausmodellen University Medical Center Hamburg-Eppendorf (UKE) on May 2, 2017. zusammen mit einer Gruppe an der Charité untersuchen. Prof. Dr. Dr. Thomas J. Jentsch wurde am 2. Mai 2017 zum Ehrendoktor der Medizinischen Fakultät des Universitätsklinikums Hamburg-Eppendorf Wie könnte denn ein therapeutischer Eingriff aussehen? (UKE) ernannt. Die Hoffnung ist, gezielt die entsprechend zusammengesetzten Molecular and Theoretical Neuroscience Molekulare und Theoretische Neurowissenschaften Molecular Cell Physiology Group leader Molekulare Zellphysiologie Dr. Alexander Matthias Walter Group leader  PD Dr. Ingolf E. Blasig PAGE 46

 PAGE 31

SECTION MOLECULAR PHYSIOLOGY AND CELL BIOLOGY

BEREICH MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE

Cellular Imaging Animal Facility Zelluläre Bildgebung Tierhaltung

Group leaders Group leader Dr. Burkhard Wiesner (Light Microscopy) Dr. Natali Wisbrun Dr. Dmytro Puchkov (Electron Microscopy)  PAGE 52  PAGE 49 CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE Physiology and Pathology of Ion Transport Physiologie und Pathologie des Ionentransports

Group leader Prof. Dr. Dr. Thomas J. Jentsch

 PAGE 20

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 Protein Trafficking Membrantransport und Group leader Zellbeweglichkeit Prof. Dr. Ralf Schülein Group leader  PAGE 28 PD Dr. Tanja Maritzen

 PAGE 37 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 across endo- / lysosomal membranes regulates intracellular trafficking. and their well-orchestrated interplay. In the case of disease, During the reporting period, many exciting discoveries have been this interplay becomes unbalanced. Research in the “Molecular made by groups of the section, often in collaborations between FMP Physiology and Cell Biology” section aims at understanding groups. For instance, the department “Molecular Pharmacology and such mechanisms in molecular detail, as well as their dysfunc- Cell Biology”, headed by Volker Haucke, has discovered a mechanism tion in disease. Cellular targets for pharmaceutical intervention, for the local conversion of phosphoinositides, minor phospholipids many of them membrane proteins such as ion channels and that couple organelle identity to membrane traffic and signaling, G-protein-coupled receptors, are identified, studied in their from phosphatidylinositol 3-phosphate to phosphatidylinositol physiological environment, and their modulation by bioactive 4-phosphate, to enable exit from the endosomal system. A defect in compounds explored. According to our mission to create a this phosphoinositide conversion at endosomes underlies X-linked broader basis for pharmacology we focus on the study of centronuclear myopathy in humans (Ketel et al., Nature 2016). The less-explored membrane proteins and of molecules of key “Physiology and Pathology of Ion Transport” department, led by importance for intracellular trafficking. To this end, we employ Thomas Jentsch, found that volume-regulated LRRC8 (VRAC) a broad arsenal of techniques, ranging from molecular and channels, only recently identified by the group, transport organic cellular biology, to biochemistry and biophysics, to whole-animal compounds including neurotransmitters and the anti-cancer drug physiology using genetically modified mice, often with links cisplatin depending on the particular subunit composition, proving to human disease. Our projects have benefitted greatly from that LRRC8 proteins form the channel pore. Downregulation of interactions with other sections of the FMP, including those LRRC8D is clinically relevant in tumor drug resistance (Planells- concerned with structural biology and modeling, drug and siRNA Cases et al., EMBO J 2015). The “Molecular Cell Physiology” screening, as well as chemical biology. research group, headed by Ingolf Blasig, discovered a novel pathway to overcome pharmacological tissue barriers that can then be targeted to Two main research topics addressed in this section concern membrane improve drug delivery, while the “Protein Trafficking” group of Ralf proteins like ion channels and transporters, receptors, and junctional Schülein identified novel inhibitors of the eukaryotic Sec / translocon proteins, as well as cellular trafficking processes, in particular exo- pathway in cell-based high-throughput screens. and endocytosis. Strong links exist between these research topics: for instance, the interplay between exocytic membrane insertion of The four junior research groups of the section contributed significantly receptors into the plasma membrane and their endocytic retrieval to the scientific output of the section. The “Behavioral Neurodynamics” determines the cellular activity of receptors; conversely, ion transport 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 (Zacchariassenet 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 Mathias Böhme mathematical modeling (Böhme et al., Nat. Neurosci. 2016). MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 17

Sophie Dithmer Dmytro Puchkov

The FMP is closely connected to the Berlin neuroscience Leben gründet sich auf komplexe zelluläre und physiologische community via the groups of Korotkova / Ponomarenko and Mechanismen und deren optimal abgestimmtes Zusammenspiel. Alexander Walter, located at the Charité in central Berlin, as well Gerät dieses aus dem Gleichgewicht, entstehen Krankheiten. as through the DFG-financed Cluster of Excellence, NeuroCure. Das Verständnis dieser Mechanismen im molekularen Detail The heads of both departments (Jentsch, Haucke) are als auch deren Störung bei Krankheit ist Ziel der Forschung im members of NeuroCure, as are the junior group leaders Bereich „Molekulare Physiologie und Zellbiologie“. Zelluläre Alexey Ponomarenko, Tatiana Korotkova, Andrew Plested, and Zielmoleküle (Targets) für eine pharmakologische Einfluss- Janine Kirstein. These junior groups receive substantial co-financing nahme, darunter viele Ionenkanäle und G-Protein-gekoppelte from NeuroCure. Additional financing has also come from Rezeptoren, werden identifiziert und in ihrer physiologischen competitive intramural grants from the Leibniz Association. An SAW Umgebung untersucht. Zudem werden bioaktive Substanzen, grant to Thomas Jentsch helped initially to establish the Korotkova / die diese Targets modulieren können, gesucht. Im Sinne unserer Ponomarenko group, and Tanja Maritzen’s group is largely financed Mission, die Basis für pharmakologische Einflussnahme zu by a competitive Leibniz program aimed at fostering female group vergrößern, ist unsere Forschung darauf ausgerichtet, wenig leaders. In 2014 Volker Haucke and Thomas Jentsch, together with charakterisierte Membranproteine und Schlüsselmoleküle des Hartmut Oschkinat and Jens von Kries from the Structural Biology intrazellulären Membrantransportes zu untersuchen. Dazu and Chemical Biology sections, respectively, jointly obtained an setzen wir eine breite Palette von Techniken und Methoden aus SAW grant for the “Role of protein homeostasis in cellular aging”, Molekular- und Zellbiologie, Biochemie, Biophysik und Physio- as part of a program aimed at creating synergies with other Leibniz logie an Tiermodellen, in der Regel genetisch veränderten Institutes (Leibniz-Institut für Neurobiologie (LIN, Magdeburg) and Mäusen, ein. Die untersuchten Tiermodelle sind oft mit the Fritz-Lipmann-Institut (FLI, Jena)), and they likewise received menschlichen Krankheiten verknüpft. Die Projekte des Berei- intramural grants for collaborative aging-related research. Most ches profitieren hier sehr von der Zusammenarbeit mit den recently, an SAW grant was awarded to Volker Haucke, together with anderen Bereichen des FMP, insbesondere mit Strukturbiologie Dorothea Fiedler (Chemical Biology Section) and Hartmut Oschkinat, und Modellierunng, Wirkstoff-, siRNA-Screening und to develop a novel methodology for the quantitative determination chemischer Biologie. of inositol phosphates. Moreover, the section has invested heavily in cellular imaging techniques, a development that was initiated by Zwei wesentliche Forschungsschwerpunkte des Bereiches sind einerseits Volker Haucke and that is supervised by Burkhard Wiesner. Membranproteine wie Ionenkanäle, Transporter, Rezeptoren und Tight Super-resolution technologies such as PALM / STORM and STED, Junction-Proteine, andererseits Vorgänge des zellulären Membrantrans- spinning disk microscopy, and high pressure freezing for electron ports. Zwischen diesen Schwerpunkten gibt es enge Verknüpfungen. microscopy are now readily available to FMP scientists. Research Beispielsweise wird die zelluläre Aktivität von membranständigen carried out in the section is thoroughly interconnected with research Rezeptoren durch das Wechselspiel von Insertion der Rezeptoren in die being done in the other sections of the FMP, especially the screening Plasmamembran durch Exozytose und Entfernung aus dieser Membran and chemical biology capacities provided by the “Chemical Biology durch Endozytose bestimmt. Im Gegenzug reguliert der Ionentransport Platform”, including the “Screening Unit”, as well as with the über endo- / lysosomale Membranen den intrazellulären Membranfluss. proteomics capabilities of the “Mass Spectrometry” group of the Im Berichtszeitraum sind etliche hochinteressante Entdeckungen Chemical Biology section. The diversity of approaches and techniques, durch Mitglieder des Bereichs veröffentlicht worden, viele in enger together with our common interest in cell biology and neurobiology, Zusammenarbeit zwischen Arbeitsgruppen des FMP. So entdeckte die provides an excellent basis for advancing our knowledge of crucial Abteilung „Molekulare Pharmakologie und Zellbiologie“ unter der mechanisms that may be amenable to pharmacological intervention. Leitung von Volker Haucke einen Mechanismus der lokalen 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 Ponomarenko zeigten mit neuartigen optogenetischen Verfahren zur von Organellen mit Membranverkehr und Signaltransduktion koppeln), Manipulation neuronaler Aktivität, dass hochfrequente elektrische der Phosphatidylinositol 3-Phosphat in Phosphatidylinositol 4-Phosphat Schwingungen im Hypothalamus und in der Hirnrinde das Nahrungs- umwandelt. Eine Störung dieser Reaktion, die für den Transport aus suchverhalten ermöglichen (Carus-Cadavieco et al., Nature 2017). Die dem endosomalen System wichtig ist, liegt der X-chromosomalen Nachwuchsgruppe „Molekulare Neurowissenschaft und Biophysik“ zentronukleären Myopathie beim Menschen zugrunde (Ketel et al., unter Leitung von Andrew Plested stellte einen Glutamatrezeptor her, Nature 2016). Die von Thomas Jentsch geleitete Abteilung „Physiologie der seine eigene Aktivierung mit einem Fluoreszenzsignal anzeigt und Pathologie des Ionentransports“ entdeckte, dass die erst kürzlich von (Zacchariassen et al., PNAS 2016), den ersten seiner Art. Dies ist ein der Gruppe identifizierten volumenregulierenden LRRC8 (VRAC) – erster Schritt hin zu einem genetisch-kodierten optischen Detektor Kanäle, abhängig von ihrer spezifischen Zusammensetzung aus LRRC8 synaptischer Übertragung. Die Nachwuchsgruppe „Membrantransport Untereinheiten, organische Verbindungen wie z. B. Neurotransmitter und Zellbeweglichkeit“ von Tanja Maritzen entdeckte, dass das bislang oder das Krebsmedikament Cisplatin transportieren – ein Befund, noch nicht charakterisierte endozytische Adapterprotein Stonin1 die der beweist, dass LRRC8 Proteine die Pore des Kanals bilden. Eine Zellmotilität durch Vermittlung der Internalisierung des Gliom- Herunterregulierung von LRRC8D ist klinisch relevant für die Resistenz assoziierten Proteoglycans NG2 reguliert (Feutlinske et al., Nature bestimmter Krebsformen gegen Zytostatika-Therapie (Planells-Cases et Commun. 2015), wodurch wahrscheinlich auch das kreberzeugen- al., EMBO J 2015). Die Arbeitsgruppe „Molekulare Zellphysiologie“, de Potenzial von NG2 eingeschränkt wird. Janine Kirstein, die die geleitet von Ingolf Blasig, entdeckte einen neuen Signaltrans- Juniorgruppe „Rolle der Proteostase beim Altern und in Krankheit“ duktionsweg, der gezielt eingesetzt werden kann, um durch Überwin- leitet, identifizierte einen neuartigen menschlichen Chaperon-Komplex, dung von Gewebebarrieren die Wirkstoffeffizienz zu verbessern. Die der die Protein-Aggregation bei der Huntington-Krankheit vollständig von Ralf Schülein geleitete Gruppe „Protein Trafficking“ identifizierte unterdrückt und die durch diese neurodegenerative Erkrankung in einem zellbasierten Hochdurchsatz-Screen neue Inhibitoren verursachenden Amyloidfibrillen auflöst. Die Liaison Gruppe des eukaryotischen Sec / Translocon Weges über die Membran des Neurowissenschaften „Molekulare und Theoretische Neurowissen- endoplasmatischen Retikulums. schaften“ unter der Leitung von Alexander Walter konnte durch eine Kombination von hochauflösender Mikroskopie, Physiologie, Die vier Nachwuchsgruppen des Bereichs haben ebenfalls signifikant Genetik und mathematischer Modellierung molekulare Mechanismen zu dessem wissenschaftlichen Erfolg beigetragen. Die Nachwuchs- ergründen, die Neurotransmitter-Freisetzung auf der Skala millionstel- gruppe „Verhaltensneurodynamik“ von Tatiana Korotkova und Alexey Millimeter räumlich präzise an der Synapse organisieren, um eine 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 (Böhmeet al., Nat. (LIN, Magdeburg) und dem Fritz-Lipmann-Institut (FLI, Jena), Neurosci. 2016). die gleichermaßen Fördermittel aus diesem Programm für die gemeinsame Forschung zum Altern erhalten. Jüngst konnte Volker Sowohl durch die Gruppen Korotkova / Ponomarenko und Alexander Haucke zusammen mit Dorothea Fiedler (Bereich Chemische Biologie) Walter, deren Labore sich an der Charité-Universitätsmedizin im und Hartmut Oschkinat eine SAW-Förderung zur quantitativen Zentrum Berlins befinden, als auch durch das DFG-finanzierte Bestimmung von Inositolphosphaten einwerben. Exzellenzcluster „NeuroCure“ ist das FMP eng in die neurowis- senschaftliche Forschungsszene Berlins eingebunden. Die Abtei- Zudem investiert der Bereich kontinuierlich und substanziell in lungsleiter beider Abteilungen des Bereiches (Thomas Jentsch, den Aufbau hochmoderner zellulärer Visualisierungstechniken, Volker Haucke) sowie die Nachwuchsgruppenleiterinnen und -leiter eine Entwicklung initiiert von Volker Haucke und geleitet von Janine Kirstein, Tatiana Korotkova, Andrew Plested und Alexey Burkhard Wiesner. Hochauflösende Lichtmikroskopietechnologien Ponomarenko sind Mitglieder dieses Exzellenzclusters. Ihre Nach- wie PALM / STORM und STED, Spinning Disc-Mikroskopie wuchsgruppen erhalten eine zum Teil substanzielle Co-Finanzierung und High Pressure Freezing für die Elektronenmikroskopie sind durch NeuroCure. Zusätzliche Finanzmittel stammen von kompetitiv nunmehr für FMP-Wissenschaftlerinnen und -Wissenschaftler vergebenen Fördermitteln innerhalb der Leibniz-Gemeinschaft. gut zugänglich. Die Forschung des Bereichs ist ausgezeichnet mit Eine SAW-Förderung für Thomas Jentsch ermöglichte so die der Forschung der anderen Bereiche des FMP vernetzt. Das trifft Einrichtung der Nachwuchsgruppe Korotkova / Ponomarenko und insbesondere auf die Nutzung von Screening und Methoden der Tanja Maritzens Nachwuchsgruppe wird weitgehend aus einem chemischen Biologie, die „Screening Unit“ und die „Chemical kompetitiven Leibniz-Programm zur Förderung von Wissenschaft- Biology Platform“ bereithalten, sowie auf das Proteomik-Portfolio lerinnen in Leitungsfunktionen finanziert. 2014 warben Volker der Arbeitsgruppe „Massenspektrometrie“ im Bereich Chemische Haucke und Thomas Jentsch zusammen mit Hartmut Oschkinat Biologie zu. Die Vielfalt der wissenschaftlichen Ansätze und (Bereich Strukturbiologie) und Jens von Kries (Bereich Chemische Techniken, zusammen mit unserem hohen Interesse an Zellbiologie Biologie) eine SAW-Förderung für das gemeinsame Projekt zur und Neurobiologie, bieten eine ausgezeichnete Grundlage um „Rolle der Proteinhomöostase für das zelluläre Altern“ ein. Das unser Wissen über grundlegende Mechanismen zu vergrößern, die Projekt ist Teil eines größeren Vernetzungsprojektes in einem pharmakologischen Eingriffen zugänglich sein könnten. Programm, das Synergien mit anderen Leibniz-Instituten schaffen soll, in diesem Fall mit dem Leibniz-Institut für Neurobiologie 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 SUMMARY

1972 – 1978 Studied Medicine, We aim to understand ion transport processes from the molecular to the subcellular and 1974 – 1980 Studied Physics, cellular levels, up to the level of the organism. The latter levels are addressed through an Free University of Berlin 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+ 1981 – 1985 Staff scientist, Institute of channels, KCC cation-chloride cotransporters, anoctamin Ca2+-activated Cl- channels, and Clinical Physiology (Prof. Wiederholt), the volume-regulated VRAC channel. Key research areas are structure / function analysis, Free University of Berlin cellular neurobiology, extracellular signaling, volume regulation, and the endosomal-lysosomal 1982 Ph.D. in Physics, Fritz-Haber-Institute system. We study many organs, including the brain, inner ear, olfactory epithelium, skin (Prof. Block), Berlin 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 1984 M.D., Institute of Clinical Physiology structure / function and physiology of this ‘new’ channel. We are particularly excited by its (Prof. Wiederholt), Free University of Berlin ability to transport organic signaling molecules and drugs and are generating and analyzing 1986 – 1988 Postdoctoral fellow, Whitehead knock-out mouse models for each of its five subunits. These mice have begun to provide novel Institute (Harvey F. Lodish, MIT), Cambridge MA biological, and medically important, insights.

1988 – 1993 Research group leader, ZUSAMMENFASSUNG Center for Molecular Neurobiology Hamburg (ZMNH), Hamburg University Unser Ziel ist es, Ionentransportprozesse von der molekularen über die subzelluläre und zelluläre Ebene bis zur Rolle im gesamten Organismus zu verstehen. Letzteres versuchen 1991 “Habilitation” in Cell Biochemistry, wir durch Untersuchung der Phänotypen von knock-out (KO)- und knock-in (KI)-Mäusen Medical School of Hamburg University und die Analyse humangenetischer Erkrankungen zu erreichen. Unser Schwerpunkt liegt 1993 – 2006 Full professor (C4) of Molecular dabei auf CLC Cl- Kanälen und -Transportern, KCNQ K+ Kanälen, KCC-Kation-Chlorid- Neuropathology, ZMNH, Hamburg University; Kotransportern, Anoctamin Ca2+-aktivierten Cl- Kanälen und in letzter Zeit vor Allem Director of the Institut für Molekulare Volumen-regulierten VRAC Kanälen. Wir befassen uns mit Struktur / Funktions-Analyse Neuropathobiologie dieser Kanäle, ihrer Rolle im Nervensystem, bei extrazellulärer Signaltransduktion, in der Volumenregulation und in Endosomen und Lysosomen. Dabei untersuchen wir eine 1995 – 98 & 2001 – 2003 Director of the Center Reihe von Organen wie das Gehirn, Innenohr, Mechanorezeptoren der Haut, Riechepithelien, for Molecular Neurobiology Hamburg (ZMNH) Niere und Hoden. Nachdem uns 2014 der Durchbruch mit der molekularen Identifizierung Since 2006 Head of department, FMP and des schwell-aktivierten Anionenkanals VRAC gelungen ist, entwickeln wir breite MDC, Berlin (joint appointment), Full Professor Forschungsprogramme zur Aufklärung der Struktur-Funktions-Beziehungen und (W3), Charité – University Medicine Berlin physiologischen Rollen dieses „neuen“ Kanals. Uns fasziniert insbesondere, dass er auch organische Signalmoleküle und Medikamente transportiert, und wir haben eine Reihe von Since 2007 Member of NeuroCure Cluster Mausmodellen für alle seine fünf Untereinheiten erzeugt. Ihre Analyse erlaubt schon jetzt of Excellence wichtige Einblicke in bisher unbekannte physiologische Prozesse und ergibt medizinisch Since 2009 Deputy Director, FMP 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: www.leibniz-fmp.de/tjj-awards

Since 2000 elected member of EMBO and four Academies of Science

2011, 2017 ERC Advanced Grants MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 21

DESCRIPTION OF PROJECTS

Properties and roles of the long-sought volume-regulated anion We have previously shown that disruption of the plasma membrane channel VRAC Cl- channel ClC-2 leads to leukodystrophy, testicular degeneration, Cells must regulate their volume, for instance during growth or and retinal degeneration. We are now generating and analyzing cell when exposed to osmotic challenges. A key player in this process is type-specific ClC-2 KOs to better understand these pathologies that the volume-regulated anion channel VRAC that has been known are also found in patients with CLCN2 mutations. biophysically for more than 20 years but whose molecular identity had remained obscure. Using a genome-wide siRNA screen at Anoctamin (TMEM16) Ca2+-activated chloride channels the FMP screening facility, we have identified heteromers of We have shown that Ano2 is the Ca2+-activated Cl- channel of olfactory LRRC8 proteins, containing four membrane spans and C-terminal sensory neurons, but surprisingly our Ano2-/- mice showed that Ano2 leucine-rich repeats, as crucial VRAC components (Voss et al., is dispensable for olfaction. Whereas the main olfactory epithelium Science 2014). LRRC8A is required for VRAC activity, but needs at expresses only Ano2, the vomeronasal organ (VNO), which is least one other isoform (LRRC8B to -E) to form channels. VRACs relevant for social interactions, also expresses Ano1. We are now are probably hexamers of up to five different LRRC8 isoforms. We studying conditional double KOs of both Ca2+-activated Cl- channels have shown that VRAC also conducts various organic compounds, in the VNO. including neurotransmitters and modulators, suggesting it has a role in extracellular signal transduction and diseases such as stroke. KCNQ potassium channels We discovered that the subunit composition determines VRAC’s We previously cloned and characterized the K+ channels KCNQ2-5, permeation properties, with inclusion of LRRC8D enhancing the showed that mutations in KCNQ2 and 3 cause neonatal epilepsy, and transport of various compounds, and LRRC8E that of glutamate. that mutations in KCNQ4 cause a form of deafness. In collaboration Our findings demonstrate that LRRC8 proteins form the pore of with A. Ponomarenko and T. Korotkova we analyzed Kcnq5dn/dn mice VRACs. Excitingly, LRRC8D-containing VRACs also transport the in which channels containing KCNQ5 are inactivated. These studies anti-cancer drug cisplatin and down­regulation of LRRC8D confers showed that KCNQ5 is important for controlling synaptic inhibition tumor drug resistance. By impairing apoptotic cell volume decrease, and network activity. We have recently shown that KCNQ3 is also ex- disruption of VRAC impairs drug-induced apoptosis. The LRRC8 pressed in extremely sensitive D-hair mechanoreceptors in the skin and subunit composition determines the inactivation of VRAC currents modulates their sensitivity, complementing our previous work showing and we identified relevant residues for this inactivation. In a major a similar role of KCNQ4 in rapidly adapting skin mechanoreceptors. effort to uncover the physiological and pathological roles of VRACs, we generate and analyze multiple mouse models for various LRRC8 Potassium-chloride cotransporters subunits. We disrupt Lrrc8a in a cell- and tissue-specific manner We have previously analyzed constitutive KOs of the KCC K+-Cl-- as the KO of LRRC8A, which completely abolishes VRAC function, cotransporters KCC1 – KCC4 and discovered unexpected roles in is lethal. various tissues. Neuronal KCC2 lowers cytosolic Cl- concentration, a process needed for the inhibitory response to the neurotransmitters CLC chloride channels and transporters GABA and glycine. Using a mitral cell-specific Kcc2 KO we have Proteins of the CLC gene family, discovered by us in 1990, reside in now shown that KCC2-dependent synaptic inhibition in the olfactory the plasma membrane and intracellular vesicles. We have generated bulb is crucial for discriminating closely related odors. 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. 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, 2993-3008 (2015). Figure adapted from Jentsch, Nature Rev. Mol. Cell Biol. 17, 293-307 (2016).

GROUP MEMBERS COLLABORATIONS

Dr. Kathrin Gödde International Gerd Krause, Dr. Maja Hoegg-Beiler Piet Borst, Leibniz-Forschungsinstitut für Molekulare Dr. Anna Oliveras Martínez Netherlands Cancer Institute, Amsterdam, Pharmakologie (FMP), Berlin Dr. Ian Orozco The Netherlands Jens von Kries, Dr. Rosa Planells-Cases Alan Carleton, Leibniz-Forschungsinstitut für Mole­kulare Dr. Sonali Saha Université de Genève, Pharmakologie (FMP), Berlin Dr. Tobias Stauber Switzerland Trese Leinders Zufall, Dr. Janis Vogt Dominique Eladari, Universität des Saarlandes, Homburg / Saar Dr. Felizia Voss Faculté de Médecine, Marc Nazaré, Dr. Stefanie Weinert Université Paris-Descartes, France Leibniz-Forschungsinstitut für Molekulare Dr. Joanna Ziomkowska Sven Rottenberg, Pharmakologie (FMP), Berlin Dr. Pingzheng Zhou Netherlands Cancer Institute, Amsterdam, Alexei Ponomarenko, Dr. Norma Nitschke (research coordinator) The Netherlands Charité – Universitätsmedizin Berlin and Sebastian Albrecht (doctoral student) Francisco Sepúlveda, FMP, Berlin Anja Blessing (doctoral student) CECS, Valdivia, Chile Dmytro Puchkov, Andreia Cruz e Silva (doctoral student) Guillermo Spitzmaul, Leibniz-Forschungsinstitut für Mole­kulare Tony Daubitz (doctoral student) INIBIBB, Bahía Blanca, Argentina Pharmakologie (FMP), Berlin Deborah Elger (doctoral student) Chris de Zeeuw, Christian Rosenmund, Corinna Göppner (doctoral student) Erasmus MC, Rotterdam and Netherlands Charité – Universitätsmedizin Berlin Karen López Cayuqueo (doctoral student) Institute for Neuroscience, Amsterdam, Dietmar Schmitz, Jennifer Lück (doctoral student) The Netherlands Charité – Universitätsmedizin Berlin Carmen Ludwig (doctoral student) Bernd Wollnik, Darius Lutter (doctoral student) National Universität Göttingen Jonas Münch (doctoral student) Ulrich Dirnagl, Frank Zufall, Karina Oberheide (doctoral student) Charité – Universitätsmedizin Berlin Universität des Saarlandes, Homburg / Saar Maya Polovitskaya (doctoral student) Maik Gollasch, Werner Zuschratter, Sebastian Schütze (doctoral student) Charité – Universitätsmedizin Berlin Leibniz-Institut für Neurobiologie (LIN), Till Stuhlmann (doctoral student) Hans-Jürgen Holdt, Magdeburg Florian Ullrich (doctoral student) Universität Potsdam Carolin Backhaus (technical assistant) Christian Hübner, Anyess von Bock (technical assistant) Universitätsklinikum Jena Karolin Fuchs (technical assistant) Vera Kalscheuer, Petra Göritz (animal care taker) Max-Planck-Institut für Molekulare Genetik, Anika Günther (technical assistant) Berlin Johanna Jedamzick (technical assistant) Uwe Kornak, Janet Liebold (technical assistant) Charité – Universitätsmedizin Berlin and Antje Maluck (technical assistant) Max-Planck-Institut für Mole­kulare Genetik, Ruth Pareja-Alcaraz (technical assistant) Berlin Katrin Räbel (technical assistant) Tatiana Korotkova, Patrick Seidler (technical assistant) Charité – Universitätsmedizin Berlin and Andrea Weidlich (technical assistant) FMP, Berlin Eberhard Krause, Staff employed within the reporting period Leibniz-Forschungsinstitut für Mole­kulare Pharmakologie (FMP), Berlin MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 23

Ethyl valerate versus ethyl tiglate a b Fig. 2: Mitral cell-specific lox/lox + - Kcc2 MC-∆Kcc2 90% Criterion disruption of the K Cl cotransporter KCC2 in Kcc2lox/lox the olfactory bulb (a) MC-∆Kcc2 entails the inability to Chance level Correct responses (%) responses Correct distinguish closely relat- ed odors in behavioral Blocks of 20 trials olfactometry experiments

0.6/0.4% ethyl valerate/ethyl tiglate versus (b). Taken from Gödde 0.4/0.6% ethyl valerate/ethyl tiglate et al., Nature Communi- cations 7, 12043 (2016). DAPI Kcc2 reelin Correct responses (%) responses Correct

Blocks of 20 trials

SELECTED PUBLICATIONS

Planells-Cases R, Lutter D, Guyader C, Gerhards N M, Ullrich F, Elger Deutsche Forschungsgemeinschaft, „Strukturelle Grundlagen und D A, Kucukosmanoglu A, Xu G, Voss F K, Reincke S M, Stauber T, physiologische Funktion des Cl- / H+-Gegenaustausches bestim- Blomen V A, Vis D J, Wessels L F, Brummelkamp T R, Borst P, Rottenberg mter CLC-Chloridtransportproteine”, ZD 58 / 1-1, with A. Zdebik, S*, Jentsch T J * (2015). VRAC channel composition determines its 07.2006 – 10.2010. Continued as: JE 164 / 9-2, 01.2011 – 07.2015, 320.500 € substrate specificity and cellular resistance to Pt-based anti-cancer Deutsche Forschungsgemeinschaft, „Der CIC-7 / Ostm1 Chlorid- drugs. EMBO J. 34, 2993 – 3008. transporter in Lysosomen und Osteoklasten”, JE 164 / 7-1, Fidzinski P, Korotkova T, Heidenreich M, Maier N, Schuetze S, 01.2007 – 08.2010. Continued as: JE 164 / 7-2, 07.2011 – 12.2015, 669.000 € Kobler O, Zuschratter W, Schmitz D, Ponomarenko A *, Jentsch T J * Deutsche Forschungsgemeinschaft, „Funktionelle Charakterisierung (2015). KCNQ5 K+ channels control hippocampal synaptic inhibition and ausgewählter Mitglieder der Anoctamin-Kanalfamilie”, JE 164 / 10-1, fast network oscillations. Nature Commun. 6, 6254. 05.2014 – 11.2018, 391.000 € Schütze S, Orozco I J, Jentsch T J * (2016). KCNQ potassium Deutsche Forschungsgemeinschaft, „Volumen-regulierter Anionen- channels modulate sensitivity of skin D-hair mechanoreceptors. J. Biol. Kanal VRAC und seine Rolle im Gehirn“, JE 164 / 12-1, 01.2016 – 12.2018, Chem. 291, 5566 – 5575. 433.500 € Ullrich F, Reincke S M, Voss F K, Stauber T, Jentsch T J * (2016). Deutsche Forschungsgemeinschaft (Aufbau internationaler Inactivation and anion selectivity of volume-regulated VRAC channels Kooperationen), „Untersuchung der Funktion der Kaliumkanäle KCNQ depend on carboxy-terminal residues of the first extracellular loop. J. in den Augen anhand genetischer Mausmodelle“, JE 164 / 13-1, Biol. Chem. 291, 17040 – 17048. 07.2015 – 01.2017, 9.383 € Gödde K, Gschwend O, Puchkov D, Pfeffer C K, Carleton A *, Jentsch Europäischer Forschungsrat (7. Forschungsrahmenprogramm), “Ion T J * (2016). Disruption of Kcc2-dependent inhibition of olfactory bulb homeostasis and volume regulation of cells and organelles (CYTOVO- output neurons suggests its importance in odour discrimination. Nature LION)”, ERC-2011-ADG_294435, 04.2012 – 03.2017, 2.096.800 € Commun. 7, 12043. Bundesministerium für Bildung und Forschung (ERA-NET: E-Rare), “CLC chloride channels and Megalencephalic Leucoencephalopathy: EXTERNAL FUNDING molecular mechanisms and therapeutics”, 01GM1403, 06.2014 – 01.2018, 401.419 € Prix Louis Jeantet, 04.2000 – 12.2016, 308.008 € Leibniz-Gemeinschaft (Leibniz Wettbewerb 2014), “Role of proteostasis Deutsche Forschungsgemeinschaft, Exzellenzinitiative an der Humboldt- in cellular aging”, SAW-2014-FMP-2, with V. Haucke, H. Oschkinat, Universität zu Berlin, Projekt NeuroCure: “Towards a better outcome of J.-P. von Kries, 06.2014 – 05.2017, 158.555 € (pro rata) central nervous system disorders”, - Innovation Project 2015: “Role of the volume-regulated anion channel EMBO Long-Term Fellowship, Sonali Saha, 02.2016 – 01.2018, 75.816 € VRAC in hearing and deafness”, 01.2015 – 12.2015, 20.000 €; Alexander von Humboldt Research Fellowship, Pingzheng Zhou, - Innovation Project 2016: “The volume-regulated anion channel VRAC 08.2016 – 07.2018, 82.800 € and the ventricular system”, 01.2016 – 12.2016, 20.000 €

Deutsche Forschungsgemeinschaft, SFB 740, C05, “Protein modules involved in vesicular acidification and trafficking: focus of CIC-6”, 01.2007 – 12.2010. Continued as: SFB 740 / 2, C05, “Funktionale Module in der endosomal-lysosomalen Ionenhomöostase und ihre Funktion” 01.2011 – 12.2014, 703.100 €. Continued as: SFB 740 / 3, C05, 01.2015 – 12.2018, 490.800 €. FMP authors Group members * corresponding authors 24 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

MOLECULAR PHARMACOLOGY AND CELL BIOLOGY

MOLEKULARE PHARMAKOLOGIE UND ZELLBIOLOGIE

GROUP LEADER PROF. DR. VOLKER HAUCKE

BIOGRAPHY SUMMARY

1989 – 1994 Studied Biochemistry, Membrane dynamics within the endocytic and endosomal system play crucial roles in cell Free University of Berlin and Biozentrum, physiology and membrane homeostasis, cell signaling and development, the functioning of University of Basel 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 1994 – 1997 PhD (summa cum laude), of techniques that range from in vitro approaches to the in vivo analysis of cellular systems. Department of Biochemistry (Prof. G. We are particularly interested in the cycling of synaptic vesicles at neuronal synapses and Schatz), Biozentrum, University of Basel its role in brain function and disease and in the physiological functions of inositol lipids. An 1997 – 1999 Postdoctoral fellow, important aspect of these studies is to determine how events at the molecular and sub- Yale University School of Medicine and cellular levels translate into the functions of individual cells and of cellular networks within HHMI (Prof. P. De Camilli), New Haven 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 2000 – 2003 Independent group leader, genetic, biochemical, and pharmacological approaches, and by further developing super- Center for Biochemistry and Molecular resolution imaging techniques. The long-term goal of our work is to unravel the molecular Cell Biology, University of Göttingen basis of endocytic and endosomal function and dysfunction, thereby opening new avenues for 2003 – 2005 Professor of Membrane pharmacological interference. Biochemistry, Free University of Berlin ZUSAMMENFASSUNG 2005 – 2011 Full Professor and Chair (W3), Department of Membrane Biochemistry, Dynamische Membranprozesse des endozytotischen und endosomalen Systems spielen eine Free University of Berlin entscheidende Rolle in der Zellphysiologie und Membranhomöostase, bei der Signalüber- tragung zwischen Zellen und der Zellentwicklung, für die Aktivitäten des Nervensystems since 2007 Member of Neurocure Cluster und bei Krankheiten wie Krebs. Der Forschungsschwerpunkt unserer Abteilung liegt auf den of Excellence molekularen Mechanismen des endozytotischen und endosomalen Membrantransports, die 2008 – 2010 Speaker, Collaborative wir mit einem breiten Spektrum an Techniken, von in vitro Ansätzen bis zur in vivo-Analyse Research Center (SFB) 449 zellulärer Systeme, untersuchen. Besonders interessiert sind wir am Zyklus synaptischer Vesikel an neuronalen Synapsen und dessen Rolle bei der Gehirnfunktion und Erkran- 2011 – 2012 Speaker, Collaborative kungen des Nervensystems sowie der physiologischen Funktion von Inositol-Lipiden. Ein Research Center (SFB) 958 wesentlicher Aspekt dieser Untersuchungen besteht darin, zu ermitteln, wie Ereignisse since 2012 Director of the FMP, Head of the auf molekularer und subzellulärer Ebene in Funktionen einzelner Zellen und zellulärer Department of Molecular Pharmacology & Netzwerke innerhalb eines Gesamtorganismus übersetzt werden. Um dies zu erreichen, Cell Biology at the FMP, Full Professor of entwickeln wir mit Hilfe genetischer, biochemischer und pharmakologischer Ansätze und Molecular Pharmacology (S-W3), Institute of durch Weiterentwicklung hochauflösender bildgebender Verfahren molekulare Werkzeuge Pharmacy, Free University of Berlin zur Analyse und Manipulation des Exo-Endozytose Zyklus synaptischer Vesikel sowie der endosomalen Membrandynamik. Das langfristige Ziel unserer Arbeit ist es, die molekularen 2014 Elected Member of the European Grundlagen der endozytotischen und endosomalen Funktion und Dysfunktion zu enträtseln Molecular Biology Organization (EMBO) und dabei neue Wege für pharmakologische Eingriffe zu eröffnen. 2016 Reinhart Koselleck-Grant Award of the Deutsche Forschungsgemeinschaft (DFG)

2017 Avanti Award of the American Society for Biochemistry & Molecular Biology (ASBMB) MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 25

DESCRIPTION OF PROJECTS

Research within the department is conducted within two subgroups Regulation of endocytic and endolysosomal membrane (led by V. Haucke and M. Krauss) and covers three major areas: (i) homeostasis and cell signaling by phosphoinositides the role of exo-endocytic and endolysosomal membrane dynamics Eukaryotic cells internalize nutrients, antigens, growth factors, in synapse function and neuronal development; (ii) the regulation pathogens, ion channels and receptors via endocytosis. We are interested of membrane homeostasis and cell signaling by phosphoinositides, in determining the exact function of endocytic adaptors and scaffolds, in- and (iii) the function of septins scaffolds in membrane dynamics cluding lipid kinases and phosphatases as well as Bin / Amphiphysin / Rvs and organelle contacts. Together with the Core Facility “Cellular homology (BAR) domain proteins, in the spatiotemporal regulation of Imaging”, we also develop and use super-resolution light (e. g. clathrin-mediated endocytosis. We have addressed the question of how multi-color STORM and 3D-gSTED, TIRF-SIM) and electron membrane deformation in endocytosis is coupled to dynamin-mediated microscopy approaches for studying these processes. fission and how dynamin assembly is coupled to endocytic vesicle formation through regulated synthesis and turnover of phosphoinositide Exo-endocytic and endolysosomal membrane dynamics in the lipids (PIs). Other projects aim at unraveling mechanisms of clathrin- functioning of synapses and in neuronal development independent endocytosis such as macropinocytosis in dendritic cells of Neurotransmission involves the calcium-triggered exocytic release of the immune system. Genetics, RNA interference, and acute chemical and neurotransmitters from synaptic vesicles (SVs) at presynaptic active optogenetic perturbations are used to designate the roles of specific PIs zones, followed by their endocytic recycling. Using mouse knockout during the progression of endocytosis and to elucidate how PI conversion technology, Drosophila mutants (with Stephan J. Sigrist, FU Berlin), along the endolysosomal pathway is linked to cell signaling processes, and RNA interference in combination with optical imaging including as well as how these processes are regulated. In recent studies we optogenetics and electrophysiology, we aim to dissect the pathways discovered a mechanism for endosomal exocytosis mediated by the and molecular mechanisms of SV recycling, regeneration, and axonal PI(3)P phosphatase MTM1, an enzyme whose loss of function leads to transport of SV precursor organelles. A key question in this regard is X-linked centronuclear myopathy in humans. Removal of endosomal how exo- and endocytosis are coupled. PI(3)P by MTM1 is accompanied by generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. We have discovered that the endocytic protein AP180 acts as a Our data show that defective PI conversion at endosomes underlies X-linked governess that oversees sorting of the essential vesicular SNARE centronuclear myopathy caused by mutation of MTM1 in humans. synaptobrevin 2. Loss of AP180 impairs neurotransmission and leads As many PI-metabolizing enzymes are implicated in cancer as well as to excitatory / inhibitory imbalance and fatal epilepsies due to reduced hereditary disorders such as Charcot-Marie-Tooth disease we also seek copy numbers of synaptobrevin 2 in SVs. Further studies have shown to identify novel pharmacological and chemical inhibitors of select PI- that endocytic adaptors such as AP180 and its close relative CALM, a metabolizing enzymes. protein implicated in Alzheimer's disease, limit the diffusional spread of newly exocytosed SV proteins to prevent their loss into the axon. Function of septins scaffolds in membrane dynamics and In our most recent work, we found that at physiological temperature organelle contacts SV endocytosis occurs on several timescales, ranging from less than Recent work from our lab has identified key connections between the a second to several seconds, and largely occurs via formin-mediated endosomal system and the microtubule-based, as well as the actin, endocytosis independent of clathrin, whereas clathrin / AP-2 are cytoskeleton. Septin GTPases are another element of the cytoskeleton required for SV reformation from internal structures. and play key roles in regulating a variety of cellular functions, ranging from membrane traffic and signaling to cell motility. By employing Other ongoing studies have revealed unexpected novel endocytosis- biochemical, cell biological, and optical techniques we dissect the role of independent roles in neuronal development for endocytic proteins septin-based membrane scaffolds, adaptor proteins and PI-metabolizing such as AP-2 and intersectins, which regulate key signaling processes. enzymes in regulating cytoskeletal dynamics, the formation of organelle Our studies have implications for the understanding and treatment of contact sites, and protein sorting through the Golgi-endosomal interface neurological disorders and for neurodegeneration. during cell signaling and migration, as well as polarized secretion. 26 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014

CONTROL CELL 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 integrin integrin MTM1 (right image). Inset images are magnified views. Taken from Ketel et al. (2016). vesical vesical

GROUP MEMBERS COLLABORATIONS

Prof. Dr. Michael Krauß (group leader) International National Dr. Jan Schmoranzer (group leader) Sandra M. Bajjalieh, Oliver Daumke, Dr. Caroline Bruns Univ. of Washington, Seattle, USA Max-Delbrück-Center for Molecular Dr. Gaga Kochlamazashvili Daniel F. Cutler, Medicine, Berlin Dr. Natalia Kononenko University College London, London, UK Christian Freund, Dr. Marijn Kuijpers Emilio Hirsch, Freie Universität Berlin Dr. Martin Lehmann University of Torino, Italy Gary R. Lewin, Dr. Tania Lopez-Hernandez Jocelyn Laporte, Max-Delbrück-Center for Molecular Dr. Marta Maglione (joint postdoc with IGBMC, Strasburg, France Medicine, Berlin S. J. Sigrist, FU Berlin) Adam McCluskey, Tobias Moser, Dr. Mouhannad Malek University of Newcastle, Australia Georg-August-Universität, Göttingen Dr. Andrea Lynn Marat Phillip J. Robinson, Silvio O. Rizzoli, Dr. Christoph Ott Children's Medical Research Institute (CMRI), Georg-August-Universität, Göttingen Dr. Tolga Soykan Sydney, Australia Christian Rosenmund, Dr. Domenico Azarnia Tehran Takeshi Sakaba, Charité – Universitätsmedizin Berlin Dr. Anna Wawrzyniak Kyoto University, Japan Dietmar Schmitz, Dr. Mirjana Weimershaus Oleg Shupliakov, Charité – Universitätsmedizin Berlin Dr. Haibin Wang Karolinska Institute, Stockholm, Sweden Stephan J. Sigrist, Gala Claßen (doctoral student) Freie Universität Berlin Katrin Diesenberg (doctoral student) Carsten Schultz, Fabian Feutlinske (doctoral student) EMBL, Heidelberg 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)

Staff employed within the reporting period 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, Deutsche Forschungsgemeinschaft, SFB 958, Z02, “Super-resolution Puchkov D, Kononenko N L, Haucke V (2017) Synaptic vesicle light microscopy to resolve nanoscale molecular structures”, to endocytosis occurs on multiple timescales and is mediated by J. Schmoranzer, 07.2011 – 06.2015 403.600 € formin-dependent actin assembly. Neuron 93, 854 – 866. Deutsche Forschungsgemeinschaft, SFB 740 / 3, C08, „Funktionelle Ketel K, Krauss M, Nicot A S, Puchkov D, Wieffer M, Müller R, Organisation und Dynamik PI-Kinase-basierter Module für die Protein- Subramanian D, Schultz C, Laporte J, Haucke V (2016) sortierung an endosomalen Membranen”, 01.2015 – 12.2018, 544.320 €

A phosphoinositide conversion mechanism for exit from endosomes. Deutsche Forschungsgemeinschaft, SFB 765, B04, „Multivalente Nature 529, 408 – 412. Modulation der Clathrin vermittelten Rezeptorendozytose”, Koo S Y, Kochlamazashvili G, Rost B, Puchkov D, Gimber N, 01.2012 – 12.2015, 414.400 €

Lehmann M, Tadeus G, Schmoranzer J, Rosenmund C, Haucke V#, Deutsche Forschungsgemeinschaft, TRR 186, A08, “Phosphoinositide- Maritzen T# (2015) Vesicular synaptobrevin / VAMP2 based switches in endocytic membrane traffic and signaling”, levels guarded by AP180 control efficient neurotransmission. to V. Haucke (w / C. Schultz, EMBL) 07.2016 – 06.2020, 555.200 € Neuron 88, 330 – 344 (#co-corresponding authors).

Deutsche Forschungsgemeinschaft, TRR 186, A09, “Phosphoinositide- Gimber N, Tadeus G, Maritzen T, Schmoranzer J, Haucke V. (2015) based switches in polarized sorting and signaling”, Diffusional spread and confinement of newly exocytosed synaptic to V. Haucke (w / S. Boulant, Heidelberg) 07.2016 – 06.2020, 441.600 € vesicle proteins. Nature Communications 6, 8392.

Deutsche Forschungsgemeinschaft, Excellence Initiative, EXC-257 Reubold T, Faelber K, Plattner N, Posor Y, Ketel K, Curth U, Schlegel J, NeuroCure “Towards a better outcome of central nervous system Roopsee A, Manstein D J, Noé F, Haucke V, Daumke O, Eschenburg S. disorders”, 01.2012 – 12.2016, 385.750 € (2015) Crystal structure of the dynamin tetramer. Nature 525, 404 – 408. European Commission, Horizon 2020, ITN, H2020-MSCA-ITN-2015, "Deciphering PI3K biology in health and disease”, to V. Haucke, 11.2015 – EXTERNAL FUNDING 10.2019, 498.433 €

Bundesministerium für Forschung und Technologie (BMBF), „Die European Commission, Horizon 2020, H2020-MSCA-IF-2014, “The role neurobiologischen Grundlagen Polyamin-induzierter Protektion gegen of autophagy in presynaptic protein turnover (SYNPT)”, to M. Kuijpers, altersassoziierte Einschränkungen der Gedächtnisfunktionen 03.2015 – 04.2017, 159.460 €

(SMARTAGE)”, to V. Haucke, 07.2015 – 06.2020, 438.924 € Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Deutsche Forschungsgemeinschaft, Reinhart Koselleck-Award Forschung und Innovation, “Role of proteostasis in cellular aging”, SAW (HA 2686 / 13-1), 1.2017 – 12.2021, 750.000 € 2014-FMP-2, to Volker Haucke, Thomas Jentsch, and Hartmut Oschkinat,

06.2014 – 05.2017, 649.465 € Deutsche Forschungsgemeinschaft, “Functional characterization of the SNARE adaptors AP180 and CALM in synaptic exo- and endocytosis in Alexander von Humboldt Foundation, Mirjana Leona Weimershaus, vivo”, HA 2686 / 8-1, with T. Maritzen, 11.2013 – 12.2017, 194.790 € 02.2015 – 01.2018, 82.800 €

Deutsche Forschungsgemeinschaft, SFB 958, A01, “Structural and University of Newcastle, International Collaboration Award (ICA), functional organization of endocytic scaffolds within the periactive 01.2014 – 12.2016, 13.440 € zone”, with T. Maritzen, 07.2015 – 06.2019, 487.400 €

Deutsche Forschungsgemeinschaft, SFB 958, A07, “Regulation of SH3 domain-containing scaffolds in synaptic vesicle clustering”, with C. Freund, 07.2015 – 06.2019, 455.400 €

Deutsche Forschungsgemeinschaft, SFB 958, A11, “Structural and functional analysis of septin scaffolds mediating endosomal membrane FMP authors trafficking”, to M. Krauss (w / O. Daumke, MDC Berlin), Group members 07.2015 – 06.2019, 395.400 € 28 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

PROTEIN TRAFFICKING

GROUP LEADER PROF. DR. RALF SCHÜLEIN

BIOGRAPHY SUMMARY

1982 – 1989 Biology studies, University G protein-coupled receptors (GPCRs) are arguably the most important of drug targets. These of Würzburg 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 1989 Diploma in Biology signal sequence-mediated insertion of the receptors into the membrane of the endoplasmic 1990 – 1993 PhD thesis on toxin transport reticulum (ER) by the translocon complex (Sec translocon pathway). The aim of the Protein in E. coli in the laboratory of Werner Goebel, Trafficking group is to find novel substances that influence ER insertion of GPCRs and other Department of Microbiology, University of integral membrane proteins at the level of the Sec translocon pathway. For pharmacological Würzburg 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 1995 – 1997 Postdoc in the laboratory this pathway (type 1 inhibitors; potential tumor drugs), and those that block specific signal of Walter Rosenthal, Department of sequences and thereby inhibit the biosynthesis of specific proteins (type 2 inhibitors; potential Pharmacology, University of Gießen; work alternatives to classical antagonists in the case of closely-related proteins). on the vasopressin V2 receptor since 1997 Group leader, FMP; work on ZUSAMMENFASSUNG the trafficking mechanisms of GPCRs G-Protein-gekoppelte Rezeptoren (GPCR) sind die wichtigsten Zielproteine für 2002 “Habilitation” in Pharmacology and Arzneimittel. Um korrekt zu funktionieren, müssen die Rezeptoren in das richtige Toxicology, Charité University Medicine Zielkompartiment der Zelle transportiert werden, normalerweise in die Plasmamembran. Berlin Dieser Transport wird über den sekretorischen Weg ermöglicht und startet mit einem Signalsequenz-vermittelten Einbau des Rezeptors in die Membran des endoplasmatischen 2014 Adjunct professorship, Charité Retikulums (ER) über den Translokon-Komplex (Sec-Translokon-Mechanismus). University Medicine Berlin 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 gene- rell 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). 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. Identification of novel type 1 and type 2 inhibitors of the Sec Selectivity of cotransin, an inhibitor of the Sec61 complex translocon pathway by high-throughput screening. The basic mechanism of the Sec translocon pathway for an integral Setting up a high-throughput screening assay for small molecule membrane protein is outlined in Figure 1. In the beginning, the inhibitors of the Sec translocon pathway, in particular for Sec61α, protein is synthesized at cytosolic ribosomes. The signal sequence is notoriously difficult. Sec61α has no enzymatic activity, is is bound by the signal recognition particle (SRP) and translation of expressed only in the ER membrane, and its isolation and functional the nascent chain is arrested. The resulting complex is targeted to reconstitution is problematic in large quantities. Our novel whole cell the SRP receptor of the translocon machinery at the ER membrane, screening assay included two steps, the first of which was a primary which consists mainly of the heterotrimeric Sec61 complex (Sec61α, screen using the GFP-tagged corticotropin-releasing factor

Sec61β, Sec61γ; Sec61α represents the protein-conducting channel). receptor type 1 (CRF1R.GFP) as a target, a GPCR which uses the Sec The ribosome binds to Sec61 and the signal sequence destabilizes the translocon pathway. Following pre-treatment of the cells with the closed conformation of the protein-conducting channel. Translation library of compounds, CRF1R.GFP expression was induced and then resumes and the transmembrane domains are released into the receptor expression quantified by measuring the GFP fluorescence

ER membrane. Extracellular domains are translocated into the ER signals. Hit compounds of the primary screen reduce CRF1R. lumen while cytosolic domains remain at the opposite site. GFP expression which means that they could inhibit trancription, translation or the Sec translocon pathway. To specifically identify The cyclodepsipeptide cotransin was described as a Secc61α inhibitor inhibitors of the Sec translocon pathway, a secondary screen was that acts in a signal sequence-discriminatory manner (mixed type performed using the unfused cytosolic GFP protein, a target 1 / type 2 inhibitor). Originally, cotransin was shown to inhibit the which does not use this pathway. Compounds behaving as inhibitors biosynthesis of only a small subset of proteins. However, cotransin in the primary screen, but not in the secondary screen, were selectivity was unknown, nor was it known which properties of a signal considered as real hits. These remaining substances were rated sequence were responsible for its cotransin sensitivity. To address according to their IC50 value (cut off = 10 µM) and tested for these questions, we performed a proteomic study using biosynthesis inhibition of various target proteins in a cellular cotransin-treated cells and the stable isotope labeling by amino acids selectivity assay. We also used a cell-free assay to assess for inhibition in cell culture (SILAC) technique, in combination with quantitative of the reconstituted Sec translocon pathway (in cooperation with mass spectrometry (in cooperation with the mass spectrometry Kurt Vermeire, University of Leuven, Belgium). Four compounds group of the FMP). Using a saturating concentration of cotransin, were found to inhibit the reconstituted Sec translocon pathway. One we found that the biosynthesis of almost all secreted proteins was of these, 503533, seems to be specific for the screening target CR- cotransin-sensitive. In contrast, the biosynthesis of the majority of F1R.GFP and may represent a novel type 2 inhibitor for the signal the integral membrane proteins was cotransin-resistant. Moreover, sequence of CRF1R (Figure 2). Another of these compounds, 401319, we were able to identify the first conformational consensus motif seems to block the Sec translocon pathway in general (Figure 2). in signal anchor sequences mediating cotransin sensitivity. Protease protection experiments showed that 401319 does indeed Although cotransin has a very interesting mechanism of action, its block co-translational translocation at the level of the Sec61 complex pharmacological application seems to be precluded due to its mixed (data not shown in Fig. 2). 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). 30 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

Fig. 2: Compounds 401319 and 503533 behave as type 1 and type 2 inhibitors 503533 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 CRF1R.GFP (red column) and various GFP-tagged integral membrane proteins. Cells were

treated with the compounds (concentration = 5×IC50) or DMSO for 19 hours and the total GFP fluorescence of the cells was analyzed using flow cytometry as a measure of biosynthesis. Compound 401319 affects the biosynthesis of all target proteins and behaves as a type 1 inhibitor, whereas compound 503533

impairs only biosynthesis of CRF1R.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 CRF1R.GFP was fused to a pre- prolactin sequence. A truncated PCR fragment without stop codon (78mer) was transcribed and translated using a reticulocyte lysate and [35S]Met 401319 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 auto- radiography. Note that the mature, processed construct without signal sequence (red arrow) was strongly reduced upon treatment with compounds 401319 and 503533, indicating that they inhibit the Sec translocon pathway (although obviously by different mechanisms; see above). Protein

abbreviations: AQP2, aquaporin2; CRF1R, corticotropin-releasing factor

receptor type 1; CRF2(a)R, corticotropin-releasing factor receptor type 2a, ETBR, endothelin B receptor; LHR, luteinizing hormone receptor; PAR1, protease-

activated receptor 1; TSHR, thyrotropin receptor; V2R, vasopressin 2 receptor.

GROUP MEMBERS COLLABORATIONS

Dr. Jens Furkert International National Dr. Claudia Rutz Ulrike Steckelings, Heike Biebermann, Arthur Gibert (doctoral student) University of Southern Denmark, Odense, Charité University Medicine Berlin Wolfgang Klein (doctoral student) Denmark Duska Dragun, Bettina Kahlich (technical assistant) Giovanna Valenti, Charité University Medicine Berlin University of Bari, Italy Mathias Dreger, Staff employed within the reporting period Kurt Vermeire, Caprotec Bioanalytics GmbH, Berlin University of Leuven, Belgium Gunnar Kleinau, Charité University Medicine Berlin

SELECTED PUBLICATIONS

Gibert A, Wiesner B, Schülein R (2017) The monomer / homodimer Hinz K M, Meyer K, Kinne A, Schülein R, Köhrle J, Krause G (2015) equilibrium of G protein-coupled receptors: formation in the secretory Structural insights into thyroid hormone transport mechanisms of the pathway and functional significance. Curr Mol Pharmacol. (Epub ahead L-type amino acid transporter 2. Mol Endocrinol 29: 933-942. of print) doi, 10.2174 / 1874467210666170224094915.

Vezzoli V, Duminuco P, Vottero A, Kleinau G, Schülein R, Minari R, Bassi EXTERNAL FUNDING I, Bernasconi S, Persani L, Bonomi M. (2015) A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects Deutsche Forschungsgemeinschaft, „Ableitung von Struktur- / Funktions- receptor biogenesis causing leydig cell hypoplasia. Hum Mol Genet 24, beziehungen spezifischer Hemmstoffe der Biosynthese G-Protein- 6003 – 6012. gekoppelter Rezeptoren“, SCHU 1116 / 2-1, 01.2011 – 12.2015. 235.000 €

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, 267 – 287.

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 FMP authors sequences: a proteomic and site-directed mutagenesis study. PLoS One Group members 10, e0120886. MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 31

MOLECULAR CELL PHYSIOLOGY

MOLEKULARE ZELLPHYSIOLOGIE

GROUP LEADER PD DR. INGOLF E. BLASIG

BIOGRAPHY SUMMARY

1970 – 1974 Studied biology and The group focuses on the structure, function, and modulation of cell-cell contacts to explore biochemistry in Leipzig, diploma thesis on tight junctions (TJs) in tissue barriers with the aim of revealing pathological mechanisms cancer research, Robert- Rössle-Hospital in and improving therapies. One intended outcome of this work is to propose new strategies for Berlin-Buch manipulating neurological and other barriers in order to improve drug delivery or to enhance drug efficacy, and to prevent barrier dysfunction. 1984 Dissertation on the pharmacology of myocardial infarction, Academy of Sciences, Membrane proteins such as claudins, TAMPs (occludin, tricellulin), and associated Berlin scaffolding proteins (e. g. ZO-1) determine the paracellular tightness of endothelial and 1992 Habilitation for investigations on epithelial barriers. As the complex of the TJs is not well defined it is unclear how the reactive species in myocardial dysfunction structure, regulation, and interactions among these proteins control the barrier properties. and venia legendi for biochemical Our work concentrates on exploring the TJ proteome and interactome, the detailed processes pharmacology, University of Halle-Wittenberg that underlie the oligomerization of key proteins in tissue barriers, and mechanisms to open and / or reconstitute TJs after injury. since 1992 Head of the independent research group for Molecular Cell Physiology, FMP, We organize the annual international symposium “Signal Transduction at the Blood- and teaching of pharmacology, functional Brain Barriers” to disseminate news of progress in the field, with the aim of stimulating biochemistry, neurochemistry, universities in collaborations in order to generate publications and new joint projects. Potsdam and Berlin

1993 – 1995 Awarded project leader, NIH, ZUSAMMENFASSUNG Washington DC, USA 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 Wirkstofftrans- port 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 Gewebs- barrieren 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. 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). 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 New regulatory role of tight junction proteins: TJ proteins. (A) Scheme of protein composition in TJs at the blood-brain barrier The molecular function of TAMPs is still unknown. A new concept with special consideration of members of the claudin protein family and the was developed and proven for how occludin and tricellulin are TJ-associated marvel proteins (TAMPs) occludin and tricellulin (tetraspanning). Single membrane-spanning JAMs (junctional adhesion molecules), as well as involved in redox-dependent signal-transduction mechanisms and how membrane-associated zonula occludens proteins (ZO), or multi-PDZ domain redox-sensitive domains of TAMPs are crucial in the redox regulation protein 1 (MUPP-1), are not directly involved in paraendothelial tightening. of protein interactions at TJs. In addition, it was found that occludin Amongst others, signaling via membrane receptors, e. g., adenylyl cyclase (AC), acts as an oxidase under reducing conditions, which is a new regulatory second messengers, e. g., cyclic AMP or inositol-3-phosphate (IP 3), and protein mechanism. These data are highly relevant for diseases related to kinases (PKs), may regulate TJs. (B) Ultrathin section transmission electron micros- oxidative stress and for possible pharmacological interventions (Castro copy of two adhering CECs visualizing TJ elements sealing the intermembrane et al., 2016; Marko et al., 2016; collaboration with the University of gap, arrows indicating TJ. (C) Freeze-fracture transmission electron microscopy Miami and the MDC/Charité Berlin, respectively). 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, New modulators of tight junctions: exoplasmic and protoplasmic face of intramembranous TJ strands. 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 intracellular 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 extracellular (see Figure 2). Intravenous injection in mice facilitated the uptake of C5C2C1C2 small molecules into the brain (Dithmer et al., 2017 under revision). In addition, a new peptidomimetic based on the second extracellular loop of cell membrane tricellulin, named trictide, was designed that is able to open epithelial intracellular barriers for molecules up to 10 kDa and that affects not only tricellulin, but mmclaudin-5claudin-1 also occludin (Arzlan et al., 2017 under revision). In conclusion, novel tools drug were developed to improve the delivery of pharmaceutical agents through neurological barriers.

no paracellular paracellular Investigations for a better understanding of neuropathologies no paracellular paracellular drug deliverydelivery drugdrug delivery The identification of biomarkers is a field of growing importance for the pre-incubationpre-incubation diagnosis and treatment of neurological diseases. In collaboration with withwith C5C2C1C2 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 Figure 2. Scheme of how a peptidomimetic agent based on an extracellular identification of potential biomarkers, methodological experiments were loop of a tightening claudin can cause transient opening of the paracellular performed to assess the transferability of data obtained from human and cleft in tissue barriers to improve drug delivery. Example: Peptide C5C2 from dog CSF (Günther et al., 2015). murine claudin-5. MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 33

GROUP MEMBERS COLLABORATIONS

Dr. Christian Bellmann International National Dr. Rosel Blasig European consortium ‚Brains4brain’ Jörg Piontek, Dr. Reiner Haseloff Charite Berlin Anuska Andjelkovic, Dr. Lars Winkler Heike Rittner, University of Michigan Medical School, Basak Arslan (doctoral student) Universitätsklinikum Würzburg Ann Arbor, USA Philipp Berndt (doctoral student) Kai Schmidt-Ott, Maria Deli, Olga Breitkreutz-Korff (doctoral student) MDC / Charite Berlin Biological Research Centre, Jimmi Cording (doctoral student, postdoc) Hartwig Wolburg, Szeged, Hungary Sophie Dithmer (doctoral student) Eberhard-Karls-Universität Tübingen Jean-Michel Heard, Nora Gehne (doctoral student) Institut Pasteur, Paris, France Sebastian Pfeil (Dabrowski) Zhihai Qin, (doctoral student) Chinese Acad. Sci., Beijing, PR China Christian Staat (doctoral student, postdoc) Britta Engelhardt, Ramona Günther (technical assistent) Universität Bern, Schweiz Heike Meyer (technical assistent) Birger Brodin, Katrin Schildele (technical assistent) University Copenhagen, Denmark Michal Toborek, Staff employed within the reporting period University of Miami, USA

SELECTED PUBLICATIONS EXTERNAL FUNDING

Markó L, Vigolo E, Hinze C, Park J-K, Roël G, Balogh A, Choi M, VIP Projekt, BMBF, EASYPERM, „Modulatoren der Blut-Hirnschranke Wübken A, Cording J, Blasig I E, Luft F C, Scheidereit C, Schmidt-Ott als Drugenhancer für ZNS-Pharmaka“, 01.11.2013 – 30.11.2016, K M, Schmidt-Ullrich R, Müller D N (2016) Tubular epithelial NF-κB 1.133.925,00 € activity regulates acute ischemic kidney injury. J. Am. Soc. Nephrol. 27, 2658 – 2669.

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, 1 – 14.

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: Redox- sensitive cysteine sites in tricellulin regulate both tri- and bicellular junctions in tissue barriers as shown in hypoxia and ischemia. Antioxid. Redox Signal. 23, 1035 – 1049.

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, 9 – 20.

FMP authors Group members 34 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

MOLECULAR NEUROSCIENCE AND BIOPHYSICS

MOLEKULARE NEUROWISSEN- SCHAFTEN UND BIOPHYSIK

GROUP LEADER DR. ANDREW J.R. PLESTED

BIOGRAPHY SUMMARY

1994 – 1998 M.Sci. 1st Class Hons, Physics, Our principal research interests are glutamate receptors and excitatory synapses. These Imperial College, University of London, UK receptors are essential for brain function, being necessary components of excitatory synaptic transmission. Synapses themselves are implicated in cognition and numerous complex brain 1998 – 2002 Ph.D. Imperial College diseases. We aim to understand the molecular basis of fast excitatory transmission, and to (Prof. Franks and Prof. Lieb), University of develop methods to observe and alter synapse activity. To achieve these goals, we manipulate London, UK receptors with molecular and chemical biology, including deploying unnatural amino acids in 2003 – 2005 Post-Doc, Dept. of mammalian cells. We examine receptor gating with advanced electrophysiological methods Pharma­cology (Prof. Colquhoun), University such as ultra-rapid perfusion and single-channel recording. We complement these approaches College London, UK with investigations of receptor structure and composition using X-ray crystallography and biochemistry. Combining these approaches with fluorescence microscopy has enabled us 2005 – 2008 Visiting Research Fellow, to visualize receptor activity directly. Through collaborations, we employ computational laboratory of Cellular and Molecular approaches to analyze and build novel insights into receptor activation. A second aspect of our Neurophysiology (Dr. Mayer), NICHD, NIH, research is to extend these studies to other important components of fast signaling in neurons, Bethesda, USA such as enzymes and other channels. since 2008 Junior group leader, FMP, Berlin ZUSAMMENFASSUNG since 2008 Member, Cluster of Excellence NeuroCure Der Schwerpunkt unserer Forschung liegt auf Glutamatrezeptoren exzitatorischer Synapsen. Diese Rezeptoren sind für die Funktion unseres Gehirns essentiell und spielen eine 2015 ERC Consolidator Grant 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. 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 swer is that both theories are true. The different activators drive the Bilobed ligand-binding domains (LBDs), such as those found in receptor into overlapping sets of geometries, which they occupy for ionotropic glutamate receptors (iGluRs), are common architectural different lengths of time on average. What is surprising is that most elements found in hundreds of small-molecule receptor proteins. of the arrangements are inactive. We suggest that this plethora of Despite the ubiquity of these domains, processes essential to inactive states allows the receptor activation to be rapid and selective. receptor activation, such as pathways along which a ligand is guided This idea will be the subject of further tests during the project into its binding site, metastable protein-ligand interactions, and GluActive. This work was a combined study of structural biology and the coupling of ligand binding to protein conformational changes, functional experiments and is in press in Nature Communications are poorly understood. In our work, we took advantage of long (Salazar et al. 2017). molecular simulations performed on custom hardware that show the process of glutamate binding to the AMPA-type iGluR LBD for the Gating the transmembrane domain of glutamate receptors first time. Charged sidechains on the surface of the LBD are found We have continued our investigation of AMPA receptor gating with to bind glutamate transiently and funnel it into its recessed binding unnatural amino acids that are sensitive to UV light. We performed pocket. Electrophysiological recordings show that eliminating these an unbiased screen of 23 sites in the transmembrane region with two transient binding sites reveals a selective and unique kinetic signature different unnatural amino acids, azido-phenylalanine and benzoyl- of slowed activation and deactivation. These results suggest that phenylalanine. This work has revealed details of the gating preferential binding pathways have evolved to optimize rapid responses of mechanism, and has produced mutants that offer very potent glutamate receptors at central nervous system synapses. control of receptor activity with light. In particular, we see that helical contacts at the periphery of the ion channel are critical for controlling Structural dynamics of glutamate receptor activation gating and desensitization. We now aim to exploit these mutants to A recent major achievement has been understanding how certain control receptors in neurons. molecules weakly activate the glutamate receptor. When we consider the maximum activation of the receptor – i. e. when it is continuously Fluorescence studies of AMPA receptor complexes in the “on” state – we expect the receptor to adopt a certain geometry We have developed the first FRET pair between the AMPA receptor (or set of geometries). However some molecules, called partial and an auxiliary protein, stargazin. This tool employs fluorescent agonists, bind to the receptor in a way that causes non-maximal proteins fused to the subunits of interest and enables us to monitor activation. These partial agonists can be synthetic, but some also conformational changes in the complex during receptor activation occur in nature. We believe that the reasons for non-maximal by glutamate. It also allows us to detect optically if the complex activation occurring in the first place probably relate to how the dissociates, which is a controversial topic in glutamate receptor receptor fundamentally works. One explanation for this might be biology. With this tool, we are able to see that fast conformational that the receptor spends some of its time activated and some of its changes occur during gating, but we do not detect any dissociation time resting. In this interpretation, the receptor cycles between active of the receptor from the auxiliary protein on the timescale of normal and resting states and the agonist is unable to prevent this cycling gating (seconds). 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 an- 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 COLLABORATIONS

Dr. Jelena Baranovic International National Dr. Anna Carbone Daniel Choquet, Oliver Daumke, Dr. Clarissa Eibl Interdisciplinary Institute for Neuroscience, Max-Delbrück-Center for Molecular Dr. Valentina Ghisi Bordeaux, France Medicine, Berlin Dr. Ljudmila Katchan Teresa Giraldez, Peter Hegemann, Dr. Hector Salazar University of La Laguna, Spain HU-Berlin Sonja Minniberger (PhD Student) Eric Gouaux, Jana Kusch, Yuchen Hao (PhD Student) Howard Hughes Medical Institute & Oregon Jena Sebastian Opfermann (PhD Student) Health Sciences University, USA Philip Selenko, Irene Riva (PhD Student) Albert Lau, FMP-Berlin Anahita Poshtiban (PhD Student) Johns Hopkins University, Baltimore, MD, Antje Maluck (Technician) USA Ronny Schaefer (Technician) Marcus Wietstruk (Technician)

Staff employed within the reporting period

SELECTED PUBLICATIONS EXTERNAL FUNDING

Salazar H†, Eibl CE†, Chebli M & Plested AJR (2017) Mechanism 2016 – 2020 DFG SFB / TRR 186 A07 “Optical Control of Calcium Switches of Partial Agonism in AMPA-type glutamate receptors. Nature that Orchestrate Fast Signaling in the Brain” (with Peter Hegemann, Communications 8, 14327. HU Berlin) 295.000 € † These authors contributed jointly. 2015 – 2017 DFG PL619 / 2 “Optical control of glutamate receptors using Zachariassen LG†, Katchan L†, Jensen AG, Pickering DS, Plested AJR* & genetically encoded unnatural amino acids“ 299.000 € Kristensen AS* (2016) Structural rearrangement of the intracellular domains 2015 – 2020 ERC CoG 647895 “GluActive” 1.980.000 € during AMPA Receptor activation. PNAS 113, E3950-9. † 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, 896-911. † These authors contributed jointly. FMP authors Carbone AL and Plested AJR (2016) Superactivation of glutamate Group members receptors by auxiliary proteins. Nature Communications 7, 10178. 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 SUMMARY

1997 – 2002 Biochemistry and Molecular In order to interact with the extracellular environment cells rely to a large extent on cell Biology studies, University of Hamburg surface-localized proteins. The binding of extracellular ligands to these cell surface receptors triggers diverse intracellular signaling cascades that determine, for instance, 2002 – 2005 Ph.D. student, Center for whether a cell starts to proliferate, to differentiate, to migrate, or to trigger an immune Molecular Neurobiology Hamburg response. Accordingly, the number of these signal receptors at the cell surface has to be (Prof. T.J. Jentsch), Dr. rer. nat. (summa tightly regulated in each situation to achieve the right level of responsiveness to a specific cum laude), University of Hamburg extracellular cue. Endocytosis, in conjunction with intracellular membrane transport, 2006 Hans-Dietrich Herrmann- constitutes a highly effective way in which to regulate the levels of diverse proteins at the Promotionspreis for Molecular Medicine 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. 2005 – 2006 Postdoctoral Research Associate, However, for many cell surface proteins it is still unclear whether they are in fact internalized Center for Molecular Neurobiology Hamburg by endocytosis, how they interact with the endocytic machinery, and how their (Prof. T.J. Jentsch) internalization is regulated. By employing cell biological and optical techniques in 2006 – 2012 Postdoctoral Research conjunction with mouse genetics, our group dissects the role of endocytic and endosomal Associate, Freie Universität Berlin scaffold and adaptor proteins in the regulation of cell surface-localized proteins, especially (Prof. V. Haucke) in the context of cellular motility, immune functions, and neurotransmission. since 2011 Co-Project Leader in the ZUSAMMENFASSUNG Collaborative Research Center SFB958 Zellen interagieren mit ihrer Umgebung im Wesentlichen über Proteine, die an der since 2012 Junior Group Leader at the FMP Zell­oberfläche lokalisiert sind. Die Bindung solcher Zelloberflächenrezeptoren an since 2016 Privatdozent following extrazelluläre Liganden löst eine Vielzahl intrazellulärer Signalkaskaden aus, die habilitation at Freie Universität Berlin 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 Membran- transportprozessen, 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 inter­agieren und wie ihre Internalisie- rung 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. 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 actin- nucleating 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.

Diverse proteoglycans modulate cell motility by interacting with extracellular ligands and triggering intracellular signaling cascades that modulate actin dynamics. However, their endocytosis-based Fig. 1: The endocytic adaptor protein Stonin1 (green) localizes specifically to regulation is poorly understood. We identified the proteoglycan endocytic structures directly behind focal adhesions (red) at the leading edge of a mouse embryonic fibroblast (arrow indicates direction of migration; cell NG2 / CSPG4 as cargo of the so-far orphan endocytic adaptor nucleus is stained in blue). 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.

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. Fig. 2: Migration parameters such as speed and directionality of movement They have to be retrieved to regenerate SVs for sustained neurotrans- are measured by tracking cells as they move along a chemotactic gradient. mission. 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 DESCRIPTION OF PROJECTS adaptor protein Stonin2 mediates the sorting of the SV Ca2+ sensor Membrane transport in cell motility synaptotagmin1, while AP180 is crucial for internalizing the SV Cell motility is not only crucial during human development, but protein synaptobrevin2. Currently, we are dissecting the neuronal also thereafter for immunity in the healthy adult. Its dysregulation role of the AP180-related adaptor protein CALM, a protein that is involved in autoimmune disorders and cancer, and cell migration has been implicated in Alzheimer’s disease. We also analyze the inhibitors hold promise for the treatment of inflammatory mechanisms by which the endocytic scaffold intersectin affects brain conditions. Cell motility relies on coordinated membrane transport function. To elucidate the physiological relevance of these proteins to regulate the cell surface levels of the proteins that mediate cell in an organismic context we employ mouse genetics in conjunction adhesion and migratory signaling such as integrins and proteoglycans. with live cell imaging approaches and behavioural studies. MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 39

GROUP MEMBERS COLLABORATIONS

PD Dr. Tanja Maritzen (group leader) International National Dr. Domenico Azarnia Tehran Daniel Legler Volker Haucke, Eberhard Krause Dr. Hannah Schachtner BITg at the University of Konstanz, FMP, Germany Marietta Browarski (doctoral student) Kreuzlingen, Switzerland MinChi Ku, Sonia Waizcies, Thoralf Niendorf Fabian Feutlinske (doctoral student) Laura M. Machesky MDC, Berlin, Germany Marine Gil (doctoral student) Beatson Institute for Cancer Research, Uta Hoepken Fabian Lukas (doctoral student) Glasgow, UK MDC, Berlin, Germany Dennis Vollweiter (doctoral student) William B. Stallcup Matthias Selbach Lennart Hoffmann (student) Sanford-Burnham Medical Research MDC, Berlin, Germany Claudia Schmidt (technical assistant) Institute, CA, USA Christian Rosenmund Nicolas Chevrier Charité Universitätsmedizin Berlin, Germany Staff employed within the reporting period FAS Center for Systems Biology, Harvard Annette Schürmann University, MA, USA 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 EXTERNAL FUNDING

Feutlinske F, Browarski W, Ku MC, Trnka P, Waiczies S, Niendorf T, Deutsche Forschungsgemeinschaft, „Untersuchung der Bedeutung Stallcup W B, Glass R, Krause E, Maritzen T (2015) Stonin1 mediates von Stonin1 für die Dynamik fokaler Adhäsionen und die Unterdrückung endocytosis of the proteoglycan NG2 and regulates focal adhesion von Tumoren”, MA 4735 / 2-1, 2017-2019, 428.330 € dynamics and cell motility. Nat Commun. 6, 8535. Leibniz-Gemeinschaft, Leibniz Vorhaben im Rahmen des Pakts für Koo S J, Kochlamazashvili G, Rost B, Puchkov D, Gimber N, Lehmann Forschung und Innovation, “Regulation of cell motility by membrane-­ M, Tadeus G, Schmoranzer J, Rosenmund C, Haucke V *, Maritzen T * associated endosomal adaptors”, SAW-2013-FMP-5, 2013-2017, (2015) Vesicular Synaptobrevin / VAMP2 levels guarded by AP180 con- 647.052 € trol efficient neurotransmission. Neuron. 88(2), 330-44. Deutsche Forschungsgemeinschaft, SFB 958, A01, „Structural and Schachtner H, Weimershaus M, Stache V, Plewa N, Legler D F, functional organization of endocytic scaffolds within the periactive Höpken U E, Maritzen T (2015) Loss of Gadkin affects dendritic cell zone“, with V.Haucke, 2015-2019, 467.200 € migration in vitro. PLoS One. 10(12), e0143883. Deutsche Forschungsgemeinschaft, „Funktionelle Charakterisierung Jung S *, Maritzen T *, Wichmann C *, Jing Z, Neef A, Revelo N H, der SNARE Adaptoren AP180 und CALM bei der synaptischen Exo- und Al-Moyed H, Meese S, Wojcik S M, Panou I, Bulut H, Schu P, Ficner R, Endozytose in vivo”, MA 4735 / 1-1, with V. Haucke, 2014-2017, 194.825 € 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), 2686 – 702.

Maritzen T *, Schachtner H, Legler D F * (2015) On the move: Endocytic trafficking in cell migration. Cell Mol Life Sci. 72(11), 2119 – 34.

FMP authors Group members * contributed equally 40 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

PROTEOSTASIS IN AGING AND DISEASE

PROTEINHOMÖOSTASE IM ALTERN UND IN KRANKHEITEN

GROUP LEADER DR. JANINE KIRSTEIN

BIOGRAPHY SUMMARY

2003 Diploma / MSc., University of Our research goal is to advance our understanding of the protein-folding problem. We want to Greifswald 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 2007 Ph.D., Free University Berlin the ribosome, they facilitate folding of unfolded or denatured protein stretches, they prevent (summa cum laude) misfolding and aggregation, and they also disaggregate aggregated proteins. Our group studies 2007 – 2008 Postdoc, Free University Berlin the contribution of molecular chaperones in the context of aging and neurodegenerative diseases (supervisor: K. Turgay) 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 2008 – 2013 Postdoc, Northwestern proteins in cells and the nematode C. elegans. We have also established a number of in vitro University, IL, USA (supervisor: R. Morimoto) assays that allows us to monitor the activity of chaperones and chaperone complexes in amyloid 2008 Postdoc fellowship of DFG and EMBO fibrilization and disaggregation of disease-associated proteins. awarded, but declined ZUSAMMENFASSUNG 2008 – 2011 Postdoc fellowship of Human Frontiers in Science (HFSP) Das Ziel unserer Forschung ist es, ein besseres Verständis zur Protein-Faltungs Problematik zu gewinnen. Wir wollen mechanistische Erkenntnisse zur Komplexität, Funktion und 2009 – 2013 Instructor, Northwestern Dynamik molekularer Chaperone erlangen. Molekulare Chaperone helfen bei der Faltung University, IL, USA von neusynthetisierten Proteinen am Ribosom, ermöglichen die Faltung von ungefalteten und Since 2013 Group Leader in the Molecular denaturierten Proteinen und können auch Proteinaggregate resolubilisieren. Unsere Gruppe Physiology and Cell Biology Section of the erforscht die Funktion der Chaperone während des Alterns und in neurodegenerativen FMP Krankheiten, die durch eine Anhäufung von amyloiden Proteinfibrillen gekennzeichnet sind. Wir stellen diese Szenarien in Zellkultur und im Nematoden C. elegans durch gezielte Since 2013 Member of NeuroCure Cluster Synthese dieser krankheitserzeugenden Peptide und Proteine künstlich nach. Zusätzlich of Excellence haben wir eine Reihe von in vitro Assays etabliert, die es uns erlauben, die Aktivität einzelner 2015 / 2016 DFG NeuroCure Innovation Chaperone oder Chaperonkomplexe auf Fibrilisierung der amyloiden Proteine und deren Award Disaggregation zu untersuchen. 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 Aβ1-42.

DESCRIPTION OF PROJECTS

The trimeric chaperone complex Hsp70 / Hsp110 / J-protein solubility of HttQn and related polyQ proteins on an organismal suppresses Htt amyloid formation and resolubilizes preformed level in C. elegans. To do so, we employed nematode lines that express

Htt fibrils HttQn and SCA3Qn proteins and depleted the chaperone expression Huntington’s disease (HD) is a neurodegenerative disorder that using RNAi. Knockdown of the chaperone genes lead to a substantial is caused by expanded CAG trinucleotide repeats within the increase in aggregation of the polyQ proteins. To confirm the huntingtin gene (HTT). Molecular chaperones have been vital role of molecular chaperones for the maintenance of the implicated in suppressing or delaying the aggregation of mutant solubility of polyQ proteins we overexpressed a specific chaperone Htt. The first observations of chaperones being able, to a certain (the J-protein DNAJB1) in HEK cells expressing robustly extent, to decrease the aggregation propensity of HttQn in in vitro aggregating HttExon1Q97. This J-protein is the rate-limiting assays were obtained using bacterial or yeast Hsp70 and Hsp40 chaperone in the in vitro chaperone assays and in vivo it is the least (J-protein) chaperones. In vivo, overexpression of Hsp70, J-protein, abundant protein of the three chaperones. Importantly, over- Hsp110, or TRiC reduces the aggregation toxicity of Htt in cul- expression of the J-protein results in a pronounced decrease of tured cells, flies, and mice HD models. It has also been observed HttExon1Q97 aggregation. Currently we are investigating the that expression of two chaperones (Hsp70 / J-protein or Hsp110 / functional spectrum of this trimeric chaperone complex. We have J-protein) synergistically suppress Htt aggregation. These findings already demonstrated that the same chaperone complex disaggregates are in agreement with previous reports that these chaperones form fibrils formed byα -synuclein, suggesting that this chaperone functional complexes, and suggest that they cooperate in vivo to complex has a broad substrate spectrum. prevent or reverse polyQ aggregation. Despite these efforts many unanswered questions remain. Do chaperones interfere with Interplay between disaggregation and proteolytic clearance nucleation events of beta-sheet formation and subsequent pathways elongation into amyloids or seeding activities? Can chaperones The cell has three strategies to cope with protein aggregates: (i) resolubilize Htt once it is assembled into amyloid fibrils? Are there deposition of aggregates into cellular assemblies such as INQ, specific chaperones or chaperone complexes that recognize distinct CytoQ and IPOD, (ii) clearance via disaggregation by molecular moieties of misfolded and aggregated Htt? The diversity within chaperones, and (iii) proteolytic clearance by the ubiquitin the chaperone families has increased over the course of evolution. proteasome system (UPS) or autophagy. Little is known regarding A pronounced expansion in the number of distinct chaperones if and how the clearance pathways communicate with each other occurred, e. g. within the J-protein family, suggesting an increased to maintain a balanced proteostasis with the progression of aging functional specialization of chaperones. or in disease. We set out to address this question by analyzing the disaggregation, UPS, and autophagy capacity under control By using a novel FRET-based in vitro assay to monitor the amyloid conditions and upon impairment of one of the aggregate clearance formation of Htt proteins and the effect of molecular chaperones pathways. We developed tools to monitor all three activities in on the fibrilization kinetics, we have demonstrated that a trimeric vitro and in living animals. Interestingly, depletion of chaperones chaperone complex composed of a member each of the Hsp70, involved in disaggregation lead to an induction of autophagy. Hsp110, and type II J-protein families can completely suppress the Autophagy appears to compensate for the lack of disaggregation formation of amyloid fibrils by HttQn. We also demonstrated for the activity as a means to cope with the accumulation of misfolded first time a disaggregation of HttExon1Q48 fibrils by this trimeric and aggregated proteins. It is noteworthy that the UPS activity chaperone complex. The composition of the chaperone complex is and the abundance of 20S subunits decreased upon knockdown of very dynamic. The combination of different HSP-70 and J-protein chaperones that mediate disaggregation. We observed the same chaperones, together with HSP-110, forms distinct chaperone response upon expression of aggregation-prone disease-causing complexes that exhibit different suppression and disaggregation peptides and proteins such as Aβ1-42 and polyQ that lead to an activities. Depletion of these chaperones in HD patient-derived induction of autophagy and a reduction of UPS capacity. We are now neuronal progenitor cells (NPCs) lead to a pronounced increase aiming to elucidate the mechanism of the interplay between these in aggregation of the endogenous Htt protein (Q44). We have clearance pathways in the context of aging and neurodegeneration. confirmed the importance of these chaperones for maintaining the 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 toα -synuclein- fibrils independently of other chaperones and probably recruits the cooperating chaperones HSP-70 and HSP-110 to initiate the disaggregation process.

GROUP MEMBERS COLLABORATIONS

International Bernd Bukau, Dr. Katrin Jünemann Harm Kampinga, ZMBH-DKFZ Alliance, Heidelberg Dr. Annika Scior UMCG, Groningen, Netherlands Baris Tursun, Kristin Arnsburg (doctoral student) Funda Sar, MDC, Berlin Diogo Feleciano (doctoral student) Koc University, Istanbul, Turkey Ralf Schülein, Christian Gallrein (doctoral student) Ansgar Siemer, FMP, Berlin Manuel Iburg (doctoral student) University of Southern California, USA Eberhard Krause, Lucia Pigazzini (doctoral student) FMP, Berlin Marc Bohlmann (MSc student) National Hartmut Oschkinat, Linda Bergemann (MSc student) Erich Wanker, FMP, Berlin Kalina Tosheva (MSc student) MDC, Berlin Phil Selenko, Christina Hildebrandt (BSc student) Alessandro Prigione, FMP, Berlin Wolfram Thielicke (BSc student) MDC Dmytro Puchkov, Kerstin Steinhagen (technical assistant) Josef Priller, FMP, Berlin Charite, Berlin Christian Hackenberger Staff employed within the reporting period Britta Eickholt, FMP, Berlin Charite, Berlin

SELECTED PUBLICATIONS EXTERNAL FUNDING

Lechler M C, Crawford E D, Groh N, Widmaier K, Jung R, Kirstein J, DFG EXC 257 NeuroCure (2013-2017) 100.000 € Trinidad J C, Burlingame A L, David D C (2017) Reduced Insulin / IGF-1 DFG EXC 257 NeuroCure Innovation Award (2015+2016) 40.000 € Signaling Restores the Dynamic Properties of Key Stress Granule Proteins during Aging. Cell Reports 18, 454 – 467. DFG SFB740 B8 (01 / 2015-12 / 2018) 222.000 €

Scior A, Juenemann K, Kirstein J (2016) Cellular strategies to cope with DFG SPP1623 (01 / 2016-12 / 2018) 217.500 € protein aggregation. Essays in Biochemistry 60, 153 – 161. DFG KI1988 / 3-1 (01 / 2015-12 / 2018) 203.200 € Feleciano D R, Arnsburg K, Kirstein J (2016) Interplay between redox Daimler und Benz Stiftung (Annika Scior; 02 / 2015-01 / 2017): 40.000 € and protein homeostasis. Worm 5. AXA Research Fund (Katrin Jünemann; 10 / 2016-09 / 2018): 130.000 € 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, EU-COST STSM Fellowship (Diogo Feleciano; 02-03 / 2016): 1.000 € Morimoto R I, Mayer M P, Bukau B (2015) Crucial HSP70 co-chaper- one complex unlocks metazoan protein disaggregation. Nature 524, 247 – 251.

Kirstein J *, Morito D, Kakihana T, Sugihara M, Minnen A, Hipp M S, FMP authors Nussbaum-Krammer C, Hartl F U, Nagata K, Morimoto R I (2015) Group members Proteotoxic stress and ageing triggers the loss of redox homeostasis * co-corresponding author across cellular compartments. EMBO J 34, 2334 – 2349. MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 43

BEHAVIOURAL NEURODYNAMICS

VERHALTENSNEURODYNAMIK

GROUP LEADERS DR. TATIANA KOROTKOVA DR. ALEXEY PONOMARENKO

BIOGRAPHY SUMMARY

Tatiana Korotkova Our aim is to reveal the contribution of individual neuronal membrane conductancies to the excitability and synchronization of neuronal networks in vivo. Brain synchronization 1995 – 2000 Studied biology and physiology, regimes are instrumental for cognitive functions such as learning and memory and play Lomonosov Moscow State University, Russia a key role in disorders such as epilepsy. Using genetic mouse models, we are focusing on 2000 – 2003 Ph.D., Heinrich-Heine-University, the role of voltage-gated channels and GABAA receptors in the operation of hippocampal Düsseldorf (Prof. H.L. Haas and Prof. J.P. networks. We also investigate how cortico-subcortical communication is organized Huston) 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. 2003 – 2004 Post-Doc, We study the activity and interactions of specific neurons in the hypothalamus across Heinrich-Heine-University,­ Düsseldorf vigilance states and homeostatic challenges using high-density electrophysiological (Prof. H.L. Haas) recordings and optogenetics in freely behaving mice. We aim to gain insights into the 2004 – 2009 Post-Doc, University Clinic for neural basis and molecular determinants of vital functions and their pathologies, Neurology, Heidelberg (Prof. H. Monyer) particularly in obesity and sleep.

2009 – 2012 Post-Doc, FMP, NeuroCure Fellow ZUSAMMENFASSUNG since 2012 Junior group leader, Unser Ziel ist es, den Beitrag, den die Leitfähigkeit einzelner neuronaler Membranen FMP / NeuroCure zur Erregbarkeit und Synchronisation neuronaler Netzwerke in vivo leistet, aufzudecken. Für kognitive Funktionen wie Lernen und Gedächtnis ist Synchronisation im Gehirn Alexey Ponomarenko unabdingbar, Sie spielt auch bei Erkrankungen wie Epilepsie eine Schlüsselrolle. Der 1995 – 2000 Studied biology and physiology, Fokus unserer Forschung liegt auf der Untersuchung der Rolle spannungsabhängiger Lomonosov Moscow State University, Russia Kanäle und GABAA-Rezeptoren auf die Arbeitsweise hippokampaler Netzwerke an genetische Mausmodelle. Außerdem untersuchen wir, wie die kortiko-subkortikale 2000 – 2003 Ph.D., Heinrich-Heine-­University, Kommunikation durch Synchronisation organisiert wird. Ein weiteres Ziel ist es, Düsseldorf (Prof. H.L. Haas and Prof. J.P herauszufinden, wie das Gehirn molekulare metabolische Signale wahrnimmt und Huston) diese umsetzt, um verschiedene Vitalfunktionen wie das Essverhalten und den Schlaf / 2003 – 2004 Post-Doc, Wach-Rhythmus zu koordinieren. Mit Hilfe paralleler elektrophysiologischer Ableitungen Heinrich-Heine-University, Düsseldorf und Optogenetik an sich frei bewegenden Mäusen untersuchen wir die Aktivität und (Prof. H.L. Haas) Interaktionen spezifischer Neurone im Hypothalamus quer über Vigilanzzustände und homöostatische Herausforderungen. Wir wollen auf diese Weise Einblicke in die neuronalen 2004 Internship, Rutgers University Grundlagen und molekularen Determinanten von Vitalfunktionen und ihrer patholo- (Prof. G. Buzsaki), Newark, USA gischen Veränderungen, insbesondere bei Fettleibigkeit und Schlafstörungen, gewinnen. 2004 – 2009 Post-Doc, University Clinic for Neurology, Heidelberg (Prof. H. Monyer) since 2009 Junior group leader, FMP / NeuroCure­ 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 inhibitory (halorhodopsin) opsins were expressed locally in the and 5 channels hippocampus or in the medial septum in wild-type and parvalbumin- In collaboration with Prof. T. J. Jentsch, we have investigated the Cre mice. Hippocampal theta oscillations were controlled by role of KCNQ channels in the control of neuronal excitability optostimulation of septo-hippocampal projections. The peak of the in vivo. This project focuses on channel proteins known as power spectral density of optogenetically-paced theta oscillations KCNQ3 and KCNQ5, which permit a passage of potassium ions in matched the stimulation frequency. This allowed us to interfere with neurons. Mutations in KCNQ3 cause neonatal epilepsy. While these the rhythm generation in vivo while monitoring mouse behavior, channels are expressed in the hippocampus and affect the excitability as well as neuronal network and single cell activity. We use this of neurons in vitro, their functions in vivo had been unknown. We approach to study the role of hippocampal theta oscillations in spatial found that the two channels play distinct and important roles in navigation. We also study functions of theta-rhythmic coordination controlling neuronal excitability in the hippocampus of mice. Their of the hippocampus and its output regions. (Bender et al., Nature functions include shaping the discharge mode of hippocampal Communications, 2015). neurons, determining the efficacy of fast network synchronization and the precision of neural representations of space, and preventing GABAergic cells in the lateral hypothalamus adaptively hyperexcitability and pathological forms of neural synchronization. coordinate innate behaviors Our results (Fidzinski et al., Nature Communications, 2015) show The lateral hypothalamus (LH) is crucial for the regulation of innate that KCNQ5 controls excitability of hippocampal networks and behaviors, including food intake and the sleep-wake cycle, yet influences cognitive processes. temporal coordination of hypothalamic neuronal populations remains elusive. We used a combination of high-density electro- Optogenetic control of hippocampal theta oscillations reveals physiological recordings and optogenetics in behaving mice to study their function in spatial navigation the function of GABAergic cells in the LH. Excitatory (ChETA) or The activity of large neuronal populations at various time scales is inhibitory (halorhodopsin, eNpHR3.0) opsins were expressed in the organized by hippocampal network oscillations that are important LH of VGAT-Cre mice to ensure selective targeting of GABAergic for cognitive processes. Hippocampal theta oscillations (5-12 Hz) in cells. Recordings of neuronal activity and optostimulation were rodents occur during exploration and REM sleep, organize neuronal performed in various behavioral paradigms for assessing innate discharge, and are implicated in spatial navigation and memory. Yet behaviors. We found that optogenetic stimulation of GABAergic the role of theta oscillations in shaping spatial behavior remains LH cells at various frequencies, as well as stimulation of projections elusive because until recently theta rhythm could not be manipulated of these neurons, changed transitions between innate behaviors. in behaving animals. In this study, we combined optogenetic control Activation or inhibition of GABAergic neurons in the LH also of theta rhythm generators (the medial septum and hippocampus) affected feeding behavior. Furthermore, neuronal activity of LH with electrophysiological monitoring of hippocampal network neurons was behavior- and state-dependent (Herrera et al., Nature oscillations and neuronal activity. Excitatory (ChR2, ChETA) or Neuroscience, 2016; Carus-Cadavieco et al., Nature, 2017). MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 45

GROUP MEMBERS COLLABORATIONS

Dr. Alexey Ponomarenko International National (principal investigator) Antoine Adamantidis, Thomas J. Jentsch, Dr. Tatiana Korotkova McGill University, Montreal, Canada Leibniz-Forschungsinstitut für Molekulare (principal investigator) Christoph Börgers, Pharmakologie (FMP), Berlin Franziska Bender (doctoral student) Tufts University, Boston, USA Lisa Marshall, Marta Carus-Cadavieco (doctoral student) Denis Burdakov, University of Lübeck Xiaojie Gao (doctoral student) National Institute for Medical Research, Dietmar Schmitz, Maria Gorbati (doctoral student) London, UK Charité – Universitätsmedizin Berlin Tugba Ozdogan (doctoral student) Karl Deisseroth, Achim Schweikard, Franziska Ramm (undergraduate student) Stanford University, USANancy Kopell, University of Lübeck Suzanne van der Veldt (graduate student) Boston University, Boston, USA Emmanouela Volitaki (graduate student) Genela Morris, University of Haifa, Israel Staff employed within the reporting period

SELECTED PUBLICATIONS EXTERNAL FUNDING

Carus-Cadavieco M *, Gorbati M *, Ye L, Bender F, van der Veldt S, The Human Frontier Science Program, “Neural basis of behavioural Kosse C, Börgers C, Lee S Y, Ramakrishnan C, Hu Y, Denisova N, multitasking and coordination by specific hypothalamic circuits”, Ramm F, Volitaki E, Burdakov D, Deisseroth K, Ponomarenko A * §, 10. 2012-04. 2016; 350.000 $ Korotkova T * § (2017) Gamma oscillations organize top-down signaling Die Deutsche Forschungsgemeinschaft, Priority Program 1665 to hypothalamus and enable food seeking. Nature, 542(7640), 232 – 236. (Schwerpunktprogramm), “Resolving and manipulating neuronal Herrera C G, Carus-Cadavieco M, Jego S, Ponomarenko A, Korotkova T, networks in the mammalian brain – from correlative to causal analysis”; Adamantidis A (2016) Hypothalamic feed-forward inhibition of 09.2013-08.2019; 361.200 € thalamocortical network controls arousal and consciousness. Nature Die Deutsch-Israelische Stiftung für wissenschaftliche Forschung und Neuroscience, 19(2), 290 – 8. Entwicklung (GIF). Midbrain dopamine and GABA inputs onto Stempel A V, Stumpf A, Zhang H-Y, Özdogan T, Pannasch U, hippocampus and medial prefrontal cortex: function in motivation, Theis A-K, Otte D M, Wojtalla A, Rácz I, Ponomarenko A, Xi Z-X, spatial learning and memory. 01.2016-12.2018; 90.000 € Zimmer A and Schmitz D (2016) Cannabinoid Type 2 Receptors Mediate Die Deutsche Forschungsgemeinschaft, Cluster of Excellence a Cell Type-Specific Plasticity in the Hippocampus. Neuron, 90(4), NeuroCure: “Towards a better outcome of central nervous system 795 – 809. disorders”, PI position A. Ponomarenko, 10.2009-10.2017; 440.000 € Bender F *, Gorbati M *, Carus-Cadavieco M, Denisova N, Gao X, Deutsche Forschungsgemeinschaft, Cluster of Excellence NeuroCure: Holman C, Korotkova T * §, Ponomarenko A * § (2015) Theta “Towards a better outcome of central nervous system disorders”, oscillations regulate speed of locomotion via hippocampus to lateral Habilitationsgrant für Nachwuchswissen-schaftlerinnen (including PI septum pathway. Nature Communications, 6, 8521. position), T. Korotkova, 01.2011-10.2017, 243.400 € 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, 6254.

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 46 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

MOLECULAR AND THEORETICAL NEUROSCIENCE

MOLEKULARE UND THEORETISCHE NEUROWISSENSCHAFTEN

GROUP LEADER DR. ALEXANDER MATTHIAS WALTER

BIOGRAPHY SUMMARY

2001 – 2004 University Course in Chemistry, We aim to understand the molecular mechanisms of synaptic transmission. This University of Göttingen, Germany 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 2004 – 2006 Master’s in Neuroscience, particularly interested in understanding how synaptic molecules function together to Max Planck Research School Göttingen, produce the complex features of neurotransmission, which is not only optimized for speed Germany & Karolinska Institute, Stockholm, but which can also plastically adapt to evade interference or to store information. To do Sweden so we combine experimental and theoretical approaches. Our experimental system is the 2006 – 2009 Dr. rer. nat., Department of neuromuscular junction of the fruit flyDrosophila melanogaster, a powerful genetic model Membrane Biophysics, Max Planck Institute system. Despite its simplicity, human and Drosophila synaptic genes are remarkably similar for Biophysical Chemistry (Prof. Erwin Neher, and the majority of genes implicated in neurological disorders are conserved from flies to Prof. Jakob Sørensen, Prof. Reinhard Jahn) 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 2010 – 2011 Post-Doc, Center for molecular machinery driving transmission on the nanometre scale. To arrive at a conceptual Neurogenomics and Cognitive Research, framework of how synaptic molecules enable, control, and adapt synaptic transmission, we Free University Amsterdam, generate mathematical models based on parameters derived from our experiments. These The Netherlands (Prof. Matthijs Verhage) are used to generate hypotheses which are then tested experimentally. Iterating between 2011 – 2013 Post-Doc, Institute for experiments and theoretical work allows us to constantly refine our models and to increase Neuro­science and Pharmacology, their predictive value, furthering our understanding of molecular synapse function. Copenhagen University, Denmark (Prof. Jakob Sørensen) ZUSAMMENFASSUNG

2013 – 2015 Post-Doc, Department of Wir möchten die molekularen Mechanismen synaptischer Signalübertragung aufklären. Molecular Pharmacology and Cell Biology, Diese Kommunikation zwischen Nervenzellen ist Grundlage von Überleben, Kognition FMP Berlin, Germany (Prof. Volker Haucke) und Verhalten. Fehlerhafte Synapsenfunktionen sind der Grund vieler neurologischer Erkrankungen. Insbesondere versuchen wir zu verstehen, wie einzelne synaptische Since 2015 Group Leader, FMP Berlin Moleküle gemeinsam komplexe Eigenschaften neuronaler Kommunikation bedingen. 2015 – 2020 Emmy Noether fellow Diese Kooperation ist nicht nur auf unglaubliche Geschwindigkeiten ausgelegt, sondern of the DFG 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 FruchtfliegeDrosophila 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 experimen- teller 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. 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 embryo pupa three consecutive larval stages. Before the late third instar, when larvae crawl Drosophila 1st instar larva out of the food and pupate, they are collected for experiments using several life cycle existing techniques employed in the lab, ranging from optical methods such as prepupa 2nd instar larva confocal, super-resolution and functional imaging of synaptic transmission, to 3rd instar larva electrophysiological methods. Our experimentally obtained data are then used for computational assays to model and simulate synaptic mechanisms. (Modified from http://flymove.uni-muenster.de and adapted from Kononenko super-resolution confocal analysis et al., 2014).

electrophysiology activity tracking mutant ctrl local synapse synaptic activity transmission

DESCRIPTION OF PROJECTS mathematical modeling exocytosis endocytosis exo- endocytosis 1 Single synapse sub-architecture and its plastic modulation experiment simulation Neurotransmitters are released from a specialized sub-synaptic region known as the active zone (AZ), where evolutionarily 0 conserved proteins orchestrate synaptic vesicle (SV) fusion. kCME kCIE 0.5 Ca2+ channels open in response to action potential (AP)-induced ∆ F/F depolarisation and trigger rapid, locally confined Ca2+ elevations that activate the SV Ca2+ sensor synaptotagmin to induce fusion. FV FVm 2+ 2+ 0 Owing to the steep Ca concentration gradient from the Ca Syt-pHluorin clathrin FV fused vesicle k clathrin dependent rate CME 0 50 100 source, its distance to the SV release site is a major determinant m cooperativity kCIE clathrin independent rate Time [s] 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 super- resolution STED microscopy we have found that residence times molecular requirements on PI(4,5)P2 binding proteins are currently and nanoscale distribution of AZ-proteins differ vastly. While some being identified in the laboratory usingDrosophila mutants. proteins specifically and very stably (on the scale of hours) localize to sub-AZ regions where SVs typically fuse, others show broad Mathematical Modeling of synaptic transmission distribution and high fluctuation (on the scale of minutes). Using theoretical methods, we seek to develop a realistic model of Strikingly, these characteristics are not static, but change with the SV release at the synapse. As reactions at the synapse are typically functional status of the synapse. For instance, induction of synaptic governed by few SVs and Ca2+ channels, deterministic approaches plasticity that increases SV release greatly increases the localisation may fail to viably reproduce these processes. Our computational specificity and lifetime of proteins which are unstable and approach employs stochastic Ca2+ channel gating and vesicle promiscuously localised when at rest. Identifying the molecular positioning based on experimentally determined distributions. We principles of this modulation and how they influence SV coupling have found that current models fail to predict synaptic physiology distances to Ca2+ channels is a subject of ongoing investigation. when taking the actual spatial distribution of SVs into account as they predict rapid fatigue of transmission upon repetitive activation. Characterising lipids as regulators of synaptic transmission We are currently exploring which molecular mechanisms ensure While we possess considerable knowledge about how AZ proteins performance during continued activity. contribute to SV release, much less is known about membrane lipids. However, we do know that the key step in transmission concerns Molecular basis of synaptic heterogeneity SV fusion, where SV and plasma membrane lipids merge. The Although core components driving SV fusion are conserved, AZ phosphoinositide PI(4,5)P2 is enriched at synapses, known to activity, even among AZs of the same neuron, is largely heterogeneous. influence secretion, and directly interacts with proteins of the release This heterogeneity is the neural basis of information processing. machinery, such as synaptotagmin. This makes a direct function We are currently investigating the molecular principles involved of PI(4,5)P2 in SV fusion likely. Classical methods of altering using live cell imaging, where synaptic transmission is visualized by 2+ PI(4,5)P2 levels rely on long-lasting changes which may affect postsynaptic expression of Ca -sensitive fluorescence reporters. As upstream reactions or change the levels of PI(4,5)P2 metabolites. To postsynaptic glutamate receptors of the fly neuromuscular junction 2+ investigate direct PI(4,5)P2 function on the timescale relevant for (NMJ) are permeable to Ca , this allows for the tracking of AZ synaptic transmission, we use caged PI(4,5)P2 variants, generated activity with high temporal and spatial resolution. As a developmental by our collaborator Carsten Schultz from the EMBL in Heidelberg, system, the larval NMJ contains a mixture of AZs in various states which are loaded into the presynapse. An intense burst of UV light of maturation. By comparing the activity of single AZs with their uncages PI(4,5)P2 in the plasma membrane within milliseconds. molecular composition we identified proteins that locally set Electrophysiological characterisation has revealed that increasing the functional status. Identifying local regulators of synaptic

PI(4,5)P2 rapidly augments synaptic transmission by increasing the transmission will allow us to understand how developmental likelihood of SV fusion, implying a direct action of PI(4,5)P2. The trajectories generate functional AZs and optimize transmission. 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 Ca2+ source (Cac; blue). Unc13B is localized at a larger distance of 120 nm. The color transition from dark to light blue indicates the different Ca2+ concentrations the vesicles sense. This precise vesicle positioning is essential to control synaptic transmission.

GROUP MEMBERS COLLABORATIONS

Dr. Mathias Böhme Carsten Schulz, Susanne Ditlevsen, Andreas Till Grasskamp (doctoral student) EMBL Heidelberg, Heidelberg, Germany University of Copen­hagen, Copenhagen, Anthony McCarthy (doctoral student) Synthesis and analysis of signalling lipids Denmark Mathematical modeling of synaptic Meida Jusyte (student) André Nadler, transmission Sabine Hahn (technical assistant) MPI for Mol. Cell Biology and Genetics, Jakob Sørensen, Gabriela Pimenta Dos Reis (research intern) Dresden, Germany / Investigating the role of University of Copenhagen, Copenhagen, signalling lipids in exocytosis Denmark / Investigating the molecular Staff employed within the reporting period Stephan Sigrist, mechanisms of neurosecretion Free University Berlin, Berlin, Germany / Matthijs Verhage, Neurogenetics and ultrastructural analysis Free University of Amsterdam, Amsterdam, of the synapse The Netherlands / Mathematical modeling of Stefan Hell, synaptic transmission Max Planck Institute for Biophysical Nils Brose, Chemistry, Göttingen, Germany / Max Planck Institute for Experimental Superresolution (STED) microscopy Medicine, Göttingen, Germany / Mathematical modeling of neurosecretion

SELECTED PUBLICATIONS

Bohme M A, Beis C, Reddy-Alla S, Reynolds E, Mampell M M, Walter A M, Kurps J, de Wit H, Schoning S, Toft-Bertelsen T L, Lauks J, Grasskamp A T, Lutzkendorf J, Bergeron D D, Driller J H, Babikir H, Ziomkiewicz I, Weiss A N, Schulz A, Fischer von Mollard G, et al. (2014). Gottfert F, Robinson I M, O'Kane C J, Hell S W, Wahl M C, Stelzl U, Loll B, The SNARE protein vti1a functions in dense-core vesicle Walter A M *, Sigrist S J * (2016) Active zone scaffolds differentially biogenesis. The EMBO Journal 33, 1681-1697. accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling. Walter A M * , Pinheiro P S, Verhage M, Sorensen J B (2013). A sequential Nat Neurosci. 19, 1311 – 1320. vesicle pool model with a single release sensor and a ca(2+)-dependent *: equally contributing senior author priming catalyst effectively explains ca(2+)-dependent properties of Muhammad K, Reddy-Alla S, Driller J H, Schreiner D, Rey U, Bohme M A, neurosecretion. PLoS Comp Biol 9, e1003362. Hollmann C, Ramesh N, Depner H, Lutzkendorf J, Matkovic T, Gotz T, *: corresponding author 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. EXTERNAL FUNDING Nat Commun. 6, 8362. Deutsche Forschungsgemeinschaft, TRR 186 “Molecular switches Schotten S *, Meijer A *, Walter A M *, Huson V, Mamer L, Kalogreades L, in the spatio-temporal regulation of cellular signal transduction”, ter Veer M, Ruiter M, Brose N, Rosenmund C, Sorensen J B, Verhage M, 06.2016 – 05.2020, 197.600 € Cornelisse L N (2015) Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate. eLife 4, Deutsche Forschungsgemeinschaft, Emmy Noether funding, e05531. „Investigating how the active zone cytomatrix orchestrates neuronal *: equally contributing first author exo- and endocytosis“, 03.2015 – 02.2020, 1.522.250 €

FMP authors Group members 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 intra- cellular 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 Ca2+ 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 Bereit- stellung 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 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 electron microscopy, and quantitative live cell imaging to investigate Methoden gehören die Weitfeld-, konfokale und hochauflösende the role of the BAR protein SNX9 in the late stages of CME. A Mikroskopie. FRET (Fluoreszenz Resonanz Energie Übertragung), variety of collaborators make use of our multicolor STED setup to FRAP (Fluoreszenz-Rückgewinnung nach Photobleichung), investigate the molecular organisation of tight junctions (AG Blasig), FLIM (Fluoreszenz-Lebensdauer Mikroskopie), TIRFM (Totale Golgi transport (AG Krauss), neuronal synapses (AG Haucke), and Interne Reflexion Fluoreszenz-Mikroskopie), FCS (Fluoreszenz- active zone proteins in the fly neuromuscular junction (AG Sigrist, Korrelation-Spektroskopie) sowie Ca2+-Messungen und das Arbeiten FU Berlin). With Dmytro Puchkov we are currently establishing mit „caged Coumpounds“ (Verbindungen, die durch das Einbringen correlative light and electron microscopy for cells and tissue samples. einer sogenannten Schutzgruppe, Käfig, biologisch inaktiv sind) sind ebenfalls gut etablierte Methoden in unserer Gruppe. Darüber The heptahelical G protein-coupled receptors (GPCRs) are important hinaus geben wir Unterstützung bei der Bildanalyse und entwickeln drug targets. Following activation by their ligands, they function via neuartige Algorithmen für die biophysikalische Datenanalyse. the binding of G proteins and activation of specific signal transduction pathways. Our focus is on the analysis of GPCR oligomerization, Elektronenmikroskopie whose functional significance is not completely understood. It is known Die FMP-Service-Gruppe Elektronenmikroskopie unterstützt die that for some GPCRs oligomerization modulates receptor transport Visualisierung zellulärer Ultrastrukturen und die Lokalisierung and / or the dynamics of receptor activation. Most importantly, einzelner Proteine auf subzellulärer Ebene. Das Labor stellt it is not clear for most of the GPCRs whether they exist exclusively as Probenpräparationstechniken in Standard- und gehobener Ausfüh- oligomers or in a certain monomer-dimer ratio (M / D), or whether a rung, mikroskopische Aufnahmen, quantitative Analysen und Inter- given ratio is dynamic. In our group, the oligomerization of GPCRs pretationshilfen für alle biologische Applikationen bereit. Immuno- is analysed using fluorescence resonance energy transfer (FRET), gold-Markierungen, Korrelative Licht- und Elektronenmikroskopie fluorescence-lifetime imaging microscopy (FLIM) and fluorescence (KLEM), tomographische 3D-Rekonstruktionen werden bei Projekten cross-correlation spectroscopy (FCCS). mit vielversprechenden Phenotypen angewendet. Zusätzlich zu unserem Hauptfokus in der Zellbiologie, helfen wir bei der Visuali- The electron microscopy core facility sierung von in vitro Strukturen bzw. Proteinen, fibrillären Strukturen The major interest of the group is the regulation of intracellular und Liposomen mit Negativfärbung sowie als Kryoprobe. 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) DESCRIPTION OF PROJECTS we investigate the contribution of various adaptor proteins such as The light microscopy core facility AP2, AP180, and Stonin 2, scaffolding and accessory proteins such as In neuronal synapses and in clathrin-mediated endocytosis (CME), clathrin and intersectin, and different phosphatidylinositol kinases membranes and proteins are organized on the nanometer scale, which and phosphatases, to endocytosis, endosomal sorting and organelle is below the diffraction limit of light (< 250 nm). Recently developed maturation. We conduct these investigations in cells and, in super-resolution microscopy techniques like STED and STORM particular, in neuronal synapses. In cooperation with Thomas Jentsch overcome this diffraction barrier and achieve molecular resolutions (Physiology and Pathology of Ion Transport, FMP) we investigate down to 20 nm. In collaboration with Jan Schmoranzer (AG Haucke) mice lacking various ion transporters that also exhibit significant we screened more than 30 organic fluorophors and optimized alterations in membrane trafficking and organelle dynamics in a imaging conditions to establish robust multicolor STORM and variety of tissues and organs. In cooperation with Martin Lehmann STED imaging. We used multicolor STORM to visualize the (light microscopy) we develop correlative light and electron nanoscale organisation of CME proteins, the actin cytoskeleton, microscopy approaches for understanding organelle dynamic identity. intracellular organelles, and synaptic proteins in mouse brain slices. In addition to our work on cell biological projects, we provide support In collaboration with Volker Haucke and Frank Noe (FU Berlin) we with negative staining for groups studying protein structures. combined multicolor STORM imaging with mathematical modeling, 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 COLLABORATIONS

Light Microscopy International Max-Delbrück Center Dr. Martin Lehmann Thomas Walther, for Molecular Medicine, Berlin: Jenny Eichhorst (technical assistant) Hull University, Hull, UK Enno Klussmann, Dominique Müller Pavel Nedvetsky, Matthew Poy, Bettina Purfürst, Electron Microscopy Catholic University Leuven, Leuven, Belgium Anje Sporbert, Alessandro Prigione, Svea Hohensee (research assistant) Thomas Wiglenda Martina Ringling (technical assistant) National Dorothea Eisenhardt, Leibniz-Forschungsinstitut für Staff employed within the reporting period Freie Universität Berlin Molekulare Pharmakologie (FMP): Stephan Sigrist, Ingolf Blasig, Margitta Dathe, Frei Universität Berlin Volker Haucke, Thomas Jentsch, Karin Müller, Gerd Krause, Michael Krauß, Leibniz Institute for Zoo and Wildlife Ronald Kühne, Tanja Maritzen, Research, Berlin Jan Schmoranzer, Ralf Schülein Alexander Wenig, Charité – Universitätsmedizin Berlin Maria Maares, Technische Universität Berlin

SELECTED PUBLICATIONS

Opitz R, Müller M, Reuter C, Barone M, Soicke A, Roske Y, Piotukh K, Ketel K, Krauss M, Nicot A S, Puchkov D, Wieffer M, Müller R, Huy P, Beerbaum M, Wiesner B, Beyermann M, Schmieder P, Subramanian D, Schultz C, Laporte J, Haucke V (2016) A phosphoinositide Freund C, Volkmer R, Oschkinat H, Schmalz HG, Kühne R (2015) conversion mechanism for exit from endosomes. Nature 529, 408 – 412. A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions. Proc Natl Acad Sci U S A. 112. 5011 – 5016.

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 Self- assembly Properties. ACS Chem Biol. 10, 2733 – 2742.

Lehmann M, Lichtner G, Klenz H, Schmoranzer J (2016) Novel organic dyes for multicolor localization-based super-resolution microscopy. J Biophotonics. 9, 161 – 170.

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, 149 – 163. FMP authors Group members 52 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

CORE FACILITY

ANIMAL FACILITY TIERHALTUNG

GROUP LEADER DR. NATALI KRISTINA WISBRUN

BIOGRAPHY SUMMARY

Until 1999 Studied Veterinary Medicine, The animal facility manages the housing and breeding of laboratory animals for use in University Zagreb, Croatia and Free scientific projects. Animal welfare legislation and the highest scientific standards are enforced University of Berlin to guarantee reliable, ethically obtained scientific results. We support and give advice to scientists on all matters regarding planning and performing experiments involving animals. 1999 Veterinary degree, Faculty of Veterinary We also provide practical support to FMP scientists, for instance by taking samples and in Medicine, Free University of Berlin keeping proper documentation on their behalf. Furthermore, we organize the export and 2006 Dissertation, Cloning and Expression import of laboratory animals and embryonal stem cells. of Enzymes involved in the salvage pathway of L-fucose ZUSAMMENFASSUNG

2009 Graduate in Veterinary Medicine, Die Tierhaltung beschäftigt sich mit dem Management und der Organisation der Zucht und specializing in laboratory animal science Haltung von Versuchstieren für deren Einsatz in der Bearbeitung wissenschaftlicher Frage- stellungen. Dies erfolgt unter Berücksichtigung der Anforderungen des Tierschutzgesetzes PROFESSIONAL BACKGROUND zur Erzielung von relevanten wissenschaftlichen Ergebnissen. Wissenschaftler werden bei allen Fragen zur Planung und Durchführung von Tierversuchen unterstützt und beraten. Sie Since 2013 Group leader and animal welfare erhalten zudem praktische Unterstützung, z. B. bei der Probengewinnung und der Führung officer, FMP Berlin der Dokumentation. Zudem organisiert die Tierhaltung den Export und Import von Tieren 2012 – 2013 Head of animal experimental und Embryonalzellen weltweit. facilities, Philipp University of Marburg, Marburg

2009 – 2012 Head of an animal experimental facility and Animal Welfare Officer, Max Delbrück Center for Molecular Medicine, Berlin Buch

2006 – 2009 Research Fellow and animal welfare officer, Central Animal Laboratory of the University Clinic Essen, Essen

2001 – 2006 Research Fellow, Institute for Biochemistry and Molecular biology, Charité-Universitätsmedizin Berlin GROUP MEMBERS COLLABORATIONS

National Dr. Nadja Heinrich (Animal Welfare Officer) Animal Facility Nadja Daberkow-Nitsche (veterinarian) Max-Delbrück-Center for Molecular Eva Lojek (animal keeper) Medicine, Berlin Sina Scholz (animal keeper) Animal Facilities Jannette Unnasch (animal keeper) Charité, Berlin Elisabeth Lettau (studentische Hilfskraft / AG Blasig student of molecular bioscience) AG Haucke Marco Walther (studentische Hilfskraft / AG Jentsch student of biology) AG Maritzen AG Plested Staff employed within the reporting period AG Schröder MOLECULAR PHYSIOLOGY AND CELL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE 53

DESCRIPTION OF PROJECTS INTERESTS AND FOCUS

We provide FMP scientists with animal welfare officers who ensure The Animal Facility oversees all research at the FMP that involves that experiments using animals are in compliance with the Animal animals, including: Welfare Act.  Managing the breeding of a variety of genetically modified mice, frogs, and rats in compliance with animal welfare regulations. The facility supplies animal housing and manages all genetically  Organizing veterinary care and health monitoring in accordance modified mice, frogs, and rats, including overseeing performance with FELASA guidelines. standards, veterinary care, qualified animal care staff, and the use  Implementing European guidelines on animal welfare in of standard operating procedures and their related documentation. high-quality research. Our service also includes the education and training of personnel  Organizing imports and exports of animals, embryos, stem cells, responsible for animal care and of personnel carrying out experimental and related materials. procedures (research technicians), along with all scientists involved in  Consulting for scientists who are designing experiments that use experimental trials using animals. animals.  Supporting scientists in implementing experiments 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.

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, 1085 – 1093. NMR Biomed. 27, 1085 – 1093.

FMP authors Group members 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 Molecular Imaging Lösungs-NMR Molekulare Bildgebung

Group leader Group leader Dr. Peter Schmieder Dr. Leif Schröder

 PAGE 68  PAGE 79 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 56 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

SECTION STRUCTURAL BIOLOGY

BEREICH STRUKTURBIOLOGIE

Molecular pharmacology requires three-dimensional via peptide-based Kinase Activity Reporters (KARs), which allow representations of supramolecular arrangements within the dynamic changes of cellular kinase activities to be measured under a cell, which are controlled in vivo by temporal and spatial variety of physiological and pathophysiological conditions. Investigation coordination of protein expression, degradation, and of the structure of α-synuclein within cells aims at a molecular post-translational modification. The dynamic nature of understanding of Parkinson’s disease. The ‘Molecular Imaging’ junior these phenomena challenges static structure determination group of Leif Schröder works at the physiological level using magnetic techniques and offers a strong incentive to employ Nuclear resonance imaging (MRI) as a major technique for exploring the Magnetic Resonance (NMR) spectroscopy. With this in mind, the possibility of a biosensor-based NMR imaging approach, employing ‘Structural Biology’ section develops and applies solution and hyperpolarisation to achieve unprecedented sensitivity. In the course solid-state NMR techniques to investigate pharmacologically of this work, Leif Schröder and his colleagues devised a highly efficient relevant proteins in their native environments, or even in intact combination of hyperpolarization and controlled depolarization of biological systems such as cells or functional modules. Beyond xenon, which is then used to detect membrane-embedded and receptor- this, the insights gained into molecular interactions are used to attached probes with hyperpolarised nuclei. In this way, enhanced MRI develop NMR reporters for diagnostic imaging purposes where can detect specific cell markers, raising the possibility of tissue- or fluorescence detection fails. For example, the departments of molecule-specific contrasting that could be used for non-invasive Adam Lange and Hartmut Oschkinat apply solid-state NMR detection of various diseases at an early stage. The cheminformatics / to membrane proteins in native lipid bilayers, Philipp Selenko bioinformatics groups utilize structural information to derive protein detects proteins and their modifications in live cells by solution interaction inhibitors, and to understand protein function, especially NMR, and Leif Schröder images cells or organisms by means of G-protein-coupled receptors (GPCRs), via pharmacological of molecule-specific contrast agents. In concert with molecular interference. The ‘Drug Design’ group led by Ronald Kühne modeling and structure-function studies provided by the accomplished the impressive task of developing a set of small- groups of Gerd Krause and Ronald Kühne, atomic-resolution molecule fragments that inhibit protein-protein interactions involving structural data are derived that are indispensable to potential proline-rich motifs, yielding efficient inhibitors of EVH1 and WW pharmacological interference, a process typically supported by protein domain interactions. The ‘Structural Bioinformatics and the ‘Solution NMR’ group of Peter Schmieder. With regards to Protein Design’ group headed by Gerd Krause generated a new the design of bioactive molecules, the development of protein web-based system that assists in evaluating the molecular effects of interaction inhibitors is a major theme of this section. genetic variations in GPCRs. The structure-function relationship of two receptors, LHR and TSHR, were deciphered to derive molecular The department of Adam Lange uses solid-state NMR methods in details of activation and inactivation patterns, and to understand the combination with complementary techniques, such as scanning activity of agonists and antagonists. Both computational groups tested transmission electron microscopy (STEM) and cryo-EM, to investigate the compounds developed in their biological laboratories, thereby protein structure and dynamics. One focus of the group is on bacterial closing the gap between structural studies and computational supramolecular assemblies involved in infection processes. Recent modeling, as well as biochemical and cell physiological analyses. examples include the Shigella type-III secretion needle, the type-I With the arrival of the group of Dorothea Fiedler and the new pilus of uropathogenic E. coli, and cytoskeletal bactofilin filaments. department of ‘Chemical Biology I’, the demand for solution-state Furthermore, the group is interested in solid-state NMR methods NMR support has grown considerably and the services provided by development. In the department of Hartmut Oschkinat, the application the NMR spectroscopy core facility have been extended. This has of microwave-based hyperpolarisation (DNP) has recently offered a also lead to an increase in the collaborations between the group of picture of the nascent polypeptide chain growing inside the ribosome. Peter Schmieder and the Chemical Biology section. Further applications of the technique yielded well-resolved spectra of proteins at temperatures around 200 K. The ‘Solution NMR’ group In der molekularen Pharmakologie ist die Kenntnis der of Peter Schmieder continued to investigate protein dynamics in drei-dimensionalen Struktur supramolekularer Komplexe MHC complexes to obtain information that will help to explain the innerhalb der Zelle, die in vivo durch die zeitliche und interactions between T-cell receptors and MHC complexes. In räumliche Koordination von Proteinexpression, -abbau addition, several projects seeking to clarify the constitution of small und posttranslationaler Modifikation kontrolliert wird, molecules (natural products or products of chemical synthesis) were von entscheidender Bedeutung. Die diesen Prozessen initiated. The ‘In-cell NMR’ junior group headed by Philipp Selenko innewohnende Dynamik ist eine große Herausforderung für explores novel NMR-based methodologies that allow monitoring of statische Strukturbestimmungsmethoden und legt es nahe, proteins inside cells. Their projects include profiling kinase activities Kernspinresonanzmethoden (NMR) anzuwenden. Daher STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 57

Veniamin Chevelkov Jonas Protze, Katrin M. Hinz (in front) and students

entwickeln und nutzen die Wissenschaftler des Bereichs Die Abteilung von Adam Lange verwendet Festkörper-NMR Methoden „Strukturbiologie“ Lösungs- und Festkörper-NMR-Techniken, in Kombination mit komplementären Techniken wie der Scanning um pharmakologisch relevante Proteine in ihrer natürlichen Transmission Electron Microscopy (STEM) und der Cryo-EM Umgebung oder sogar in intakten biologischen Systemen um die Struktur und Dynamik von Proteinen zu untersuchen. Ein wie Zellen oder funktionellen Modulen zu untersuchen. Schwerpunkt der Forschung der Arbeitsgruppe liegt auf bakteriellen Darüber hinaus werden die Erkenntnisse über molekulare supramolekularen Strukturen, z. B. Typ-3 Sekretionsnadeln, dem Wechselwirkungen eingesetzt, um daraus NMR-Reporter Typ I-Pilus und Bactofilinfilamenten, einem Bestandteil des für solche diagnostische Bildgebungszwecke zu entwickeln, bakteriellen Zytoskeletts. Gleichzeitig beschäftigt die Gruppe sich bei denen Fluoreszenz-Bildgebung nicht anwendbar ist. Die mit der Weiterentwicklung von Festkörper-NMR-Methoden. Durch Abteilungen von Hartmut Oschkinat und von Adam Lange die Abteilung von Hartmut Oschkinat wurde kürzlich die Struktur nutzen beispielsweise Festkörper-NMR für Membranproteine der naszierenden Polypeptidkette innerhalb des Ribosoms aufge- in nativen Lipidmembranen, und Philipp Selenko detektiert klärt. Dies geschah mittels Mikrowellen-gestützter Hyperpolarisa- Proteine und Proteinmodifikationen in lebenden Zellen. tion (DNP). Weitere Anwendungen dieser Technik erbrachten gut- Leif Schröder arbeitet an der Bildgebung von Zellen und aufgelöste Spektren von Proteinen bei Temperaturen um 200 K. Die Organismen mittels spezifischer Kontrastmittel. Zusammen Gruppe „Lösungs-NMR“ von Peter Schmieder hat ihre Arbeiten zur mit molekularem Modelling und Struktur-Funktions-Unter- Untersuchung der Dynamik in MHC-Komplexen fortgesetzt, um suchungen der Arbeitsgruppen von Gerd Krause und Ronald Informationen zu erhalten, die helfen werden die Wechselwirkung Kühne werden Strukturdaten mit atomarer Auflösung zwischen T-Zell-Rezeptoren und MHC-Komplexen besser zu erarbeitet, die unverzichtbar sind, um Wege zu einer pharma- verstehen. Daneben wurden mehrere Projekte begonnen, die die kologischen Einflussnahme auf Zielproteine zu finden. Diese Konstitutionsbestimmung kleiner Moleküle (Naturstoff oder Untersuchungen werden gewöhnlich von der „Lösungs-NMR“- Syntheseprodukte) zum Ziel haben. Die Nachwuchsgruppe Gruppe unter der Leitung von Peter Schmieder unterstützt. „NMR in Zellen“ von Philipp Selenko erforscht neuartige NMR- Hinsichtlich des Designs von biologisch aktiven Wirkstof- Methoden, die eine Beobachtung von Proteinen in lebenden Zellen fen ist insbesondere die Entwicklung von Inhibitoren von ermöglichen. So erarbeitet seine Gruppe beispielsweise ein Profil Protein-Wechselwirkungen ein Leitthema des Bereichs. von Proteinkinaseaktivitäten mit Peptiden als Kinaseaktivitäts- 58 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

Reportern (KARs). Diese ermöglichen es, dynamische Änderungen von Ronald Kühne gelang die Lösung einer ausgesprochen zellulärer Kinaseaktivitäten unter verschiedenen physiologischen anspruchsvollen Aufgabe. Sie entwickelte eine Gruppe von und pathophysiologischen Bedingungen zu messen. Ein weiteres Fragmenten kleiner Moleküle, die Protein-Protein-Wechselwirkungen Projekt, die Untersuchung der Struktur von α-Synuklein, zielt von Prolin-reichen Motiven hemmen. Resultat waren effiziente auf ein molekulares Verständnis der Parkinson-Krankheit. Die Inhibitoren für die Interaktion von EVH1 und WW-Proteindomänen. Nachwuchsgruppe „Molekulare Bildgebung“ von Leif Schröder Die Gruppe „Strukturelle Bioinformatik und Proteindesign“ nutzt die Magnetresonanztomographie (MRT) zur Bildgebung auf unter Gerd Krause entwickelte ein neues Web-basiertes System, das der physiologischen Ebene. Die Arbeitsgruppe entwickelt einen Wissenschaftler darin unterstützt, molekulare Effekte genetischer neuartigen, auf Biosensoren beruhenden Ansatz zur Bildgebung, Unterschiede bei GPCRs zu analysieren. Die Struktur-Funktions- der Hyperpolarisation nutzt um eine bislang unerreichte Sensitivität Beziehungen zweier Rezeptoren, LHR und TSHR, wurden zu erzielen. Es gelang den Wissenschaftlern, eine sehr effiziente entschlüsselt und so die Muster für Aktivierung oder Inaktivierung Kombination aus Hyperpolarisation und gezielter Depolarisation von dieser Rezeptoren durch Liganden im molekularen Detail aufgeklärt. Xenon zu entwickeln, die dann benutzt wird, um membranständige Dies ermöglicht es, die Wirkungsweise von Agonisten und Antago- oder Rezeptor-gebundene Sondenmoleküle mit hyperpolarisierten nisten zu verstehen. Beide Gruppen testen die cheminformatisch / Atomkernen zu detektieren. So gelang es durch signalverstärkte bioinformatisch als aktiv vorhergesagten Substanzen in biologischen MRT, spezifische Zellmarker darzustellen. Damit steht der Weg für Experimenten. Sie schließen damit den Kreis zwischen Strukturun- eine Weiterentwicklung in Richtung gewebe- oder molekülspezifischer tersuchungen, computergestütztem Modelling, biochemischen und Kontrastmittel offen, die zum Beispiel für eine nicht-invasive zellphysiologischen Ansätzen. Durch die Etablierung der Gruppe Früherkennung von verschiedenen Krankheiten eingesetzt werden von Dorothea Fiedler als Abteilung „Chemische Biologie I“ hat die könnten. Die Cheminformatik- / Bioinformatik-Gruppen nutzen Nachfrage nach Lösungs-NMR-Unterstützung stark zugenommen Strukturdaten um Inhibitoren für Proteinwechselwirkungen und der von der Core Facility „NMR-Spektroskopie“ angebotene abzuleiten und um Proteinfunktionen, insbesondere von G-Protein- NMR-Service wurde ausgedehnt. Das hat auch zu einer verstärkten gekoppelten Rezeptoren (GPCRs), mittels pharmakologischer Zusammenarbeit der Gruppe von Peter Schmieder mit dem Bereich Beeinflussung zu verstehen. Der Arbeitsgruppe „Wirkstoff-Design“ 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 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) 60 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

MOLECULAR BIOPHYSICS

MOLEKULARE BIOPHYSIK

GROUP LEADER PROF. DR. ADAM LANGE

BIOGRAPHY SUMMARY

10 / 1997 – 11 / 2002 Studied Physics, We study protein structure and dynamics using nuclear magnetic resonance in the solid state Georg-August-University, Göttingen (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 11 / 2002 – 09 / 2006 Ph.D. / Postdoctoral information for systems which are insoluble or do not crystallize easily. For instance, membrane studies, Max Planck Institute for Biophysical proteins in a lipid bilayer environment or supramolecular assemblies such as the needle of the Chemistry, Göttingen type three secretion system (T3SS) – composed of multiple copies of a single small protein – 10 / 2003 – 04 / 2004 Research visit, National can be readily studied. For solid-state NMR investigations, samples are placed in a strong Institutes of Health, Bethesda, USA 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 2006 Ernst Award of the German Chemical around an axis that is inclined to the magnetic field by a “magic” angle of 54.7° emulates the Society conditions of fast and freely tumbling molecules in solution. By means of magic-angle spinning, 2006 Otto-Hahn Medal of the Max Planck NMR spectra with high resolution and sensitivity can be achieved for solid-state protein Society 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 10 / 2006 – 08 / 2008 Postdoctoral fellow, in solid-state NMR methods development. Laboratory of Physical Chemistry, ETH Zürich, Switzerland, EMBO long-term ZUSAMMENFASSUNG fellowship Wir untersuchen mittels Festkörper-Kernspinresonanz (Festkörper-NMR) und anderen 09 / 2008 – 03 / 2014 Independent Group biophysikalischen Methoden die Struktur und Dynamik von Proteinen. In den vergangenen Leader, Max Planck Institute for Biophysical zehn Jahren hat sich die Festkörper-NMR zu einer wichtigen Technik in der Strukturbiologie Chemistry, Göttingen, Emmy Noether entwickelt, die Zugang zu Strukturinformationen an solchen Systemen ermöglicht, die fellowship unlöslich oder nicht zu kristallisieren sind. Membranproteine in ihrer Lipidbilayer- 2013 ERC Starting Grant “3D structures Umgebung beispielsweise oder supramolekulare Strukturen wie die Nadel des Typ-3- of bacterial supramolecular assemblies by Sekretionssystems (T3SS), die aus vielen Kopien eines einzelnen kleinen Proteins aufgebaut solid-state NMR" 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. 400.000- Since 04 / 2014 Department Head, FMP, mal so stark wie das Magnetfeld der Erde) und in eine schnelle Rotation versetzt (bis zu Berlin and W3-S Professor, Humboldt 100.000 Umdrehungen pro Sekunde), was die Situation von sich frei und schnell bewegenden University of Berlin Molekülen in Lösung simuliert. Die Rotationsachse der Probe ist gegenüber dem Magnetfeld 2016 ICMRBS Founders’ Medal 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. STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 61

DESCRIPTION OF PROJECTS

The type III secretion system needle The structure of bactofilin, an element of the Homo-oligomeric supramolecular protein assemblies such as the bacterial cytoskeleton needle of the type III secretion system (T3SS) are ideal targets for For the study of the cytoskeletal bactofilin protein BacA from solid-state NMR investigations. The T3SS is a nanomachine used Caulobacter crescentus, a uniformly 13C- and 15N-labeled sample was by many gram-negative bacteria to inject effector proteins into host prepared and investigated by solid-state NMR spectroscopy. From cells. Once injected, these substances manipulate essential metabolic the resulting NMR spectra we were able to determine essentially processes and disable the immune defence of the infected cells. A complete resonance assignments. Additional information was schematic representation of the T3SS is provided in Figure 1. The provided by STEM measurements that yielded a restraint for the base of the T3SS is firmly anchored in the inner and outer bacterial mass-per-length of the assembly. In combination with homology membranes. A channel through the base ends in a hollow extra- modeling, a β-helical fold with six windings per subunit was cellular “needle”. This needle is formed by the self-assembly of proposed [Vasa et al., PNAS, 2015]. A β-helical fold such as this has around 100-150 copies of a single, small-sized protein; in the case of never been observed for any other cytoskeletal filament. Subsequently, Salmonella typhimurium it is the 80-residue protein PrgI. we determined a high-resolution solid-state NMR structure of BacA [Shi et al., Science Advances, 2015]. In this work, which was based on We have previously shown that T3SS needles can be produced in numerous distance restraints from sparsely 13C-labeled samples and the lab by self-association of recombinantly expressed PrgI and that 4D HN-HN data from a deuterated sample, we were able to confirm solid-state NMR spectra of such in vitro needles exhibit exceptionally and refine our initial model and determine unambiguously that the high spectral resolution [Loquet et al., Nature, 2012]. As a result of β-helix is right-handed. the excellent data quality we could determine an atomic model of the needle. For this purpose solid-state NMR data were combined with The structure of the type I pilus of uropathogenic E. coli results from electron microscopy (EM) and computer modeling. Using solid-state NMR we also studied uniformly and sparsely labeled The resulting atomic model [Loquet et al., Nature, 2012] represents samples of polymerized FimA, the main subunit of the type I pilus one of the largest and most complex protein structures determined of uropathogenic E. coli. As with the bactofilin study, we obtained to date by solid-state NMR. The structure reveals an inner needle complementary STEM data. Based on the combination of solid-state diameter of only 2 nm. The secreted proteins thus have to pass NMR data, STEM data, and a solution NMR structure of a soluble through the needle in an unfolded state and then refold again in FimA construct (called FimAa) we determined a hybrid model of the the host cell before performing their tasks. The solid-state NMR FimA assembly [Habenstein et al., Angewandte Chemie – International structure also showed that the conserved C-terminus of the needle Edition, 2015]. protein faces the lumen and that the more variable N-terminus is found on the outer needle surface. This variability might reflect a Solid-state NMR method development strategy of the bacteria to evade immune recognition by the host. The focus of our solid-state NMR method development work is on new experiments for efficient sequential resonance assignment. For High-resolution solid-state NMR structures of secretion needles instance, we have introduced a set of proton-detected 3D experiments More recently, we have determined high-resolution solid-state NMR based on dipolar out-and-back transfers and applied them to structures of needles from Salmonella [Loquet et al., J Am Chem Soc, deuterated T3SS needles [Chevelkov et al., J Magn Reson, 2014]. By 2013] and from Shigella [Demers et al., Nat Commun, 2014], which means of band-selective homonuclear cross polarization (BSH-CP) have allowed us to determine the handedness of the needle. This for efficient CO-CA transfer [Chevelkovet al., J Magn Reson, 2013], parameter was not unambiguously defined in our previous study we also assembled a set of carbon-detected 3D experiments for [Loquet et al., Nature, 2012]. We found that the T3SS needle adopts sequential backbone assignment [Shi et al., J Biomol NMR, 2014]. a right-handed helical structure with approximately eleven subunits Furthermore, we exploit sparse isotope-labeling schemes that per two turns, similar to the arrangement observed in the related enhance spectral resolution and facilitate the detection of long-range flagellar filament. distance restraints [Loquet et al., J Am Chem Soc, 2011]. 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, Bakterielle Menschl. Zelle 2015] are relatively difficult to perform. In order to facilitate the Proteine 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].

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 Bakterielle Bakt. Zelle microscopy and computer modeling [Loquet et Proteine al., Acc Chem Res, 2013].

GROUP MEMBERS COLLABORATIONS

Adam Lange (PI) International National Dr. Veniamin Chevelkov David Baker, Martin Thanbichler, Dr. Matthias Herrera Glomm University of Washington, Seattle, USA Philipps-Universität Marburg Dr. Oxana Krylova Nikolaos Sgourakis, Stefan Becker, Dr. Sascha Lange UC Santa Cruz, USA Max Planck Institute for Biophysical Chemis- Dr. Stamatios Liokatis Yusuke Nishiyama, try, Göttingen Dr. Chaowei Shi RIKEN, Kobe, Japan Michael Kolbe, Dr. Yong-hui Zhang Sophie Zinn-Justin, Helmholtz Centre for Infection Research, Claudia Bohg (Student) CEA, Gif-Sur-Yvette, France Hamburg Jean-Philippe Demers (Ph.D. Student) Changlin Tian, Michael Habeck, Pascal Fricke (Ph.D. Student) University of Science and Technology of Max Planck Institute for Biophysical Songhwan Hwang (Ph.D. Student) China, Hefei, China Chemistry, Göttingen 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)

Staff employed within the reporting period STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 63

A C

D B

E

Fig. 2: A new methodological approach to determine the structure of the T3SS needle and other F 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].

SELECTED PUBLICATIONS EXTERNAL FUNDING

Vasa S, Lin L, Shi C, Habenstein B, Riedel D, Kühn J, Thanbichler M, and Emmy Noether Grant (DFG) “Solid-state NMR characterization of tau in Lange A (2015) β-Helical architecture of cytoskeletal bactofilin filaments paired helical filaments and bound to microtubules as well as of toxic revealed by solid-state NMR. Proceedings of the National Academy of and non-toxic oligomers of a-synuclein and tau.” (DFG GZ.: LA 2705 / Sciences, 112, E127 – E136. 1-1); Duration: 10 / 2009-09 / 2015; Total amount: 977.000 €

Habenstein B, Loquet A, Hwang S, Giller K, Vasa SK, Becker S, Habeck ERC Starting Grant “3D structures of bacterial supramolecular assemblies M, and Lange A (2015) Hybrid structure of the type I pilus of uropatho- by solid-state NMR” (Project acronym: assemblyNMR; Grant agreement genic Escherichia coli. Angewandte Chemie-International Edition, 54, no.: 337490); Duration: 60 months, starting date: May 1, 2014; Total 11691 – 11695. amount: 1.456.000 €

Shi C, Fricke P, Lin L, Chevelkov V, Wegstroth M, Giller K, Becker S, DFG Eigene Stelle (Dr. Stamatios Liokatis) „Post-translationale Protein Thanbichler M, and Lange A (2015) Atomic-resolution structure of Modifikationen, Querregulierung und Auswirkungen auf Histone H3 cytoskeletal bactofilin by solid-state NMR. Science Advances 1, e1501087. Tail Struktur und Dynamik: Eine strukturelle und mechanistische Studie an intakten Nukleosomen mittels hochauflösender Kernresonanzspek- Fricke P, Mance D, Chevelkov V, Giller K, Becker S, Baldus M, and troskopie“ (DFG GZ.: LI 2402 / 2-2); Duration: 04 / 2017-09 / 2019; Total Lange A (2016) High resolution observed in 800 MHz DNP spectra of amount: 281.000 € extremely rigid type III secretion needles. Journal of Biomolecular NMR 65, 121 – 126. Kekulé Ph.D. Scholarship to Pascal Fricke

Fricke P, Chevelkov V, Zinke M, Giller K, Becker S, and Lange A, Alexander-von-Humboldt Fellowship to Dr. Yong-hui Zhang (former Backbone assignment of perdeuterated proteins by solid-state NMR Postdoc) using proton-detection and ultrafast magic-angle spinning. Nature Protocols, in press.

FMP authors Group members 64 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

NMR-SUPPORTED STRUCTURAL BIOLOGY

NMR-UNTERSTÜTZTE STRUKTURBIOLOGIE

GROUP LEADER PROF. DR. HARTMUT OSCHKINAT

BIOGRAPHY SUMMARY

1975 – 1976 Chemistry degree, Magic-angle-spinning (MAS) solid state NMR provides high-resolution structural information University of Frankfurt 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 1978 – 1983 Chemistry Diploma, as proteins embedded in lipid bilayers or attached to the cytoskeleton. In the long run, we aim to University of Frankfurt carry out structural investigations within the ‘real space’ of a cell, capitalizing on a 20-100-fold 1983 – 1984 Visit to the laboratory of increase in the signal-to-noise ratio afforded by the use of dynamic nuclear polarization (DNP). Prof. Dr. Ray Freeman, Oxford, England 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 1983 – 1985 Completion of dissertation as well as we refinal esomerisation in channelrhodopsin. In addition, we study membrane in Prof. Kessler's Laboratory, proteins in native (or native-like) lipid environments, with a focus on bacterial membrane University of Frankfurt proteins such as OmpG and YadA. A new research area of ours involves high-frequency magic- 1986 Graduate thesis: “Analysis of the angle spinning for studying biological samples. At high MAS frequencies, high-resolution conformation of Cyclosporin in solution proton spectra can be obtained using a minimal amount of sample. We have been systematically using NMR-spectroscopy: development optimizing conditions and performing pilot studies on various proteins (SH3 domain, ABC and use of new methods" transporter, bacterial biofilms). Finally, we investigate protein systems involved in protein homeostasis, including small heat shock proteins. 1986 – 1987 Postdoctoral work with Prof. Dr. Bodenhausen, ZUSAMMENFASSUNG University of Lausanne, Switzerland Die „Magic-Angle-Spinning“ (MAS)-Festkörper-NMR-Spektroskopie ermöglicht die 1987 – 1991 NMR-spectroscopist, Bestimmung der dreidimensionalen Struktur von Proteinen in komplexen Proben mit Max-Planck-Institute for Biochemistry, atomarer Auflösung, unabhängig vom Molekulargewicht der Biomoleküle und ohne vorherige Martinsried, Germany, first in the Clore / Kristallisation. Sie stellt damit eine attraktive Methode zur Bearbeitung schwierig zu Gronenborn group and later independently untersuchender Systeme dar. Dazu gehören beispielsweise Proteine, die in Lipid-Doppelschichten in the department of Prof. Huber eingebaut oder mit dem Zytoskelett verbunden sind. Unser langfristiges Ziel ist es, 1992 Habilitation in Biophysical Chemistry, Strukturuntersuchungen innerhalb von Zellen oder Organellen durchzuführen, was insbesondere Technical University of Munich durch die Anwendung der dynamischen Kernpolarisation (dynamic nuclear polarization, DNP) und der damit verbundenen 20-100-fachen Verbesserung des Signal-Rausch-Verhältnisses 1992 – 1998 Group leader, EMBL, Heidelberg ermöglicht wird. Zu diesem Zweck haben wir DNP-Methoden an biologischen Proben getestet, Since 1998 Head of the department optimiert und diese Methodik zur Untersuchung von am Ribosom wachsenden Proteinketten “NMR-supported Structural Biology”, sowie des Protonentransports in Kanalrhodopsin eingesetzt. Darüber hinaus wenden wir Leibniz-Forschungsinstitut für Molekulare besonders schnelle Rotation, um den magischen Winkel (> 60 kHz) zur Strukturbestimmung Pharmakologie, Professor of Structural von Membranproteinen in möglichst nativer Lipidumgebung an, wobei unser Schwerpunkt Chemistry, Free University of Berlin auf bakteriellen Membranproteinen wie OmpG und YadA liegt. Als neues Forschungsgebiet hinzugekommen ist die Hochfrequenz-MAS zur Erforschung biologischer Proben. Bei 1998 Elected member of the European hohen MAS-Frequenzen können mit minimalen Probenmengen Protonenspektren mit hoher Molecular Biology Organization (EMBO) Auflösung erzielt werden. Wir haben die Bedingungen systematisch optimiert und bereits 2013 Elected Member of National Magnetic an verschiedenen Proteinen (SH3-Domäne, ABC-Transporter, OmpG, bakterielle Biofilme) Resonance Society of India Pilotstudien durchgeführt. Schließlich bearbeiten wir Proteinsysteme, die an der Proteinhomöostase beteiligt sind, u. a. kleine Hitzeschockproteine. 2014 Günther Laukien Prize STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 65

DESCRIPTION OF PROJECTS

Dynamic Nuclear Polarization we found that two loops (3 and 4) appear well ordered over much of Methods that enable structural studies on membrane-integrated their length, and we speculate that these represent a binding site. receptor systems without the necessity of protein purification or on proteins bound to the cytoskeleton are attractive prospects We have used solution and solid state NMR to investigate proton for structural biology. Dynamic nuclear polarization magic angle transport in channelrhodopsin, an important protein used in spinning NMR allows the investigation of such systems by delivering neurobiology to probe the light-induced activation of nerve cells. the required sensitivity. In this method, the very strong In these studies the use of DNP was indispensible for showing that polarization of electron spins is transferred to nuclear spins, which in the fully dark-adapted state, the retinal chromophore resides can then be detected at a much higher signal-to-noise ratio. With exclusively in the all-trans configuration. DNP, we have studied crystalline preparations of soluble proteins (SH3 domain), membrane proteins (neurotoxin II bound to the Chaperone systems and their functional complexes nicotinic acetyl choline receptor, OmpG, mistic), functional Small heat shock proteins (Hsp) such as αB-crystallin, or larger amyloid fibrils (Het-s, curli), and even selectively labeled ribosomes. chaperone systems such as Hsp40 / 70 / 110, are attractive but little Initial results from these studies include the first chemical shift explored drug targets. Following structural work on αB-crystallin assignments of residues in the nascent protein chain emanating we are now investigating its mechanism of activation and from the ribosome and analysis of its secondary structure. interaction with substrates such as β- and γ-crystallins. Our initial investigations aimed at the characterization of the binding site for Special emphasis has been placed on making DNP a routine γS-crystallin and a mutant of βB-crystallin. method for structural investigations of proteins by adopting a special approach that is termed ‘high temperature DNP’. Although Interfering with protein-protein interactions higher enhancements of signals are observed at a measurement In a third line of projects, we search for small-molecule inhibitors temperature of 100 K, spectra are much better resolved when of protein-protein interactions, using PDZ (PSD-95, Dlg, ZO-1) measured at 200 K. We developed a methodology that enabled domains as an example. They play important roles in cellular us to record sufficiently resolved spectra of protein samples while signaling pathways and are structurally characterized by a hydrophobic retaining an appreciable signal enhancement of around 20 to 40 in pocket surrounded by a conserved sequence motif, G-L-G-F. This the temperature range of 180 to 200 K. Future applications of DNP pocket binds the C-termini of target proteins, in most cases receptors comprise the interaction of ribosome-bound signal peptides with and ion channels. Their functional diversity and characteristic, interaction partners as well as studies of the chromophore states of relatively small binding pocket make them attractive targets for channelrhodopsin, during the photocicle. the design of small-molecule inhibitors which may, in the long run, allow for the treatment of several PDZ-related human disorders Structures of membrane proteins in native lipids such as neuropathic pain, congenital diseases, psychiatric disorders, by solid-state NMR and cancer. A large number of high-resolution structures of PDZ- We have determined the structure of OmpG reconstituted in ligand complexes provide an excellent basis for rational design. native lipids by a new solid-state NMR structure determination methodology. Using a combination of 13C-detected experiments We identified inhibitors with low to medium affinity for several on fully protonated samples with different 13C labeling schemes PDZ domains and collected members of the respective substance and 1H-detected experiments on deuterated samples for which the classes in a ‘PDZ library’. We intend to exploit our results further exchangeable sites were protonated, we were able to assign most by targeting three PDZ domains (AF6, DVL, Shank3), as we seek atoms in the membrane spanning β-sheets. A combination of an understanding of the biology of the respective proteins. 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, 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 COLLABORATIONS

Dr. Umit Akbey International Melanie Rosay, Dr. Ying Wing Chow Burkhard Bechinger, Bruker BioSpin, Billerica, USA Dr. Anne Diehl University of Strasbourg / CNRS, France Paul Tordo, Dr. Trent Franks Lyndon Emsley, Aix-Marseille Université, France Dr. Matthias Hiller ENS Lyon, France Shimon Vega, Dr. Madhu Nagaraj Lucio Frydman, The Weizmann Institute of Science, Rehovot, Dr. Andrew Nieuwkoop The Weizmann Institute of Science, Rehovot, Israel Dr. Shakeel Ahmad Shahid Israel Dr. Barth van Rossum Daniela Goldfarb, National Michel Andreas Geiger (doctoral student) The Weizmann Institute of Science, Rehovot, Bernd Bukau, Johanna Münkemer (doctoral student) Israel Universität Heidelberg Joren Retel (doctoral student) Robert G. Griffin, Michael Habeck, Mahsheed Sohrabi (doctoral student) Massachusetts Institute of Technology, Max Planck Institut für Intelligente Systeme, Daniel Stöppler (doctoral student) Cambridge, USA Tübingen Anja Voreck (doctoral student) Angela Gronenborn, Sandro Keller, Arndt Wallmann (doctoral student) Elena MateiUniversity of Pittsburgh, USA Technische Universität Kaiserslautern Natalja Erdmann (technical assistant) Victoria A. Higman, Dirk Linke, Liselotte Handel (technical assistant) University of Oxford, Oxford, UK Max-Planck-Institut für Martina Leidert (technical assistant) Rachel Klevit, Entwicklungsbiologie, Tübingen Kristina Rehbein (technical assistant) University of Washington, Seattle, USA Thomas Müller, Nils Cremer (technical assistant) Rachel W. Martin, Universität Würzburg UC Irvine, Irvine, USA Thomas Prisner, Staff employed within the reporting period Niels Chr. Nielsen, Johann-Wolfgang-Goethe Universität, Center for Insoluble Protein Structures Frankfurt (inSPIN), Interdisciplinary Nanoscience Center Bernd Reif, (iNANO) and Aarhus University, Denmark Technische Universität München Michael Nilges, Hans-Günther Schmalz, Institut Pasteur, Paris, France Universität zu Köln 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 EXTERNAL FUNDING

Nieuwkoop AJ, Franks WT, Rehbein K, Diehl A, Akbey Ü, Engelke F, Deutsche Forschungsgemeinschaft, SFB 765 / C4, „Multivalente Emsley L, Pintacuda G, Oschkinat H (2015) Sensitivity and resolution of Protein-Protein-Interaktionen zwischen WW-Domänen und Prolin- proton detected spectra of a deuterated protein at 40 and 60 kHz reichen Segmenten“, with Christian Freund, 01.08 – 12.15, 676.800 € magic-angle-spinning. J Biomol NMR 61(2), 161 – 71. Deutsche Forschungsgemeinschaft, SFB 740 / B07-2, „Untersuchungen Bruun S, Stoeppler D, Keidel A, Kuhlmann U, Luck M, Diehl A, Geiger an Komplexen kleiner Hitzeschockproteine mit Substraten mittels MA, Woodmansee D, Trauner D, Hegemann P, Oschkinat H, Festkörper-NMR-Spektroskopie und dynamischer Kernpolarisation“, Hilde­brandt P, Stehfest K (2015) Light-Dark Adaptation of 01.15 – 12.18, 515.520 € Channelrhodopsin Involves Photoconversion Between the all-trans and Deutsche Forschungsgemeinschaft, SFB 1078 / B01, „Strukturelle 13-cis Retinal Isomers. Biochem 54, 5389 – 5400. Dynamik von Kanalrhodopsinen“, 01.13 – 12.16, 310.587 €; 01.17 – 12.20, Nagaraj M, Franks TW, Saeidpour S, Schubeis T, Oschkinat H, Ritter 699.121 € C, van Rossum BJ (2016) Surface binding of TOTAPOL assists structural Deutsche Forschungsgemeinschaft, “DIP – Dynamic Nuclear Polarization: investigations of amyloid fibrils by dynamic nuclear polarization NMR Integrating fundamentals and new applications“, OS 106 / 12-1, spectroscopy. ChemBioChem 17(14), 1308 – 11. 01.2011 – 12.2015, 331.000 € Lange S, Franks WT, Rajagopalan N, Döring K, Geiger MA, Linden A, Deutsche Forschungsgemeinschaft, “Bacterial adhesin CsgA: A structural van Rossum BJ, Kramer G, Bukau B, Oschkinat H (2016) Structure basis of nucleator-mediated amyloid formation and host protein analysis of a signal peptide inside the ribosome tunnel by DNP MAS interactions studied by solid-state NMR”, RO 3496 / 3-104.13-03.16, NMR. Sci Adv 2(8), e1600379. 149.760 € Geiger MA, Orwick-Rydmark M, Märker K, Franks WT, Akhmetzyanov Alexander von Humboldt-Stiftung, Forschungskostenzuschuss als D, Stöppler D, Zinke M, Specker E, Nazaré M, Diehl A, van Rossum BJ, Gastgeber von Dr. Andrew Nieuwkoop, 03.13 – 02.15, 38.600 €; Aussenac F, Prisner T, Akbey Ü, Oschkinat H (2016) Temperature 01.16 – 03.16, 56.964 € dependence of cross-effect dynamic nuclear polarization in rotating solid: advantages of elevated temperatures. PCCP, 18(44), 30696 – 30704. iNEXT, Infrastructure for NMR, EM and X-rays for Translational Research, GA 653706, 09.15 – 08.19, 438.609 €; 05.12 – 08.19, 283.236 € (Access Costs) FMP authors UCB PHARMA S.A, Protofibril-drug interaction investigated by Group members solid-state NMR / Research Services Agreement, 08.15 – 12.17, 86.989 € 68 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

SOLUTION NMR

LÖSUNGS-NMR

GROUP LEADER DR. PETER SCHMIEDER

BIOGRAPHY SUMMARY

1982 – 1987 Studied Chemistry, The group focuses on applying solution state NMR-spectroscopic techniques to the investigation Johann Wolfgang Goethe University, of the structure and dynamics of biomolecules at atomic resolution. The full repertoire of Frankfurt / Main multidimensional NMR techniques is used in combination with appropriate labeling schemes and other biophysical techniques. 1988 Diploma thesis (Prof. Kessler), Johann Wolfgang Goethe University, Recently we have been interested in a quantitative determination of the role of dynamics in Frankfurt / Main biomolecules. We have been exploring the role of mobility in the presentation of peptides by major 1989 – 1992 Ph.D.(Prof. Kessler), histocompatibility complex (MHC) class I molecules and their recognition by T-cell receptors, Technical University of Munich 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 1990 Hans-Fischer-Preis der TU München will help in understanding immunological processes, and in applications such as vaccine design. 1992 – 1995 Post-Doc (Prof. Wagner), Harvard Medical School, Boston MA 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 since 1995 Group leader small molecules and biologically relevant natural products. One example is a collaboration “Solution NMR spectroscopy”, FMP 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. 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, β2m 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 β2m, are highlighted. The region of β2m 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 micropolymorphism- NMR-spectroscopic analysis of ProM constitution and dependent dynamics of human major histocompatibility antigens EVH1-interaction MHC class I molecules accommodate small peptide fragments of Protein-protein interactions are of major importance in many process- 8 to 12 amino acids within a binding groove and present them to es within cells and the ability to interfere with these interactions in a T-cell receptors on cytotoxic T-cells. T-cells constitute a necessary controlled manner would consequently be of pharmaceutical interest. component of normal adaptive immune responses, but can also be The group of Ronald Kühne has succeeded in developing small involved in autoimmunity. Over the years it has become clear that molecule inhibitors of the interaction between poly-Pro sequences and the static picture resulting from crystallographic studies cannot fully EVH1 domains using a rational approach based on modeling and X-ray explain the interaction between MHCs and T-cell receptors. crystallography. The compounds were synthesized by the group of Prof. Schmalz in Köln. The result was a library of proline mimetics (ProMs) NMR spectroscopy is able to provide information on dynamic features with promising activity and the ability to interfere with the binding and we employ heteronuclear techniques to investigate the dynamics of the FPPPP motif to the EVH1 domain. An example for one of the of MHC complexes of two HLA-B27 subtypes that differ only by compounds (Ac-Cl-Phe-ProM2-ProM1-NH2) is shown in Figure 2a. a single amino acid, but which are differentially associated with an autoimmune disease. Figure 1a shows the structure of one of the MHC NMR spectroscopy was important within this project in two respects: molecules with the amino acid (116) highlighted. We have determined the Several ProM-based molecules were tested for binding to different differences between the dynamics of the two subtypes when in complex with EVH1 domains using a 1H,15N-HSQC-based assay. As a prerequisite different peptides. for this assay the proteins had to be assigned using standard triple resonance experiments. An example for a titration of an-EVH1 domain

While we focused initially on β2m, recently we have performed a full with a ProM-based molecule is shown in Figure 2b. We were able to “Lipari-Szabo” analysis of the relaxation times of the heavy chain. not only measure the strength of binding but could also determine the Four different complexes with the two types of heavy chain (B27*05 binding site based on the available NMR assignments. and B27*09), and two different peptides (pVIPR and TIS), have been investigated. While the α3 domain exhibits no large differences, variations In addition, since the ProM-based molecules are the result of an can be found in the α1 / α2 domain in a subtype- and peptide-dependent elaborate synthesis, proof of their constitution after the complete manner. Dynamics occur on different timescales, as can be seen from peak assembly of the molecules was necessary. Moreover, purification usually doubling and disappearance (msec timescale) and the order factors of a yielded side products that needed to be identified. This was done using Lipari-Szabo analysis (nsec timescale). Figure 1b-e shows the results of NMR spectroscopy with the molecules dissolved in methanol, where the latter, where the magnitude of the order parameters is indicated by the molecule exhibits two sets of signals that are most likely based on a different colors. One can see that not only are the loop regions of the cis / trans-isomerization of the Phe-ProM2-peptide bond. The two sets proteins (which might be expected to be rather mobile) dynamic, but so of signals are quite similar, rendering the analysis more difficult. A full too are the two helices that form the binding groove for the peptide, assignment was nevertheless possible and the correct constitution of the exhibiting a differential mobility not only between different complexes, molecules was confirmed. Smaller amounts of side products could also but also within the same complex. be characterized. Figure 2c shows a part of the 1H,13C correlation of the ProM-based molecule shown in Figure 2a. 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-NH2. (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) 1H,13C 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 COLLABORATIONS

Dr. Peter Schmieder (group leader) National FMP-intern Martin Ballaschk (doctoral student) A. Ziegler und B. Uchanska-Ziegler Anne Diehl Monika Beerbaum (technical assistant) Charité Universitätsmedizin Berlin Ronald Kühne Brigitte Schlegel (technical assistant) T. Niedermeyer Michael Krauß Nils Trieloff (technical assistant) U Tübingen Volker Haucke T. Müller Dorothea Fiedler Staff employed within the reporting period U Würzburg Christian Hackenberger C. Rademacher Marc Nazare MPI für Kolloid und Grenzflächenforschung A. Weng FU Berlin

SELECTED PUBLICATIONS

Boschert V, Frisch C, Back J W, van Pee K, Weidauer S E, Muth E M, Preisitsch M, Heiden S E, Beerbaum M, Niedermeyer T H, Schneefeld M, Schmieder P, Beerbaum M, Knappik A, Timmerman P, Mueller T D (2016) Herrmann J, Kumpfmuller J, Thurmer A, Neidhardt I, Wiesner C, Daniel R, The sclerostin-neutralizing antibody AbD09097 recognizes an epitope Muller R, Bange F C, Schmieder P, Schweder T, & Mundt S (2016) Effects adjacent to sclerostin's binding site for the Wnt co-receptor LRP6. Open of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. Biol, 6(8). CAVN2. Marine Drugs 14(1), 21.

D'Andrea E D, Diehl A, Schmieder P, Oschkinat H, Pires J R (2016) Chemical shift assignments and secondary structure prediction for EXTERNAL FUNDING Q4DY78, a conserved kinetoplastid-specific protein from Trypanosoma cruzi. Biomol NMR Assign 10(2), 325 – 328. Deutsche Forschungsgemeinschaft “NMR spectroscopic investigation of micropolymorphism-dependent dynamics of human Hanske J, Aleksic S, Ballaschk M, Jurk M, Shanina E, Beerbaum M, major histocompatibility antigens“, SCHM 880 / 9-1, Peter Schmieder, Schmieder P, Keller B G, Rademacher C (2016) Intradomain Allosteric 01.01.2011 – 31.03.2015, 142.000 € (davon 1xE13 67 %) Network Modulates Calcium Affinity of the C-Type Lectin Receptor Langerin. J Am Chem Soc 138(37), 12176 – 12186.

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 FMP authors toolkit to inhibit proline-rich motif-mediated protein-protein interactions. Group members Proc Natl Acad Sci U S A 112(16), 5011 – 5016. STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 71

COMPUTATIONAL CHEMISTRY / DRUG DESIGN

WIRKSTOFF-DESIGN

GROUP LEADER DR. RONALD KÜHNE

BIOGRAPHY SUMMARY

1969 – 1973 Studied Biochemistry, The development of compounds that bind to biologically important target proteins is the Martin-Luther-Universität Halle Wittenberg major task of the Drug Design group. It requires strong interdisciplinary collaborations, integrating in silico ligand design and bioinformatics, as well as experimentally driven 1973 Diploma in Biochemistry disciplines like structure biology, biophysics, molecular biology, and cell biology. In recent 1973 – 1976 Research associate, years we have focused on targeting protein-protein interactions mediated by protein domains Zentralins­titut für Molekularbiologie und specifically recognizing proline-rich motifs. These domains are involved in many disease- Medizin der Akademie der Wissenschaften relevant signal transduction cascades and in cytoskeleton remodelling.

1976 – 1992 Research associate, ZUSAMMENFASSUNG Institut für Wirkstofforschung der Akademie der Wissenschaften Die Entwicklung von Substanzen, die an biologisch wichtige Zielproteine binden, ist das zentrale Forschungsziel der Arbeitsgruppe Wirkstoffdesign. Forschungen auf diesem Gebiet 1980 Doctorate degree sind vor allem durch starke Vernetzung unterschiedlicher wissenschaftlicher Disziplinen 1993 Group leader, wie computergestützter Chemie und Bioinformatik, Biophysik, Struktur- und Zellbiologie Leibniz-Forschungsinstitut für Molekulare gekennzeichnet. Zentrales Forschungsthema der Gruppe ist die Entwicklung neuartiger Pharmakologie 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. 72 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

DESCRIPTION OF PROJECTS

Molecular dynamics simulations of K2P channels homology 1 (EVH1) domains. We developed a modular strategy to Two-pore domain (K2P) K+ channels are major regulators of obtain an extendable toolkit of chemical fragments (ProMs) designed excitability that endow cells with an outwardly rectifying background to replace pairs of conserved prolines in recognition motifs. These ‘leak’ conductance. In some K2P channels, strong voltage-dependent rationally designed chemical fragments mimic dipeptide motifs activation has been observed despite the channels not containing (e. g. PP, xP, Px), adopting backbone angles typical of a left-handed a canonical voltage-gating domain. In our study, we showed that polyproline II helix (PPII). At this point more than 15 new chemical voltage-dependent gating is common to most of the K2P channels. entities have been synthesized using an innovative modular synthesis Experimental mutagenesis, as well as molecular dynamics simulation concept developed at the Universität zu Köln (Inst. Organische of permeation, revealed that the voltage-dependent gate is located Synthese, Prof. H.G. Schmalz). As a proof of concept we were able within the selectivity filter. Based on the experimental and to develop, for the first time, low molecular weight inhibitors of theoretically calculated gating charge that is coupled to pore opening, Ena / VASP EVH1 domains. 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 The optimization of EVH1 inhibitors was supported by detailed (see Figure 1). Overall, this ion-flux gating mechanism generates a insights into their binding modes (Fig. 2). Based on X-ray structures one-way ‘check valve’ within the filter because outward movement of (resolutions in the range of 1.0 Å to 2.5 Å) of different ligand-EnaH- / K+ induces filter opening, whereas in inward permeation it promotes EVL-EVH1 complexes we identified an additional binding epitope inactivation. on the EVH1 surface. The combination of experimental structural biology data and computer simulation yielded further optimization of Development of inhibitors to block proline-rich mediated low molecular weight EVH1 inhibitors. protein-protein interactions Small-molecule competitors of protein-protein interactions are Functional studies using the new EVH1 inhibitors urgently needed for functional analysis of large-scale genomics and The Ena / VASP protein family is involved in modulating the actin proteomics data. Particularly abundant, yet so far undruggable, cytoskeleton, a complex and highly regulated process that is the targets include domains specialised in recognizing proline-rich driving force of directed cell migration and which plays important segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila roles in disease-relevant processes like tumor metastasis. We found enabled (Ena) / vasodilator-stimulated phosphoprotein (VASP) 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. STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 73

of Ena / VASP-mediated protein-protein interactions at the focal in-house library design toolbox utilizes an innovative fragment-based adhesions, the leading edge, and the tips of filopodia. Consequently, concept to select chemically diverse screening compounds with our novel EVH1 inhibitors block cell migration and motility of sufficient solubility, low toxicity, and low chemical reactivity. invasive tumor cells. We have shown that our compounds were able to Our library design toolbox provides a core module to select the inhibit tumor metastasis in a zebrafish metastasis model. EU-OPENSCREEN screening library. Within the Helmholtz- initiative “Wirkstoffforschung” we developed a biannually updated, Cheminformatics, Library design, and Molecular Modelling Web-based database of commercially available compounds (DACS) Cheminformatics, bioinformatics, and molecular modeling are containing more than 80 million compounds. The combination of disciplines important for supporting the rational design of chemical our toolbox and DACS allows fast design of hit- or target-focused probes. In combination with experimental chemical biology, these in libraries, as well as of new screening libraries. Target-based ligand silico tools improve the probability of success for ligand development. optimization is another focus of our work. In particular, we support We have established a wide range of methods covering library design, selected screening projects of the FMP Screening Unit. ligand optimization, and calculation of ADMET parameters. Our

GROUP MEMBERS COLLABORATIONS

Dr. Michael Lisurek FMP National Dr. Matthias Müller Dr. P.Schmieder Prof. H.-G. Schmalz, Dr. Robert Opitz Dr. G. Krause Inst. Organische Chemie, Universität zu Köln Dr. Kiril Piotoukh Dr. B. Wiesner Prof. Chr. Freund, Dr. Bernd Rupp Dr. E. Krause FU Berlin Dr. Han Sun Dr. M. Dathe Prof. R. Hiesinger, Matthias Barone (PhD student) FU Berlin Hafiza Nayab (PhD student) International Prof. U. Stein, Raed Al-Yamori (Techn. Informatics) Prof. P. van Dijke, MDC Berlin Kathrin Motzny (Techn. Ass.) Univ. Leiden, Netherlands Prof. U. Heinemann, MDC Berlin Staff employed within the reporting period

SELECTED PUBLICATIONS EXTERNAL FUNDING

Schewe M, Nematian-Ardestani E, Sun H, Musinszki M, Cordeiro S, Innovative Inhibitoren Polyprolin-Motiv-erkennender Protein- Bucci G, de Groot BL, Tucker SJ, Rapedius M, Baukrowitz (2016 ) A Protein-Interaktionsdomänen als Ausgangspunkt zur Validierung Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P neuer pharmakologisch relevanter Zielproteine und zur Entwicklung K(+) Channels. Cell 164, 937 – 949. neuartiger Ansätze in der Krebstherapie, BMBF 03V0475, 07 / 2013 – 12 / 2016, Förderumfang: 606.691 € 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, 5011 – 6.

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, 8464 – 70.

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, 90 – 101.

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 Myco- FMP authors bacterium tuberculosis Cytochrome P450 CYP126A1. J Biol Chem. 292, Group members 1310 – 1329. 74 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

STRUCTURAL BIOINFORMATICS AND PROTEIN DESIGN

STRUKTUR-ORIENTIERTE BIOIN- FORMATIK UND PROTEINDESIGN

GROUP LEADER DR. GERD KRAUSE

BIOGRAPHY SUMMARY

1970 – 1975 Studied chemistry, The group focuses on analyzing the relationship between the sequences and structures of University of Leipzig membrane proteins using structural bioinformatics, combined with experimental studies of the functions of altered sequences. Our aim is to reveal the structure-function relationships 1982 Ph.D. in biochemistry, of proteins and their potential interaction partners. A major activity of ours is developing Martin Luther University, Halle bioinformatic tools for investigating such structure-function relationships; another is to 1981 – 1986 Researcher at the Institute apply these to particular molecular biological projects of protein-ligand, protein-substrate, of Drug Design, Berlin on behalf of or protein-protein interactions. To verify our structure-function hypotheses we use our pharmaceutical industry models to guide site-directed mutagenesis of specific residues and analyze available mutation data. Bioinformatic tools / database development and molecular biology applications 1986 – 1991 Research Position, mutually support one another. Institute of Drug Design, Berlin

1991 – 1992 Visiting Scientist, The main aims of the group are to achieve: (1) A detailed understanding of the intramolecular Washington University (Prof. Marshall), mechanisms of membrane proteins; (2) The rational discovery of molecular mechanisms and St. Louis, MO, USA 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 1992 – 1997 Project leader at research pharmacological interventions. institute of molecular pharmacology, FMP, Berlin since 1998 Group Leader of Structural ZUSAMMENFASSUNG Bioinformatics and Protein Design, FMP, Die Arbeitsgruppe beschäftigt sich mit der Analyse der Beziehung zwischen Sequenz Berlin 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 Entwick- lung 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. 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 Structural bioinformatics webserver as GPHR research resource Clostridium perfringens enterotoxin (Piontek, Protze) and GPCR modeling platform (Kreuchwig, Sargent) Claudins (Cld) – the main constituents of tight junctions (TJ) – Of central importance for several GPHR-related projects studying regulate paracellular permeability across epithelial and endothelial structural-functional properties of the wild-type receptor is the fact monolayers and maintain their tightness. In our project we utilize the that amino acid side-chain substitutions often modify phenotypes. ability of Clostridium perfringens enterotoxin (CPE) to bind to certain Therefore, our web-based research resource (www.ssfa-GPHR. members of the Cld family. We have shown that modifications of the de) compiles a huge amount of functional data gathered from both non-cytotoxic C-terminal domain of CPE (cCPE) allow a targeted naturally occurring and designed mutations (> 1500) of the GPHRs, along interaction with distinct Cld-subtypes for potential pharmacological with structural information for detailed structure-function analyses. alterations of TJ. We have also shown that cCPE-WT modulates the epidermal barrier in vivo via interaction with an ortholog Despite recent advances in crystallizing GPCRs, it is still difficult of Cld4 in Zebrafish. (Zanget al., Exp. Dermatol 2015). Using to obtain their crystal structures. For this reason, a web server for structure-guided mutagenesis we created cCPE-mutants that bind to fragment-based homology modeling of class A GPCRs has been Clds differently than cCPE-WT; e. g. cCPE-Y306W / S313H (Fig.1) developed (http://www.ssfa-7tmr.de/ssfe2/). This server uses a binds to Cld5 (Protze et al., CMLS 2015), which is strongly expressed finger­print correlation scoring strategy to select the best template per in the blood-brain-barrier (BBB). cCPE-Y306W / S313H opens transmembrane helix based on the presence or absence of fingerprint the BBB transiently and reversibly in vivo and in vitro (Liao et al., features. Currently, over 1000 models of human, mouse, and rat Neuroscience 2016). Several Cld structures were solved recently and GPCRs have been pre-calculated and the results made available to users. confirmed our predicted structural determinants that are important for CPE-Cld interactions (Krause et al., Sem Cell Dev. Biol. 2015). Structural determinants of transmembrane transporter MCT8 and LAT2 for thyroid hormone import and export (Kinne, Hinz, Modulation of G-protein-coupled receptors (GPCRs) Protze) (Marcinkowski, Hoyer, Kreuchwig) Thyroid hormones (TH) are transported via specific transmembrane Glycoprotein hormone receptors (GPHRs) are a type of GPCRs that (TM) transporters into and out of the cell. We utilize homology play an important role in pharmacology. Both the lutropin (LH) / models for two TH-transporters, the monocarboxylate transporter choriogonadotropin (CG) receptor (LHCGR) and the thyroid- 8 (MCT8) and the L-type amino acid transporter 2 (LAT2), and stimulating hormone receptor (TSHR) are GPHRs. LHCGR is combined them with directed mutagenesis to delineate molecular activated by two closely-related gonadotrophic glycoprotein hormones. mechanisms of TH transport. Both transporters show a secondary The binding event of both hormones to the receptor's extracellular structure with 12 TM helices but differ in their overall fold and leucine-rich-repeat domain (LRRD) is similar, however a detailed substrate specificity. Structural and molecular determinants common study combining homology modeling with successive experimental for import and export of T3 and T4 have been elucidated in MCT8 characterization (in cooperation with B. Wiesner and R. Schülein) (Fig. 2) (Protze et al., CMLS 2017). We have shown (in cooperation revealed that the functional differences in signal activation between with R. Schülein) that LAT2 imports amino acids and specific THs, hLH and hCG are mediated by structural changes in the extracellular e. g. 3,3´-T 2 and T3, but not rT3 and T4 (Kinne et al., Eur. Thyroid hinge region (Grzesik et al., Front Endocrin. 2015). Pathogenic J. 2015), and provided initial structural insights into TH transport activation of human TSHR by autoantibodies results in dysregulation mechanisms of LAT2 (Hinz et al., Mol. Endocrinol. 2015). Based on of the thyroid hormone status. Allosteric small molecule antagonists the LAT2 models, we selected several mutants for structure-function identified by HTS in the FMP screening unit (in cooperation analysis in Xenopus leavis oocytes. Just one pocket-widening mutation with J von Kries) that were synthetically modified (by E. Specker) enabled T4, and increased T3, import, but did not alter their export. are able to block pathogenic TSHR activation (Krause G, Hoyer In addition, we have shown that LAT2 allows only the unidirectional

I, Marcinkowski P, Specker E, Kries J von et al. 2016, EU Patent, import of T2 into the cells. In fact LAT2 exports amino acids, but EP16167304). Structure-function studies of other GPCRs revealed not any TH, out of the cell (Hinz et al., Cell Mol. Endocrinol. 2017). the ligand-binding site of the peptidic urotensin-II receptor agonist (Bandholtz et al. J Med Chem 2016). Momentan ist kein Platz für Fig. 2! 76 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

Fig. 2: The monocarboxylate trans- porter 8 (MCT8) mediates thyroid hormone (TH) transport across the plasma membrane of the cells. Our MCT8 molecular models of extra- cellular 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 COLLABORATIONS

Dr. Inna Hoyer International J. Gromoll Dr. Anita Kinne T. Visser Universität Münster Dr. Annika Kreuchwig Uni Rotterdam, The Netherlands R. Schülein, B. Wiesner, P. Schmieder, Dr. Anna Piontek (Veshnyakova) Furkert J, Rutz C, A. Diehl, H. Oschkinat, National Dr. Jonas Protze J.vKries, T. Jentsch S. Kaufmann Dr. Catherine Sargent FMP MPI für Infektionsbiologie, Berlin Miriam Eichner (doctoral student) J. Piontek, M. Fromm, H. Biebermann, Katrin M. Hinz (doctoral student) G. Kleinau, J. Köhrle Patrick Marcinkowski (doctoral student) Charité-Universitätsmedizin Berlin Dominik Neef (student) U. Schweizer Carolin Scheffner (student) Wilhelm Friedrich Universität, Bonn E. Klussmann Staff employed within the reporting period MDC, Berlin

SELECTED PUBLICATIONS EXTERNAL FUNDING

Hinz K M, Neef D, Rutz C, Furkert J, Koehrle J, Schuelein R, Krause G Deutsche Forschungsgemeinschaft (DFG), SPP 1629, – Thyroid Trans (2017) Molecular features of the L-type amino acid transporter 2 determine Act- Role of L-type amino acid transporter Lat2 in transport of thyroid different import and export profiles for thyroid hormones and amino acids hormones KR1273 / 5-1, KI1751 / 1-1, 11 / 2012 – 10 / 2015 445.330 € Mol Cell Endocrinology Mar 5, 443, 163 – 174. Mundi Pharma Research GmbH: Proteininteraktionen 9 / 2014 – 10 / 2015 Hinz K M, Meyer K, Kinne A, Schülein R, Köhrle J, Krause G (2015) 90.000 € Structural insights into thyroid hormone transport mechanisms of Lat2 DFG, SPP1629, -Molekulare Determinanten unterschiedlicher Mechanismen Mol Endocrinology Jun 29(6), 933 – 42. des Schilddrüsenhormon-Imports / -Exports der L-type Aminosäuretrans- Protze J, Eichner M, Piontek A, Dinter S, Rossa J, Blecharz K G, Vajcoczy porter KR1273 / 5-2; 6 / 2016 – 9 / 2019, 193.850 € P, Piontek J, Krause G (2015), Directed structural modifications of DFG, Modulatoren für den Thyrotropin Rezeptor: Molekulare Mechanismen Clostridium perfringens enterotoxin to enhance binding to claudin-5, Cell allosterischer Bindung und Wirkungsweise kleiner Moleküle, KR1273 / 4-2,1 Mol Life Sci. Apr 72:7,1417 – 1432. 0 / 2015 – 9 / 2018 310,650 € Grzesik P, Kreuchwig A, Rutz C, Furkert J, Wiesner B, Schülein R, DFG: Molekulare Architektur Claudin-basierter Tight Junction Stränge Kleinau G, Gromoll J, Krause G (2015), Differences in signal activation und parazelluläre Ionenkanäle, KR1273 / 8-1, 4 / 2016 – 3 / 2019 157.000 € mediated by L H and hCG are triggered by the LH / CG receptor`s extracellular hinge region, Frontiers in Endocrinology Sep 22, 6, 140. Wilhelm Sander Stiftung: 2015.112.1, Targeting Claudin überexprimierender Moura-Alves P, Fae K, Houthuys E, Dorhoi A, Kreuchwig A, Furkert J, Lungen und korektal Karzinome mittels modifiziertem Clostridium Perfringens Barison N, Diehl A, Munder A, Constant P, Guhlich-Bornhof U, Klemm Enterotoxin, 2 / 2016 – 1 / 2018 110.500 € 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, DFG, Searching for transport proteins for TRIAC or DITPA acting as Krause G, Kaufmann SHE (2014) AhR sensing of bacterial pigments T3 /TH substitutes, KR1273/9-1; PR1616/2 – 1(eigene Stelle J. Protze) regulates anti-bacterial defences, Nature Aug 28, 512(7515), 387 – 92. 4/2017 – 4/2020, 352.825 €

FMP authors Group members STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 77

IN-CELL NMR

NMR IN ZELLEN

GROUP LEADER DR. PHILIPP SELENKO

BIOGRAPHY SUMMARY

2002 Ph.D., European Molecular Biology We employ high-resolution in-cell NMR spectroscopy to study the structural and functional Laboratory (EMBL), Heidelberg 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. 2003 – 2008 Post-Doc, By doing so, we obtain novel mechanistic insights into previously unknown cellular aspects Harvard Medical School of protein functions in health and disease, thereby opening new routes for drug discovery 2003 – 2004 EMBO fellowship and therapeutic intervention.

2004 – 2006 Human Frontiers in ZUSAMMENFASSUNG Science (HFSP) fellowship Wir untersuchen strukturelle und funktionelle Eigenschaften von Proteinen in lebenden 2006 – 2007 Max Kade fellowship Zellen mittels hochauflösender NMR-Spektroskopie an lebenden Zellen. Diese Methode by the Austrian Academy of Science erlaubt eine unmittelbare „Visualisierung“ von Proteinen bei der Arbeit in ihren natürlichen Since 2007 Group Leader in the zellulären Umgebungen. Dadurch gewinnen wir neuartige mechanistische Einsichten in Structural Biology Section, FMP, Emmy bisher unbekannte zelluläre Aspekte von Proteinfunktionen. Unsere Untersuchungen an Noether fellowship by the Deutsche gesunden und an kranken Zellen erlauben so die Entwicklung neuer Ansätze für das Forschungsgemeinschaft (DFG) Aufspüren potentieller neuer Pharmaka und neue pharmakologische Therapien.

2015 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. 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 COLLABORATIONS

Dr. Andres Binolfi International National Dr. Cedric Eichmann Daniella Goldfarb Wolfgang Fischle Dr. Reeba Jacob Weizmann Institute of Science, Rehovot, Max Planck Institute of Biophysical Dr. Antonio Limatola Israel Chemistry, Göttingen Dr. Francois-Xavier Theillet Richard Treisman Jonas Kosten (PhD Student) The Crick Research Institute, London, UK Marchel Stuiver (technical assistant) Marleen van Rossum (technical assistant)

Staff employed within the reporting period

SELECTED PUBLICATIONS EXTERNAL FUNDING

Thongwichian R, Kosten J, Benary U, Rose H M, Stuiver M, Theillet F X, 2015 – 2020 ERC Consolidator Grant, NeuroInCellNMR 2.000.000 € Dose A, Koch B, Yokoyama H, Schwarzer D, Wolf J, Selenko P (2015) A multiplexed NMR-reporter approach to measure cellular kinase and phos- phatase activities in real-time. J Am Chem Soc. 137(20), 6468 – 71.

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), 12402 – 7.

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), 247 – 59.

Müntener T, Häussinger D, Selenko P, Theillet F X (2016) In-cell protein structures from 2D NMR experiments. J Phys Chem Lett. 7(14), 2821 – 5.

Mylona A, Theillet F X, Foster C, Cheng T M, Miralles F, Bates P A, FMP authors Selenko P, Treisman R (2016) Opposing effects of Elk-1 phosphorylation Group members shape its response to ERK activatio. Science 354(6309), 233 – 237. STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 79

MOLECULAR IMAGING

MOLEKULARE BILDGEBUNG

GROUP LEADER DR. LEIF SCHRÖDER

BIOGRAPHY SUMMARY

1995 – 1997 Studies in Physics and Chemistry, Targeted imaging reporters have revolutionized our understanding of many biological Georg-August Universität Göttingen 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 1997 – 2001 Studies in Physics and Astronomy, fails as a non-invasive modality in larger whole organisms. Nuclear Magnetic Resonance Ruprecht-Karls Universität Heidelberg, (NMR) and its imaging modality, MRI, have the potential to address this need, but it Diploma in Physics requires a fundamentally new approach to overcome the intrinsic sensitivity limitations that 2001 – 2003 PhD student, Deutsches accompany conventional NMR / MRI protocols. By optimizing the steps of preparation, Krebsforschungszentrum and Ruprecht-Karls manipulation, and encoding of spin magnetization, our group strives to design ultra-sensitive Universität Heidelberg, Dr. rer. nat. 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 2003 – 2005 Research Assistant, Deutsches of particular molecular interactions. Xenon biosensors have an outstanding potential to Krebsforschungszentrum, Heidelberg improve early disease detection and for the visualization of drug delivery response, which is 2005 – 2007 Emmy Noether Fellow of the critical for individualized therapy. To explore this potential, our group engages in programs DFG, University of California, Berkeley supporting highly innovative research, including the Koselleck Program of the DFG and the European Research Council. We develop methodologies to obtain MRI contrast from 2005 Philips Research Prize for Medical molecular targets at extremely low concentrations and sense molecular interactions within a Physics, awarded by the German Society for few minutes, where conventional MRI protocols would theoretically require thousands of years. Medical Physics (DGMP)

2007 – 2009 Research Fellow, Lawrence ZUSAMMENFASSUNG Berkeley National Laboratory Zielgerichtete Kontrastmittel haben unser Verständnis vieler biologischer Prozesse 2007 Gorter Award of the International revolutioniert. Die Fluoreszenz-Bildgebung ist das beste Beispiel für die Umsetzung Society for Magnetic Resonance in Medicine biochemischen Wissens in Methoden zur Untersuchung von Prozessen in lebenden Zellen, (German Chapter) aber sie hat nichtsdestotrotz eine fundamentale Einschränkung: für größere, komplette Organismen ist sie als nicht-invasive Modalität nicht einsetzbar. Die Kernspinresonanz 2008 Dr. Emil-Salzer-Award for Cancer (NMR) mit ihrer Bildgebungs-Modalität (MRT) hat das Potenzial, diesen Bedarf zu Research of the Federal State of decken. Dabei erfordert diese Herausforderung einen grundsätzlich neuen Ansatz, um Baden-Württemberg, awarded by the die geringe intrinsische Empfindlichkeit, die mit herkömmlichen NMR / MRI-Protokollen German Cancer Research Center einhergeht, zu überwinden. Durch die Optimierung der Schritte zur Vorbereitung, 2009 Emmy Noether Fellow of the DFG, Manipulation und Kodierung der Spin-Magnetisierung zielt unsere Gruppe auf die Group Leader at FMP Entwicklung ultra-sensitiver NMR-Reporter ab. Diese sind essentiell für die Darstellung der räumlichen Verteilung von Krankheitsmarkern in der personalisierten Medizin, 2009 – 2014 ERC Starting Grantee at sowie für die Verfolgung von Wirkstoffträgern und den Nachweis bestimmter molekularer FMP (first grant of its kind in the Leibniz Wechselwirkungen. Xenon-Biosensoren haben ein herausragendes Potenzial zur frühzeitige Association) Diagnose und Visualisierung der Therapieantwort, was entscheidend für individuelle 2009 Young Scientist Prize for Medical Therapien ist. Um dieses Potenzial zu erforschen, engagiert sich unsere Gruppe in Physics of the International Union of Pure Programmen für hochinnovative Forschung wie dem Koselleck-Programm der DFG und and Applied Physics (IUPAP) dem Europäischen Forschungsrat. Wir entwickeln Methoden, um MRT-Kontrast von molekularen Markern in extrem geringer Konzentration zu erzielen und molekulare since 2014 Independent Group Leader, FMP Wechselwirkungen innerhalb weniger Minuten zu detektieren, wo herkömmliche 2015 Reinhart Koselleck Grant Award of MRT-Protokolle andernfalls theoretisch tausende von Jahren erfordern würden. the DFG (first grant of its kind in the Leibniz Association) 80 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

DESCRIPTION OF PROJECTS

Cell Surface Glycan Imaging with Ultra-Sensitive MRI Reporters CEST Image Contrast Modeling We demonstrated the first case of NMR mapping of a diagnostic Xenon biosensors represent an emerging class of contrast agents with target that is inaccessible with existing MRI reporters. Cell surface detection limits down to the nano- and picomolar regimes. However, glycans are an excellent example of this. Their modification through build-up of the measured Hyper-CEST effect is complex and relies metabolic oligosaccharide engineering has not been able to be on the specific Xe-host system properties and the applied saturation combined with MRI in previous studies. We designed a multimodal pulse strength and duration. In this project, we introduced optimal biosensor for both fluorescent and xenon MRI detection that is targeted saturation pulse parameters for a maximum, but still spectrally to metabolically labeled sialic acid through bioorthogonal chemistry narrow, Hyper-CEST effect. The parallel development of sensors (see Fig. 1). While conventional relaxivity agents require micromolar and their respective detection technique will accelerate the target concentrations, this xenon biosensor could be detected at transition to the firstin vivo studies, where knowledge about efficient nanomolar concentrations. This first demonstration of live cell signal build-up is critical. glycan-targeted MRI presents an excellent test bed for moving forward with xenon biosensors as these reporters continue to progress MRI Visualization of Enzymatic Activity in vivo to investigate unsolved diagnostic questions, including glycome Based on the promising potential of cucurbit[n]urils (CB) for analysis. reversible binding of Xe and their established use in switchable fluorescence detection, we devised an enzyme activity reporter Optimized Xenon Host-Guest Complexes for MRI purposes. Our design relies on hyperpolarized Xe NMR The expanding use of the Hyper-CEST technique (chemical exchange spectroscopy, in which we 1) use CB hosts as contrast agents, 2) saturation transfer with hyperpolarized nuclei) for MR applications rationally exploit the molecular recognition properties of these requires quantitative characterization of the exchange dynamics and hosts, and 3) for the first time apply an optimized magnetization optimizing the physico-chemical behaviour of xenon hosts. We could transfer (MT) experiment for the Xe MRI of enzyme activity. achieve the first quantitative comparison of various hosts, including This extends the use of biosensors by introducing a signal switch cryptophane cages, cucurbit[n]urils, and genetically engineered capability in which Xe is gradually displaced from its host as an bacterial gas vesicles. By introducing the gas turnover rate as a novel enzymatic reaction progresses (see Fig. 2). Critically, this approach quantitative parameter, our work has demonstrated, for instance, that circumvents the shortcomings in previous attempts by others where the fast exchanging host cucurbit[6]uril is approximately 100-fold more the enzyme activity was detected as a minor shift in resonance potent as an MRI reporter than cryptophane-A. frequency of caged Xe.

ular tar olic labe ical sel cell get etab lin hem ecti m g oc vit A bi y xenon host

cells + glycan

Ac4ManNAz N3 scaffold

azide N3 label chemoselective modified sugar N in situ coupling N N N cells + 3 3 N N Man 3

g l N yc 3 ob ios ynt hesis

ced magn led depolar I detectio du eti rol iza MR n B -in za nt tio er tio o n s n c RF on la Rb polarized cells + Ac ManNAz Rb photons 4 Rb Xe Xe + sensor 50 Xe

Xe 40 Hyper-CEST

Rb effect [%] Xe signal 30 Xe contrast magnetization Xe 20 build-up RF off 10 × 10’000 Xe Xe Xe Xe 0 cells + sensor

tra le sp g nsfer to samp atial encodin

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. 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 cucurbit[7]uril (CB7) and displaces Xe from this molecular container. (B) LDC The absence of enzymatic activity maintains free exchange access for Xe into CB7 and yields an unchanged, high magnetization transfer (MT) signal. The enzymatic conversion Xe displacement 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 change in switchable signal contrast. B Xe Xe@CB7 Xenon Magnetization Cad@CB7 Transfer MR Imaging Xe Lys

− enzyme 2 mm + enzyme

GROUP MEMBERS COLLABORATIONS

Dr. Jabadurai Jayapaul International National Dr. Jan Oliver Jost Mikhail Shapiro, Caltech, USA Andreas Hennig, Jacobs University Bremen Dr. Martin Kunth Alexander Pines, UC Berkeley, USA Gil Westmeyer, TU München Dr. Honor Rose David Wemmer, UC Berkeley, USA Jörg Piontek, Charité, Berlin Dr. Christopher Witte Matthew Francis, UC Berkeley, USA Lorenz Mitschang, PTB, Berlin Jörg Döpfert (doctoral student) Ville-Veikko Telkki, University of Oulu, Finland Salim Seyfried, MDC, Berlin Stefan Klippel (doctoral student) Yoram Cohen, Tel Aviv University, Israel Hartmut Kühn, Charité, Berlin Ursula Pfeiffer (doctoral student) Xin Zhou, WIPM, China Daniel Messroghli, Deutsches Herzzentrum, Federica Rossella (doctoral student) Paul Beer, University of Oxford, UK Berlin Matthias Schnurr (doctoral student) Jörg Matysik, Universität Leipzig Nils Bogdanoff (HiWi)

Staff employed within the reporting period

SELECTED PUBLICATIONS EXTERNAL FUNDING

Korchak S, Kilian W, Schröder L, Mitschang L (2016) Design and International Human Frontiers Science Program Organization, Comparison of Exchange Spectroscopy Approaches to Cryptophane- “Cell Profiling with Xenon Biosensors”, Long-Term Postdoctoral Xenon Host-Guest Kinetics. J. Magn. Reson. 265, 139 – 145. Fellowship for Christopher Witte, 04.2010 – 04.2015; 142.836 €

Schnurr M, Sloniec-Myszk J, Döpfert J, Schröder L, Hennig A (2015) International Human Frontiers Science Program Organization, Supramolecular Assays for Mapping Enzyme Activity by Displacement-­ “Imaging Cellular Function Non-invasively with genetically Engineered Triggered Change in Hyperpolarized 129Xe Magnetization Transfer NMR. Reporters for Hyperpolarized MRI”, Program Grant with Mikhail Angew. Chem. Int. Ed. 54, 13444 – 13447. Shapiro, Chemical Engineering, California Institute of Technology 05.2016 – 04.2019; 750.000 $, FMP share: 375.000 $ Kunth M, Witte C, Hennig A, Schröder L (2015) Identification, Classification, and Signal Amplification Capabilities of High-Turnover Deutsche Forschungsgemeinschaft, “Multivalent Hosts for Hyperpolarized Gas Binding Hosts in Ultra-Sensitive NMR. Chem. Sci. 6, 6069 – 6075 Xenon Enabling in vivo MRI Visualization of Tumor Cell Surface (highlighted as cover article). Glycans”, Reinhard Koselleck-Förderung (first of its kind in the Leibniz Association); GZ SCHR 995 / 5 – 1; 04.2017 – 03.2022; 1.525.000 € Kunth M, Witte C, Schröder L (2015) Continuous-wave Saturation Considerations for Efficient Xenon Depolarization. NMR Biomed. 28, Michael J. Fox Foundation for Parkinson‘s Research, “Developing a 601 – 606. Molecular Imaging Tool That Binds to Alpha-Synuclein and Inhibits Its Formation”, Porgram for Improved Biomarkers & Clinical Outcome Witte C, Martos V, Rose H M, Reinke S, Klippel S, Schröder L, Measures; Grant ID: 12549; 10.2016 – 09.2019; 349.500 $ Hackenberger C P R (2015) Live-cell MRI of Xenon Hyper-CEST Biosensors Targeted to Metabolically-labeled Cell-surface Glycans. Angew. Chem. Deutsche Forschungsgemeinschaft, Internationale Kooperationsan- Int. Ed. 54, 2806 – 2810 (highlighted as VIP and inside back cover article; bahnung mit Prof. X. Zhou, State Key Laboratory of Magnetic Resonance recommended by F1000Prime). and Atomic Molecular Physics, Wuhan Institute of Physics and Mathe- matics, The Chinese Academy of Sciences; “Live Animal MRI of Hyper- polarized Xenon for Hyper-CEST Applications”; GZ SCHR 995 / 6 – 1; 03.2016 – 03.2017; 2.860 €

Deutsche Forschungsgemeinschaft, Research Training Group “BIOQIC – Biophysical Quantitative Imaging Towards Clinical Diagnosis“; administered through Prof. I. Sack, Department of Radiology, Charité – FMP authors Universitätsmedizin Berlin; GRK 2260 / 1; 04.2017 – 09.2021; 4.497.810 €, Group members FMP share: 225.885 € 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 31P-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 STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 83

Fig. 1: 1H- and 13C-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 supra- resolution spectroscopy. The site of attachment of the proton transferred by NaBH4 was clarified using multidimensional NMR spectra. 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 1H,15N HSQC spectra of 15N-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. STRUCTURAL BIOLOGY STRUKTURBIOLOGIE 85

SELECTED PUBLICATIONS

Bertran-Vicente J, Penkert M, Nieto-Garcia O, Jeckelmann J M, Lehmann M, Gottschalk B, Puchkov D, Schmieder P, Schwagerus S, Schmieder P, Krause E, Hackenberger C P (2016) Chemoselective Hackenberger C P, Haucke V, Schmoranzer J (2015) Multicolor Caged synthesis and analysis of naturally occurring phosphorylated cysteine dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain. peptides. Nat Commun 7, 12703. Angew Chem Int Ed Engl 54(45), 13230 – 13235.

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), 6839 – 6843.

Diesenberg K, Beerbaum M, Fink U, Schmieder P, & Krauss M (2015) SEPT9 negatively regulates ubiquitin-dependent downregulation of FMP authors EGFR. Journal of Cell Science 128(2), 397 – 407. Group members 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 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 88 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014

SECTION CHEMICAL BIOLOGY

BEREICH CHEMISCHE BIOLOGIE

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

Robert Puschmann CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 89

The “Peptide-Lipid Interaction” group, led by Margitta Dathe, has RNAi libraries. Scientific highlights of research within the Screening a long-standing expertise in the development of peptide-modified Unit include the identification of approved drugs to rescue heart liposomal drug carriers. In cooperation with the University of Münster development in zebrafish embryos and work led by Thomas Jentsch they developed an enzyme substitution therapy for the rare skin using genome-wide RNA interference that led to the identification of disorder transglutaminase 1-deficient ichthyosis, granted orphan the gene-encoding VRAC that is essential for regulation of cellular designation by the European Commission in 2013. Current efforts are water content in cells. The “Screening Unit” has also served as a focused on the development of a pathogenesis-based therapy for the central anchoring point for projects within the Leibniz research peeling skin disease. network “Drug Research and Biotechnology”, the Helmholtz consortium “Drug Research Initiative”, the Berlin Institute of Health An important aim within the “Chemical Biology Platform” at the FMP (BIH), and the neighboring Max-Delbrück-Center for Molecular is the chemical optimization and validation of first hits discovered in the Medicine. In addition, the “Screening Unit” provides a focal point small molecule screening of pharmacological targets. The “Medicinal of the “Chemical Biology Platform”, as a key partner of the EU- Chemistry” group led by Marc Nazaré is developing new chemical tools OPENSCREEN initiative. using strategies like fragment growing, re-scaffolding approaches, and structure-based design to improve the initial screening hit rate. Overall, the “Chemical Biology” section has contributed important These efforts have led to new small molecule probes for kinase chemical methods and discoveries to research at the FMP, thus PI3KC2α, the phosphatase SHP2, tryptophanhydroxylase TPH1, providing privileged access to pharmaceutically active substances and the Poly-ADP-ribosyltransferase tankyrase, with some of these through the synthesis, identification / screening, and optimization of now being profiled in pharmacological modelsin vivo. Moreover, the new (small) molecules for studies of underexplored protein targets. FMP small molecule screening collection was considerably enhanced These activities support the core mission of the FMP to develop new (it now stands at 66,000 compounds) through academic donations, principles at the molecular level for pharmacological intervention in a newly approved drug sub-set, and its own synthetic libraries. The biological processes that ultimately shall lead to new possibilities in platform’s research activities are deeply interconnected with most the treatment of diseases. 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

Michael Schümann Kristina Siebertz and Oliver Reimann 90 RESEARCH REPORT FORSCHUNGSBERICHT 2013 / 2014

Liudmila Perepelittchenko and María Pascual López-Alberca

Forschungsprojekte aus diesem Bereich dienen der Protein-Wechselwirkungen und PTMs in der Signalweiterleitung Untersuchung der biologischen Funktion zellulärer in der Zelle liegt. Die beiden Labore trugen zur ersten Synthese Zielmoleküle (Targets) mit innovativen synthetischen und und MS-Analyse von ortsspezifisch phosphoryliertem Lysin sowie diagnostischen Techniken der Chemie. Sie sollen den Zugang Cystein-Peptiden bei. zu neuartigen Ansätzen in Pharmazie und Medizin eröffnen. Die Arbeiten der beteiligten Gruppen in diesem Bereich Die Gruppe „Massenspektrometrie“ arbeitet auch eng mit der widmen sich der Synthese und Identifizierung neuartiger Arbeitsgruppe von Dorothea Fiedler zusammen, mit dem Ziel, eine biologisch aktiver Moleküle mit hohem pharmakologischen labile PTM, die Pyrophosphorylierung, nachweisen zu können. Potential sowie der Entwicklung neuer chemischer und Dorothea Fiedler kam im Jahr 2015 zum FMP und leitet dort die analytischer Werkzeuge zur funktionellen Analyse biologisch Abteilung „Chemische Biologie I“. Sie wurde an die Humboldt- relevanter Proteine. Universität zu Berlin berufen und ist eine der Direktoren am FMP. Die Forschung in der Fiedler-Gruppe konzentriert sich auf die Christian Hackenberger, der als Leibniz-Humboldt-Professor Verwendung von chemischen Ansätzen zur Erkennung von berufen ist, fungiert als Sprecher des Bereichs Chemische Biologie. Signal- und Stoffwechselnetzen und deren Deregulierung bei Die Forschung in seiner Abteilung „Chemische Biologie II“ hat Krankheiten. Im Fokus ihres Forschungsprogramms stehen dabei ihren Fokus auf der Synthese funktioneller Peptide und Proteine sogenannte Inositol-Pyrophosphate, eine Gruppe von Botenstoffen, durch Kombination modernster Techniken der organischen Chemie die bei der Regulierung des Körpergewichts, der Lebenserwartung mit biochemischen und biophysikalischen Methoden. Die so zum und der Fruchtbarkeit eine Rolle spielen. Um deren spezifische Einsatz kommenden chemischen Werkzeuge haben in den letzten Signalfunktionen zu entschlüsseln, setzt die Gruppe synthetische zwei Jahren entscheidend dazu beigetragen, neue pharmakologisch Chemie, Wirt-Gast-Chemie und chemische Genetik in aktive Biopolymere zu generieren und die Bedeutung posttrans- Kombination mit Geneditierungstechniken und Proteomik ein. lationaler Modifikationen (PTMs) für die Proteinfunktion zu So ist es der Gruppe z.B gelungen, mit immobilisierten, nicht- verstehen. Beispiele sind hier der Transport funktionaler Proteine hydrolysierbaren Analoga Protein-Bindungspartner der Inositol- in lebende Zellen und die Identifizierung neuer Targets für die Pyrophosphat-Botenstoffe aus Bäckerhefe zu identifizieren. Neben medizinisch-chemische Forschung im Kontext viraler Infektion bona fide Protein-Bindungspartnern führte diese Analyse zur und neurodegenerativer Erkrankungen. Des Weiteren werden Isolierung von Substraten der Protein-Pyrophosphorylierung, neu entwickelte ortspezifische Konjugationsmethoden in der einer ungewöhnlichen durch Inositol-Pyrophosphate vermittelten Herstellung pharmakologisch relevanter Antikörper-Konjugate Modifikation. Um die endogen pyrophosphorylierten Proteine für einen gezielten Wirkstofftransport („Targeted Drug näher zu charakterisieren, wurden in ihrem Labor Reagenzien zur Delivery“) verwendet. Diese Methoden dienen als Grundlage für eine Anreicherung von Peptiden, die Pyrophosphatgruppen enthalten, Firmengründung, die in naher Zukunft vollzogen wird. Ein weiteres entwickelt. Und gemeinsam mit der Arbeitsgruppe von Eberhard neues Highlight wurde in enger Zusammenarbeit mit der Gruppe von Krause soll mithilfe der Massenspektrometrie eine robuste Strategie Eberhard Krause durchgeführt, der die Massenspektrometrie-Gruppe zum Nachweis dieser Modifikation implementiert werden. Durch am FMP leitet und sich auf hochauflösende Proteomstudien Analyse der Signalübertragungseigenschaften der Inositol- konzentriert, wobei der Schwerpunkt auf der Rolle von Protein- Pyrophosphate in gesundem und erkranktem Zustand möchte die CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 91

Edgar Specker, Martin Neuenschwander and Silke Radetzki

Gruppe herausfinden, wie der Inositolpyrophosphat-Stoffwechsel für von Gerd Krause und die „Wirkstoff-Design“-Gruppe von Ronald therapeutische Zwecke genutzt werden kann. Kühne zu erwähnen. Erhebliche Anstrengungen wurden unternommen, um hochprofessionelle Core Facilities, insbesondere Die Arbeitsgruppe „Peptid-Lipid-Interaktion“ von Margitta Dathe die international anerkannte „Screening Unit“ unter Leitung von besitzt seit Langem große Expertise in der Entwicklung von peptid- Jens von Kries, aufzubauen. Die „Screening Unit“ unterstützt modifizierten liposomalen Carriern. Die Arbeitsgruppe entwickelte Hochdurchsatz-Screening von kleinen Molekülen und RNAi- in Kooperation mit der Universität Münster eine Enzymsubsti- Bibliotheken. Wissenschaftlich herausragend ist hierbei die tutionstherapie für die seltene Hautkrankheit „Transglutaminase Identifizierung von zugelassenen Arzneimittel-Wirkstoffen, die 1-deficient Ichthyosis“, der 2013 durch die Europäische Kommission Defekte in der Entwicklung des Herzens von Zebrafischembryos der „Orphan Drug“-Status zuerkannt wurde. Gegenwärtige beheben können und somit für entsprechende humane Krankheiten gemeinsame Aktivitäten richten sich auf die Entwicklung einer eingesetzt werden könnten. Darüber hinaus wurde in der Unit – Therapie zur Behandlung der „Peeling Skin Disease“. durch RNA-Interferenz – in Zusammenarbeit mit Thomas Jentsch das Gen für den Ionenkanal identifiziert, der in jeder Zelle den Ein sehr wichtiges Ziel der „Chemical Biology Platform“ des FMP Wasserhaushalt reguliert. Die „Screening Unit“ dient weiterhin als ist die chemische Optimierung und die Validierung von biologisch Dreh- und Angelpunkt für Forschungskooperationen im Leibniz wirksamen kleinen Molekülen (Hits), die initial durch Screening Forschungsverbund „Wirkstoffforschung und Biotechnologie“, im kleiner Moleküle gegen pharmakologische Targets identifiziert werden. Helmholtz-Konsortium „Wirtstoffforschung“, mit dem Berliner Instituts für Gesundheitsforschung (BIH) und mit dem benach- Die Arbeitsgruppe „Medizinische Chemie“ von Marc Nazaré barten Max-Delbrück-Centrum für Molekulare Medizin (MDC). entwickelt neue chemische Werkzeuge und nutzt dabei zur Gleichzeitig ist die „Screening Unit“ mit der „Chemical Biology Optimierung der Screening-Hits Strategien wie Fragmentwachstum, Platform“ des FMP ein zentraler Partner der EU-OPENSCREEN- Neuanordnung von Molekülgerüsten und strukturgeleitetes Design. Initiative. Die Arbeiten der Gruppe resultierten in neuen kleinen Molekülen, die als Sonden zur Beeinflussung der PI3KC2α Kinase, der Insgesamt hat der Bereich „Chemische Biologie“ wichtige chemische Phosphatase SHP2, der Tryptophanhydroxylase TPH1 oder der Methoden entwickelt und Entdeckungen gemacht, die einen Poly-ADP-Ribosyltransferase Tankyrase wirken. Einige dieser einzigartigen Zugang zu pharmakologisch aktiven Substanzen durch Substanzen werden gegenwärtig in pharmakologischen Tiermodellen Synthese, Identifikation / Screening und die chemische Optimierung in vivo evaluiert. Zudem wurde die Substanzsammlung des FMP für neuer (kleiner) Moleküle für Studien an wenig erforschten zellulären das Small Molecule Screening durch die Integration von akademischen Targets ermöglicht. Durch seine Ergebnisse unterstützt der Bereich Substanz-Spenden, zugelassenen Medikamenten und eigenen den Kernauftrag des FMP in optimaler Weise, neue molekulare synthetischen Verbindungen auf nunmehr 66.000 Substanzen Prinzipien der pharmakologischen Beeinflussung biologischer Vor- erheblich erweitert. Die Forschungsaktivitäten des Bereiches gänge zu erarbeiten, die letztendlich neue Wege in der Behandlung „Chemische Biologie“ sind eng mit vielen Gruppen der anderen von Krankheiten eröffnen werden. Bereiche verzahnt. Besonders sind hier aus dem Bereich Struktur- biologie die „Strukturelle Bioinformatik und Proteindesign“-Gruppe 92 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

CHEMICAL BIOLOGY II

CHEMISCHE BIOLOGIE II

GROUP LEADER PROF. DR. CHRISTIAN P.R. HACKENBERGER

BIOGRAPHY SUMMARY

1996 – 1998 Undergraduate studies and In the densely packed world of a cell, protein modifications control many signaling pathways prediploma in Chemistry, that support healthy functioning and that are disrupted in disease. Common examples Albert-Ludwigs-Universität Freiburg include protein phosphorylation and glycosylation, but increasingly other post-translational modifications (PTMs) such as acetylation, ubiquitylation, and methylation, are being 1998 – 1999 Graduate studies and M.Sc. in identified as important “toggle switches” in health and disease. Chemical biologists want to Chemistry with Prof. Samuel H. Gellman, control these protein modifications in the cell, both to study the biological role of PTMs and University of Wisconsin / Madison, USA to decorate proteins with fluorescent moieties that permit their visualization. 2000 – 2003 Ph.D. research with Prof. Carsten Bolm (summa cum laude), The Hackenberger laboratory aims to identify new chemical reactions that allow the modi- RWTH-Aachen fication 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 2003 – 2005 Postdoctoral scholar (DAAD and reactions to study the functional consequences of natural protein modifications, as well as DFG) work with Prof. Barbara Imperiali, Mas- to generate novel peptide- and protein-conjugates, in particular antibody-drug conjugates sachusetts Institute of Technology, USA (ADCs), for pharmaceutical and medicinal applications (Figure 1). 2004 Research stay with Prof. Sheena E. Radford, University of ZUSAMMENFASSUNG Leeds, UK In der dicht gepackten Welt einer Zelle werden viele Signalwege, die normales Leben steuern 2005 – 2006 Junior group leader as und bei Krankheit gestört sind, durch Veränderungen an Proteinen reguliert. Am häufigsten Liebig-Scholar (FCI), Free University of Berlin sind hierbei Phosphorylierungen und Glycosylierungen zu beobachten; vermehrt werden aber auch andere solcher posttranslationaler Modifikationen wie Acetylierung, Ubiquitylierung 2006 – 2011 Emmy-Noether-Group (DFG) und Methylierung identifiziert, die wie „Wechselschalter“ zwischen Gesundheit und leader, Free University of Berlin Krankheit wirken können. Wissenschaftler / innen in der Chemischen Biologie versuchen 2011 – 2012 Habilitation and Associate deshalb zunehmend die Modifizierung von Proteinen in der Zelle zu kontrollieren, um Professor (W2) for Bioorganic Chemistry, entweder die biologische Rolle solcher posttranslationalen Modifikationen zu erforschen oder Free University of Berlin um Proteine mit fluoreszierenden Gruppen zu versehen, die ihre Visualisierung ermöglichen.

2013 Margaret and Harlan Goering Visiting Das Labor von Christian Hackenberger hat sich zum Ziel gesetzt neue chemische Reaktionen Professor, University of Wisconsin / Madison, zu entwickeln, um Peptide und Proteine sowohl in isolierter Form als auch in lebenden Zellen USA oder Organismen gezielt funktionalisieren zu können. Das Hauptaugenmerk liegt dabei auf Since 2008 Speaker of the graduate college der Entwicklung hochselektiver organisch-chemischer Methoden für die Biokonjugation, “Multivalency in Chemistry and Biochemistry” welche die Auswirkungen natürlich vorkommender Modifikationen auf die Funktion von within the SFB 765; member of the SFB 765 Proteinen untersuchen und neue medizinische und pharmakologische Anwendungen, beispielsweise durch Antikörper-Wirkstoff-Konjugate (antibody-drug-conjugates, ADCs), Since 2011 Speaker DFG priority program ermöglichen (Figure 1). SPP 1623 “Chemoselective Reactions for the synthesis and application of functional proteins”

Since 2012 Leibniz-Humboldt Professor (W3) for Chemical Biology funded by the Einstein Foundation Berlin CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 93

DESCRIPTION OF PROJECTS

Bioorthogonal Staudinger Reactions: study to glycosylated Tau to evaluate whether a perturbed balance chemical phosphorylation, PEGylation, and more between phosphorylation and glycosylation (O-GlcNAc) is Over the years, we have introduced the Staudinger-phosphite associated with Tau aggregation. Other studies (with Caroline reaction as a bioorthogonal reaction for the modification of Smet-Nocca, CNRS Lille), in which synthetic phosphorylated azide-containing biomolecules. In a first biological application peptides were subjected to enzymatic transformations, have of this reaction we engineered a chemoselective phosphorylation already identified two new O-GlcNAc glycosylation sites in Tau of proteins, which allowed the site-specific incorporation of a and, furthermore, demonstrated the reciprocal relationship phospho-Tyr mimetic into full-length proteins. Recently, we between phosphorylation and O-GlcNAcylation. extended this concept to the site-specific phosphorylation of Lys-peptides, which represents a very labile PTM and which we Chemical approaches to modulate sialylation in vivo were able to analyse using electron transfer dissociation tandem In collaboration with Prof. Stephan Hinderlich (Beuth- mass spectrometry (ETD / MS / MS) in collaboration with Eberhard Hochschule) we recently expanded the repertoire of unnaturally Krause (FMP Berlin). Furthermore, the use of nucleophilic modified sialylated glycoproteins by metabolic oligosaccharide phosphites also allowed us to obtain site-specifically engineering. For this purpose C4-substituted ManNAc-derivatives phosphorylated Cys-peptides, which were again analysed by were synthesized and incorporated into novel C7-modified sialic ETD / MS / MS (Figure 2). With this protocol in hand we detected acids in glycoproteins in cells and in living zebrafish. These novel the phosphorylation of a Cys-residue in the glycolysis pathway. biopolymers are of interest for the development of new diagnostic and therapeutic glycoproteins and potentially can be applied to In subsequent studies, we used an unsymmetrical version of the in vivo modification of living animal models. Furthermore, in the Staudinger-phosphite, as well as a Staudinger-phosphonite, collaboration with the late Werner Reutter (Charité Berlin), we reaction for the chemical lipidation, biotinylation, and glycosylation have identified mannosamine-based diselenides as highly potent of proteins, as well as polymeric materials. We demonstrated inhibitors of the ManNAc-kinase, a key enzyme in the biosynthesis that Staudinger-reactions are efficient transformations even in of sialic acid, and showed that addition of these inhibitors to a cell lysates, and further employed these reactions for an efficient and model system reduced sialylation. metal-free peptide or protein PEGylation to deliver a new class of branched oligoethylene glycol scaffolds for the stabilization against Site-specific functionalization of proteins for the acquisition degradation in plasma or the cytosol. of multivalent glycoconjugates In a combined effort with Prof. Budisa (TU Berlin), we employ a Probing the impact of post-translational modifications on combination of classical site-directed mutagenesis, genetic code peptide and protein aggregation: semi-synthesis of the engineering, and bioorthogonal reactions to deliver chemically Alzheimer’s disease-relevant Tau protein modified proteins with carbohydrates installed at specific residues. In this project we are studying the structural consequences of These protein conjugates are employed in multivalent binding post-translational modifications on pharmacologically relevant studies within the collaborative research center 765, which proteins, in particular intrinsically unstructured proteins. A support the use of proteins as structurally defined scaffolds for the prime example is the neuronal Tau protein, which exists in an presentation of an exact number of multivalent ligands. unstructured soluble form, a microtubule bound state of unknown structure, and a hyperphosphorylated aggregated state found in Chemoenzymatic labeling and cellular delivery of proteins neurofibrillar tangles in Alzheimer’s disease. and antibodies In collaboration with Prof. Leonhardt (LMU Munich) and Currently, we are investigating the complex relationship between Prof. Cardoso (TU Darmstadt) we aim to develop powerful new phosphorylation and aggregation of Tau by generating otherwise methods for the chemoenzymatic modification of proteins as well inaccessible homogeneously phosphorylated proteins with the as antibodies. Our long-term goal within this project is to generate recent first semi-synthesis of a homogeneously phosphorylated new antibody-drug conjugates (ADCs) as well as cell-permeable functional Tau protein. Furthermore, we have extended this antigen-recognizing proteins. To reach this aim we have recently 94 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

engineered so-called ‘Tub-tag labeling®’, which allows the modular of the protein with cyclic cell-penetrating peptides (cCPPs). Next C-terminal modification of proteins by the enzyme tubulin tyrosine we will combine these approaches for the fluorescent labeling of ligase and which will be the basis of a new start-up company in nanobodies, small antigen-binding proteins that remain active the near future (Tubulis Technologies). Additionally, in a proof of within the reductive milieu inside living cells, functionalized with concept study we were able to transport a functional full length cCPPs to directly detect intracellular targets after cellular uptake. protein to the cytosol and the nucleus of living cells by conjugation

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 bioconjugation reactions for the synthesis of functional peptides and proteins)

GROUP MEMBERS COLLABORATIONS

Dr. Debasish Bhowmick International Janine Kirstein, Dr. Wenyi Li Caroline Smet-Nocca, Leibniz-Forschungsinstitut für Mole­kulare Dr. Olaia Nieto Université des Sciences et Technologies Pharmakologie (FMP) Lukas Artner (doctoral student) de Lille, France Rainer Haag, Lorenzo Assennato (doctoral student) Roland Brock, Freie Universität Berlin Alice Baumann (doctoral student) Radboud University Nijmegen Medical Bettina Keller, Jordi Bertran (doctoral student) Centre, The Netherlands Freie Universität Berlin Maria Glanz (doctoral student) Eric Strieter, Roland Netz, Marc-André Kasper (doctoral student) University of Massachusetts-­Amherst, Freie Universität Berlin Simon Klenk (doctoral student) MA, USA Bernd Lepenies, Michaela Mühlberg (doctoral student) Ron Raines, Leibniz-Universität Hannover Nicole Nischan (doctoral student) Massachusetts Institute of Andreas Herrmann, Martin Penkert (co-supervised with Technology (MIT), MA, USA Humboldt Universität zu Berlin Dr. Eberhard Krause) Cristina Cardoso, Oliver Reimann (doctoral student) National Technische Universität Darmstadt Simon Reiske (doctoral student) Nedjliko Budisa, Heinrich Leonhardt, Tom Sauer (doctoral student) Technische Universität Berlin Ludwig-Maximilians-Universität München Dominik Schumacher (doctoral student) Jens Dernedde, Anselm Schneider (doctoral student) Charité – Universitäts­medizin Berlin Sergej Schwagerus (doctoral student) Werner Reutter, Kristina Siebertz (doctoral student) Charité – Universitätsmedizin Berlin Kristin Kemnitz-Hassanin (technical assistant) Stephan Hinderlich, Dagmar Krause (technical assistant) Beuth-Hochschule, Berlin Inez Kretzschmar (technical assistant, Eberhard Krause, Peptide service facility) Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Staff employed within the reporting period Jens-Peter von Kries, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) 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 EXTERNAL FUNDING

Bertran-Vicente J, Penkert M, Nieto-Garcia O, Jeckelmann J-M, Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623 Schmieder P, Krause E, Hackenberger C P R * (2016) Chemoselective “Chemoselective reactions for the synthesis and application of functional synthesis and analysis of naturally occurring phosphorylated cysteine proteins”, funds for the coordination of the priority programme, peptides. Nature Comm. 7, Article number, 12703. 2012 – 2019, 612.500 €

Nieto-Garcia O, Wratil P R, Nguyen L D, Böhrsch V, Hinderlich S, Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623, Reutter W*, Hackenberger C P R * (2016) Inhibition of key enzyme of “Site-specific functionalization of nanobodies: From labelling to cellular sialic acid biosynthesis by C6-Se modified N-acetylmannosamine uptake”, jointly with H. Leonhardt (LMU München) and C. Cardoso (TU analogs. Chem. Sci. 7, 3928 – 3933. Darmstadt), 2012 – 2019, 412.800 €

Schumacher D, Helma J, Mann F A, Pichler G, Natale F, Krause E, Deutsche Forschungsgemeinschaft, Priority Programme SPP 1623, Cardoso M C, Hackenberger C P R *, Leonhardt H* (2015) Versatile and “Chemoselective Staudinger-induced Michael-additions to antibodies efficient site-specific protein functionalization by tubulin tyrosine ligase. to analyze protein homeostasis in C.elegans”, jointly with J. Kirstein Angew. Chem. Int. Ed. 54, 46: 13787-13791. Vielseitige, effiziente (FMP Berlin), 2016 – 2019, 217.150 € und ortsspezifische Proteinfunktionalisierung durch das Enzym Tubulin Boehringer-Ingelheim Stiftung, “Chemoselective Staudinger-reactions Tyrosin Ligase. Angew. Chem. 127, 46, 13992 – 13996. for the modification of peptides and proteins“,“Plus 3”- Programme, Nischan, N., Herce, H.D., Natale, F., Bohlke, N., Budisa, N., Cardoso, 2010 – 2014, 838.000 € M.C.,* Hackenberger C P R * (2015) Covalent Attachment of Cyclic Deutsche Forschungsgemeinschaft, SFB 765 B05, “Synthesis of TAT Peptides to GFP Results in Protein Delivery into Live Cells with multivalent ligand binding systems via chemoselective Immediate Bioavailability. Angew. Chem. Int. Ed. 54, 6: 1950-1953. saccharide- and peptide-ligations” (1st funding period 2008 – 2011), Kovalente Verknüpfung cyclischer TAT-Peptide mit GFP resultiert in der “Site-specific functionalization of proteins for the acquisition of direkten Aufnahme in lebende Zellen mit sofortiger biologischer multivalent glycoconjugtes“ (2nd and 3rd funding period 2012 – 2019), Verfügbarkeit. Angew. Chem. 127, 6, 1972 – 1976. 2008 – 2019, jointly with N. Budisa (TU Berlin), 802.200 € Reimann O, Smet-Nocca C, Hackenberger C P R * (2015) Traceless Deutsche Forschungsgemeinschaft, SFB 765 (1st and 2nd funding Purification and Desulfurization of Tau Protein Ligation Products. Angew. period 2008 – 2015), Integriertes Graduiertenkolleg des SFB, 2008 – 2015, Chem. Int. Ed. 54, 1: 306-310. Spurlose Aufreinigung und Desulfurierung 879.400 € von Ligationsprodukten des Tau-Proteins. Angew. Chem. 127, 1, 311 – 315.

FMP authors Group members 96 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

CHEMICAL BIOLOGY I

CHEMISCHE BIOLOGIE I

GROUP LEADER PROF. DR. DOROTHEA FIEDLER

BIOGRAPHY SUMMARY

1996 – 2001 Undergraduate studies Our group seeks to develop a better understanding of the multiple ways in which nature and Diploma in Chemistry, utilizes phosphate in both protein signaling cascades and metabolic networks. Signaling and Ludwigs-Maximilians-Universität­ Würzburg 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. 2000 – 2001 Diploma thesis with As deregulation of cellular information transfer is associated with a wide range of diseases, Prof. John Arnold, University of California a detailed annotation of signaling events in healthy and diseased states can help to highlight at Berkeley, USA new avenues for therapeutic intervention. 2001 – 2005 Graduate studies in Inorganic and Supramolecular Chemistry One group of densely phosphorylated messengers of particular interest to our group are the with Prof. Kenneth N. Raymond and inositol pyrophosphates (PP-InsPs). These molecules have emerged as central regulators Prof. Robert G. Bergman, University of cell homeostasis, and genetic studies in mice and humans implicate PP-InsPs in a host of California at Berkeley, USA 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”, 2006 – 2010 Postdoctoral research in in which the temporally and spatially controlled production of a particular PP-InsP dictates Molecular Biology / Chemical Biology specific downstream signaling events. However, how these molecules exert their effects at the with Prof. Kevan M. Shokat, University molecular level is not well understood. Using a multi-disciplinary approach that employs of California at San Francisco, USA techniques from inorganic and organic chemistry, chemical genetics and genetics, molecular 2009 NIH Pathway to Independence Award 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, 2010 – 2015 Assistant Professor of diabetes, and obesity. Chemistry, Princeton University, USA

2013 NIH Director´s New Innovator Award ZUSAMMENFASSUNG

2013 Rita Allen Scholar Award Unsere Gruppe versucht, ein besseres Verständnis zu entwickeln, wie die Natur Phosphat in Protein-Signalkaskaden und metabolischen Netzwerken verwendet. Signal- und Stoffwechsel- Since 2015 Full Professor (W3-S) of Chemical wege sind sehr komplex, aber die einzelnen Schritte innerhalb dieser Kaskaden hängen von Biology, Humboldt University of Berlin, and einfachen chemischen Reaktionen ab und beinhalten oft die reversible Addition von Phosphoryl- Director at the Leibniz-Forschungsinstitut für gruppen. Da die Deregulierung des zellulären Informationstransfers mit einer Vielzahl Molekulare Pharmakologie (FMP), Berlin von Krankheiten einhergeht, kann eine ausführliche Erläuterung von Signalereignissen in gesunden und kranken Zuständen dazu beitragen, neue Wege für therapeutische In- terventionen 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. CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 97

DESCRIPTION OF PROJECTS

New analytical approaches for the detection of cellular inositol metabolic networks that maintain cellular energy homeostasis. poly- and pyrophosphate species Notably, we also isolated several known and novel substrates of protein Despite their essential functions in cell physiology, the analysis of pyrophosphorylation, a unique posttranslational modification inositol polyphosphates (InsPs) and PP-InsPs remains a formidable mediated by PP-InsPs. Our findings not only demonstrate that PP- challenge. As a result, information on the production, local con- InsPs provide a central line of communication between signaling and centrations, biosynthetic pathways, and exact chemical nature of InsP metabolic networks, but also highlight the unusual ability of these messengers is sparse and sometimes controversial. molecules to access two distinct modes of action.

In collaboration with H. Oschkinat (FMP) and V. Haucke (FMP) we Chemical tools for annotating the pyrophosphoproteome are seeking to adress these questions by using 13C-labeled inositol, in In addition to the traditional binding mechanism, a covalent protein combination with NMR spectroscopy, to enable the detection and modification termed ‘protein pyrophosphorylation’ has been proposed quantification of InsPs and PP-InsPs in complex samples. Through for PP-InsPs. Efforts to characterize protein pyrophosphorylation chemical synthesis of 13C-labeled InsPs and PP-InsPs, we have have exclusively relied on in vitro labeling strategies; consequently, established the relevant reference spectra, and metabolic labeling of many questions about the regulation of pyrophosphorylation in vivo cells with 13C-inositol proceeds smoothly without the side-effects of still linger. To address these shortcomings, our group has implemented using radioactive materials (which is the standard procedure). In a a set of complementary methods with the goal of comprehensively parallel effort, we have also developed a derivatization strategy for the annotating in vivo pyrophosphorylated proteins. We have developed highly charged InsPs / PP-InsPs to enable their detection using mass a convenient synthetic approach to obtain pyrophosphorylated spectrometry with high sensitivity. In the long term, these convenient peptides. These peptides are currently being used to investigate the bioanalytical approaches will find widespread applications in the fields feasibility of mass spectrometry-based detection in complex mixtures of membrane traffic and inositol signaling, and which help elucidate (in collaboration with E. Krause, FMP). In parallel, we devised a method the multifaceted effects of these central molecules in a wide range of for the incorporation of a stabilized pyrophosphoserine moiety biological contexts. into peptide sequences. With the goal of generating antibodies against the pyrophosphoserine group. To complement these antibodies, Characterization of inositol pyrophosphate binding proteins which may display sequence sensitivity, an affinity reagent for the It is the general consensus that inositol phosphates regulate protein enrichment of pyrophosphoserine-containing proteins from cell function by binding to their targets, but the literature on PP- lysates was developed. This reagent contains a cavity that allows for InsP interacting proteins is sparse. Therefore, our group set out to selective recognition of pyrophosphate esters. A combination of these develop affinity reagents to identify PP-InsP binding partners newly developed techniques is now on the verge of providing the first on a proteome-wide scale. To achieve this goal we have designed, comprehensive analysis of endogenous pyrophosphoproteins. 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.

The analogs were converted to affinity reagentsvia 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 98 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

PP-InsP binding proteins Pyrophosphorylation substrates

P P P P P P P P PCP P PCP P P P P P PCP P

5PP-InsP5 beads

Nucleotide Polyphosphate Protein Carbohydrate Ribosome metabolism metabolism phosphorylation metabolism 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 COLLABORATIONS

Dr. Barbara Dul International National Dr. Anastasia Hager Adam Resnick, Henning Jessen, Dr. Sarah Hostachy University of Pennsylvania, USA Universität Freiburg Dr. Javier Moreno Solomon Snyder, Thomas Schrader, Dr. Florence Williams Johns Hopkins University, USA Universität Duisburg-Essen John Conway (Ph.D. student) Rashna Bhandari, Eberhard Krause, Jeffrey Bratz (Ph.D. student) CDFD, India Leibniz-Forschungsinstitut für Molekulare Nathaniel Brown (Ph.D. student) Adolfo Saiardi, Pharmakologie (FMP) Robert Harmel (Ph.D. student) University College London, Great Britain Volker Haucke, Alan Marmelstein (Ph.D. student) Roberto Docampo, Leibniz-Forschungsinstitut für Molekulare Cesar Perez Ramirez (Ph.D. student) University of Georgia, USA Pharmakologie (FMP) Robert Puschmann (Ph.D. student) Stephen Shears, Hartmut Oschkinat, Mingxuan Wu (Ph.D. student) NIH, Triangle Park, North Carolina, USA Leibniz-Forschungsinstitut für Molekulare Lisa Yates (Ph.D. student) Christopher Barker, Pharmakologie (FMP) Natascha Heinsohn (Master student) Karolinska Institutet, Sweden Lucy Chong (Visiting scholar) Andreas Mayer, Katy Franke (technical assistant) University of Lausanne, Switzerland Lena von Oertzen (technical assistant) Michael Hothorn, University of Geneva, Switzerland Staff employed within the reporting period Sebastian Hiller, University of Basel, Switzerland Robbie Loewith, University of Geneva, Switzerland Julianne Djordjevic, University of Sydney, Australia Nicolas Veiga, University of Montevideo, Uruguay 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 EXTERNAL FUNDING

Wu M, Chong L S, Perlman D H, Resnick A C, Fiedler D (2016) The National Institute of Health, NIH Director’s New Innovator Award inositol polyphosphates intersect with protein signaling and metabolic Program. “Understanding phosphate metabolism in cancer and networks via two distinct mechanisms. Proc. Nat. Acad. Sci. USA 113, metastasis” 2013 – 2015, 1.100.000 US $ E6757 – E6765. Sidney Kimmel Foundation for Cancer Research, Kimmel Scholar Brown N W, Marmelstein A M, Fiedler D (2016) Chemical tools for Program. “Does inorganic phosphate promote metastasis to bone?” interrogating inositol pyrophosphate structure and function. Chem. 2013 – 2015, 200.000 US $ Soc. Rev. 45, 6311 – 6326. Rita Allen Foundation, Rita Allen Scholars Program. “Understanding Hager A, Wu M, Wang H, Brown Jr. N W, Shears S B, Veiga N, phosphate metabolism in cancer and metastasis” 2013-2016, Fiedler D (2016) Cellular cations control conformational switching of 300.000 US $ inositol pyrophosphate analogs. Chem. Eur. J. 22, 12406 – 12414. Swiss National Science Foundation, Sinergia. Joint with A. Mayer, Williams F J, Fiedler D (2015) A fluorescent sensor and gel stain for S. Hiller, M. Hothorn. “Discovery and mechanistic dissection of novel detection of pyrophosphorylated proteins. ACS Chem. Biol. 10, signaling pathways controlling phosphate homeostasis in eukaryotes” 1958 – 63. 2016 – 2020, 2.405.000 CHF

Conway J H, Fiedler D (2015) An affinity reagent for recognition of Leibniz Gemeinschaft, Leibniz Wettbewerb. Joint with H. Oschkinat, pyrophosphorylated peptides. Angew. Chem. Int. Ed. 54, 3941 – 5 V. Haucke. “Systems level analysis of inositol messengers in nutrient signaling” 2017 – 2020, 1.063.000 €

FMP authors Group members 100 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

PEPTIDE-LIPID INTERACTION / PEPTIDE TRANSPORT

PEPTID-LIPID-INTERAKTION / PEPTIDTRANSPORT

GROUP LEADER DR. MARGITTA DATHE

BIOGRAPHY SUMMARY

1974 Diploma thesis in Physics, Our group is interested in the interaction of cell-penetrating and cell-permeabilizing peptides Humboldt-University Berlin, GDR 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 1978 Ph.D, Academy of Sciences of the GDR therapeutic drug molecules. Currently, our efforts are focused on targeting the blood-brain 1979 – 1992 Research Associate, Institute of barrier and delivering bioactive compounds via the skin. Furthermore, we are attempting Drug Research of the Academy of Sciences to elucidate the mode of action of small cyclic arginine and tryptophan-rich antimicrobial of the GDR peptides. Unlike the common membrane-permeabilizing mechanism of antimicrobial peptides, these compounds activate a novel mode of action against prokaryotic cells and are 1992 – 1999 Team Leader of the able to penetrate the membrane of eukaryotic cells. In this way they might offer new ways to Conformational Analysis Group, FMP generate antimicrobial compounds for selected applications against intracellular pathogens. since 1999 Team Leader of the Peptide Lipid Interaction / Peptide Transport Group, FMP ZUSAMMENFASSUNG

2015 “Leibniz Drug of the Year” Award Uns interessiert die Zell-penetrierende und Membran-permeabilisierende Wirkung von of the Leibniz Research Alliance Peptiden auf menschlichen Zellen und Bakterien. Zell-penetrierende Peptide werden genutzt, “Bioactive Compounds and Biotechnology“ 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 Aufnahme- vermittlung bioaktiver Substanzen in die Haut gerichtet. Ein weiterer Forschungsschwer- punkt 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. CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 101

DESCRIPTION OF PROJECTS

Small cyclic antimicrobial peptides Optimizing the activity and bacterial selectivity of antimicrobial Fig. 1: cWFW-triggered formation of lipid domains and segregation of peptides (AMPs) requires an understanding of their mechanism membrane proteins (a) Phase contrast, GFP-protein fluorescence, Nile of action. With its amphipathic structure and high content of ar- red staining of lipid domains and fluorescent color overlays for cells ginine residues, the synthetic cyclic hexapeptide cWFW (cyclo expressing different integral membrane proteins (upper panels) and (RRRWFW)) is highly membrane-active. However, in contrast different peripheral membrane proteins (lower panels) in the presence of cWFW (20 min incubation with 12 μM). Whereas there is uniform to most antimicrobial peptides, cWFW neither permeabilizes the membrane fluorescence of GFP-proteins and Nile red in non-treated cells membrane nor translocates into the cytoplasm of bacteria. In a (not shown), the formation of domains of different fluidity is observed collaboration with the Centre for Bacterial Cell Biology, Newcastle after incubation with cWFW. Integral proteins accumulate in Nile red-free University, UK, we have shown that cWFW instead triggers a regions whereas peripheral proteins co-localize with Nile red, the regions rapid reduction of membrane fluidity, both in live Bacillus subtilis in which cWFW also accumulates. Strains used: (a) B. 816 subtilis BS23 cells and in bacterial model membranes. This immediate activity is (AtpA-GFP), B. subtilis HS41 (YhaP-GFP), B. subtilis HS64 (WALP23-GFP), accompanied by the formation of distinct membrane domains 817 (b) B. subtilis KR318 (SpoVM-GFP), B. subtilis HS65 (GFP-MinDMTS) which differ in local membrane fluidity and which severely disrupt and B. subtilis HS208 818 (SepFMTS-GFP), Reference: Scheinpflug K. membrane protein organization by segregating peripheral and et al. (2017) Antimicrobial peptide cWFW kills by combining lipid phase integral proteins into domains of different rigidity (Fig. 1). We separation with autolysis. Scheinpflug et al. (2017) Scientific Reports 7:44332. 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.

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 quantification of the signal molecule (Fig. 2). Thus, the micelles antimicrobial compounds for the treatment of intracellular pathogens. combine a high selectivity for human brain capillary endothelial cells with the great sensitivity of Xe Hyper-CEST MRI and might be a Peptide-modified micellar and liposomal carriers potential tool in MRI-based brain diagnostics. Cell-recognising and membrane-translocating peptides provide promising tools for the development of efficient drug delivery Ongoing activities with industrial partners to advance the preclinical systems. Early observations showed that small micelles, formed of an development of a P2A2 peptide-modified liposomal preparation of arginine- and lysine-rich lipopeptide (P2A2), selectively internalise recombinant Transglutaminase 1 (in cooperation with University into endothelial cells of the blood-brain barrier (BBB). Hospital, Münster) to treat autosomal recessive congenital ichthyosis (ARCI) underline the huge therapeutic and economic potential of Chemical exchange saturation transfer with hyperpolarized xenon this formulation. nuclei (Hyper-CEST) allows sensitive detection of supramolec- ular cages such as cryptophane-A (CrA) in non-invasive Magnetic Further studies using liposomes decorated with an oligo-arginine Resonance Imaging (MRI). We generated a lipopeptide, PCrAA2, lipopeptide showed that they are highly suitable for transporting with a covalently attached CrA. Studies in cooperation with L. Corneodesmosin to its site of action, the membrane of skin Schröder (FMP) confirmed cell selectivity of PCrAA2 micelles and keratinocytes, and provide a promising basis for the development allowed us to distinguish BBB endothelial cells from control aortic of a pathogenesis-based therapy for Peeping Skin Disease (in endothelial cells based on high local cage concentration and reliable cooperation with University Hospital, Münster). 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; 1.5×106 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 COLLABORATIONS

Dr. Margitta Dathe (group leader) International National Dr. Oxana Krylova Henrik Strahl, Heiko Traupe, Kathi Scheinpflug (doctoral student, Newcastle University, UK University Münster, Germany postdoc) Marina Rautenbach, Vincence Oji, Heike Nikolenko (technical assistant) University Stellenbosch, South Africa University Münster, Germany Sven Richter (master student) Sonia Waiczies, Ines Volk (master student) MDC Berlin, Germany Alfred Blume, Staff employed within the reporting period 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 Schnurr M, Sydow K, Rose HM, Dathe M, Schröder L (2015). Brain (2017) Antimicrobial peptide cWFW kills by combining lipid phase Endothelial Cell Targeting via a Peptide-functionalized Liposomal Carrier separation with autolysis. Scientific Report 7, 44332. for Xenon Hyper-CEST MRI. Adv Healthcare Mat 4, 40 – 45.

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, 130 – 139.

Rautenbach M, Troskie A M, Vosloo J A, Dathe M (2016) Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi. Biochimie 130, 122 – 131.

Scheinpflug K, Krylova O, Nikolenko H, Thurm C, Dathe M (2015) FMP authors Evidence for a novel mechanism of antimicrobial action of a cyclic R-, Group members W-rich hexapeptide. PlosONE 10(4) e0125056. CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 103

MASS SPECTROMETRY

MASSENSPEKTROMETRIE

GROUP LEADER DR. EBERHARD KRAUSE

BIOGRAPHY SUMMARY

1975 Diploma degree in Physical Our group focuses on the development and application of proteomic methods to investigate Chemistry, Humboldt University, Berlin cellular signaling processes. Our main topics of research have been protein-protein inter- actions and post-translational modifications and their functional consequences. In this context, 1982 Dr. rer. nat., Humboldt University we are interested in T cell receptor (TCR)-associated protein-protein interactions that are 1984 – 1986 Research Group Leader mediated by specific protein phosphorylations which may function as modulators of cellular ‘Drug Development’ in the Pharmaceutical adhesion and migration processes. Moreover, we have contributed to various proteomic Industry studies, developed new mass spectrometry-based methods for the discovery of uncommon protein modifications, and improved approaches for the identification and quantification of 1987 – 1991 Research Associate, proteins in affinity-purification mass spectrometry experiments. Institute of Drug Research, Berlin since 1992 Senior Scientist and Head of ZUSAMMENFASSUNG Mass Spectrometry Group, FMP 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 inter- essieren 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 Protein- modifikationen entwickelt. 104 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

DESCRIPTION OF PROJECTS

Serine phosphorylation-dependent protein-protein interactions Analysis of ‘uncommon’ protein phosphorylation. of the T cell adaptor protein ADAP. Reversible phosphorylation is the most widespread post- Reversible protein phosphorylation is an important feature of T cell translational protein modification and is a key regulatory signaling, regulating many of the signal transduction pathways required mark in most cellular processes. While the majority of protein for proper T cell functioning. The human adhesion and degranulation phosphorylation research has been focused on hydroxyl amino acids promoting adaptor protein (ADAP) plays a central role in T cell signaling. such as serine, threonine, and tyrosine, amino acid side-chains of Upon T cell receptor stimulation ADAP is strongly tyrosine- histidine, arginine, cysteine and lysine residues may also undergo phosphorylated and serves as a hub for SH2 domain-containing phosphorylation. However, because of the instability of the P-N effector proteins. In addition, phosphoproteomics revealed several and P-S bonds, these modifications most often remain undetected serine and threonine phosphorylation sites within the N-terminal in conventional phosphoproteomics studies by mass spectrometry domain of ADAP. However, the role of these phosphorylation (MS), even though recent studies have indicated their potential events in T cell signaling events has not been investigated. importance in various biological signaling processes. We performed Using quantitative SILAC-based peptide and protein MS studies of phosphocysteine and pyrophospholysine peptides pull-down approaches, we demonstrate for the first time that two that very recently became synthesizable via new synthetic routes distinct phosphorylated serine residues in the N-terminal region of established by the Hackenberger group at the FMP. The ADAP bind specifically to 14-3-3 isomers (Figure 1). Since 14-3-3 preparation of well-characterized, site-specifically modified proteins are known for playing an important role in T cell signaling, peptides enables a systematic study of the behavior of such peptides this new phosphorylation-dependent interaction between ADAP and using various mass spectrometric fragmentation techniques. Our 14-3-3-proteins may yield new insights into T cell signaling pathways. data indicate that the stability of both modifications during electron-

A B Fig. 1: Results of SILAC-based pull-down experiments with site-specifically serine- phosphorylated 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 14-3-3 isomers can be considered as phosphorylation-dependent interaction partners.

GROUP MEMBERS COLLABORATIONS

International Hans G. Börner, Annika Manns (doctoral student) Remigiusz Serwa, Humboldt-Universität, Berlin Martin Penkert (doctoral student) Imperial College London, UK Jörg Rademann, Michael Schümann (technical assistant) Edward W. Tate, FU Berlin Heike Stephanowitz (technical assistant) Imperial College London, UK Volker Haucke, Dorothea Fiedler, FMP Staff employed within the reporting period Princeton University, USA Christian Hackenberger, FMP National Dorothea Fiedler, Ria Baumgrass, FMP DRFZ, Berlin Christian Freund, Beate Braun, FU Berlin IZW, Berlin Dirk Schwarzer, Kurt Engeland, Universität Tübingen Universität, Leipzig Ralf Schülein, U. Benjamin Kaupp, FMP Center of Advanced European Studies and Michael Veit, Research, Bonn FU Berlin CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 105

transfer dissociation (ETD) mass spectrometry is high, keeping the these processes has not been elucidated and remains a controversial modified side-chain completely intact during fragmentation and topic of debate. MS analysis of a set of synthesized, site-specifically making ETD particularly suitable for proteomic studies as an modified peptides which were very recently accessible via a new essential tool for evaluating the biological relevance of these synthetic approach developed by the Fiedler group at Princeton uncommon protein modifications. To demonstrate the use of University (now at the FMP) have shown that using conventional, ETD MS-based proteomics in resolving biological questions, we collision-based MS / MS methods such as CID and HCD, identified an endogenous Cys phosphorylation site in IICBGlc, pyrophosphorylated peptides exhibit a characteristic neutral which is known to be involved in carbohydrate uptake in the loss pattern of 98, 178 and 196 Da, preventing the identification bacterial phosphotransferase system (Betran-Vicente et al. Nature of the modification site. In contrast, ETD combined with higher Commun. 2016). energy collision dissociation (EThcD) provides useful tandem MS spectra for direct and unambiguous assignment of the site Another labile ‘phospho-modification’ that has eluded detection by of pyrophosphorylation. Based on the specific fragmentation conventional MS methods is protein pyrophosphorylation. Located behavior of pyrophosphorylated peptides during collision-induced close to polyacidic amino acid stretches, this modification occurs dissociation, we developed a data-dependent neutral-loss-triggered on phosphorylated serine residues and is mediated by inositol EThcD acquisition method (Figure 2) that allows for the reliable pyrophosphate messengers. While perturbation of inositol characterization of protein pyrophosphorylations (Penkert et al. pyrophosphate biosynthesis has revealed a wide range of functions Anal. Chem. 2017) and, in combination with a selective enrichment for these messengers, including insulin signaling and central energy procedure, uncovered the first pyrophosphorylation sitesin vivo. metabolism, the role of protein pyrophosphorylation in regulating

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 high- resolution 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, Kuropka B, Witte A, Sticht J, Waldt N, Majkut P, Hackenberger C P R, Krause E (2017) Unambiguous identification of serine and threonine Schraven B, Krause E, Kliche S, Freund C (2015) Analysis of pyrophosphorylation using neutral-loss-triggered EThcD mass phosphorylation-dependent protein interactions of ADAP reveals novel spectrometry. Anal. Chem. 89, 3672 – 3680. interaction partners required for chemokine-directed T cell migration. Mol. Cell. Proteomics 14, 2961 – 2972. 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, 12703.

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, 6990 – 6994.

Kuropka B, Royla N, Freund C, Krause E (2015) Sortase A-mediated site-specific immobilization for identification of protein interactions in affinity FMP authors purification-mass spectrometry experiments. Proteomics 1, 1230 – 1234. Group members 106 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

MEDICINAL CHEMISTRY

MEDIZINISCHE CHEMIE

GROUP LEADER DR. MARC NAZARÉ

BIOGRAPHY SUMMARY

1986 – 1995 Studies in Chemistry, Small molecule tools allow one to probe protein functions and elucidate mechanisms and signal University of Karlsruhe 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 1995 Diploma in Chemistry with serve as starting points for new therapeutic approaches and drugs. Our aim is to discover and Prof. H. Waldmann, University of Karlsruhe develop highly active and selective chemical probes for the specific modulation of protein-ligand 1995 – 1999 Ph.D. research with and protein-protein interactions. Together with our collaboration partners, we are currently Prof. H. Waldmann, University of Karlsruhe 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 1999 – 2013 Medicinal chemist and study the structure-activity relationship (SAR) of protein-ligand interactions by synthesizing project leader in drug discovery, Sanofi, distinct, small molecule derivatives or small focused libraries of the initial screening hit structure. Frankfurt am Main Iterative cycles of biological testing, design, and synthesis of new analogues based on the data 1999 – 2005 Medicinal Chemistry obtained yield optimized chemical tools for proof-of-concept studies. A second field of interest is Department, Sanofi, Frankfurt am Main 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 2005 – 2009 Therapeutic department research. Careful expansion of this library should guarantee optimal coverage of the biological “Thrombosis and Angiogenesis”, Sanofi, space being screened, along with high hit rates as well as suitable starting points for chemical Frankfurt am Main optimization. The Medicinal Chemistry group closely collaborates with the Screening Unit and the 2009 – 2013 Therapeutic Strategic Computational Chemistry group on work for the FMP library and biological profiling in SAR studies. Unit “Age-Related Diseases”, Sanofi, Frankfurt am Main ZUSAMMENFASSUNG

Since 2013 Group leader, Kleine Moleküle lassen sich als Forschungswerkzeuge einsetzen, mit denen Proteinfunktionen Leibniz-Forschungsinstitut für Molekulare untersucht, molekulare Mechanismen aufgeklärt und Signaltransduktionswege durch direkte Pharmakologie (FMP), Berlin 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 Wechsel- wirkungen. 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 Da- ten 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 60.000 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. 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 (HGF)-stimulated canine MDCK-C cells, as well as human disruption of clathrin-endocytic protein interactions with pancreatic tumor cells for epithelial-mesenchymal transition (EMT), Pitstop derivatives a hallmark of cancer cell dissemination. Clathrin-mediated endocytosis (CME) regulates many key physiological processes such as the internalization of growth Tryptophan hydroxylase (TPH) inhibitors factors and receptors, entry of pathogens (e. g. HIV-1), and synaptic The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] transmission by formation of so-called ‘clathrin-coated vesicles’. An is causally involved in multiple aspects of mood control in the ELISA-based high-throughput screen (HTS) using 17,000 small central nervous system, such as regulating sleep, anxiety, drug abuse, molecules from the ChemBioNet library at the Screening Unit of and food intake. In peripheral tissues, serotonin regulates vascular the FMP resulted in the identification of two hit compounds that tone, gut motility, primary hemostasis, and cell-mediated immune inhibit complex formation between the clathrin terminal domain responses, and is associated with diseases like irritable bowel (TD) and amphiphysin B / C (V. Haucke et al. Cell, 2011). Start- syndrome and carcinoid syndrome. The biosynthesis of serotonin ing from these hits, and in collaboration with the group of Volker is a highly regulated two-step process, starting with the essential Haucke (FU & FMP, Berlin), we synthesized focused libraries of amino acid L-tryptophan (Trp), while tryptophan hydroxylase around 150 compounds, providing a structure-activity relationship (TPH) is the initial and rate-limiting enzyme in the biosynthesis for Pitstop2. Co-crystallization of Pitstop derivatives with the of serotonin. In collaboration with the groups of Michael Bader clathrin TD guided the further design and provided insights into and Udo Heinemann (both MDC), and Jens von Kries (FMP), we the key interactions between these Pitstop ligands and the clathrin have identified and further developed highly active TPH inhibitors TD. Surprisingly, X-ray structure determination (Haydar Bulut, that are able to modulate physiological serotonin levels. The X-ray FU Berlin) of six nearly equipotent inhibitors derived from one core co-crystal structures obtained with our inhibitors allowed us to scaffold showed four different binding modes. These non-canonical elucidate the binding mode and to reveal the structural determi- binding modes of the novel Pitstop analogues revealed several new nants for the remarkably efficient protein-ligand interaction of these aspects of the structural basis for the disruption of clathrin TD- inhibitors. Several inhibitors are currently undergoing in vivo efficacy endocytic protein interactions. For cellular studies we are developing studies in mice. a new photocleavable protecting group (photocage) that allows for the spatio-temporally controlled intracellular release of the non-cell Shaping the FMP library with novel scaffolds permeable Pitstop1. The optimal design of a chemical library to provide the best possible coverage of chemical space is crucial for the successful outcome of a Development of specific inhibitors of the tyrosine HTS. The selection of the central scaffold for a drug molecule is a phosphatase Shp-2 conceptually challenging and decisive task in the design of new small In stark contrast to their validated significance in signal transduction molecule modulators. Whereas the peripheral side-chain decoration and disease pathology, phosphatases are notoriously difficult to of a given hit structure is the first and obvious starting point for inhibit using small molecules. The protein tyrosine phosphatase variation, the exchange of the scaffold core is inherently more Shp-2 plays a critical role in growth factor-mediated processes, difficult. Availability, i. e. ease of synthetic accessibility, is an primarily by promoting the activation of the RAS / ERK signaling important yet often underestimated factor in the successful pathway. Aberrant gain-of-function mutations are associated with optimization of a tool compound. Therefore, we have investigated several metastatic cancers. In collaboration with Walter Birchmeier, and developed new and efficient routes for the synthesis of privileged a re-scaffolding approach that involves replacing the former scaffolds like 2H indazoles and pyrazolo-triazoles for generating framework of a tyrosine phosphatase Shp2 inhibitor (W. Birchmeier libraries, and we have included them in our FMP compound et al., PNAS, 2008), led to the discovery of novel structural classes collection. and eliminated several chemical liabilities, i. e. unfavorable structural features. These novel compounds are not only active in a sub- micromolar range in the Shp2-enzyme assay, but are also effective in the low micromolar range on hepatocyte growth factor 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 COLLABORATIONS

Dr. Upendra Anumala International National Dr. Isabel Fernández Bachiller Uwe Grether, Michael Bader, Dr. Hassen Bel Abed F.Hoffmann-La Roche, Basel, Switzerland MDC, Berlin Dr. André Horatscheck Haiyu Hu, Walter Birchmeier, Dr. María Pascual López-Alberca Institute of Materia Medica, MDC, Berlin Dr. Vera Martos Chinese Academy of Medical Sciences & Udo Heinemann, Dr. Lioudmila Perepelittchenko Peking Union Medical College MDC, Berlin Dr. Edgar Specker Stefan Krauss, Hans-Jürgen Holdt , Benjamin Jakob Brennecke University Hospital, Oslo, Norway University of Potsdam, Potsdam (doctoral student) Christoph Rademacher, Thais Gazzi (doctoral student) MPI, Potsdam Jens Schöne (doctoral student) K. Lenhard Rudolph, Keven Mallow (technical assistant) FLI, Jena Sandra Miksche (technical assistant) Claus Scheidereit, Jessica Przygodda (technical assistant) MDC, Berlin David W. Will, Staff employed within the reporting period EMBL, Heidelberg

SELECTED PUBLICATIONS EXTERNAL FUNDING

Bel Abed H, Schoene J, Christmann M, Nazare M (2016) Helmholtz Wirkstoffforschung Organophosphorus-mediated N-N bond formation: facile access Berlin Institute of Health (BIH) to 3-amino-2H-indazoles. Org. Biomol. Chem. 14, 8520 – 8528. SAW “DNA damage responses in aging” P21 collaboration with FLI Kozian D H, von Haeften E, Joho S, Czechtizky W, Anumala U R, Roux P, Jena 2014 – 2016 111.735.0 € Dudda A, Evers A, Nazare M (2016) Modulation of Hexadecyl-LPA-Mediated Activation of Mast Cells and Microglia by a Chemical Probe for LPA5. VIP „Validierung neuartiger Wirkstoffe zur Behandlung von Depressionen ChemBioChem 17, 861 – 865. durch pharmakologische Aktivierung der Tryptophan-Hydroxylase 2“ together with Michael Bader, MDC. 2012 – 2015 1.200.000 € Aretz J, Kondoh Y, Honda K, Anumala U R, Nazaré M, Watanabe N, Osada H, Rademacher C (2016) Chemical fragment arrays for rapid druggability MDC PreGo-Bio “Novel compounds for the targeted therapy of assessment. Chem. Commun. 52, 9067 – 9070. prostate cancer” together with Udo Heinemann, MDC. 2013 – 2016 480.000 € 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 DFG, NA 1274 / 1-1 “Development and characterization of specific small N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active molecule inhibitors of class II phosphatidylinositol 3-kinase C2alpha and Selective SGK1 Inhibitors. ACS Med. Chem. Lett., 6, 73 – 78 function” together with Volker Haucke, FMP. 2015 – 2018 194.325 €

Hu H-Y, Lim N-H, Juretschke H-P, Ding-Pfennigdorff D, Florian P, Kohlmann Sino-German Center for Research Promotion, GZ 1271, M, Kandira A, von Kries J, Saas J, Rudolphi K A, Wendt K U, Nagase H, “Tumor-­targeting SMART Imaging Agents” together with Haiyu-Hu, Plettenburg O, Nazare M, Schultz C (2015) In vivo visualization of Institute of Materia Medica, Chinese Academy of Medical Sciences & osteoarthritic hypertrophic lesions. Chem.Sci. 6, 6256 – 6261. Peking Union Medical College, Beijing, China. 2016 – 2019 207.845 €

FMP authors Group members CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 109

CORE FACILITY

SCREENING UNIT

GROUP LEADER DR. JENS PETER VON KRIES

BIOGRAPHY SUMMARY

1987 – 1995 Diploma & Ph.D. in biology, The Screening Unit serves as an open access technology platform for automated screening, University Hospital Hamburg-Eppendorf using either compound libraries such as the ChemBioNet and other collections (Prof. Strätling) (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 1995 – 2000 Research on proteins involved in possible the use of drugs in academic research for analysis of molecular mechanisms in origin of cancer in the lab of W. Birchmeier disease and development. Besides supporting assay development, process automation, screening 2000 – 2003 Head of Screening Unit, and automated data analysis, the Unit engages in the identification of novel screening Semaia Pharmaceuticals technologies that may prove useful in its services. The Unit currently supports compound screening projects in assay development and optimization of High-Throughput Screening since 2003 Head of Screening Unit, FMP; (HTS, Silke Radetzki), and in process automation (Martin Neuenschwander), including set up and technology development automated data documentation and analysis. Genome-wide RNAi screening (Katina Lazarow) 2005 Core facility for Helmholtz-Initiative has been established as a service complementing the identification of cellular targets through für Wirkstoffforschung similar cellular phenotypes that have been generated either by compounds or RNA-interference.

2015 Core facility for Chemical Biology, The Unit is building a central core facility for drug screening on the Campus Berlin-Buch for Berlin Institute of Health the Helmholtz-Initiative für Wirkstoffforschung, the Berlin Institute of Health (BIH), and 2017 Core facility for EU-OPENSCREEN EU-OPENSCREEN.

2011 – 2015 Chairman of Gemeinsame ZUSAMMENFASSUNG Fachgruppe Chemische Biologie, DECHEMA Die Screening Unit ist eine frei zugängliche Technologieplattform für automatisierte 2014 Advisory Board SFICAST, systematische Testungen („Screenings“); verwendet werden entweder Substanzbibliotheken University Oslo wie die ChemBioNet- und andere Sammlungen (60.000 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-Wirkungs- beziehungen (SAR) zusammen. 110 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

DESCRIPTION OF PROJECTS

Therapeutic targeting of Hodgkin lymphoma touch can cause pain, and here STOML3 inhibitors can reverse Classical Hodgkin lymphoma (cHL) reflects a clinical challenge mechanical hypersensitivity. In this way, small molecules applied when presenting as primary refractory or relapsed disease. locally to the skin may be used to modulate touch and could serve Interestingly, cHL is an archetypical example of malignant plasticity. as peripherally-acting drugs to treat tactile-driven pain following cHL is characterized by a virtual lack of gene products whose neuropathy. expression constitutes the B-cell phenotype. Restoring the B-cell phenotype may render cHL susceptible to clinically established Wnt inhibitors for suppression of self-renewal of cancer stem antibody therapies, targeting B-cell surface receptors, or small cells and tumorigenesis compounds interfering with B-cell receptor signaling. We supported Wnt / b-catenin signaling is a highly conserved pathway essential a high-throughput pharmacological screening based on more than for embryogenesis and tissue homeostasis. However, deregulation 28,000 compounds in cHL cell lines carrying a CD19 reporter to of this pathway can initiate and promote human malignancies, identify drugs that promote re-expression of the B-cell phenotype. especially of the colon, head, and neck. With Walter Birchmeier Three compounds were identified that robustly enhanced CD19 (MDC) we performed a high-throughput screen using purified transcription. Subsequent chromatin immune precipitation-based proteins in AlphaScreen and ELISA techniques to identify small analyses indicated that the action of two of these compounds was molecules that disrupt the critical interaction between β-catenin associated with lowered levels of the transcriptionally repressive and the transcription factor TCF4. We found a 4-thioureido- lysine 9-trimethylated histone H3 mark at the CD19 promoter. benzenesulfonamide derivative that robustly inhibits this interaction Moreover, the anti-leukemia agents all-trans retinoic acid and in colon cancer cells. Remarkably, the self-renewal capacity of arsenic trioxide (ATO) were found to reconstitute the silenced B-cell cancer stem cells was also blocked by the inhibitor, as demonstrated transcriptional program and reduce viability of cHL cell lines. by sphere formation of colon, head, and neck cancer stem cells under Furthermore, restoration of the B-cell phenotype also rendered nonadherent conditions. (Cancer Res; 76(4); 891 – 901. 2015 AACR). cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we Genome-wide RNAi for identification of the Volume-Regulated identified a conceptually novel, redifferentiation-based treatment Anion Channel (VRAC) strategy for cHL. (Figure 1, Blood. 2017;129(1):71-81). The group of Thomas Jentsch and the FMP Screening Unit identified a long-sought channel that helps cells to reduce their STOML3 inhibitors affecting tactile-driven chronic pain. volume. For this purpose a human genome-wide RNA-interference The skin is equipped with specialized mechanoreceptors that allow library was tested with a cellular reporter system for iodide influx perception of the slightest pressure. Indeed, some mechanoreceptors and intracellular quenching of an iodide-sensitive yellow fluorescent can detect even nanometer-scale movements. Movement is protein. The messenger RNA of 21,687 genes was targeted by transformed into electrical signals via the gating of mechanically ~130,000 transfections with interfering RNA. The Volume- activated ion channels at sensory endings in the skin. The Regulated Anion Channel (VRAC) was stimulated by a change of sensitivity of Piezo mechanically gated ion channels is controlled by cell culture medium from isotonic to hypotonic conditions. The stomatin-like protein-3 (STOML3), which is required for normal inhibition of quenching provided a read out using high-speed kinetic mechanoreceptor function. We identified small-molecule inhibitors imaging (FLIPR Tetra-system, Molecular Devices). Eighty-seven of STOML3 oligomerization that reversibly reduce the sensitivity genes were identified as candidates and were used in a secondary of mechanically gated currents in sensory neurons and silence screen with newly designed silencer RNAs for identification of mechanoreceptors in vivo. Under pathophysiological conditions LRRC8 heteromers as essential components of VRAC. following nerve injury or diabetic neuropathy, even the slightest

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) CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 111

GROUP MEMBERS COLLABORATIONS

Dr. Katina Lazarow (RNAi) International Thomas F. Meyer, Dipl. Ing. Romy Leu (HCT) Andrew W. Munro, MPIIB, Berlin Dr. Martin Neuenschwander Manchester University Walter Birchmeier, (Process Automation, HTS-Analysis) Stefan Krauss, MDC, Berlin Dr. Silke Radetzki University Oslo Claus Scheidereit, (High Content Screening, HCT) MDC, Berlin National Sabrina Kleißle (MDC) Michael Bader, Salim Seyfried, (RNAi, cellular test systems) MDC, Berlin Universität Potsdam & MHH Andreas Oder Udo Heinemann, Clemens Schmitt, (protein-ligand interactions surface plasmon MDC, Berlin Charité Berlin resonance, FRET, AlphaScreen) Lenhard Rudolph, Karoline Krause, Carola Seyffarth FLI, Jena Charité Berlin (capillary electrophoresis, enzyme screens) Ulrich Martin, Björn Schuhmacher, M.Sc. Marc Wippich (HTS) MHH, Hannover Universität Köln Stefan Kubick, Staff employed within the reporting period IZI Fraunhofer Potsdam-Golm

SELECTED PUBLICATIONS EXTERNAL FUNDING

Du J, Neuenschwander M, Yu Y, Däbritz JHM, Neuendorff N-R, EU-OPENSCREEN, BMBF Schleich K, Bittner A, Milanovic M, Beuster G, Radetzki S, Specker E, Berlin Institute of Health, BMBF Reimann M, Rosenbauer F, Mathas S, Lohneis P, Hummel M, Dörken B, von Kries JP, Lee S, Schmitt CA (2017) Pharmacological restoration CCMCURE, E-RARE 2014; BMBF; 01.06.2015 – 31.05.2018, Coordinator and therapeutic targeting of the B-cell phenotype in classical Hodgkin Prof. Salim Seyfried; 102.040,80 € lymphoma. Blood 129, 71 – 81. Helmholtz Wirkstoffforschung, BMBF; 2011 – 2015 Wetzel C, Pifferi S, Picci C, Gök C, Hoffmann D, Bali KK, Lampe A, Deutsche Forschungsgemeinschaft, “Synthesis, optimisation, and Lapatsina L, Fleischer R, Smith ESJ, Bégay V, Moroni M, Estebanez L, screening of small molecule libraries targeting protein-protein inter- Kühnemund J, Walcher J, Specker E, Neuenschwander M, von Kries actions”, FOR 806, TP Z1 (RA 895 / 5 – 1), with J. Rademann, M. Beyer- JP, Haucke V, Kuner R, Poulet JFA, Schmoranzer J, Poole K, Lewin GR mann, 05.2007 – 05.2010, 120.814 € (2017) Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity. Nat. Neurosci 20, 209 – 218. Bundesministerium für Bildung und Forschung, “Screening Unit: Assay development screening for lead identification and optimization”, Chenge JT, Le DV, Swami S, McLean KJ, Kavanagh ME, Coyne AG, 01GU0514 – KR, with C. Freund, R. Kühne, 01.2006 – 05.2009, 543.976 € Rigby SEJ, Cheesman MR, Girvan HM, Levy CW, Rupp B, von Kries JP, Abell C, Leys D, Munro AW (2016). Structural Characterization and Europäische Kommission, (6. Forschungsrahmenprogramm, PL018771, Ligand / Inhibitor Identification Provide Functional Insights into the with J. Rademann, 08.2006 – 01.2010, 389.800 € Mycobacterium tuberculosis Cytochrome P450 CYP126A1. J. Biol. EU-ANTIFLU FP7, Coordinator: Thomas F. Meyer, (MPI-IB, Berlin) Chem. 292, 1310 – 1329. Juni 2011 – Juni 2015, 400.000 € Fang L, Zhu Q, Neuenschwander M, Specker E, Wulf-Goldenberg A, EU-SFMET FP7-HEALTH-2007-A, HGF / SF and MET in metastasis, Weis WI, Kries von JP, Birchmeier W (2016) A Small-Molecule EU-SFMET, Coordinator: Ermanno Gherardi, (MRC-Cambridge, UK), Antagonist of the β-Catenin / TCF4 Interaction Blocks the Self-Renewal 04.2008 – 03.2011, 223.750 € of Cancer Stem Cells and Suppresses Tumorigenesis. Cancer Res. 76, 891 – 901. MDC, Screening Unit, 04.2008 – 03.2011, 223.750 €

Khatri Y, Ringle M, Lisurek M, Kries von JP, Zapp J, Bernhardt R PAKT (FLI-Jena), with W. Rosenthal, 01.2011 – 12.2012, 180.000 € (2016) Substrate Hunting for the Myxobacterial CYP260A1 Revealed ECRC, with MDC, 10.2008 – 2011, 360.000 € New 1α-Hydroxylated Products from C-19 Steroids. Chembiochem 17, 90 – 101. BMBF / MDC, RNAi & REMP, with MDC, J. Rademann, 11.2008 – 2009, 800.000 €

NGFNplus, 5 Projektförderungen á 30.000 €, 06.2008 – 05.2013

FMP authors Group members 112 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

CORE FACILITY

PEPTIDE SYNTHESIS PEPTIDSYNTHESE

GROUP LEADER DR. RUDOLF VOLKMER

BIOGRAPHY SUMMARY

1969 – 1972 Industrial training as a The ‘Peptide Synthesis’ group was formed in February 2013 as a service unit for peptide laboratory assistant, Hoechst AG, synthesis and is part of the department of Chemical Biology II, headed by Prof. Dr. Christian Frankfurt / Main 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 1975 – 1984 Studied Chemistry, standard solid phase peptide synthesis methods are used, resulting in linear peptides with University of Frankfurt / Main and FU Berlin or without side chain and / or termini-modifications. More challenging peptides can also be 1977 – 1978 Freelancer, German Trade Union synthesized following the relevant consultations. For peptide synthesis a small reimbursement Federation, Frankfurt / Main 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 1984 Diploma thesis, FU Berlin applied to the mapping and screening of protein-protein binding sites. (Prof. Rewicki)

1991 Ph.D., FU Berlin (Prof. Rewicki) The service unit for peptide synthesis is a collaborative and service partner in ongoing research projects at the FMP and at the Charité. 1991 Teacher at the DRK-nurse’s training school ZUSAMMENFASSUNG 1992 – 1999 Post-Doc, Charité – University Unserer Gruppe hat ihre Tätigkeit als „Serviceeinheit Peptid Synthese“ im Februar 2013 Medicine Berlin (Prof. Schneider-Mergener) aufgenommen. Sie ist Teil der Chemischen Biologie II welche von Prof. Dr. Christian Since 1999 Group leader “Molecular Hackenberger geleitet wird. Die Aufgabe der Serviceeinheit ist es allen Arbeitsgruppen des Libraries and Recognition Group”, FMP Zugang zu synthetischen Peptiden anzubieten. In der Regel werden diese über die Charité – University Medicine Berlin, Technik der Festphasensynthese hergestellt wobei das Standartrepertoire jener Technik Institute of Medical Immunology eingesetzt wird. Somit können lineare Peptide mit oder ohne Seitengruppen- und / oder Terminus-Modifizierung hergestellt werden. Kompliziertere Peptidstrukturen können nach Since 2013 Guest scientist and group leader Rücksprache ebenfalls hergestellt werden. Für die Peptidsynthese wird ein Unkostenbeitrag “Peptide Synthesis”, FMP für die benötigten Materialien erhoben. Seit Oktober 2016 bietet die Servicegruppe zusätzlich die Möglichkeit der SPOT Synthese an. Diese Technologie eignet sich hervor- ragend um Protein-Protein Kontaktstellen aufzuspüren und zu kartieren.

Die Serviceeinheit ist als Kooperations- und / oder Servicepartner in laufende Forschungs- projekte am FMP und an der Charité eingebunden. CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE 113

A B Hoechst

MPG-iCAL42, 1μM, 1h Pen-iCAL42, 1μM, 1h GFP-CAL

Tamra -MPG-iCAL36

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 Reducing the pathogenicity of cystic fibrosis: stabilizing the The peptide synthesis service unit at the FMP has been operating CFTR at the apical membrane using CAL-PDZ inhibitors since 2013. At this time three FMP labs, namely those of Blasig, The cystic fibrosis transmembrane conductance regulator (CFTR) Haucke and Kühne, were our first customers for peptide synthesis. is an ion channel that is mutated in patients with cystic fibrosis Four years later, we are periodically receiving peptide synthesis (CF), disrupting fluid and ion balance in multiple epithelial tissues. orders from 12 FMP groups. These orders include not only simple Most CF patients carry one or two copies of the ΔF508 allele, which linear peptides (on a milligram to gram scale), but also dye-labeled encodes a protein that is inefficiently folded, shows limited channel peptides, head-to-tail cyclic peptides with and without dye-labeling, activity, and is rapidly degraded. Compounds have been identified peptides without gene-encoded amino acids, building blocks or that address folding and channel defects. Neither provides significant proline-mimetics, D-amino acids or polyethylene glycol molecules, benefits as a monotherapy, but in combination they produce and phosphorylated peptides. In its early years, the peptide service significant improvement in lung function (ΔFEV1 >10 %) in 25 % of unit synthesized approximately 140 peptides per year. In 2016, Ines ΔF508 homozygous patients. To reach more patients and increase Kretzschmar synthesized 255 peptides, an increase of almost 75 %. the functional response, we have proposed the early-stage pharma- In total, 771 peptides have been synthesized since March 2013. In cological validation of a novel translational strategy to address the addition to its services, the peptide synthesis unit is embedded in remaining defect – the breakdown of rescued ΔF508-CFTR. No several FMP research projects. In addition to standard peptide clinical trials have yet included compounds specifically designed to synthesis, the SPOT synthesis technology for preparing peptide increase CFTR stability at the apical membrane. Having identified arrays has also been offered since October 2016 . the CFTR-Associated Ligand (CAL) as a key mediator of CFTR 114 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

degradation, we have localized a critical binding interface, designed Our data confirm substantial additivity for a cell-permeable iCAL peptides (named iCAL36 and iCAL42) that block it, and have shown in concert with a known corrector, the small molecule VX-809. that they act as first-in-class ‘stabilizers’ of functional ΔF508-CFTR Figure 1 depicts the cellular internalization of modified iCAL peptides in polarized CF bronchial epithelial cells. Preclinical advancement of into Caco-2 cells, as well as the colocalization of a TAMRA-labelled our inhibitor-of-CAL (iCAL) approach is currently limited by lead iCAL36 peptide with CAL in GFP-CAL transfected Caco-2 cells. affinity, delivery, and limited data regarding the extent of additional rescue compared to combination therapies currently used in clinical trials. The project is NIH-funded and is a collaboration with Dean Madden, Hanover, USA; Rudolf Volkmer and Nico Derrichs, Charité – The project focuses on several cell-permeable iCALs, on demonstrating Universitätsmedizin, Berlin; Prisca Boisguerin, Montpellier, France; the intracellular iCAL-CAL interaction, and on the stabilizers’ effect and Hartmut Oschkinat at the FMP Berlin. on functional CFTR and ΔF508-CFTR. Cellular models are Caco-2 cells, ΔF-CFBE cells, and human biopsies of CF-patients.

GROUP MEMBERS COLLABORATIONS

Ines Kretzschmar (technical assistant) International National Dean Madden, Enno Klussmann, Staff employed within the reporting period Geisel School of Medicine at Dartmouth, Max-Delbrück-Center for Molecular Hanover, USA Medicine, Berlin Prisca Boisguerin, Karola Rück-Braun, CRBM, Montpellier, France Technische Universität Berlin Michel Steinmetz, Christian Freund, Paul Scherer Institut, Freie Universität Berlin Switzerland Jörg Höhfeld, Marius Sudol, University of Bonn Institute of Molecular and Cell Biology, Henning Mootz, Singapore University of Münster Michael Ehrmann, University of Duisburg-Essen

SELECTED PUBLICATIONS

Kathage B, Gehlert S, Ulbricht A, Lüdecke L, Tapia V E, Orfanos Z, Tapia Mancilla V E, Volkmer R (2016) Peptide Arrays on Planar Supports. Wenzel D, Bloch W, Volkmer R, Fleischmann B K, Fürst D O, Höhfeld J Methods Mol Biol 1352, 3 – 17. (2017) The cochaperone BAG3 coordinates protein synthesis and Opitz R, Müller M, Reuter C, Barone M, Soicke A, Roske Y, Piotukh K, autophagy under mechanical strain through spatial regulation of Huy P, Beerbaum M, Wiesner B, Beyermann M, Schmieder P, Freund mTORC1.Biochim Biophys Acta 1864, 62 – 75. C, Volkmer R, Oschkinat H, Schmalz H G, Kühne R (2015) A modular Eccles R L, Czajkowski M T, Barth C, Müller P M, McShane E, Grunwald S, toolkit to inhibit proline-rich motif-mediated protein-protein interac- Beaudette P, Mecklenburg N, Volkmer R, Zühlke K, Dittmar G, Selbach tions. Proc Natl Acad Sci U S A. 112, 5011 – 5016. M, Hammes A, Daumke O, Klussmann E, Urbé S, Rocks O (2016) Bimodal antagonism of PKA signalling by ARHGAP36. Nat Commun 7, 12963.

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 FMP authors 27, 3729 – 3745. Group members 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 116 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

ALL RESEARCH GROUPS ALLE FORSCHUNGSGRUPPEN

MOLECULAR PHYSIOLOGY AND CELL BIOLOGY STRUCTURAL BIOLOGY MOLEKULARE PHYSIOLOGIE UND ZELLBIOLOGIE STRUKTURBIOLOGIE

THOMAS J. JENTSCH ADAM LANGE

DEPARTMENTS DEPARTMENTS ABTEILUNGEN ABTEILUNGEN

Physiology and Pathology Molecular Pharmacology Molecular Biophysics of Ion Transport and Cell Biology Adam Lange Thomas J. Jentsch Volker Haucke

RESEARCH GROUPS RESEARCH GROUPS FORSCHUNGSGRUPPEN FORSCHUNGSGRUPPEN

Protein Trafficking Molecular Cell Physiology Solution NMR Ralf Schülein Ingolf E. Blasig Peter Schmieder

Computational Chemistry / Drug Design Ronald Kühne

JUNIOR RESEARCH GROUPS JUNIOR RESEARCH GROUPS JUNIOR FORSCHUNGSGRUPPEN JUNIOR FORSCHUNGSGRUPPEN

Molecular Neuroscience and Biophysics LIAISON GROUP NEUROSCIENCE In Cell-NMR Andrew Plested Behavioural Neurodynamics Philipp Selenko Tatiana Korotkova / Alexey Ponomarenko Membrane Traffic and Cell Motility Molecular Imaging Tanja Maritzen Leif Schröder LIAISON GROUP NEUROSCIENCE Proteostasis in Aging and Disease Molecular and Theoretical Neuroscience Janine Kirstein Alexander Walter

CORE FACILITIES CORE FACILITY

Cellular Imaging Animal Facility NMR Burkhard Wiesner / Dmytro Puchkov Natali Wisbrun Hartmut Oschkinat / Peter Schmieder APPENDIX ANHANG 117

CHEMICAL BIOLOGY CHEMISCHE BIOLOGIE

CHRISTIAN HACKENBERGER

DEPARTMENTS ABTEILUNGEN

NMR-Supported Structural Biology Chemical Biology II Chemical Biology I Hartmut Oschkinat (Leibniz-Humboldt) Dorothea Fiedler Christian Hackenberger

RESEARCH GROUPS FORSCHUNGSGRUPPEN

Structural Bioinformatics Peptide-Lipid-Interaction / Mass Spectrometry and Protein Design Peptide Transport Eberhard Krause Gerd Krause Margitta Dathe Medicinal Chemistry Marc Nazaré

CORE FACILITIES

Peptide Synthesis Screening Unit Christian Hackenberger / Rudolf Volkmer Jens Peter von Kries 118 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

CAMPUS BERLIN BUCH CAMPUS BERLIN BUCH

ROBERT-RÖSSLE-STR. 10 13125 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 Walter-Friedrich-House C 31 Max-Delbrück-House C 83 Max Delbrück Communications Center C 84 Hermann-von-Helmholtz-House A 10 Library B 63 Research services

Shared Facilities by MDC and FMP

C 84.1 Research services C 87 Timoféeff-Ressovsky-House

CLINICAL RESEARCH

COMMON FACILITIES

A 8 Gate House with Café Max A 9 Reception A 13 Life Science Learning Lab; Campus-Info-Center A 14 Cafeteria

Guesthouses of the MDC

B 54 Hans-Gummel-Guest House B 61 Kindergarten; Salvadore-Luria-Guest House

COMPANIES APPENDIX ANHANG 119 120 RESEARCH REPORT FORSCHUNGSBERICHT 2015 / 2016

ADMINISTRATIVE AND TECHNICAL SERVICES ADMINISTRATIVE UND TECHNISCHE DIENSTLEISTUNGEN

DIRECTORATE ADMINISTRATION TECHNICAL SERVICES

Prof. Dr. Dorothea Fiedler Frank Schilling Hans-Jürgen Mevert Director, Managing Director since 01 / 2017 Head Administration Marco Mussehl Prof. Dr. Volker Haucke Thomas Ellermann Holger Panzer Director, Managing Director until 12 / 2016 General Administration Michael Uschner († 03.12.2015) Dr. Henning Otto Marina Spors Dr. Elvira Rohde Personnel Management Stephanie Wendt Scientific Coordinators Christel Otto Roy Wolschke Silke Oßwald General Administration Pascal Schulz Public Relations Claudia Messing Bernd-Uwe Wagner Dr. Anne Höner General Administration EU-Liaison Officer (until 09 / 2016) Dennis Bischoff ANIMAL CARE OFFICER Dr. Franziska Ringleb General Administration EU-Liaison Officer (since 10 / 2016) Dr. Nadjeschda Heinrichs Gabriele Schumacher Dr. Birgit Oppmann Secretary Technology Transfer WORKPLACE SAFETY

Katrin Wittig COMPUTER SERVICES (IT) Dr. Jens Furkert PhD-Programme Coordinator Ronald Jäckel Alexandra Chylla Chief Information Officer (CIO), GENETIC ENGINEERING / Secretary System Administration BIOLOGICAL SAFETY

Heidemarie Petschick Ingrid Hermann Dr. Ralf Schülein Secretary System Administration

Ingo Breng OFFICES Service Engineer

Andrea Steuer Alexander Heyne Department of NMR-Supported Service Technician 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 IMPRINT IMPRESSUM

Leibniz-Forschungsinstitut für Molekulare Pharmakologie Research Report (FMP) im Forschungsverbund Berlin e.V. 2015 / 2016

Campus Berlin-Buch Editorial Board Robert-Rössle-Str. 10 Dorothea Fiedler, Christian Hackenberger, Volker Haucke, 13125 Berlin Thomas Jentsch, Adam Lange, Hartmut Oschkinat Germany Coordination Silke Oßwald Phone + 49 30 947 93 - 104 Fax + 49 30 947 93 - 109 Author Feature Articles and Interview E-mail [email protected] Beatrice Hamberger, Birgit Herden (p. 8)

Editing www.leibniz-fmp.de 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