Research Report 2003 I 2004

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2 RESEARCH REPORT I 3 0

0 2003 I 2004 2 T R O P E R H C R A E S E R . V . E N I L R E B D N U B R E V S G N U H C S R O

F FORSCHUNGSINSTITUT M

I FÜR MOLEKULARE PHARMAKOLOGIE E I

G IM FORSCHUNGSVERBUND BERLIN E.V. O L O K A M R A H P E R A L U K E L O M R Ü F T U T I T S N I S G N U H C S R O F

FORSCHUNGSINSTITUT FÜR MOLEKULARE PHARMAKOLOGIE

Campus Berlin-Buch Robert-Rössle-Str. 10 D-13125 Berlin fon: +49-(0)30-94793-100 fax: +49-(0)30-94793-109 e-mail: [email protected]

Outstation: Department of Molecular Genetics FEM Steglitz Krahmerstr. 6-10 D-12207 Berlin fon: +49-(0)30-8437-1910 fax: +49-(0)30-8437-1922

The Forschungsinstitut für Molekulare Pharmakologie (FMP) evolved out of the Institut für Wirkstoffforschung (IWF) of the Academy of Sciences of the GDR. Together with seven other research institutions, the institute is administratively organized within the Forschungsverbund Berlin e.V. The FMP is a member of the Leibniz Association.

Das Forschungsinstitut für Molekulare Pharmakologie ist aus dem Institut für Wirkstoffforschung (IWF) der Akademie der Wissenschaften der DDR hervor- gegangen. Es ist mit sieben weiteren Forschungsinstituten im Forschungs- verbund Berlin e.V. zusammengeschlossen. Das FMP ist ein Mitglied der Leibniz-Gemeinschaft.

RESEARCH REPORT 2003 / 2004 FORSCHUNGSINSTITUT Editorial Board: FÜR MOLEKULARE PHARMAKOLOGIE Walter Rosenthal, Michael Bienert, Ivan Horak, Hartmut Oschkinat IM FORSCHUNGSVERBUND BERLIN E.V.

Compilation & Editing: Björn Maul

Cover Design and Layout: Hoch3 GmbH, Berlin

Photos: Bernhard Schurian, Thomas Oberländer, Björn Maul, Dana Hausbeck FMP

Print: Druckhaus Berlin-Mitte

This Research Report is also available at www.fmp-berlin.de

Cover: The G-Protein coupled receptor GPR 109: binding site with ligand (nicotinic acid) SCIENTIFIC ADVISORY BOARD WISSENSCHAFTLICHER BEIRAT

Professor Dr. Rudolf Balling GBF - Gesellschaft für Biotechnologische Forschung mbH Mascheroder Weg 1 38124 Braunschweig

Professor Dr. Annette G. Beck-Sickinger Institut für Biochemie der Universität Leipzig Brüderstr. 34 04103 Leipzig

Professor Dr. Matthias Bräutigam Schering AG Müllerstr. 170-178 13342 Berlin

Professor Dr. Christian Griesinger Max-Planck-Institut für Biophysikalische Chemie Am Faßberg 11 37077 Göttingen

Professor Dr. Reinhard Jahn (Vorsitz) Max-Planck-Institut für Biophysikalische Chemie Am Faßberg 11 37077 Göttingen

Professor Dr. Hans-Georg Joost Deutsches Institut für Ernährungsforschung Arthur-Scheunert-Allee 114 14558 Potsdam-Rehbrücke

Professor Dr. Frauke Melchior Universitätsklinikum der Georg-August-Universität Göttingen Zentrum für Biochemie und Molekulare Zellbiologie Humboldt-Allee 23 37073 Göttingen

Professor Dr. Herbert Waldmann Max-Planck-Institut für Molekulare Physiologie The Scientific Advisory Board of the FMP formally consti- Otto-Hahn Str. 11 tuted itself at its meeting on November 6, 1992. At the 44202 Dortmund board meeting on November 16, 2000 Professor Reinhard Jahn was elected chairman.

Der Wissenschaftliche Beirat des FMP konstituierte sich anläßlich seiner Sitzung am 6. November 1992. Auf der Sit- zung am 16. November 2000 wurde Professor Dr. Reinhard Jahn zum Vorsitzenden gewählt. wissenschaftliche ArbeitamFMPistdieenge scher Biologiegeführthat.Kennzeichnendfürdie Genetik,Strukturbiologie und Chemi- duktion/Molekularer satz, derzurZusammenführungvonZellulärerSignaltrans- Das FMPverfolgteineninterdisziplinärenForschungsan- Vorfeld derEntwicklungvonArzneimittelnangesiedelt. entwickeln. DamitistdieForschungstätigkeitdesFMPim functions andinteractionsofproteins torische Chemie“geschaffen. Gleichzeitig wurdeeine der PeptidchemieeinneuerSchwerpunkt „Kombina- fung vonFMPundFreierUniversität Berlin)wurdeneben Professur fürMedizinischeChemie geprägt. DurchdieBerufungvonJörgRademannaufeine warendurchdenAusbau der Chemie Die Jahre2003/2004 MEILENSTEINE 2003/2004 men versuchtes,die grundsätzlicher Bedeutungzuerbringen.IndiesemRah- laren Pharmakologie.Dabeiistesbestrebt,Beiträgevon Das FMPbetreibtForschungaufdemGebietderMoleku- FOREWORD Rademann as the chemistrydepartment.WithappointmentofJörg The years2003/4werecharacterizedbytheexpansionof MILESTONES 2003/2004 I strives tomakecontributionsoffundamentalsignificance. pharmacology. Inaccordancewiththisobjective,theFMP The FMPundertakesresearchinthefieldofmolecular s work attheFMPisclose gy andchemicalbiology. Characteristicforthescientific genetics,structuralbiolo- signal transduction/molecular research approach,whichhasbroughttogethercellular d Thus, theresearchactivityofFMPisatforefront of new conceptsrelatingtotheirpharmacologicalimpact. nen vonEiweißen new importantareaofcompetence,combinatorialchemi- appointment bytheFMPandFreeUniversityBerlin),a fung vonChemieundBiologie zepte zuihrer n try andbiology rug development.TheFMPpursuesaninterdisciplinary this context,itisengagedinelucidatingthe Professor forMedicinalChemistry . pharmakologischen Beeinflussung (Proteinen) aufzuklärenundneueKon- Struktur, FunktionenundInteraktio- interrelationship ofchemi- . (gemeinsame Beru- and indeveloping s Verknüp- tructure, (joint zu dieses Typs verfügbar. Mittel(insge- Dieerforderlichen wicklung repräsentiert.Weltweitsind erstwenigeGeräte ragendes Ereigniswarauchdie Inbetriebnahmeeines Zentrum desFMPpersonellstarkerweitert.Einheraus- und Charité–UniversitätsmedizinBerlin)wurdedasNMR- ler Weise amAufbaueinesnationalenNetzwerks( betrachten. WissenschaftlerdesFMPsindauchinzentra- sie aberauchalsPrototypenneuartigerPharmakazu wichtige Werkzeuge fürdieForschungdar. Zugleichsind fahren identifizierenkann.DiesekleinenMolekülestellen eine biologischeWirkungentfalten,imHochdurchsatzver- die kleineMoleküle,anbestimmteProteinebindenund FMP dieersteakademischeEinrichtunginDeutschland, Jens PetervonKriesgewonnenwerden.Damitistdas und Screening-Technologie verfügt.AlsLeiterkonnte Mit derEinrichtung Screening-Technologie erhaltensollen. mische GruppenZugangzuSubstanzbibliothekenund MHz spectrometer Professor forDrugDesign NMR 0 MHz-Spektrometers 900 und derBerufungeinesLeiters,BerndReif,aufeine Screening Unit fessur fürDrugDesign With theestablishmentof stance librariesandscreeningtechnology. t standing eventwasalsothefirst-timeoperationof the NMRCenterofFMPwasgreatlyexpanded.Anout- and theCharité–UniversityMedicineBerlin),staffof P ce librariesandscreeningtechnologyavailable.Jens BioNet same timea stry, wasestablishedalongsidepeptidechemistry. Atthe national network( FMP scientistsarecentrallyinvolvedinbuildingupanew also beregardedasprototypesofnovelpharmaceuticals. important toolsforresearch.Atthesametimetheycan e that bindtospecificproteinsanddevelopabiological high throughputmethods,canidentifysmallmolecules thus thefirstacademicinstitutioninGermanywhich,using hrough whichacademicgroupscanobtainaccesstosub- ffect (pages92-95).Thesesmallmoleculesrepresent eter vonKriescouldberecruitedtodirectit.TheFMPis and theappointmentofgroupleaderBerndReifas ; www.chembionet.de) beteiligt,durchdasakade- Screening Unit aufgebaut, dieüberSubstanzbibliotheken , which representsthecurrentstate-of- ChemBioNet (gemeinsame BerufungvonFMP , Arbeitsgruppe Festkörper-NMR das denaktuellenStandderEnt- (joint appointmentoftheFMP was builtup,makingsubstan- research groupSolidState ; www.chembionet.de Chem- Pro- 900 ),

3 Foreword the-art development. Worldwide only a few devices of this type are in existence. The necessary funding (totaling 4.9 million ?) was provided by the BMBF (Federal Ministry of Education and Research) in the framework of a project grant and the Berlin Senate Department for Science, Research and Culture (EFRE funds). Thus an outstandingly well-equipped and well-staffed center for structural biology oriented NMR spectroscopy has been created that has an international reputation.

In June 2003 at a ceremony which included the governing mayor of Berlin, the cornerstone was laid for the new Medicinal Genomics Building. It is a joint initiative of the FMP and the Max Delbrück Center for Molecular Medicine (MDC). The striking building, designed by the Staab archi- tectural bureau, provides lab and office space for a staff of 120. The move into the new building will take place in autumn 2005. Of the 230 employees on the staff of the FMP, about 40 will work in the new building.

FOREWORD

samt 4,9 Mio ?) wurden vom BMBF im Rahmen der Pro- PERSPEKTIVEN jektförderung und der Berliner Senatsverwaltung für Wis- senschaft, Forschung und Kultur (EFRE-Mittel) zur Verfü- Arzneimittel entfalten ihre Wirkung durch Interaktion ihrer gung gestellt. Damit ist ein gerätemäßig und personell Inhaltsstoffe (Wirkstoffe) mit körpereigenen Strukturen. hervorragend ausgestattetes, international ausstrahlen- Bei diesen Andockstellen im Organismus (Zielstrukturen, des Zentrum für die strukturbiologisch orientierte NMR- „targets“) handelt es sich fast ausnahmslos um Proteine Spektroskopie etabliert. (> 99%). Bisher ist erst ein sehr kleiner Teil der Eiweiße des menschlichen Körpers als Zielstruktur für einen Wirkstoff Im Juni 2003 wurde gemeinsam mit dem Max-Delbrück- erschlossen (ca. 500). Sicher kommen nicht alle 100.000 Centrum für Molekulare Medizin (MDC) im Beisein des Eiweiße des Organismus als „targets“ für Wirkstoffe in Regierenden Bürgermeisters von Berlin der Grundstein für Frage. Man geht aber davon aus, dass zumindest einige den Neubau „Medizinische Genomik“ gelegt. Das mar- tausend geeignet sind. Damit stellt sich die Aufgabe, die kante, von dem Architektenbüro Staab entworfene Gebäu- derzeit schmale Basis der Arzneimitteltherapie durch de bietet 120 Mitarbeitern Labor- und Büroarbeitsplätze. Identifizierung neuer Zielstrukturen zu erweitern und so Der Bezug des neuen Gebäudes wird im Herbst 2005 erfol- bestehende Therapiekonzepte zu verbessern und neue zu gen. Von den 230 Mitarbeitern des FMP werden ca. 40 im etablieren. Hier sieht das FMP ein zentrales Tätigkeitsfeld. neuen Gebäude tätig sein. Zu den Proteingruppen, die gegenwärtig am FMP bearbei- tet werden, gehören als klassische „targets“ Membran- proteine wie Rezeptoren und Kanalproteine, aber auch Proteine, die bisher kaum als „targets“ in Betracht gezo- gen wurden. Zu dieser Gruppe gehören Transkriptions- faktoren, die Gene regulieren, Interaktionsdomänen von Proteinen und sogenannte Ankerproteine, die Proteine in belong till nowhavehardlybeenconsideredtargets.To thisgroup receptors andchannelproteins,butalsoproteinsthatup include asclassicaltargets tein groupsthatarecurrentlybeingprocessedattheFMP ones. HeretheFMPseesacentralfieldofactivity. Thepro- to improveexistingtherapyconceptsandestablishnew drug therapybyidentifyingnewtargetstructuresandthus r assumed thatatleastseveralthousandaresuitable.The- appropriate asatargetforsubstances.However, itis To besure, notall100,000proteinsoftheorganismare p drugs havebeendevelopedforonlyasmallnumberofthe without exceptioninvolvesproteins(>99%).Untilnow, action domains schläge zuneuen„targets“mit Grundlagenforschung aufzugeben.EsistkannnunVor- erweitert, ohneallerdingsseinefesteVerankerung inder Arzneimitteln erheblichinRichtungAnwendung führt, tenz innerhalbderWertschöpfungskette, die zuneuen Einrichtung derScreeningUnithatdasFMPseineKompe- kung derchemischenForschungundbesondersdurchdie tischer FirmeninsAuslandzubeobachten.DurchdieStär- Verlagerung derForschungsabteilungenpharmazeu- lung vorausgeht,zurück.Außerdemistseitlangemeine der „high-risk“Forschung,dieArzneimittelentwick- h.aus Forschung imVorfeld eines„proofofprinciple“,d. Pharmazeutische Firmenziehensichimmermehrausder heimerschen Erkrankungvermehrtauftreten). pharmazeutischen Industriezuschließen unddamitdas richtungen undderArzneimittelentwicklung inder logischer Grundlagenforschung indenForschungsein- form dazubeiträgt,dieLücke zwischenpharmako- präsentieren. Wirhoffen,dassdieErweiterungderPlatt- kologische Charakterisierungpotentieller„targets“ fügen, sowieextrazelluläre der ZellezugrößerenfunktionellenKomplexenzusammen- these dockingsitesintheorganism( t throughinteractionoftheircon- Drugs achievetheireffect P kandidaten ents (substances)withthebody’s ownstructures.At efore, thetaskistoexpandpresentnarrowbaseof roteins ofthehumanbody(approx.500). ERSPECTIVES transcription factors verbinden undevtl.aucheineerstepharma- of proteinsandso-called Fibrillen , membrane proteins which regulategenes, Vorschlägen zuWirkstoff- . dez B.beiderAlz- (die z. targets anchor proteins ), thisalmost such as inter- , nal complexes,aswellextracellular which assembletheproteinsincelltolargerfunctio- „ChemBioNet“ burg“ suggestions for Now itcancombinesuggestionsfornew“targets”with without relinquishingitsfirmanchoringinbasicresearch. that leadstonewdrugswithregardapplication,yet e blishment oftheScreeningUnit,FMPhasconsiderably ning ofchemicalresearchandparticularlyduetotheesta- nies havebeenrelocatingabroad.Duetothestrengthe- that theresearchdepartmentsofpharmaceuticalcompa- l i.e. from“high-risk”researchwhichprecedesdrugdeve- ofprinciple“, more outofresearchpreparatorytoa“proof Pharmaceutical companiesarewithdrawingmoreand are increasinglyobservedwithAlzheimer’s disease). in thepharmaceuticalindustry, andthusthattheenviron- research intheinstitutesanddrugdevelopment help closethegapbetweenpharmacologicalbasic “targets”. We hopethattheextensionofplatformwill sent afirstpharmacologicalcharacterizationofpotential das FMPkonstruktiv-kritischbegleitet hat. richtet sichauchandenwissenschaftlichenBeirat,der herzlich fürihrEngagementbedankenmöchte.MeinDank Mitarbeiter desFMPzuzuschreiben,beidenenichmich Der ErfolgderletztenJahreistdenMitarbeiterinnenund den verschiedenerDisziplinendargestellt. die BeteiligungdesFMPanderAusbildungvonStudieren- Publikationstätigkeit, dieEinwerbungvonDrittmittelnund Leistungsbilanz fürdenBerichtszeitraum.Sowirddie Erenthältaber auchdie trum diesesBerichtes2003/2004. Arbeitsgruppen überihrewissenschaftlicheArbeitimZen- Wie indenvergangenenJahrenstehenBeiträgeder werks lich beteiligtist.BeispielesinddieEtablierungeinesNetz- Netzwerkinitiativen dienen,andenendasFMPmaßgeb- zu gestalten.DiesemZielsollenauchdieverschiedenen Umfeld fürUnternehmendesPharma-Brancheattraktiver opment. Moreover, foralongtimeithasbeenobserved xpanded itscompetencewithinthevaluecreationchain „Arzneimittelentwicklung inBerlinundBranden- und diebereitserwähntenationaleInitiative . usac candidates substance and possiblypre- fibrils (which e.g.

5 Foreword ment for companies in the pharma sector will become I would like to invite readers to take an informative and more attractive. Various network initiatives in which the interesting tour through the pages of this report to see how FMP plays a major role shall also serve this goal. Exam- the FMP has developed since its founding 12 years ago. ples are the establishment of a network “Drug Develop- ment in Berlin und Brandenburg” and the already mentioned national initiative “ChemBioNet”.

As in past years, the contributions of the various research groups about their scientific work stand in the center of Walter Rosenthal, Director this research report 2003/2004. However, it also contains a performance account for the period of the report. Thus, publication activity, solicitation of third-party funds and the participation of the FMP in the education and training of students of the various disciplines are also presented.

The success of the last years is due to the colleagues of the FMP, whom I would like to thank very much for their dedication. My thanks also go to the Scientific Advisory Board for its constructive and critical support of the FMP.

FOREWORD

Ich wünsche den Lesern einen aufschlussreichen und interessanten Streifzug durch das FMP, wie es sich 12 Jahre nach seiner Neugründung darstellt.

Walter Rosenthal, Direktor April 2005 Scientific andtechnicalservices/ Section ChemicalBiology/ MolekulareGenetik Bereich Signaltransduktion/ MolecularGenetics/ / Section CellularSignalling Section StructuralBiology/ F Scientific AdvisoryBoard/ C oreword / ONTENTS Public RelationsandtheMedia/ 101 ...... DNA Sequencing 98 Microdialysis ...... 92 ...... Screening Unit 89 ...... Medicinal Chemistry 86 ...... Synthetic OrganicBiochemistry 83 ...... Mass Spectrometry 80 ...... Peptide Biochemistry 75 Transport...... Peptide LipidInteraction/Peptide 72 ...... Peptide Synthesis 70 Introduction ...... 65 ...... Cellular SignalProcessing 64 Mouse Models...... 62 ...... Molecular Myelopoiesis 59 ...... Cytokine Signaling 56 Biophysics ...... 52 ...... Biochemical Neurobiology 49 ...... Molecular CellPhysiology 46 ...... Cellular Imaging 42 ...... Anchored Signalling 39 ...... Protein Trafficking 36 Introduction ...... P 28 ...... Solid StateNMR 24 ...... Molecular Modelling 20 ...... Structural Bioinformatics 16 Solution NMR...... P 10 Introduction ...... Computer Services/ Administration / oenEgneig...... 31 ...... rotein Engineering 12 ...... rotein Structure Vorwort / INHALT ...... 3 ...... Verwaltung Computer-Service Wissenschaftlicher Beirat Bereich ChemischeBiologie Bereich Strukturbiologie ...... 106 ...... Wissenschaftlicher undtechnischerService Presse- undÖffentlichkeitsarbeit ...... 108 ...... 2 ...... 102 ......

7 Contents Appendix / Anhang Peer reviewed Articles 2003 / Originalarbeiten ...... 110 Peer reviewed Articles 2004 / Originalarbeiten ...... 114 Reviews 2003/2004 / Übersichtsarbeiten ...... 121 Contributions in Monographs 2003/2004 / Beiträge zu Sammelwerken ...... 121 Monographs 2003/2004 / Monographien ...... 122 Memberships in Editorial Boards 2003/2004 / Mitgliedschaften in Editorial Boards ...... 123 Invited Talks 2003/2004 / Eingeladene Vorträge ...... 123 External funding 2003/2004 / Drittmittel...... 128 Participation in Research Networks 2003/2004 / Beteiligung an Netzwerken und Verbundprojekten ...... 130 Cooperations with contract 2003/2004 / Vertragliche Kooperationen...... 132 Meetings, Workshops, Symposia 2003/2004 / Wissenschaftliche Veranstaltungen...... 134 Work in Panels 2003/2004 / Gremienarbeit...... 135 Review Activities 2003/2004 / Gutachtertätigkeit ...... 135 Academic Teaching 2003 / 2004 / Lehre...... 138 Calls for Appointments 2003/2004 / Rufe...... 141 Post-Doctoral Lecture Qualifications 2003/2004 / Habilitationen ...... 141 Graduations 2003/2004 / Promotionen ...... 142 Diploma theses 2003/2004 / Diplomarbeiten ...... 143 Internships 2003/2004 / Praktikanten ...... 144 Guest Scientists 2003/2004 / Gastwissenschaftler ...... 148 Lectures at the FMP 2003 / Kolloquien und Seminare am FMP ...... 149 Lectures at the FMP 2004 / Kolloquien und Seminare am FMP ...... 153 Technology Transfer 2003/2004 / Technologietransfer ...... 156 Structure of the Forschungsinstitut für Molekulare Pharmakologie (FMP) / Organigramm ...... 158 Index / Namensregister ...... 160 Maps / Lagepläne ...... 164

Flip-book Daumenkino STRUCTURAL BIOLOGY SECTION STRUCTURAL BIOLOGY Prof. Hartmut Oschkinat, Department Head: NMR-supported Structural Biology (Secretary: Andrea Steuer)

The research themes of the section cover structural and and amyloid formation. There are strong links to the functional studies of receptors and proteins involved in screening facility of the FMP through the molecular intracellular signalling, and projects on the biophysical modellling and NMR aspects of small-molecule inhibitor description of protein aggregate formation. Strong empha- development. sis is thereby put on a structural characterization of membrane-integrated receptors, channels and fibril-forming The department hosts expertise in the areas of structural systems, in combination with exploring the potential of bioinformatics, molecular biology, solution and solid-state solid-state magic angle spinning NMR for studying such NMR spectroscopy, and molecular modelling. NMR systems. As a special theme with regard to intracellular spectroscopy is used as the major tool for determining signalling, non-catalytical protein domains that mediate structures of biological macromolecules in solution, and interactions with linear peptide segments in target proof quasi-solid preparations which are difficult to crystalli- teins are analyzed with respect to specificity and affinity- ze, like amyloid fibrils and membrane proteins in native- determining sequence features. A growing part of the like lipids. It is particularly well suited for studying the research is the systematic derivation of small organic dynamics of protein structures and interactions with very compounds as modulators of protein-protein interactions weakly binding ligands. The various skills are contained in

INTRODUCTION

BEREICH STRUKTURBIOLOGIE Prof. Hartmut Oschkinat, Abteilungsleiter: NMR-unterstützte Strukturforschung (Sekretariat: Andrea Steuer)

Der Bereich Strukturbiologie erforscht Struktur und Funk- lierung und NMR-Aspekte der Entwicklung niedermoleku- tion von Rezeptoren und Proteinen, die an der intrazel- larer Hemmstoffe. lulären Signalübertragung beteiligt sind, sowie die Ent- stehung von Proteinaggregaten. Im Mittelpunkt des Der Bereich kann Expertise auf den Gebieten strukturelle Interesses steht die strukturelle Charakterisierung von Bioinformatik, Molekularbiologie, Flüssigkeits- und Fest- Rezeptoren, Kanälen und fibrillenbildenen Systemen in körper-NMR-Spektroskopie sowie Molekülmodellierung Kombination mit der Erforschung des Potentials der vorweisen. Hauptsächlich mittels NMR-Spektroskopie „magic-angle-spinning“ Festphasen-NMR für das Studium werden Strukturen biologischer Makromoleküle in Lösung solcher Systeme. Im Hinblick auf die intrazelluläre Signal- und in Präparationen an der Festkörperphase bestimmt übertragung wird besonderes Augenmerk auf die spezifi- (Amyloidfibrillen und Membranproteine in natürlichen täts- und affinitätsbestimmenden Eigenschaften nicht- Lipiden). NMR-Spektroskopie ist besonders geeignet für katalytischer Proteindomänen gelegt. Diese vermitteln die Studien zur Dynamik von Proteinen und Interaktionen mit Interaktionen mit linearen Peptidsegmenten in Zielprotei- sehr schwach bindenden Liganden. Gegenwärtig sechs nen. Zunehmend rückt die systematische Derivatisierung Arbeitsgruppen bieten wissenschaftliche Kompetenz in kleiner organischer Moleküle als Modulatoren von Pro- Protein Engineering (Christian Freund), Molekülmodellie- tein-Protein-Interaktionen und der Amyloid-Bildung in den rung (Ronald Kühne), Struktureller Bioinformatik (Gerd Mittelpunkt des Interesses. Wichtige inhaltliche Brücke Krause), Proteinstruktur (Hartmut Oschkinat), Festkörper- zum Screening-Labor des FMP sind die Molekülmodel- NMR (Bernd Reif) und Lösungs-NMR (Peter Schmieder). currently six research groups, Protein Engineering (Christian Freund), Molecular Modelling (Ronald Kühne), Structural Bioinformatics (Gerd Krause), Protein Structure (Hartmut Oschkinat), Solid State NMR (Bernd Reif) and Solution NMR (Peter Schmieder). The instrumentation includes a variety of solution and solid-state NMR spectrometers ranging from 400 to 900 MHz, and equipment for analytical ultracentrifugation and isothermal calorimetry. In the past two years, the direction of research has been shifted stronger towards solid-state NMR and its applications, resulting in the installation of a new group focused on solid-state NMR and amyloid-forming systems. Further- more, a 700 MHz wide bore NMR spectrometer was pur- chased. y g o l o i B l a r u t c u r t S 1 1

Der Bereich ist mit einer Vielzahl von Lösungs- und Fest- körper-NMR-Spektrometern (400 bis 900 MHz) sowie Gerä- ten zur analytischen Ultrazentrifugation und isothermi- schen Kalorimetrie ausgestattet. In den zurückliegenden zwei Jahren hat sich der Fokus der Forschung stärker in Richtung Festkörper-NMR und deren Anwendungen ver- schoben. Dies führte zur Ansiedlung einer neuen Arbeits- gruppe, die sich mit Festkörper-NMR und Amyloid-bilden- den Systemen befasst. Außerdem wurde ein 700 MHz-„wide bore“-NMR-Spektrometer in Betrieb genom- men. PROTEIN STRUCTURE plete ligand sequence space for the three PDZ domains Group Leader: Prof. Hartmut Oschkinat made it feasible to compare their ligand selectivity and the overlap of their recognized ligand sequence subspaces. STRUCTURAL CHARACTERIZATION OF PROTEIN-PROTEIN-INTERACTIONS NMR structure determination in a structural genomics context The main focus of our group is the structural characteri- As a part of the protein structure factory project, a num- zation of protein-protein interactions responsible for the ber of protein structures were generated, including those reception and transduction of signals in biological of the subunit B8 of complex I, of the p47 SEP domain, and systems. This research concerns protein domains which of a BRCT domain. recognise specific peptides and on the long run investigations on membrane integrated receptors and receptor- The solution structure of the subunit B8 from ubiquinone ligand complexes. It is embedded into efforts towards oxidoreductase (Complex I) (CI-B8) shows a thioredoxin structural genomics of soluble and membrane proteins fold (Fig. 1) with remarkable similarities to thioredoxin-like and supported by NMR-technical developments. The Fe2S2-ferredoxins. A detailed comparison to these pro- technical developments aim at higher throughput and teins show remarkably high similarities of surface proper- towards concepts for structure determination of membra- ties and possibly catalytically active residues. The redox- ne proteins by solid-state NMR. potential of the disulfide-bond is comparable to that of the catalytically active disulfides in thioredoxin-like proteins. This led to the hypothesis that this subunit may be invol- Structure and function of non-catalytic protein ved in the regulation of complex I. domains The BRCT domain occurs in a number of different signal Non-catalytic protein domains mediate protein-protein transduction proteins involved in transcriptional regula- interactions through the recognition of peptide segments tion, DNA repair, cell cycle progression and cancer sup- in a highly specific manner. Such interactions govern the pression. The C-terminal region of the breast cancer asso- reversible assembly of macromolecular signalling comple- ciated tumor suppressor protein BRCA1 consists of two xes which finally form a logical network of interacting pro- successive BRCT domains, separated by a short linker. We teins, thereby transmitting information. Our investigations have, in collaboration with the group of Udo Heinemann at on non-catalytic protein domains typically involve a com- the MDC, Berlin-Buch, solved the solution structure of bination of structural and functional studies, e.g. by using BRCT-c, the C-terminal BRCT domain of BRCA1. Analysis peptide libraries, NMR spectroscopy and a variety of other of the structure and comparison with other members of the biophysical techniques, aiming at an understanding of BRCT superfamily has revealed that only a small number of molecular recognition and drug design. conserved residues are necessary for the formation and We have determined the structures of a number of WW stabilization of the BRCT motif. domain-peptide complexes, and the structure of an SH3 The structure of the SEP domain of the protein p47 was domain in complex with a canonical and a non-canonical determined and a hypothesis as to its function derived and peptide revealing two different binding sites. Intense stu- verified. A particular feature of this fold is the conserva- dies with PDZ and WW domains involving NMR and pep- tion of residues in two loops, and of aromatic residues very tide library screens where set out to derive structure-acti- close to it (Fig. 2). These loops are remarkably rigid, and vity-relationships and to provide data for a theoretical the upper part of the structure in Fig. 2 resembles cystei- analysis of domain specificity. We quantitatively determin- ne protease inhibitors like cystatins and stefins. An assay ed the specificity profiles of three representative PDZ showed a weak inhibition of cathepsin L by the p47 SEP domains from the AF6, ERBIN and SNA1 proteins, with domain. respect to the complete ligand sequence space of C-terminal peptides. This quantification was achieved by combi- Toward structure determination of membrane ning efficiently dissociation constant measurements and proteins by NMR statistical analysis of synthetic peptide libraries, using an Analysis of Variance (ANOVA) approach. Predicted in vitro Major forces were concentrated on the further develop- affinities were validated by designing super-binding pep- ment of a structure determination concept for proteins tides which indeed revealed the lowest Kd values and the using magic-angle spinning solid-state NMR. Such a highest PDZ domain specificity. The coverage of the com- method would be very welcome for systems which are assignments forall mination. Atthebeginningofstudyresonance which yieldssuitableNMRspectraforstructuredeter- the various membraneproteins,amicrocrystallinesampleof proteins likeactinfilaments,forexample.Alongwith like membraneproteins,amyloids,andstructureforming difficult tocrystallizeandoutofrangeforsolutionNMR, Ribbon diagramofsubunitB8(left)fr F carbon-carbon and6 First stepstowardsmembraneproteins weretakenusing loid systemsiftheproteinscanbe expressedinbacteria. widely applicabletosmallmembrane proteinsandamy- solid-state MASNMR.Thederived procedureshouldbe ture ofthe ture calculations.We theoriginal struc- havenowrefined number oflong-rangerestrainsthatwereusedforstruc- ved fromthosesamplesshowedadramaticallyincreased The respectiveprotondrivenspindiffusionspectrarecei- either 1,3- of biosyntheticallysitedirectedlabelledsamplesbyusing determined. Akeyaspectofthisstudywasthegeneration achieved assignmentsafirststructureofproteinwas later alsoofmostexchangeableprotons.Basedonthe ved, followedbyanassignmentofnon-exchangeable,and IGURE 1 α -spectrin SH3domainisstudiedasamodelsystem 13 α C-glycerol or2- -spectrin SH3domainusing889interresidue 13 C 15 and N- 13 15 C-glycerol ascarbonsources. 15 N N om ubiquinoneoxidoreductase(ComplexI,right) restraints, allobtainedby resonances wereachie- Christian Köhler(Doctoralstudent) Mangesh Joshi(Doctoralstudent) Henrik Holtmann(Doctoralstudent) Matthias Hiller(Doctoralstudent) Michele Fossi(Doctoralstudent) Federica Castellani(Doctoralstudent) Christoph Brockmann(Doctoralstudent) Prisca Boisguerin(Doctoralstudent) Dr. BarthvanRossum Dr. RicardoPires Dr. DietmarLeitner Dr. DirkLabudde Dr. LudwigKrabben Dr. JeremyFlinders Dr. KatjaFälber Dr. AnneDiehl Dr. LindaBall Group members is underway. remarkably sharplines,andanevaluationofthespectra choline receptorasatestsystem.Thespectrashow the snaketoxinneurotoxinIIboundtonicotinicacetyl

13 Structural Biology B

C C

N N

FIGURE 2 Structural ensemble of the p47 SEP domain. The residues with side chains displayed in black and green are highly conserved.

Thien-Thi Mac (Doctoral student) Deutsche Forschungsgemeinschaft Vivien Lange (Doctoral student) „Schlüsselreaktionen der biologischen Wasserstoffakti- Doreen Pahlke (Doctoral student) vierung am Beispiel der [NiFe]-Hydrogenasen“ (Teilprojekt Ilja Poliakov (Doctoral student) C1 im Sonderforschungsbereich 498 “Protein-Kofaktor- Nikolaj Schröder (Doctoral student) Wechselwirkungen in biologischen Prozessen“) Michael Soukenik (Doctoral student) Hartmut Oschkinat, Bärbel Friedrich (Humboldt-Universi- Carolyn Vargas (Doctoral student) tät zu Berlin) Urs Wiedemann (Doctoral student) Deutsche Forschungsgemeinschaft Stefan Jehle (Student) „Bestimmung der Raumstrukturen von Rezeptor-gebunde- Heide Evers (Technical assistance) nen Agonisten und Antagonisten mittels Festkörper-NMR- Lilo Handel (Technical assistance) Spektroskopie“ (Teilprojekt B1 im Sonderforschungsbe- Martina Leidert (Technical assistance) reich 449 „Struktur und Funktion membranständiger Kristina Rehbein (Technical assistance) Rezeptoren“) Hartmut Oschkinat External funding Deutsche Forschungsgemeinschaft Deutsche Forschungsgemeinschaft „Theoriegestützte NMR-spektroskopische Analyse von „Struktur und Mechanismus der 3,4-Dihydroxy-2-butanon- Protein-Ligand-Wechselwirkungen unter Verwendung von 4-phosphat-Synthase“ (OS 106/ 4-2) Peptidbibliotheken“ (Teilprojekt in der Forschergruppe 299 Hartmut Oschkinat „Optimierte molekulare Bibliotheken zum Studium biologi- Deutsche Forschungsgemeinschaft scher Erkennungsprozesse“) „Analyse von essentiellen WW-Domänen in ß-sheet- Hartmut Oschkinat, Michael Bienert Strukturen am Beispiel der WW-Domäne mittes Einbaus nicht natürlicher Aminosäuren“ (OS 106/5-1,5-2) Hartmut Oschkinat shows atyrosineconformationthat mayexplainitsredu- The solutionstructureoftheN-terminal domainofE3L Hartmut Oschkinat glycans inarterialdisease”(BMH4-98-3289) „Interactions betweengrowthfactorsandglycosamino- European Community Hartmut Oschkinat phenotypes” (QLK3-2000-00924) to profilecancercellsandinterferewithcancer-related “Exploiting syntheticsSH2-scaffoldedrepertoirelibraries European Community Hartmut Oschkinat,JensPetervonKries zur SuchenachWirkstoffen“ tuberculosis-Zielproteinen undihrerLigandenkomplexe „Hochdurchsatz-Strukturanalyse vonMycobacterium- haben „Strukturproteomik“–KonsortiumHamburg: ten Wirkstoffdesign“(0312992J,Teilprojekt imVerbundvor- „Screening vonSubstanzbibliothekenundStruktur-basier- Bundesministerium fürBildungundForschung Hartmut Oschkinat Proteine –ProAMP“) im Verbund „ProteomweiteAnalysemembrangebundener “Festkörper-NMR-Spektroskopie“ (0312890G,Teilprojekt Bundesministerium fürBildungundForschung für dieProteomforschung“(0312832) „Aufbau einerTechnologieplattform NMR-Messtechnik B Hartmut Oschkinat strukturfabrik) relevante (01GG9812,Protein- Proteine“ BMBF-Leitprojekt „ Bundesministerium fürBildungundForschung Hartmut Oschkinat M, YG, Kahmann JD,Wecking DA,PutterV, LowenhauptK,Kim propensities of42WWdomains.ProtSci12,491-500 activity relationshipsidentifiedusingligandrecognition Selected publications schergruppe 475“BildungundStabilitätvon n Strukturen amBeispielderWW-Domäne mittelsEinbau „Analyse vonessentiellenWechselwirkungen inß-sheet- Deutsche Forschungsgemeinschaft Otte L, Mergener J, Hartmut Oschkinat Strukturanalyse mithohemDurchsatzfürmedizinisch icht natürlicherAminosäuren“(Teilprojekt inderFor- undesministerium fürBildungundForschung Schmieder P, KrauseG Schmieder P, OschkinatH Wiedemann U,SchlegelB,PiresJR,Beyermann Oschkinat H , (2003) WWdomainsequence Volkmer-Engert R,Schneider- (FMP authorsinbold) , Rich A,SchadeM(2004) β -Faltblättern) 2712-2717 ced affinitytoZ-DNAinvitro.ProcNatlAcadSciUSA101, Rudolf Volkmer-Engert, Berlin Jens SchneiderMergener, Berlin Michael Nilges,Paris Dieter Oesterhelt,Martinsried Klaus-Peter Hofmann,Berlin Udo Heinemann,Berlin Robert G.Griffin,Cambridge,USA Paul Gooley, Melbourne Alan Fersht,FRS,Cambridge,UK Adelbert Bacher, Garching Collaborations Wahl M,KühneR,OschkinatH G P B 1 Cancer-Associated ProteinBRCA1.Biochemistry43, Behavior oftheC-Terminal theBreast BRCTDomainfrom tion Structure,BackboneDynamics,andAssociation Structure 12,1645-1654 unit B8fromhumancomplexIadoptsathioredoxinfold. 5983-15995 , rockmann C,DiehlA,RehbeinK,StraussH,Schmieder aiser OJ,BallLJ,SchmiederP, LeitnerD,StraussH, Korn B, Kühne R , Oschkinat H , Heinemann U(2004)Solu- (2004) Theoxidizedsub-

15 Structural Biology SOLUTION NMR level of Pfr. The difference between the Pr and Pfr is attribu- Group Leader: Dr. Peter Schmieder ted to the Z-E-isomerization of a double bond within the chromophore, as suggested from NMR data from isolated APPLICATIONS OF SOLUTION STATE NMR chromopeptides. TO ADDRESS BIOLOGICALLY IMPORTANT With the discovery of Cph1 in the cyanobacterium Syn- QUESTIONS echocystis sp. PCC 6803 it was realized that phytochromes Peter Schmieder, Holger Strauss, Christian Appelt, Janina are also present in prokaryotes. The native chromophore Hahn, Brigitte Schlegel of Cph1 in Synechocystis as well as in many other cyanobacterial phytochromes is phycocyanobilin. Subsequent- NMR spectroscopy is an important technique to address ly, phytochromes have also been detected in other cyano- numerous questions regarding the structure of various bacteria and even non-photosynthetic bacteria, where the kinds of molecules, including their three-dimensional chromophore frequently is biliverdine. Heterologous ex- structure. Solution-state NMR is well established as a tool pression of phytochromes in E. coli yields apoproteins that to obtain the three-dimensional structure at atomic reso- are usually capable of self-assembling with the chromo- lution for soluble biomolecules. Solid-state NMR is phore to become red/farred photochromic holoproteins. A currently emerging as a technique to obtain structural deeper understanding of the photochromic mechanism of information, where solution-state NMR is becoming phytochrome proteins requires detailed knowledge of the exceedingly difficult, namely in the case of membrane pro- structure of the chromophore pocket. No structural infor- teins. mation at atomic resolution, however, is available to date. NMR spectroscopy, preferably in solution, can also provi- We want to use solution state NMR spectroscopy to obtain de valuable information on the interaction of proteins and structural information on the chromophore binding pocke ligands, either in the case of a particular ligand of interest in the two parent states (P and P ). To separate the signals or in the case of large libraries used in screening proto- r fr of the chromophore from those of the protein, deuteration cols. Last but not least, NMR is extremely important for the of the protein is one approach. Since the chromophore is determination of the constitution of smaller molecules. relatively small compared to the protein, labeling of PCB In our group we use the full repertoire of solution-state with nitrogen and carbon is also necessary. While the pro- NMR techniques in conjunction with a variety of labeling duction of proteins in isotopically-labeled form is a well- patterns to address questions of biological and pharma- established procedure and while the same is also possi- cological importance. These range from the development ble with DNA and RNA, the production of other types of of new techniques for solution-state NMR to the elucida- molecules enriched with 13C and/or 15N is usually prohibi- tion of the constitution of biologically active peptides and tively expensive. In the case of the PCB chromophore the determination of the three-dimensional structure of necessary for the present investigation, the problem is peptides and proteins. In addition, NMR spectroscopy is somewhat ameliorated by the fact that PCB is the chromo- offered as a tool to researchers and companies throug- phore in the light-harvesting antenna of photosynthetic hout the Berlin area. bacteria and that the phycobilisome which contains the chromophore molecules represents a major fraction of the soluble proteins in cyanobacteria. We therefore decided Structure of the chromophore-binding pocket of to produce isotopically-labeled PCB using Synechocystis the cyanobacterial phytochrome Cph1 sp. PCC 6803 itself (Figure 1).

Phytochrome photoreceptors control numerous develop- Using the different labeling pattern, it was possible to mental processes in all plants. Light is absorbed by a cova- obtain NMR spectra of sufficient quality to extract structu- lently bound tetrapyrrole chromophore, phytochromobilin ral information. By recording one-dimensional 15N-spectra, (PΦB), which is incorporated autocatalytically into a chro- we could solve the much-discussed question whether the mophore-binding domain in the N-terminal sensory modu- chromophore is protonated in the two parent states, which le of the protein. The physiological function of phytochro- has strong implications on the reactions taking place in the me is associated with the photochemical production of the phytochrome photocycle. The nitrogen chemical shifts thermodynamically stable Pfr signaling state following light obtained indicate that both the Pr and the Pfr form are in absorption by the Pr- ground state (λmax ~660 nm). The Pfr fact protonated. Using carbon-labeled PCB, three-dimen- form itself is photoactive (λmax ~730 nm), quantum absorp- sional edited NOESY spectra could be obtained that will tion leading to the production of Pr. In daylight the photo- yield an assignment of the chromophore resonances and cycle establishes a dynamic equilibrium with a constant will allow the selection of a most likely structure from ted usingmethanol(c). tely labeledmedium(a).Afterharvestingthecells,phycobilisomeisseparatedbyasucrosedensitycentrifugation(b).Finally, PCBisextrac- usually altertheantimicrobial activity ofthepeptides ment ofL-aminoacidswiththeir D-enantiomersdoesnot mechanism ofactionislargelyunknown. Sinceareplace- Despite agrowinginterestinthis typeofpeptides,their generation. since theyhavethepotentialtoformanentirelynewdrug stantly increasing,thesepeptideshavegainedininterest Since bacterialresistancetoexistingantibioticsiscon- cific immuneresponse,whichoccursrelativelyslowly. ple peptidesispresent,supplementingthepathogen-spe- evolutionary ancientweapons.Usuallyacocktailofmulti- bacteria andfungiisaffectedbythesepeptides,whichare organisms thatinvolvesgram-positive,gram-negative system ofmanylivingorganisms.Abroadrangemicro- Antimicrobial peptidesarepartofthenaturalimmune brane-mimicking environment Structure ofantimicrobialpeptidesinamem- residues liningthebindingpocket. interactions betweenthechromophoreandaminoacids ated aminoacidsintoadeuteratedbackgroundtodetect chromophore-binding pocketistheintroductionofproton- ofthe still inprogress,anextsteptowardsstructure several structuralmodels.Ananalysisofthosespectrais P FIGURE 1 reparation of 1 5 N and 1 3 C , 1 5 N -labeled Phycocyanobilin(PCB)from S ynechocystis nearly identical,resemblingtwo res intheneutralandnegativelychargedmicellesare the peptideisboundtoadetergentmicelle.Thestructu- structure inwater, butadistinctstructureisformedwhen peptide showsaratherflexiblebutneverthelessordered bound todetergentmicellesusingNMRspectroscopy. The lic part.Theorientationofthepeptide inthemicellewas molecule withthepeptidebackbone formingthehydrophi- of theaminoacidsidechains creates anamphipathic peptide mined thestructureofcyclic,cationicantimicrobial antimicrobial peptidesattheatomiclevel,wehavedeter- In ordertogaininsightintothemechanismofaction the selectivity. It isassumedthatthenegativechargeresponsiblefor tidylcholine andisthuschargeneutralatphysiologicalpH. of mammaliancellmembranescontainsmostlyphospha- polysaccharide, respectively. Incontrast,theouterleaflet charged, consistingmainlyofphosphatidylglycerolorlipo- positive aswellgram-negativebacteriaarenegatively microbial assault.Theoutermembraneleafletsofgram- terial membranethatisthemostlikelytargetofanti- a unless theoverallstructureisdisrupted,amechanismvia specific proteinreceptorisunlikely. Itisratherthebac- s p. PCC6803:Thecyanobacteriaaregrownontheappropria- cyc- (RRWWRF) freeinaqueoussolutionand β -turns. Theorientation

17 Structural Biology FIGURE 2 Structure of the cyclic peptide cyc-(RRWWRF) bound to DPC micelles. The molecule exhibits an amphipatic structure with the peptide backbone as the hydrophilic part (blue) and the aromatic side chains as the lipophilic part (brown). The orientation of the peptide has been determined to be parallel to the micelle surface with the longest axis, with the lipophilic side chains penetrating into the micelle.

determined using NOEs and the accessibility of the pepti- Group members de from outside the micelle as probed using paramagnetic Holger Strauss (Doctoral student) agents. The peptide is oriented mainly parallel to the Christian Appelt (Doctoral student) micelle surface in both detergents. Substitution of the argi- Janina Hahn (Doctoral student)* nine and tryptophan residues is known to influence the Brigitte Schlegel (Technical assistance) antimicrobial activity. Therefore, the structure of the micelle-bound analogs cyclo(RRYYRF), cyclo(KKWWKF) and External funding cyclo(RRNalNalRF) were determined, having remarkable similarities in backbone conformation and side-chain ori- Deutsche Forschungsgemeinschaft entation. Based on these structures, molecular dynamics „NMR-spectroscopic investigation of light-induced struc- simulations of the peptide in an explicit membrane at tural changes in protein-chromophore complexes” (Teil- various peptide-to-lipid ratio were performed. We observe projekt B6 im Sonderforschungsbereich 498 „Protein- that the NMR structure of the peptide is stable also after Kofaktor-Wechselwirkungen in biologischen Prozessen“ 100 ns simulation. At a peptide-to-lipid ratio of 2/128, the Peter Schmieder membrane is only little affected compared to a pure DPPC Fonds der Chemischen Industrie lipid membrane, but at a ratio of 12/128, the water-lipid „NMR-spektroskopische Strukturuntersuchung von pho- interface becomes more fuzzy, the water molecules can toschaltbaren Peptidliganden für die Numb-PTB-Domäne reach deeper into the hydrophobic core, and the water sowie von PTB/Ligandkomplexen“ penetration free energy barrier changes. Moreover, we Kekulé-Stipendium observe that the area per lipid decreases and the deute- Christian Appelt (Promotionsstipendium) rium order parameters increase in the presence of the peptide. We suggest that the changes in the hydrophobic core together with the changes in the head groups result in an imbalance of the membrane-stabilizing forces that will lead to the disruption of the membrane.

* part of period reported Hupfer M,RübeB, French Press.AnalBiochem321,276-277 Structure 12,1645-1654 fromhumancomplexIadoptsathioredoxinfold. unit B8 2712-2717 ced affinitytoZ-DNAinvitro.ProcNatlAcadSciUSA101, shows atyrosineconformationthatmayexplainitsredu- The solutionstructureoftheN-terminaldomainE3L bacterial phytochromeCph1.Biochemistry, 44,8244-8250 solution stateNMR-studiesofthechromophoreincyano- propensities of42WWdomains.ProtSci12,491-500 activity relationshipsidentifiedusingligandrecognition M YG, Kahmann JD,Wecking DA,PutterV, LowenhauptK,Kim s J. Hughes,JustusLiebigUniversityGiessen T. Lamparter, FUBerlin H. vonDöhren,TUBerlin P. Hildebrandt,TUBerlin K. Rueck-Braun,TUBerlin Collaborations Strauss H Wahl M,KühneR,Oschkinat H Gaiser OJ,BallLJ,SchmiederP, LeitnerD,StraussH, P, KornB,KühneR,OschkinatH Brockmann C,DiehlA,RehbeinK,StraussH,Schmieder S Selected publications 15983-15995 Cancer-Associated ProteinBRCA1.Biochemistry43, Behavior oftheC-Terminal theBreast BRCTDomainfrom tion Structure,BackboneDynamics,andAssociation nesis ofpolyphosphateinlakesediments:A Otte L, Mergener J, tudy. LimnolOceanogr49,1-10 trauss H , Schmieder P, KrauseG Schmieder P, OschkinatH Wiedemann U,SchlegelB,PiresJR,Beyermann , ( Hughes J, 2003) Adeviceforfacilitatingtheuseof Oschkinat H Schmieder P Schmieder P , (2003) WWdomainsequence Volkmer-Engert R,Schneider- (FMP authorsinbold) , , Heinemann U(2004)Solu- Rich A,SchadeM(2004) (2004) Theoxidizedsub- (2004) Originanddiage- (2005) Heteronuclear 31 P-NMR

19 Structural Biology STRUCTURAL BIOINFORMATICS protein interactions is defined by complementary shapes Group Leader: Dr. Gerd Krause and charges between the participating epitopes. In conclusion, a common molecular mechanism of forming an STRUCTURAL BIOINFORMATICS TO UNRAVEL intermolecular helical bundle between the hinge region/ MOLECULAR SIGNALING MECHANISMS GuK domain of ZO-1 and α-catenin and occludin is identified as a general molecular principle organizing the asso- The group is focused on genome and protein sequence ciation of ZO-1 at adherens and tight junctions (Schmidt et analysis by structural bioinformatics combined with com- al. 2004). The promising results of this joint project in col- parative modeling of protein-protein interaction to study laboration with the cell physiology group of I. Blasig, FMP, sequence- and structure- function relationships. The main were primarily due to the fact that the bioinformatic/ aim is rational discovery of molecular mechanisms for pro- modeling- and the experimental studies as well have been tein-protein interactions and protein-ligand interaction to carried out by the same person (S.L. Müller). We are deduce ideas for pharmacological intervention. All group currently extending the structure-function studies also to projects are characterized by close cooperation with other junctional proteins by combining structural infor- experimental partners to ensure a closed loop between mation (bioinformatics/comparative modeling) and bioche- the theoretically derived model and experimental proof as mical-, molecular biological studies. an important prerequisite for a successful application of • Prediction of protein – protein interaction network biocomputing methods. The group is concerned with two partners topics to elucidate structure - function relationships of To better understand and to predict the interactions, pre- protein-protein interactions and of G-protein coupled dictive interaction models (e.g. COMFA and profile hidden receptors. Markov model recognition activities of WW domains) were derived by U. Wiedemann from experimental screening data. Taking the sequence (Otte et al. 2003) or the Protein-protein interactions structure (Schleinkofer et al. 2004) of experimentally un- S. Müller, U. Wiedemann, G. Krause described domains or ligands, these models allowed the Protein interaction networks are mediated very often by prediction of potential binding partners. Suggested opti- non-catalytic protein domains like PDZ- and WW-domains. mal peptide sequence for binding a subfamily of WW- Narrowing down potential interacting proteins from the domains was successfully verified (Schleinkofer et al. protein interaction network, elucidation of selective pro- 2004). Additionally, some models predicted the strength tein-protein interaction patterns and subsequent identifi- (affinity) of the interaction (www.fmp-berlin.de/nmr/pdz). cation of complementary interaction sites at the levels of Using this approach, ligands with higher affinity were epitopes, residues and atoms are the main aims. designed for representative domains of both families. In particular, for the hAF6-PDZ, the hERBIN-PDZ and the • Interaction sites of junctional proteins mSNA1-PDZ so-called “super-binding” peptides were Junctional proteins connect and seal the contact sites in designed which indeed exhibited the highest affinity between endothelial cells. The exact sites, structures and achievable by combination of natural amino acids (Wiede- molecular mechanisms of interaction between junction mann et al. 2004). organizing zona occludence protein 1 (ZO-1) and the tight junction protein occludin or the adherens junction protein Molecular signal transduction mechanisms of α-catenin were unknown. Binding studies by surface plas- GPCR mon resonance spectroscopy and peptide mapping com- G. Kleinau, J. Lättig, G. Krause bined with comparative modeling utilizing crystal structu- • Selective interaction patterns for G-protein subtypes res led for the first time to a molecular model revealing the The molecular interactions at the interface between binding of both occludin and -catenin to the same bind- α G-protein-coupled receptors and the G-proteins is of high ing site in ZO-1. Our data support a concept that ZO-1 suc- functional importance, since there the decision takes cessively associates with -catenin at the adherens α place about the different specific signaling pathways (via junction and occludin at the tight junction. the G-protein subtypes G , G , and G ) into the cell. For αi αs αq Strong spatial evidence indicates that the identified occlu- the thyroid stimulating hormone receptor (cooperation din C-terminal coiled-coil domain dimerizes and interacts with the University of Leipzig and NIH Bethesda, MD, as a helix bundle with the identified structural motifs in USA), we were able to delineate residues of the second ZO-1 (Müller et al. 2005). The selectivity of both protein- intracellular loop as selective recognition patterns for investigation bythegroupofAlexanderOksche(FUBerlin). sted site-directedmutagenesis(JLättig)iscurrentlyunder tor subtypes.To tothishypothesis,sugge- support provide signalingpathwaysoftherecep- responsible fordifferent tors ledtoidentificationofaminoacidsthatmightbe complementary cognitionpatternstotheEndothelinrecep- charged residuesattheG-proteinsite.Transferring these terns. Positivechargesatligandsinteractwithnegatively revealed potentialinteractionsitesbasedonchargepat- les (lipoamines)whichdirectlyinteractwithG-proteins Our previous studiesonpeptidesandothersmallmolecu- tor mutants(CAMs)arethemolecular etiologyof>50% dism, andhypothyroidism.Constitutively activeTSHrecep- adenomas, non-autoimmuneand neonatalhyperthyroi- result inpathogenicdiseases,for examplethyroidtoxic a thyroid function.TSHplaysacriticalroleinontogeny. As TSH-Receptor (TSHR)arekeyproteinsinthecontrolof andthe Thyroid-stimulating hormone(TSH,thyrotropin) • selectively interactingwithG Site-directed mutagenesisconfirmedM527asaresidue G-protein subtypesbyusingmolecularinteractionmodels. the TSHRectodomain: Structural determinantsofTSH-receptoractivationat consequence, differentmutationsofTSHRandTSH C-terminal LRRX tail α q (Neumann etal.2005). binding site P he-Spine C ysteine Box-1 N-terminal Cysteine Box N-terminal LRRO N-terminal tail treatment of thyroid cancer by small TSH receptor ligands. treatment ofthyroidcancerbysmall TSHreceptor ment ofhyperthyroidismbyinverse agonistsorforthe requisite forestablishingnewperspectives forthetreat- receptor signalinginthelargeTSHRectodomainareapre- elucidation oftheintramolecularmechanismsTSH mone bindingattheectodomainisnotunderstood.The the signaltransductiontoserpentinedomainuponhor- N-terminal ectodomain.Theintramolecularmechanismof requires thebindingofalargehormonetowards an evenmorecomplexactivationmechanism,which brane-spanning G-protein-couplingreceptors(GPCRs)is lating hormonereceptor(FSHR)tootherseventransmem- luteinizing hormonereceptor(CG/LHR)andfollicle-stimu- mone receptors(GPHR)suchaschoriogonadotrophic/ main differencesofTSHRandtheotherglycoproteinhor- TSH belongstothefamilyofglycoproteinhormones.The modify theunderstandingofpathophysiology. pendent (constitutive)activityforTSHRwouldprofoundly relationships ofligand-dependentand-inde- inprocesses to activateG-proteins.Aconceptforstructure-function (wt) receptorandtospontaneouslyadoptastructureable to someextenttheactiveconformationofwildtype hyperthyroidism inGermany. CAMsarethoughttomimic the concaveouterface. LRR isstabilizingthefoldinsteadofmissinghelicesat entry 1OZN).A‘Phe-spine’(greenaromaticrings)insidethe β b: a: FIGURE 1 additional parallel gral structuralpartoftheLRRbycontributingwithan plate providestheN-terminalflankingCys-box1asaninte- ture ofthehNogo-66-receptorectodomain.Thenewtem- LRR modelfortheTSHR(colored)basedonX-raystruc- plate (pdbentry2BNH)andthenewgeneratedhomologous (pdb entry1XUM,grey)basedonribonucleaseinhibitortem- -sheets oftheproposedhormone-bindingsite. LRR domain:comparisonofthepreviousmodelforTSHR LRR structureoftheNogo-receptorectodomain(pdb β -stand (LRR0inred)totheconvex

21 Structural Biology FIGURE 2 TSHR ectodomain: detail of Cys-box2 (red) and Cys-box-3 Cysteine- (yellow) interaction model adopted from IL8 and IL8RA com- box-2 S281 turn/loop C284 plex structure. Aromatic interaction of F286 (C-b2) and F405 (C-b3); Disulfide bridge between C408 (C-b3) and C283 (C-b2) F 286 based on biochemical data (blue); S281-loop/turn con- Cysteine- C283 box-3 formation adopted from Malonyl Coenzyme (cyan); The subsequently predicted residues D403, E404, N406 to be involved F405 in signal transduction at the interface between ectodomain C408 and serpentine domain indeed showed constitutive acti- TM1 N406 vities upon mutations.

E404 D403

To identify determinants at the GPHR ectodomain that may The subsequent structural extracellular component Cys- be involved in signal transduction, G. Kleinau first addres- box2 is attached back-to-back to the 11th ‚-strand of the sed this issue by searching for homologous structural fea- LRR, very likely via a short turn/loop containing S281. Exten- tures at the ectodomain based on high sequence similarity. sive systematic sequence similarity searches of fragmen- ted sequence portions of the ectodomain resulted in a For the LRR motif within the large ectodomain, a new aug- homologous model based on IL8-IL8RA fragment complex mented structural model based on the Nogo-66-receptor (pdb entry 1ILQ (27) which links Cys-box 2 and Cys-box-3 ectodomain with 9+2 repeats was generated (Fig. 1a) that together (Fig. 2). resulted in a strongly enlarged radius at the hormone binding site at the inner convex surface of the LRR-arch. The Subsequently, the residues D403EFNPC408 of the C-b3 are structure is stabilized by an interior Phe-spine instead of located particularly at prominent interface positions of the helices at the concave outer face based on previous tem- ectodomain, most closely to the transmembrane domain. plates (Fig. 1b). Moreover, the sequence similarity of the The hydrophilic residues D403, E404 and N406 are therefore new template indicated that the N-terminal flanked hypothesized to participate very likely in the intramole- Cysteinbox-1 is also an integral structural part of the LRR- cular signal transduction from the ectodomain towards the arch by contributing with an additional parallel (LRR0 serpentine domain (Kleinau et al, 2004). (TSHR37-41)) anti-parallel ‚-strand to the convex ‚-sheet of Using our approach, we identified three new mutations the hormone-binding region. (D403A, E404K and N406A) in the ectodomain of the TSHR that Furthermore, this model provides for the first time a struc- are causing constitutive cAMP activity, and we suggest tural rationale for the previously observed participation of that this motif is indeed important for the transduction of residues from Cys-box1 in hormone binding, i.e. for three the signal from the ectodomain to the transmembrane residues at the additional repeat LRR0 of the FSHR29-31 and domain. According to the high sequence conservation, the of TSHR37-41. This LRR model of BHR was later on confir- results are of general relevance for the signal transduc- med by the X-ray structure of the LRR domain for FSHR tion mechanism of other glycoprotein hormone receptors. (Fan & Hendrichson, Nature 2005). crinology 146,477-485 G-Protein SelectivityoftheThyrotropin Receptor. Endo- ral DeterminantsintheSecondIntracellularLoopfor G Neumann S, Mol Biol343,703-718 Affinity andRationalDesignofSuper-Binding Peptides.J tification ofPDZDomainSpecificity, ofLigand Prediction Mol Biol344,865-881 Energetic AnalysisofWWDomain-peptideInteractions.J Schleinkofer K, propensities of42WWdomains.ProtSci12,491-500 activity relationshipsidentifiedusingligandrecognition M, Wiedemann U,BoisguerinP Schmidt A,UtepbergenovDI,MuellerSL,BeyermannM Müller SL Selected publications Gerd Krause im TSH-Rezeptor“(KR1273) „Identifizierung einesrezeptorinternenstillenTransmitters Deutsche Forschungsgemeinschaft Ingolf Blasig,GerdKrause (Bl 208/6.2) „Wechselwirkungen vonBlut-Hirnschranken-Proteinen“ Deutsche Forschungsgemeinschaft External funding Gunnar Kleinau(Doctoralstudent) Urs Wiedemann(Doctoralstudent) J Sebastian L.Müller(Doctoralstudent) Group members Chem 280,3747-3756 share acommoninteractionmechanismwithZO-1.JBiol Otte L, Cell MolLifeSci61,1354-1365 protein 1:potentialmechanismoftightjunctionregulation. din bindstotheSH3-hinge-GuKunitofzonulaoccludens Huber O, Oschkinat H Mergener J, Schneider-Mergener J, tein occludinandtheadherensjunctionprotein ens Lättig(Doctoralstudent) , Mölling K,Volkmer-Engert R, Schmieder P, KrauseG Wiedemann U,SchlegelB,PiresJR,Beyermann Blasig IE,KrauseG , Portwich M, , Krause G Wade RC(2004)ComparativeStructuraland Oschkinat H Wiedemann U , Claus M,PaschkeR(2005)Structu- Krause G,BlasigIE Schmidt A,UtepbergenovDI , (2003) WWdomainsequence Volkmer-Engert R,Schneider- (FMP authorsinbold) (2005) Thetightjunctionpro- , Leben R, , Otte L,Wang T, Oschkinat H Leitner D,Krause (2004) Occlu- (2004) Quan- α Krause G, -catenin , , mational stabilityofwild-typeandmutantvasopressinV l Oksche A(2004)Pharmacochaperonespost-translational- Hermosilla R,SchaeferM, Bethesda, MD,USA. TSHreceptor M. Gershengorn,NIH,NIDDK GPR109a receptor/ TSHreceptor Bethesda, MD,USA ligandinteraction S. Neumann,NIH,NIDDK University ofHeidelberg S. Offermanns, V2receptor Interactionofjunction O. Huber, ChariteBerlinCBF TSHreceptor, activationmechanism A. Oksche,FUBerlin Peptidemapping S. Rothemund,IZKFLeipzig University ofLeipzig R. Paschke, C receptors. JBiolChem279,47254-47263 Wüller S,WiesnerB,LöfflerA,FurkertJ,KrauseG i receptor ectodomainactingasintramolecular signalling Krause G Kleinau G nterface. JBiolChem279,51590-51600 y ollaborations enhance cellsurfaceexpressionbyincreasingconfor- (2004) IdentificationofanovelepitopeintheTSH , Jäschke H,NeumannS, Schülein R,RosenthalW proteins Lättig J , Paschke R, 2 , ,

23 Structural Biology MOLECULAR MODELLING of the receptor seems to be associated with the trans- Group Leader: Dr. Ronald Kühne location of this ion and subsequent movements of TM 3, 6, and 7. Further, we recognized a small molecule binding site COMPUTATIONAL TECHNIQUES FOR within the FSHR. Docking simulations supported by scree- LIGAND DESIGN AND PROTEIN MODELLING ning results served as a basis for lead optimization processes. Refined models of the human and rat FSHR derived The research of the Molecular Modelling/Ligand Design from molecular dynamics simulations in explicit lipid envi- Group is mainly focused on protein modeling, virtual ronment have given closer insight into the interactions of screening, and lead optimization in close collaboration the residue Histidine615 (TM7), which is specific for the with experimental partners within academia as well as human FSH receptor. We found that this residue located industry. The special expertise of the group includes a towards TM6 is establishing a hydrogen bond to a back- wide range of molecular modeling technologies, bio- bone carbonyl. In contrast to human FSHR, all known FSHR informatics tools, and computational chemistry methods. of other species contain in this position a tyrosine residue. On top of that, these methods are applied to support the In our simulations the tyrosine establishes hydrogen bonds Screening Unit and the medicinal chemistry group within to residues in TM3. These results are able to explain the the national ChemBioNet initiative. The focus in this field known mutational data and other experimental facts. The is to analyze, design, and purchase a compound databa- studies were sponsored by Schering AG. se available for academic screening projects within the framework of ChemBioNet. In addition to the already Gonadotropin-Releasing Hormone Receptor ongoing modeling projects, the group has also started (A. Söderhäll, R. Kühne) research in several new fields during the last two years: In cooperation with Zentaris AG (Frankfurt am Main), seve- structure and function of antimicrobial peptides, high ral models of the human gonadotropin-releasing hormone throughput screening evaluation and virtual screening of receptor (GnRH-R) have been derived. The models form a antigen-exchange-inducing agents targeting the major series of successively improved generations of the recep- histocompatibility complex (MHC) and inhibition of pro- tor, where each generation is based on the latest experi- tein-protein interaction in the IL6/IL-6R/gp130 complex. All mental data. The data is based on site-directed mutage- of these projects benefit from close collaborations with nesis, activity modulation based on mutagenesis of the groups within the FMP and the MDC as well as with indu- native ligand as well as modulation of peptidomimetic strial partners. ligands. The latest generation of the receptor-ligand complex was used to derive a pharmacophore pattern, which Targeting G-protein-coupled receptors was successfully used in the lead finding and optimization. G-protein-coupled receptor (GPCR) modeling and agonist As a result of this successful cooperation with Zentaris, and antagonist design is one of the key competences of two patents on new non-peptidic GnRH antagonists are the group. The applied computational tools range from currently in process. One of them could be economically quantum chemically supported ligand design to high utilized by the Forschungsverbund Berlin e.V. throughput virtual screening of millions of compounds. The GPCR's are modeled using the bovine rhodopsin x-ray Structure and function of antimicrobial peptides structure and refined using simulated annealing and mole- (A. Söderhäll, F. Eisenmenger) cular dynamics in implicit as well as explicit lipid environ- Antimicrobial peptides have recently emerged as a pro- ment. These models are exploited in virtual screening and mising new generation of antibiotics. This class of pepti- lead optimization using simulated annealing and pharma- des is generally believed to target the bacterial membra- cophore mapping. ne and destroy the chemical gradients over these, either by building transmembrane pores or by destabilizing the The follicle-stimulating hormone receptor bilayer structure of the membranes. This mechanism of (A. Schrey, R. Kühne) action is believed to make the peptides more stable We have constructed a model of the human follicle stimu- against the evolutionary pressure from bacteria develo- lating hormone receptor (FSHR) in a water-vacuum-water ping antibiotics resistance. By exploiting the in-house environment. The model is consistent with published muta- competence on peptide chemistry in Michael Bienert’s tional data. A central feature of the model is a sodium ion group, the antimicrobial peptide cyclo-(RRWWRF) was located between Asp408 transmembrane helix 2 (TM2), selected for NMR structure determination by Christian Asp581 (TM6), and Asn618 and Asn622 (TM7). Activation Appelt in the group of Peter Schmieder. Using the NMR derived model,anextendedvirtual screeningofthree the highthroughputscreeningcampaign. Basedonthis ture-activity relationships(QSAR) ofahitsubfamilyfrom tion ofapredictionmodelbasedon thequantitativestruc- zed indetail.Theanalysiswasthenusedtheconstruc- of the20,000compoundshasbeenstructuredandanaly- geting theMHC.Thedatafromhighthroughputscreen FMP databaseforantigen-exchangeinducingagentstar- The groupofOlafRötzschkeatMDChasscreenedthe (A. Söderhäll,R.Kühne) the majorhistocompatibilitycomplex High throughputandvirtualscreeningtargeting impact thishasonthemembranestructure/seeFig.1/. compound interactswiththemembranelipidsandwhat kind ofsubstances.We haveelucidatedindetail howthis formation ofexplicitpores,aswasoftensuggestedforthis tion ofthehydrophobicbarriertakesplacewithout bility ofionsaswellwater. Interestingly, theperturba- rier becomeslesseffective,andmayincreasethepermea- the membranesothatitsfunctionasahydrophobicbar- simulations wehaveconcludedthatthepeptidedisturbs us peptide-to-lipidratios.Fromthesemoleculardynamics structure wehavesimulatedrealisticmembranesatvario-

–10

–5

10 –10 –5 0 5 1 0 no detailedatomicstructure.Since mostproteinsofthis major complexoftherespiratory chainforwhichthereis NADH ubiquinoneoxidoreductase (complexI)isthelast and methodstorefineNMRderived proteinstructures. main focusinthisfieldisonNMRstructurecalculations structure elucidationofproteindomainsbyNMR.The to An importantpartofthescientificactivitiesisrelated (C. Brockmann,R.Kühne) NMR structurecalculations of actionantigenexchange. mental data,weareabletoproposeapossiblemechanism mutagenesis. Usingthesedockingstogetherwithexperi- The dockingapproachwassupportedbysite-directed to explainthemechanismsbehindantigenexchange. dockings oftheleadcompoundswerecarriedoutinorder Rötzschkes group.Withintheframeworkofthisproject, of majorhelpinthesecond,refinedscreeningroundbyO. proving thatthehitenrichmentoffocusedlibrarywas tested. Fiveoutofthesetwelvewerefoundtobeactive, A was narroweddowntoalistof5589rankedcompounds. lion compoundswascarriedout.Thismassivedataset external databasescontainingatotalofmorethanonemil- dozen ofthesewerehand-pickedandexperimentally through themembrane. i a membrane. Theamphipaticcharacterofthepeptidecreates f The figureshowstheaveragestructureofcyclo-(RRWWRF) cular dynamicssimulationsinthepresenceofexplicitlipids. w The structureoftheantimicrobialpeptidecyclo-(RRWWRF) FIGURE 1 ncreased permeabilityofwaterandmostlikelyalsoions rom ofthe thesimulationandasurfacerepresentation as determinedbyNMRandexploitedinaseriesofmole- void inthemembranesurfacethatcontributesto

25 Structural Biology A B

1,0

0,8

0,6 ] m c [ R 0,4

0,2

0,0

1E-4 1E-3 0,01 0,1 1 10 100 1000 [GSH]2 / [GSSG]

FIGURE 2 Structure and redox-potential determination of CI-B8. A: ribbon-representation of the structure of human CI-B8. Alpha-helices are depicted in green, the beta-sheet in blue. The position of the disulfide-bond is indicated in yellow. B: Determination of the redox potential. The change in Trp-fluorescence upon reduction is plotted against the relative concentrations of the redox-buffer system. From a Keq of 0.486 a redox potential of –250 mV was calculated.

highly modular complex are rather small, its components Group members make suitable targets for structural studies by NMR. In this Dr. Anna Schrey project the structure of subunit B8 (CI-B8) was investiga- Dr. Arvid Söderhäll ted by NMR. The solution structure of the oxidized subunit Dr. Jörg Wichard* shows a thioredoxin fold with remarkable similarities to Dr. Frank Eisenmenger (Unix system administration) thioredoxin-like Fe2S2-ferredoxins (Fig. 2A). A structural Christoph Brockmann (Doctoral student) comparison shows high similarities of surface properties Stefan Hübel (Unix system administration support, data- and possibly active residues. Since CI-B8 shows some base management) similarities to thioredoxins, and since the mammalian homologues may form a disulfide bridge in a similar posi- External funding tion, we determined the redox potential of oxidized CI-B8 to see if it fits into the range covered by active site disulfi- Schering AG des in thioredoxin-related proteins (Fig. 2B). The redox- Transmembranrezeptoren (Kooperationsvertrag) potential of the disulfide bond of CI-B8 compares well to Zentaris AG that of the catalytically active disulfides in other thioredo- (Kooperationsvertrag) xin-like proteins. This, in addition to the fact that CI-B8 stems from a complex that is involved in a redox-depen- Conaris AG dent reaction, leads to the idea that CI-B8 could assist in redox-associated processes or electron transfer. This Selected publications (FMP authors in bold) example of CI-B8 shows that structural studies of the indi- Brockmann C, Diehl A, Rehbein K, Strauss H, Schmieder vidual components of complexes can spark hypotheses P, Korn B, Kühne R, Oschkinat H (2004) The oxidized sub- about the function of the component within the complex. unit B8 from human complex I adopts a thioredoxin fold. Structure (Cam) 12, 1645-1654

* part of period reported 27, 143-149 covalently linkedtoaproline-richpeptide.JBiomolNMR ner G(2003)StructuralinvestigationsofaGYFdomain Walter U, family BAGdomains.FEBSLett558,101-106 static interactionsintheHSP70complexesofSODDsub- t Karges B,W, MineM,LudwigL, 278, 36810-36818 tuted peptomerligandsforEVH1domains.JBiolChem Dr. O.Roetzschke,MDC Prof. Schmalz,UniversityofCologne Dr. W. Karges,UniversityofUlm Collaborations Zimmermann J,KühneR Freund C,KühneR Brockmann C,LeitnerD,LabuddeDiehlA factor. JBiolChem279,4840-4848 p (2004) Solutionstructureofhumancofilin:actinbinding, Pope BJ,Zierler-Gould KM, associated proteinBRCA1.Biochemistry43,15983-15995 o structure, backbonedynamics,andassociationbehavior M, KühneR,OschkinatH Gaiser O,BallLJ,SchmiederP, LeitnerD,StraussH,Wahl Bussow K, nadism. JClinEndocrinolMetab88,1873-1879 conformation, causingfamilialhypogonadotropichypogo- gonadotropin-releasing hormonereceptorinitsinactive E, deRouxN(2003)MutationAla(171)Thrstabilizesthe ure oftheSODDBAGdomainrevealsadditionalelectro- H f the C-terminalBRCTdomainfrombreastcancer sensitivity, andrelationshiptoactin-depolymerizing Oschkinat H,BallLJ Kühne R,OschkinatH , Park S, , , Volkmer-Engert R,JarchauT, Thiemke K Heinemann U(2004)Solution Kühne R (2003) DesignofN-substi- (2004) Thesolutionstruc- , , Weeds AG, Reinherz EL,Wag- Kühne R , Sievert V, , Milgrom Ball LJ

27 Structural Biology SOLID STATE NMR Structural characterization of ligands targeted Group Leader: Prof. Bernd Reif against beta-amyloid fibrils (Aß) Alzheimer's disease (AD) is the most common form of age- SOLID-STATE NMR SPECTROSCOPY: related neurodegenerative disorder, and is related to PROTEIN AGGREGATION AND MEMBRANE deposition of Aβ peptides in the brains of AD patients. Aβ PROTEINS is formed by processing of APP, the Amyloid Precursor Protein, a membrane protein of yet unknown function. We We use Nuclear Magnetic Resonance (NMR) in order to use the fact that amyloid fibrils orient spontaneously in the characterize biomolecular systems which are situated at magnetic field to determine the structure of peptide inhibi- the interface between solution and solid. In this area, tors that bind to Aβ aggregates (Chen & Reif, 2004). The membrane proteins and amyloidogenic peptides and pro- alignment is based on the magnetic anisotropy of the pep- teins are the most interesting “target molecules”. By tide bond. A net alignment of amyloid fibrils is induced due nature, structural information of these systems is difficult to the arrangement of hydrogen bonds in parallel to the to obtain by means of X-ray crystallography or standard fibril axis. We expect that this approach is useful for the solution-state NMR methods. We want to address these design of diagnostic or therapeutic ligands. systems by application of a combination of modern solution state and solid state NMR methods. This requires Structural characterization of acid denatured development of especially adapted NMR techniques. So PI3K fibrils far, about 20 proteins are known for which a correlation Chris Dobson and co-workers demonstrated that not only between aggregation and disease is established. The proteins which are related to a disease can form highly most prominent examples are Alzheimer's disease, the structured aggregates, but almost any other soluble pro- prion diseases (BSE, CfJ), and Huntington disease. How- tein under certain solution conditions. One example is the ever, little is known about the mechanism which leads to SH3 domain of the phosphatidyl-inositol-3-kinase (PI3K) aggregation, as well as on the structure of the amyloid which forms fibrils under acidic pH. Solid-state NMR expe- fibrils. We would like to gain more insight into the structu- riments revealed that His25 of PI3K-SH3 is involved in ter- re of oligomeric intermediate states which are associated tiary contacts which stabilize the fibril structure (Ventura with protofibril formation. In addition, we are interested in et al., 2004). Mutational studies (H25K) confirmed this characterizing dynamic chemical exchange processes hypothesis. Most importantly, it could be shown that arti- between the soluble and aggregated state of the respec- ficial neurotoxicity is abolished in the mutated protein tive proteins. using a MTT reduction assay. We use PI3K-SH3 as a paradigm to study intermediate states as well as the aggregated state and try to obtain a better understanding of the Yeast Prions: Sup35 and Hsp104 mechanism of protein aggregation. Characterization of interactions between the molecular chaperone Hsp104 and the prion protein Sup35 in yeast by Development of MAS solid-state NMR techniques NMR spectroscopy Sup35 is a subdomain of the transla- Solid-state techniques have found great attention in the tion-termination complex in S. cerevisiae. Under specific last few years since it has turned out to be possible to conditions, Sup35 can form protein aggregates that can determine the structure of uniformly labeled (crystalline) induce a conformational change in other Sup35 proteins peptides and proteins (Rienstra et al. 2002). However, and are inherited after cell division. Hsp104 is one of the solid-state NMR experiments are intrinsically insensitive most important proteins for the thermostability in yeast and due to detection of the heteronucleus (carbon or nitrogen). is, together with two other proteins, Hsp70/Ssa1 and We address the question of sensitivity by employing pro- Hsp40/Ydj1, involved in a chaperon complex that can dis- ton detection in combination with deuteration, which che- solve protein aggregates that are produced as a heat mically eliminates the strong proton-proton dipolar coup- response. Solution-state NMR methods are employed to lings. This approach allows to detect proteins with high characterize the interactions between the aggregate and sensitivity (Chevelkov et al. 2003). In addition, long range the chaperone. We found that Hsp104 is able to disaggre- proton-proton interactions are accessible, which are gate Sup35-derived peptides in vitro, and that especially important to determine the three-dimensional fold of a pro- low oligomeric complexes of Sup35 interact with Hsp104 tein in the solid state (Reif et al. 2003). Furthermore, we (Narayanan et al., 2003). In this study, we were able to ana- explore deuterium as a source for dynamic information in lyze conformational changes of Sup35 induced by Hsp104 the solid state. by NMR in real time. Bernd Reif (Re1435/3) ters EmrEmittelsMASFestkörper-NMR-Spektroskopie“ „Strukturelle CharakterisierungdesMultidrug-Transpor- Deutsche Forschungsgemeinschaft Bernd Reif an orientiertenBiomakromolekülen“(Re1435/2) schen FlüssigkeitundFestkörper. Strukturuntersuchungen „Entwicklung NMRspektroskopischerMethodenzwi- Deutsche Forschungsgemeinschaft External funding Uwe Fink(Technical assistance) Saravanakumar Narayanan(Doctoralstudent) Muralidhar Dasari(Doctoralstudent) Veniamin Chevelkov(Doctoralstudent) Zhongjing Chen(Doctoralstudent) Dr. MaggyHologne Dr. AlexChernogolov Group members Hsp104 + S Sup35 up35 Monomeric Sup35 Intermediate 2 Intermediate 1 Sup35 States of Oligomeric Higher B Narayanan S A Chevelkov V, RossumBJv, CastellaniF, RehbeinK,Diehl Chen Z,ReifB Selected publications suppression. JAmChemSoc125,7788-7789 scopy employingpulsedfieldgradientsforresidualsolvent 530 binding topeptideamyloidfibrils.JBiomolNMR29,525- couplings inpeptidicinhibitorsweaklyalignedbytransient Bernd Reif im Sonderforschungsbereich594„MolekulareMaschinen“ im KomplexmitHsp104,Hsp40undHsp70“Teilprojekt A3 „Biochemische undStrukturuntersuchungen anSup35p Deutsche Forschungsgemeinschaft 100, 9286-9291 yeast prionsystemSup35/Hsp104.ProcNatlAcadSciUSA low oligomericweightspeciesforprionpropagationinthe , (2003) 1HdetectioninMASsolidstateNMRspectro- Hohwy M,SteuernagelS,EngelkeF, determined usingDOSYexperiments. by STDNMRexperiments.Sup35oligomericstatesare Interactions betweenSup35andHsp104arecharacterized Hsp104 interactspreferablywithlowoligomericSup35. FIGURE 1 , Bösl B,Walter S, (2004) Measurementofresidualdipolar (FMP authorsinbold) Reif B (2003) Importanceof Oschkinat H,Reif

29 Structural Biology FIGURE 2 Structure of a peptide inhibitor bound to an Aß fibril using restrained docking. The spontaneous alignment of fibrils in the magnetic field induces an ordering of the ligand peptide. Measurement of transfer residual dipolar couplings (trRDCs) yield the relative orientation of the peptide with respect to the fibril axis.

Fibril Axis Magnetic Field

Reif B, van Rossum BJ, Castellani F, Rehbein K, Diehl A, Dr. M. Sattler, EMBL Heidelberg Oschkinat H (2003) Determination of 1H 1H distances in a Prof. Dr. S. Schuldiner, Hebrew University Jerusalem, uniformly 2H,15N labeled SH3 domain by MAS solid state Israel NMR spectroscopy. J Am Chem Soc 125, 1488-1489 Dr. L. Serrano, EMBL Heidelberg Dr. S. Ventura, Universitat Autonoma de Barcelona, Rienstra CM, Tucker-Kellogg L, Jaroniec CP, Hohwy M, Spanien. Reif B, McMahon MT, Tidor B, Lozano-Pérez T, Griffin RG Dr. S. Walter, TU Munich (2002) De Novo Determination of Peptide Structure with Solid-State MAS NMR Spectroscopy. Proc Natl Acad Sci USA 99, 10260-10265

Ventura S, Zurdo J, Narayanan S, Parreño M, Mangues R, Reif B, Chiti F, Giannoni E, Dobson CM, Aviles FX, Serrano L (2004) Short amino acid stretches can mediate amyloid formation in globular proteins: Thr Src homology 3 (SH3) case. Proc Natl Acad Sci USA 101, 7258-7263

Collaborations

Dr. W. Boelens, University of Nijmegen, The Netherlands Prof. J. Buchner, TU Munich Dr. G. Gast, University of Potsdam Prof. Dr. U. Heinemann, MDC Berlin Prof. Dr. W. W. de Jong, University of Nijmegen, The Netherlands Prof. G. Multhaup, Free University Berlin the epitopeforinteraction. Pulldownexperiments binding motifsforCD2BP2-GYF, andwehavedetermined ly thecoresplicingproteinSmB/B’ containsseveral ments forbindingtotheGYFdomain ofCD2BP2.Especial- contain recognitionsequencesthatmatchtherequire- Chem. 279,28292-28297).Severalspliceosomalproteins and for theCD2BP2proteinbymeansofpeptidespotanalysis domain, wehaveidentifiednovel In additiontoitsbindingcapacityfortheCD2cytoplasmic Christian Freund Michael Kofler, MatthiasHeinze,KatharinaThiemke, and CD2BP2 cellular signaling. dies areaimedatputtingthisknowledgeinthecontextof recognition atamolecularlevel,whilebiochemicalstu- nal structureofproteindomains,wewanttounderstand mation. Basedonthedeterminationofthree-dimensio- genesis asstrategiestoderivebiologicallyrelevantinfor- laboratory implementrandomandstructure-basedmuta- research tools.ProteinEngineeringapproachesinour microscopy andscreeningofbiomolecularlibrariesas NMR spectroscopy, proteinbiochemistry, fluorescence tions isthemajortopicinourgroup.Thereforeweemploy ry mechanismsofsuchadapterdomainmediatedinterac- sequence recognitioncodeanddeterminingtheregulato- exposed regionsofintracellularproteins.Decipheringthis domains oftransmembranereceptorsorwithinsolvent tide sequencesthatarepresentinthecytoplasmic general signalingmolecules.Theytypicallyrecognizepep- proteins arepresentinmanylymphoid-specificaswell mic adapterdomainsthatguidethespatialdistributionof arrangements thattakeplaceuponstimulation.Cytoplas- patterning attheinnermembraneandcytoskeletalre- dependent onthemembraneorganization,molecular ses. Thefine-tuningoftheintracellularprocessesis immunerespon- are abletoevokedramaticallydifferent k cell receptorengagementprocess.Subtlechangesinthe response inTcellshastocopewiththehighlyadaptive tions inimmunecells,especiallyTcells.Theintracellular on adapterdomainsthatmediateprotein-proteininterac- g Our groupisinterestedinthemolecularinteractionsthat T PROTEIN ADAPTERDOMAINSINVOLVED IN Group Leader:PDDr. ChristianFreund P inetics andaffinityoftheTCR-MHC-peptideinteraction overn theassemblyofproteincomplexes.Thefocusis ROTEIN ENGINEERING CELL SIGNALING subsequent NMRanalysis(Kofleretal.2004,J.Biol. in vitro binding partners ce recognitionbetweenGYF, SH3-,WW-domains. domains andsetthestageforcomparisonofsequen- sequence requirementsforindividualsubclassesofGYF partners. Itwillalsoallowustobetterunderstandthe valuable toolfortheidentificationofinvivointeraction sequence spaceofGYFdomainsisanticipatedtocreatea that complementthepeptidebindingresults.Mapping these GYFdomains,andstructuralstudiesareunderway will allowustocomparetheligandsequencespaceof from speciesbyphagedisplay. variouseukaryotic This We arecurrentlyinvestigatingseveralotherGYF domains glycine motifforrecognitionbytheGYFdomainofCD2BP2. analysis showstheimportanceofproline-proline- within theGYFdomainbindingpocket.Peptidesubstitution thereby allowingtheprolinehelixtobeoptimallyplaced promotes akinkinthepeptidebackboneconformation, al. 2002,EMBOJ21,5985-5995).Theglycineresidue contact thehydrophobichotspotofdomain(Freundet prolines oftheligandformapolyprolinetypeIIhelixthat SHRPPPPGHRV peptidederivedfromCD2revealsthatthe structure oftheGYFdomainincomplexwith defines thebindingsiteforproline-richligand.The c is proposedtobepresentinmanyeukaryoticproteins.A The NMRstructureofthisdomaindefinesanewfoldthat Michael Kofler, KatrinMotzny, andChristianFreund G physical methodstogainanunderstanding oftheconfor- further investigatedbyNMRspectroscopy andotherbio- can berecognizedbycyclophilins. Thesepeptidesare methods fortheidentificationof peptidesequencesthat being highlysequence-specific.We willuse cyclophilins recognizepeptideconformationratherthan recognition domains.Conceptuallyitisbelievedthat sequences thatarealsoligandsforproline-richsequence potential ofcyclophilintoactonandbindproline-rich Res Rev20,452-484).We arecurrentlyinvestigatingthe within proteincomplexes(forareviewseeIvery2000,Med 478), buthavealsobeencharacterizedasbindingmodules isomerase activity(Fischeretal.1989,Nature337,476- Cyclophilins containanintrinsicpeptidyl-prolyl-cis-trans Kirill PiotukhandChristianFreund Cyclophilin their potentialtorecognizetheCD2BP2proteininvivo. for vitro resultswearenowtestingadditionalproteins n cells showsthecolocalizationoftwoproteinsin SmB/B’ proteins,andfluorescencemicroscopyoflive confirm aspecificinteractionbetweentheCD2BP2and ucleus ofJurkatandHeLacells(Fig.1).Basedonourin onserved setofhydrophobicandaromaticaminoacids YF domains in vitro

31 Structural Biology A B ADAP hSH3 FYN SH3 FIGURE 1 Comparison of the structure of the ADAP hSH3 domain with a “classical” SH3 domain. The a-helix of ADAP hSH3 contacts the SH3 fold and several residues that are important for binding the proline-rich ligand are mutated in ADAP hSH3 (compare A and B, in B the peptide ligand is shown in green). These features lead to an altered surface topology and the inability of ADAP hSH3 to bind to proline- C D rich sequences (compare C and D).

mational restraints imposed on the peptides upon complex poietic cells. From a molecular perspective, ADAP repre- formation. sents a new member of the increasingly large family of non-enzymatic, hematopoietic scaffolding proteins. In ADAP addition to tyrosine-phosphorylation sites, ADAP contains Katja Heuer, Marc Sylvester, Jürgen Zimmermann, a number of N-terminal proline-rich motifs that mediate Katharina Thiemke, and Christian Freund the interaction with the SH3 domain of SKAP55 (Liu et al. 1998, Proc Natl Acad Sci 95, 8779-8784; Marie-Cardine et ADAP (adhesion- and degranulation- promoting adapter al. 1998, J Biol Chem 273, 25789-25795). ADAP itself con- protein) was identified as an adapter protein that upon tains two regions of homology to SH3 domains and we association with the TCR complex becomes tyrosine phos- have recently solved the structure of the ADAP C-terminal phorylated and thereby creates binding sites for the SH2 domain by NMR spectroscopy (Heuer et al. 2004, Structu- domains of the Fyn kinase and of the SLP-76 protein (da re 12, 603-610). The structure reveals an extended SH3 Silva et al. 1997, Proc Natl Acad Sci 94, 7493-7498; Musci domain fold, where an N-terminal -helix contacts the SH3 et al. 1997, J Biol Chem 272, 11674-11677). ADAP has also α scaffold, thereby creating a composite surface that can- been shown to colocalize with F actin and cytoskeletal not bind to proline-rich sequences (Figure 2). We are cur- proteins such as VASP and WASP in activated Jurkat T rently investigating the binding properties of this domain, cells. A similar complex has been found enriched in the since we believe this will contribute to elucidation of phagocytic cups of activated macrophages (Coppolino et ADAP’s function within immune cell signaling. al. 2001, J Cell Sci 114, 4307-4318; Krause et al. 2000, J Cell Biol 149, 181-194). Furthermore, ADAP-deficient mice show a decrease in LFA-1 dependent adhesion of stimulated peripheral T cells (Griffiths et al. 2001, Science 293, 2260- 2263; Peterson et al., 2001, Science 293, 2263-2265) and therefore ADAP was concluded to be an important regula- tor for inside-out signaling in T cells and other hemato- C. Freund Sieger imNachwuchsgruppenwettbewerbBio-Future vermittelten Immunantwort“(Bio-Future0311879) und DesignvonAgonistenAntagonistenderT-Zell „Struktur-Funktionsbeziehung wichtigerT-Zell-Proteine Bundesministerium fürBildungundForschung External funding Katrin Motzny(Technical assistance)** Uta Ben-Slimane(Technical assistance)*** Ulrike Schneeweiß(Technical assistance)* Katharina Thiemke(Technical assistance)* Marc Sylvester(Doctoralstudent)* Matthias Heinze(Doctoralstudent) Michael Kofler(Doctoralstudent) Dr. JürgenZimmermann* Dr. KirillPiotukh Dr. KatjaHeuer Group members ** part-time * part ofperiodreported 28292-28297 sible spliceosomalfunctionofCD2BP2.JBiolChem279, Recognition sequencesfortheGYFdomainrevealapos- Kofler M,HeuerK,ZechT, ThiemkeK,FreundC Heuer K,KoflerM,LangdonThiemkeFreundC Heuer K,ArbuzovaA,StraussH,KoflerM,FreundC Selected publications adapter proteinADAP. 12,603-610 Structure Structure ofahelicallyextendedSH3domaintheTcell Biol, inpress protein ADAPisanovellipidinteractiondomain.JMol The helicallyextendedSH3domainoftheTcelladaptor C. Freund 1325/2-1) „Struktur-Funktionsbeziehung derGYF-Domäne“(FR Deutsche Forschungsgemeinschaft C. Freund Activity ofPeptidyl-Prolylcis/transIsomerases”(I/77955) “Biological FunctionofAdaptorDomainsControlledbyThe Volkswagen Foundation The surfaceofthedomainisrenderedtransparent. shown inmagentawhilethepeptideisdisplayedyellow. CD2 peptide.BindingsiteresiduesoftheGYFdomainare Binding siteoftheCD2BP2-GYFdomainincomplexwitha FIGURE 2 (FMP authorsinbold) (2004) (2005) (2004)

33 Structural Biology Kofler M, Motzny K, Freund C (2005) GYF domain proteomics reveals interaction sites in known and novel target proteins. Mol Cell Proteomics (in press)

Freund C (2004) The GYF domain. In: Modular Protein Domains, Wiley-VCH GmbH, Germany

Freund C, Kühne R, Park S, Thiemke K, Reinherz EL, Wag- ner G (2003) Structural investigations of a GYF domain covalently linked to a proline-rich peptide. J Biomol NMR 27, 143-149

Freund C, Kühne R, Yang H, Park S, Reinherz EL, Wagner G (2002) Dynamic interaction of CD2 with the GYF and the SH3 domain of compartmentalized effector molecules. EMBO J 21, 5985-5995

Collaborations

Ellis Reinherz (Dana-Farber Cancer Institute, Boston, MA, USA) Volkhard Helms (University of Saarland, Saarbrücken) Ingo Schmitz (University of Düsseldorf) Richard Kroczek (Robert Koch Institute, Berlin) CELLULAR SIGNALLING/MOLECULAR GENETICS SECTION CELLULAR SIGNALLING/ proteins are folded, and correct folding is monitored by a MOLECULAR GENETICS quality control system (QCS). Only correctly folded proteins are allowed to enter the vesicular transport via the Prof. Ivan Horak ER/Golgi intermediate compartment (ERGIC) and the Golgi Department Head: Molecular Genetics apparatus to the plasma membrane. The study of the early (Secretary: Alexandra Kiesling) stages of the intracellular transport of GPCRs is also of clinical significance. Naturally occuring receptor muta- Prof. Walter Rosenthal, tions frequently lead to misfolded proteins unable to pass Department Head: Cellular Signalling the QCS and consequently to inherited diseases. (Secretary: Heidemarie Petschick)

The aim of the Anchored Signalling group is to identify furt- Research in the FMP section “Cellular Signalling / Molecu- her proteins involved in the redistribution of the protein lar Genetics” covers some of the major aspects of euca- AQP2 that belongs to the family of water channels. It is cri- ryotic signal transduction. Increasingly, the development tically involved in the Arginine-vasopressin mediated of pharmacological strategies to interfere with cellular water reabsorption in the kidney. Arginine-vasopressin signal transduction pathways under study has become a binds to the vasopressin V2 receptor located on the sur- major point of emphasis. face of renal principal cells thereby triggering the redistri- The Protein Trafficking group is interested in the early sta- bution of AQP2 from intracellular vesicles into the urine- ges of the intracellular trafficking of G-protein-coupled facing plasma membrane. The insertion of AQP2 into the receptors (GPCRs). The intracellular transport of membra- plasma membrane introduces water-selective pores and ne proteins starts with their insertion into the membrane facilitates water entry into the cells. The studies in this of the endoplasmic reticulum (ER). During ER insertion, the group may lead to a detailed understanding of the under-

INTRODUCTION

ten in den vesikulären Transport über das „ER-cis-Golgi BEREICH SIGNALTRANSDUKTION/ intermediate Compartment“ (ERGIC) und den Golgi-Appa- MOLEKULARE GENETIK rat zur Plasmamembran ein. Das Studium der frühen Sta- Prof. Ivan Horak dien des intrazellulären Transports von GPCRs ist auch von Abteilungsleiter: Molekulare Genetik klinischer Bedeutung. Natürlich auftretende Rezeptor- (Sekretariat: Alexandra Kiesling) mutationen führen häufig zu fehlgefalteten Proteinen, die das Qualitätskontrollsystem nicht passieren können. Auf Prof. Walter Rosenthal diese Weise manifestieren sich Erbkrankheiten. Abteilungsleiter: Signaltransduktion (Sekretariat: Heidemarie Petschick) Das Ziel der Arbeitsgruppe Anchored Signalling ist es, Pro- teine zu identifzieren, die an der Umverteilung des Proteins Die Forschung im FMP-Bereich „Signaltransduktion/ AQP2 mitwirken. AQP2 gehört zur Familie der Wasserka- Molekulare Genetik“ befasst sich mit wichtigen Aspekten näle und ist an der durch Arginin-Vasopressin-vermittel- der eukaryotischen Signaltransduktion. Zunehmend liegt ten Wasserreabsorption in der Niere beteiligt. Arginin- das Augenmerk auf der Entwicklung pharmakologischer Vasopressin bindet an den Vasopressinrezeptor V2, der Strategien zur Interferenz mit den untersuchten Signal- sich auf der Oberfläche der Prinzipalzellen der Niere befin- transduktionswegen. det. Auf diese Weise wird die Umverteilung AQP2 aus Die Arbeitsgruppe Protein Trafficking interessiert sich für intrazellulären Vesikeln in die harnseitige Plasmamembran die frühen Stadien des intrazellulären Transports G-Pro- ausgelöst. Durch das Eintreten von AQP2 in die Plasma- tein-gekoppelter Rezeptoren (GPCRs). Der intrazelluläre membran wird diese mit wasserselektiven Poren ausge- Transport von Membranproteinen beginnt mit ihrer Inser- stattet, so dass Wasser in die Zellen aufgenommen wer- tion in die Membranen des endoplasmatischen Retikulums den kann. Die Untersuchungen in der Arbeitsgruppe sollen (ER). Unter der Insertion werden die Proteine gefaltet. zu einem detaillierten Verständnis des molekularen Dabei wird die korrekte Faltung durch ein Qualitätskon- Mechanismus der Wasserreabsorption führen und letzt- trollsystem überwacht. Nur korrekt gefaltete Proteine tre- lich eine Behandlung bestimmter Fälle des Diabetes insi- lying mechanism, and eventually to a treatment of certain immune system. This technology is used to generate cases of diabetes insipidus. Recently, interference of pro- mouse mutants (“knock-out mice“) that carry an experi- tein-protein interactions by the means of chemical com- mentally designed gene defect. Thus it is possible to ana- pounds has become a major aspect. lyze the consequences of a specific gene defect in the context of the whole organism. The main interest is focu- The main field of the Biophysics Group is research in mem- sed on cytokine receptor signaling, in particular inter- brane transport. Basic research projects aim to explore the ferons. These cytokines are used for treatment of human molecular mechanisms of water and proton movement. diseases, despite their unknown functional mechanisms. The activities in applied biophysics are devoted to the con- The highly pleiotropic effects of these cytokines are often trol of membrane permeability by (bio)polymers and to pho- therapeutically disadvantageous. Therefore, an exact todynamic reactions of membrane components. Peter Pohl knowledge of signaling pathways and interacting molecu- has been appointed full professor at the University of Linz les which are regulated by these cytokines could lead to in Austria and left the FMP at the end of 2004. the development of more precisely targeted therapeutic The research activity of the Department of Molecular interventions.

Genetics concerns the molecular mechanisms which con- s c i t trol development and function of cells in the blood and Cytokine receptor signaling involves STATs (Signal trans- e n e

immune system. Elucidation of these processes is an ulti- ducers and activators of transcription). Frequently, abnor- G r a l

mate requirement not only for understanding their patho- mal activity of certain STAT family members, particularly u c e logical alterations but also for the development of rational Stat3 and Stat5, is associated with a wide variety of human l o M diagnostic and therapeutic procedures. We have been malignancies, including haematological, head and neck, / g n i l using targeted mutagenesis to analyze genes thought to breast and prostate cancers. The EMBO research group l a n g have important regulatory functions in the blood and of Uwe Vinkemeier is investigating the relationship bet- i S r a l u l l e C 7 3 pidus möglich machen. Aktuell hat sich insbesondere die im Blut und Immunsystem haben. Mit Hilfe dieser Metho- mittels chemischer Verbindungen auslösbare Interferenz de werden experimentell Mäuse mit einem Gendefekt mit den Protein-Protein-Interaktionen zu einem wesent- erzeugt (Knockout-Mäuse). Auf diese Weise ist es mög- lichen Aspekt der Forschungstätigkeit entwickelt. lich, die Auswirkungen eines spezifischen Gendefekts im Kontext des Gesamtorganismus zu analysieren. Das Der Hauptforschungsgegenstand der Arbeitsgruppe Bio- Hauptinteresse der Abteilung gilt dem Zytokinrezeptor- physik ist der Transport an Membranen. Forschungspro- Signaling, insbesondere aber den Interferonen. Diese jekte zielten auf die molekularen Mechanismen der Was- Zytokine werden zur Behandlung von Erkrankungen des ser- und Protonenbewegung. Die Aktivitäten auf dem Menschen eingesetzt, man kennt jedoch ihre Wirkmecha- Gebiet der angewandten Biophysik sind darauf gerichtet, nismen nicht genau. Der stark pleiotrope Effekt der Inter- die Durchlässigkeit der Membran für (Bio-)Polymere und ferone ist meist von Nachteil für die Therapie. Deshalb die photodynamischen Reaktionen von Membrankompo- können eine genaue Kenntnis der Signaltransduktionswe- nenten zu erforschen. Der Leiter der Gruppe, Peter Pohl, ge und der Molekülinteraktionen, die durch die Zytokine folgte Ende 2004 einem Ruf auf einen Lehrstuhl an der Uni- reguliert werden, zur Entwicklung präziserer therapeu- versität Linz in Österreich. tischer Interventionen führen. Die Abteilung Molekulare Genetik befasst sich mit den molekularen Mechanismen, die die Entwicklung und die Das Zytokinrezeptor-Signaling verläuft über STATs (Signal Funktion von Zellen des Blutes und des Immunsystems transducers and activators of transcription). Eine abnor- kontrollieren. Die Erforschung dieser Prozesse ist eine male Aktivität bestimmter Mitglieder der STAT-Familie, ins- unabdingbare Voraussetzung nicht nur für das Verständnis besondere von Stat3 und Stat5, steht im Zusammenhang ihrer pathologischen Veränderungen sondern auch für die mit einer Vielzahl bösartiger Krebserkrankungen. Die Entwicklung rationaler diagnostischer und therapeuti- EMBO-Forschungsgruppe von Uwe Vinkemeier untersucht scher Verfahren. Die Wissenschaftler nutzen zielgerichte- die subzelluläre Lokalisation der STATs und die damit te Mutagenese, um Gene zu analysieren, von denen anzu- zusammenhängenden Konsequenzen für die Gen Induk- nehmen ist, dass sie wichtige regulatorische Funktionen tion. Eine der Herausforderungen ist es, herauszufinden, ween the subcellular localization of STATs and the resul- pounds) from which the active biomolecules are liberated ting consequences on gene induction. One of the challen- by UV light. These compounds (caged cAMP, caged ges is to determine how target gene access is regulated cGMP) have proved extremely useful in elucidating intra- and how this might influence the execution of transcrip- cellular signal transduction chains. tional programs. The goal is a complete description of the The Cellular Physiology Group is concentrating on signal mechanisms underlying STAT subcellular trafficking by transduction in the blood brain barrier (BBB), using co-cul- focusing on the biochemical basis of nuclear accumula- tures of endothelial and glial cells as model systems. Cur- tion as well as phosphorylation-independent nucleocyto- rent emphasis is on the structure and function of tight plasmic shuttling. The group will also investigate how junction proteins sealing the interendothelial cleft. The these activities modulate the transcriptional functions of group is also investigating the role of protein kinases in the the STATs and influence complex phenotypes such as regulation of the permeability of the BBB. The long-term growth or antiviral protection. goal is to elucidate the molecular basis of such permeabi- The Microscopic Technology Group offers a range of con- lity changes under physiological and pathological condi- ventional and advanced light and electron microscopic tions. methods as well as electrophysiology and microinjection The Biochemical Neurobiology Group is studying bioche- to all interested research groups in the institute. The group mical and molecular interactions of peptide degrading is available for all types of collaboration and the proces- enzymes and their neuropeptide substrates under patho- sing of common research projects. So, e.g. in terms of cha- logical conditions. The research is e.g. focused on the racterizing cyclic nucleotide-gated cation channels, it has function of angiotensin-converting enzyme (ACE) with long standing collaborations with the Synthetic Organic alcohol reward in the CNS. This process involves angio- Biochemistry Group. This group has continued the deve- tensin receptors and their signal transduction pathways. lopment of photolabile inactive compounds (caged com-

wie der Zugriff auf die jeweiligen Zielgene reguliert ist und dungen (caged cAMP, caged cGMP) haben sich als außer- wie dies die Tanskriptionsprogramme beeinflusst. Das Ziel ordentlich nützlich für die Untersuchung intrazellulärer der Gruppe ist die umfassende Beschreibung der Mecha- Signalketten erwiesen. nismen, nach denen der subzelluläre Transport der STATs Die Arbeitsgruppe Zellphysiologie fokussiert ihre For- abläuft. Dabei legt sie besonderes Augenmerk auf die bio- schung auf die Signaltransduktion in der Blut-Hirn-Schran- chemischen Grundlagen der Kernakkumulation und des ke (BHS). Dabei verwendet sie gemeinsam kultivierte phosphorylierungsabhängigen nukleozytoplasmatischen Endothel- und Gliazellen als Modellsystem. Gegenwärtig Transports. Zusätzlich wird untersucht, wie diese Aktivitä- stehen insbesondere Struktur und Funktion von Tight- ten die Transkriptionsfunktionen der STATs modulieren und junction-Proteinen, die den interendothelialen Spalt ab- komplexe Phänotypen, wie zum Beispiel Wachstum oder dichten, im Vordergrund des Interesses. Die Gruppe antiviralen Schutz, beeinflussen. erforscht auch die Rolle von Proteinkinasen bei der Regu- lation der Durchlässigkeit der BHS. Ziel ist es, die moleku- Die Arbeitsgruppe Mikroskopische Techniken bietet allen lare Basis der Veränderungen zu erforschen, denen die interessierten FMP-Gruppen ein breites Spektrum konven- Durchlässigkeit der BHS unter verschiedenen physiolo- tioneller und spezialisierter licht- und elektronenmikrosko- gischen und pathologischen Bedingungen unterliegt. pischer Methoden sowie Elektrophysiologie und Mikroin- jektion an. Die Gruppe ist offen für Kooperationen und Die Arbeitsgruppe Biochemische Neurobiologie unter- gemeinsame Forschungsprojekte. Sie charakterisiert zum sucht die biochemischen und molekularen Interaktionen Beispiel seit einigen Jahren zusammen mit der Arbeits- peptidabbauender Enzyme und ihrer Neuropeptidsubstra- gruppe Synthetische Organische Biochemie erfolgreich te unter pathologischen Bedingungen. Die Forschung zielt durch zyklische Nukleotide getriebene Kationenkanäle. unter anderem auf die Rolle des Angiotensin-konvertieren- Dabei kommen in dieser Arbeitsgruppe entwickelte, pho- den Enzyms (ACE) und seiner Substrate beim Alkohol- tolabile chemische Verbindungen (caged compounds) zum Reward im Belohnungssystem des ZNS. Dieser Prozess Einsatz, aus denen auf Anregung mit ultraviolettem Licht verläuft unter Einbeziehung von Angiotensinrezeptoren hin aktive Biomoleküle freigesetzt werden. Diese Verbin- und deren nachgeschalteten Signalketten. tides ofGPCRs. addition, westudythefunctionofcleavablesignalpep- caused bytransport-deficientmembraneproteins.In to identifynewdrugtargetsforthetreatmentofdiseases ry pathwayandtoidentifytheproteinsinvolved.Ouraimis mechanisms ofGPCRqualitycontrolintheearlysecreto- We useNDI-causingV retained. ne, whereasinothercases,stillfunctionalproteinsare mutant receptorsaretransportedtotheplasmamembra- the QCSmayworkfaultily. Sometimes,non-functional across theERmembrane.We havenowstudiedthesignal necessary forthetranslocation ofthereceptor’s Ntail it doesnotinfluencereceptorexpression, butratheris tide ofthehumanendothelinBreceptor andshowedthat We studiedthefunctionalsignificanceofsignalpep- other membraneproteins. res additionalsignalpeptideswasnotclearforGPCRsand ses oftheER.Thereasonwhythissecondsubsetrequi- ved offduringtheinsertionprocessbysignalpeptida- group possessesadditionalsignalpeptidesthatareclea- arethat partsequences of the maturesecond receptors.A signal sequences:onegroupcontainsanchor ER insertionofGPCRsismediatedbytwodifferenttypes Significance ofcleavable signalpeptidesof 1. of thevasopressinV2receptor(V insipidus (NDI),whichiscausedbymutationsinthegene diseases. AnexampleisX-linkednephrogenicdiabetes unable topasstheQCSandconsequentlyinherited receptor mutationsfrequentlyleadtomisfoldedproteins of GPCRsisclinicalsignificance.Naturallyoccuring The studyoftheearlystagesintracellulartransport plasma membrane. diate compartment(ERGIC)andtheGolgiapparatusto interme- to enterthevesiculartransportviaER/Golgi s ded, andcorrectfoldingismonitoredbyaqualitycontrol reticulum arefol- (ER).DuringERinsertion,theproteins with theirinsertionintothemembraneofendoplasmic The intracellulartransportofmembraneproteinsstarts l The groupisinterestedintheearlystagesofintracel- G-PROTEIN-COUPLED RECEPTORS QUALITY CONTROLANDERINSERTIONOF Group Leader:PDDr. RalfSchülein P ular trafficking of G-protein-coupled receptors (GPCRs). ular traffickingofG-protein-coupledreceptors ystem (QCS).Onlycorrectlyfoldedproteinsareallowed ROTEIN TRAFFICKING GPCRs 2 R mutants tocharacterizethe 2 R). Itisnoteworthythat ensured byN-glycosylation. lular location.Thesolubilityofthelatterhelixseemstobe transmembrane helicesandaneighthhelixwithextracel- t that thecell-penetratingpeptide penetratinrescuesthe (ERGIC-reaching; classB)ofthe V L62P mutant(ER-retained;class A) andtheY205Cmutant QCS actinginacompartment-specific way. Byusingthe been described.We havenowfoundnovelinhibitorsofthe inefficiently detectedattheERlevel. net withintheQCSrecognizingthoseproteinsthatareonly its foldingstate.TheERGICmayrepresentasecondsafety the ERGICisnotdeterminedbyitsexpressionlevelbut over, wecouldshowthattheabilityofamutanttoreach system.More- ted totheERviaretrogradetransport recognized inthiscompartmentandaremostlikelyrerou- Fig. 1),incontrast,reachtheERGIC.Thesemutantsare Class Bmutants(e.g.R143P, Y205C,R337X,V226E,InsQ292; exclusively intheERandneverleavethiscompartment. A results showthatthesemutantsfallintotwoclasses.Class question usingmisfoldedNDI-causingV2Rmutants.Our Golgi apparatusarealsoinvolved.We haveaddressedthis teins isrestrictedtotheERorwhetherERGIC It wasunknownwhetherqualitycontrolofmembranepro- 2.1. QualitycontrolintheERGIC 2 V nents mayallowtransportandleadtoafunctionalrescue. ing butpreventtheinteractionwithqualitycontrolcompo- still functional.Here,substancesthatdonotinfluencefold- times overprotectiveandretainsmutantproteinsthatare second strategyisbasedonthefactthatQCSsome- to passtheQCS(“pharmacologicalchaperones”).The ding ofthemutantproteinsandconsequentlyallowthem involves thedevelopmentofligandsthatfavorcorrectfol- Two strategiesarebeingpursuedatthemoment.Thefirst mutant membraneproteinsareofclinicalsignificance. Pharmacological strategiestorescuetransport-deficient 2.2. ofNDI-causingV Compartment-specific rescue ing V peptide oftheCRF b the Ntailofmaturereceptor. TheCRF However, itisnotcleavedafterERinsertionandremainsat tide oftheCRF tors type1(CRF peptides oftheratcorticotropin-releasingfactorrecep- he firstGPCRcontainingeighthydrophobichelices:seven ut stronglypromotesreceptorexpression.Thesignal . 2 mutants (e.g.L62P, DL62-R64,S167L;Fig.1)areretained R the V Analysis ofqualitycontrolmechanisms mutants antagonists mediatingtherescueofsomeNDI-caus- 2 R mutants aspharmacologicalchaperoneshave 2 R 1 R 1 R) andtype2 is notnecessaryforNtailtranslocation 2 α R also promotesreceptorexpression. α (CRF 2 R 2 as amodel,weshow α 2 R). Thesignalpep- α R thus represents 2 R

39 Cellular Signalling/ Molecular Genetics FIGURE 1 Two-dimensional model of the V2R. The positions of class A mutants (L62P, DL62-R64, S167L) are indicated by open rectangles, the positions of class B mutants (R143P, Y205C, R337X, V226E, InsQ292) are indicated by black rectangles. The following posttranslational modifications of the V2R are depicted: N-glycosylation at position N22, palmitoylation at positions C341 and C342 and a disulfide bond formed between residues C112 and C192.

transport of the Y205C mutant but not that of the L62P Deutsche Forschungsgemeinschaft mutant. We have also shown that the peptides exert their “ER-Insertion und Qualitätskontrolle von G-Protein-gekop- function by displacing the BiP chaperone from the mutant pelten Rezeptoren” (Teilprojekt A11 im Sonderforschungs- receptor in the ERGIC. bereich 366 “Zelluläre Signalerkennung und Umsetzung”) Ralf Schülein, Walter Rosenthal Group members Selected publications (FMP authors in bold) Dr. Ute Donalies Dr. Claudia Rutz** Oueslati M, Hermosilla R, Oorschot V, Donalies U, AnjaThielen (Doctoral student)* Schönenberger E, Beyermann M, Oehlke J, Wiesner B, Martina Alken (Doctoral student)* Oksche A, Klumperman J, Rosenthal W, Schülein R (2005) Morad Oueslati (Doctoral student) Compartment-specific rescue of nephrogenic diabetes Dagmar Michl (Technical assistance)** insipidus-causing vasopressin V2 receptor mutants by cell-penetrating peptides. J Cell Biol, in revision External funding Thielen A, Oueslati M, Hermosilla R, Krause G, Oksche A, Deutsche Forschungsgemeinschaft Rosenthal W, Schülein R (2005) The hydrophobic amino “Struktur und Funktion von Transportsignalen des Vaso- acid residues in the membrane-proximal C tail of the G pro- pressin V2-Rezeptors” (Teilprojekt A3 im Sonderfor- tein-coupled vasopressin V2 receptor are necessary for schungsbereich 449 “Struktur und Funktion membranstän- transport-competent receptor folding. FEBS Lett, in press diger Rezeptoren”) Alken M, Rutz C, Köchl R, Donalies U, Oueslati M, Furkert Ralf Schülein, Walter Rosenthal J, Wietfeld D, Hermosilla R, Scholz A, Beyermann M, Rosenthal W, Schülein R (2005) The signal peptide of the rat corticotropin-releasing factor receptor 1 promotes

* part of period reported ** part-time mosilla R,SchaeferM, Engelsberg A, Hermosilla R,KarstenU, stin interactionandsequestration.BiochemJ379,573-585 EP4 receptorforagonist-inducedphosphorylation‚arre- Ser/Thr residuesintheC-terminaldomainofhuman Rönnstrand L,PüschelG(2004)Requirementofdifferent Wüller S,WiesnerB Krause E Hermosilla R,OueslatiM,DonaliesU,SchönenbergerE, functional receptor. BiochemJ390,455-64 receptor expressionbutisnotessentialforestablishinga Y205C L62P Traffic 5,993-1005 pathway.different compartmentsoftheearlysecretory in Disease-causing V2vasopressinreceptorsareretained Neuschäfer-Rube F, RehwaldM,HermosillaR, J stability ofwild-typeandmutantvasopressinV2receptors. ce cellsurfaceexpressionbyincreasingconformational A antigens. JBiolChem278,22998-3007 surface expressionoftumor-associated O-linkedglycan ken B,RehmA(2003)TheGolgiprotein RCAS1controlscell Biol Chem279,47254-47263 (2004) Pharmacochaperonespost-translationallyenhan- PE PE , Oksche A, , Löffler A, Rosenthal W, SchüleinR Schülein R,RosenthalW Furkert J,KrauseG Schülein R Schülein R 0 2 0 2 , M/I M/I Oksche (2004) , , Dör- Her- , G. Püschel,UniversityofPotsdam Berlin A. Rehm,MaxDelbrückCenterforMolecularMedicine R. Hermosilla,Charité–UniversityMedicineBerlin A. Oksche,Charité–UniversityMedicineBerlin R.S. Hedge,NIHBethesda,MD,USA Collaborations PE PE by penetratintreatment. only thetransportofY205Cmutantisrescued SD(n=30cells).Notethatin(A)and(B) values ± lization atthecellmembrane.Columnsshowmean of thereceptorsandratios>1apredominantloca- indicate apredominantlyintracellularlocalization cence signals(M/I)wasdetermined.Ratios<1 ratio ofcellmembraneandintracellularfluores- Quantitative analysisofthepeptideeffect.The on isindicatedbyyellow).Scalebars,5µm.(B) signals wascomputed(rightpanels;co-localizati- Trypan blue(red,centralpanels)andoverlayofthe left panels),plasmamembraneswerestainedwith ce signalsofthereceptorswererecorded(green, (Pe, 1µM)orwithvehicle(-).TheGFPfluorescen- microscopy. withpenetratin Cellsweretreated transfected HEK293cells.(A)Laserscanning L62P (classA)andY205CB)intransiently lar distributionoftheGFP-taggedV Influence ofpenetratintreatmentonthesubcellu- FIGURE 2 2 R mutants

41 Cellular Signalling/ Molecular Genetics ANCHORED SIGNALLING plasma membrane constitutes an exocytosis-like process. Group Leader: PD Dr. Enno Klussmann A better understanding of this mechanism will also yield a better understanding of other cAMP-dependent exocytic WATER REABSORPTION IN THE KIDNEY processes such as renin secretion from juxtaglomerular cells, insulin secretion from pancreatic βcells or proton A major function of the kidney is the production of urine. A secretion from gastric parietal cells. Dysregulations of human kidney generates 180 l of primary urine per day, these processes cause various diseases including hyper- most of which is water. It is obvious that excretion of such tension, diabetes mellitus and gastric ulcers. vast amounts of fluid would cause dehydration and death. Therefore, mechanisms have evolved which carefully AKAP18δ anchors PKA to AQP2-bearing vesi- control body water balance by regulating water reabsorp- cles tion from primary urine. Most of the water (90%) is reab- The above mentioned phosphorylation of AQP2 is not the sorbed by passive diffusion along an osmotic gradient sole prerequisite for its redistribution. We have shown that established between the primary urine and the surround- the tethering of PKA to subcellular compartments by so ing tissue. Reabsorption of the remaining 10% of water is called A-kinase anchoring proteins (AKAPs) is also essen- regulated by antidiuretic hormone (Arginine-vasopressin, tial for the AQP2 shuttle to occur. During the search for AVP) in particular cells of the kidney, the principal cells. AKAP(s) involved in the shuttle, a new splice variant of Principal cells line the collecting duct, the terminal part of AKAP18, AKAP18δ, was identified. Biochemical and cell the tubular system transporting urine to the bladder. Loss biological approaches revealed that AKAP18δ functions of responsiveness to the hormone results in a disease as an AKAP in vitro and in vivo. Immunofluorescence known as diabetes insipidus (DI). DI is characterized by microscopy showed that in the kidney, AKAP18δ is mainly a massive loss of water (up to 20 l per day if untreated). expressed in principal cells of the terminal section of the collecting duct, closely resembling the distribution of The molecular mechanism underlying AVP-mediated AQP2. AKAP18 was identified on the same intracellular water reabsorption in the kidney involves several key pro- δ vesicles as AQP2 and PKA. AVP not only recruited AQP2, teins including the vasopressin V2 receptor, and aquapo- but also AKAP18 to the plasma membrane (Fig. 2). AVP rin-2 (AQP2). The latter belongs to the family of water δ caused the dissociation of AKAP18 and PKA. The data channels whose discovery yielded Peter Agre the Nobel δ suggest that AKAP18 is involved in the AQP2 shuttle by Prize in Chemistry in 2003. AVP binds to the vasopressin δ anchoring PKA in close proximity to AQP2 (Henn et al. V2 receptor located on the surface of renal principal cells 2004). thereby triggering the redistribution of AQP2 from intracellular vesicles into the apical (urine-facing) plasma The small GTPase RhoA mediates the diuretic membrane. This process is also known as the AQP2 effects of prostaglandin E2 shuttle. The small GTPases of the Rho family (Rho, Rac, Cdc42) participate in the regulation of the F-actin cytoskeleton. In The insertion of AQP2 into the plasma membrane introdu- previously published papers we have demonstrated that ces water-selective pores and facilitates water entry into the small GTPase RhoA, a particular member of the Rho the cells. Water exits the cells through aquaporins-3 and family, in its active state (GTP-bound) induces the forma- -4 constitutively located in the basolateral (tissue-facing) tion of F-actin-containing stress fibers in primary cultured plasma membrane. The driving force is again the osmotic principal cells,and prevents the AQP2 shuttle. We obser- gradient established between primary urine and the tis- ved that AVP causes a decrease of F-actin-containing sue. On the molecular level, AVP stimulates the elevation stress fibers, and inhibition of RhoA. of the second messenger cAMP followed by activation of protein kinase A (PKA). PKA, in turn, transfers a phospha- Prostaglandin E2 (PGE2) antagonizes AVP-induced water te group to AQP2. This phosphorylation is a prerequisite reabsorption. Using primary cultured rat inner medullary for the redistribution of AQP2. The mechanism is depicted collecting duct principal (IMCD) cells, we have shown that in Figure 1. stimulation of prostaglandin EP3 receptors induced RhoA activation and formation of F-actin-containing stress fibers The aim of our group is to identify further proteins involved in resting IMCD cells, but did not modify the intracellular in the redistribution of AQP2. This will lead to a detailed localization of AQP2. However, the AVP-induced AQP2 understanding of the underlying mechanism, and may translocation was strongly inhibited. In addition, stimula- eventually lead to a treatment of certain cases of DI. The tion of EP receptors inhibited the AVP-induced RhoA in- translocation of AQP2 from intracellular vesicles to the 3 ** part-time Christian Hundsrucker(Doctoral student) Christopher Blum(Doctoralstudent) Eduard Stefan(Doctoralstudent) Viola Weber (Doctoralstudent) Katja Santamaria(Doctoralstudent) Dr. PavelNedvetsky Dr. Volker Henn Dr. TheresaMcSorley Dr. DorotheaLorenz** Group members cAMP andcytosolicCa receptor stimulationwasindependentofincreasesin F-actin-containing stressfibers.TheinhibitoryeffectofEP activation andtheAVP-induced depolymerizationof A underlying thediureticeffectsofPGE together, thedatasuggestthatsignalingpathway by PKA,whichisknowntoinhibitthisGTPase.Taken elevation ofcAMPresultsinthephosphorylationRhoA the G-proteinsG12/13.Furtherexperimentsshowedthat 2003a, 2003b). dent RhoAactivationandF-actinformation(Tamma etal. of otherdiureticagents)includecAMP-andCa c Schematic representationofthevasopressin-mediatedwaterreabsorption.Inrestingprincipalcells(A)aquaporin-2(AQP2)islocatedinintra- FIGURE 1 ylyl cyclase;G a ma membranefacingthelumenofrenalcollectingduct.Water from theprimaryurineenterscellsthroughAQP2alonganosmoticgradient cade whichcausesthephosphorylationofAQP2(phosphorylatedAQP2,p-AQP2)anditstranslocationfromintracellularvesiclesintoplas- nd exits the cells through aquaporin-3 and aquaporin-4 (AQP3 und AQP4) constitutively present inthebasolateralplasmamembrane.AC,aden- nd exitsthecellsthroughaquaporin-3andaquaporin-4(AQP3undAQP4)constitutivelypresent ellular vesicles.B.Thebindingofarginine-vasopressin(AVP) tothevasopressinV2receptor(V s , stimulatory G-Protein; C and R, catalytic and regulatory subunits of protein kinaseA(PKA),respectively., stimulatoryG-Protein;CandR,catalyticregulatorysubunitsofprotein 2+ and ismostlikelymediatedby 2 (and probablythose 2+ -indepen- 3 B E. Klussmann,W. Rosenthal human disease”(QLK3-CT-2002-02149) “Anchored cAMPsignalling-implicationsfortreatmentof European Community, 5thFrameworkprogramme W. Rosenthal, E.Klussmann Funktion“ (Ro597/9-1) Ht31 undRt31Untersuchungenzuihrerbiologischen „Charakterisierung derProteinkinaseA-Ankerproteine Deutsche Forschungsgemeinschaft E. Klussmann,W. Rosenthal renaler Hauptzellen“(Kl1415/1-1) ten Translokation vonAquaporin-2indiePlasmamembran „Die RolledesZytoskelettsbeiderVasopressin-induzier- Deutsche Forschungsgemeinschaft External funding Michael Gomoll(Trainee) Andrea Geelhaar(Technical assistance) 2 R ) activatesacAMP-dependentsignallingcas-

43 Cellular Signalling/ Molecular Genetics AQP2 AKAP18δ overlay FIGURE 2 The effect of AVP on the subcellular distribution of AQP2 and AKAP18δ in primary cultured rat inner -AVP medullary collecting duct principal (IMCD) cells. IMCD cells were left untreated (-AVP) or incubated with AVP (100 nM, 15 min), fixed and permeabilized. AQP2 was detected by incubation with goat anti-AQP2 and secondary Cy3-conjugated antibodies, AKAP18δ with affinity-purified rabbit anti-AKAP18δ A18δ4 and secondary Cy5-conjugated antibodies. Immunofluo- +AVP rescence signals were detected by laser scanning microscopy. The overlay of Cy3 and Cy5 fluorescence signals is shown in the right panel. Scale bars, 20 µm.

Selected publications (FMP authors in bold) Tamma G, Klussmann E, Procino G, Svelto M, Rosenthal W, Valenti G (2003) cAMP-induced AQP2 translocation is Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F, associated with RhoA inhibition through RhoA phosphory- Schmitt R, Vossebein L, Tamma G, Beyermann M, Krause lation and interaction with RhoGDI. J Cell Sci 116, 1519- E, Herberg FW, Valenti G, Bachmann S, Rosenthal W, 1525 Klussmann E (2004) Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in Storm R, Klussmann E, Geelhaar A, Rosenthal W, Maric K the vasopressin-induced aquaporin-2 shuttle in renal prin- (2003) Osmolality and solute composition are strong regu- cipal cells. J Biol Chem 279, 26654-26665 lators of AQP2 expression in renal principal cells. Am J Physiol 284 (Renal Section), F189-198 Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl P, Maric K (2003) Cyclic AMP is sufficient for trigger- Collaborations ing the exocytic recruitment of aquaporin-2 in renal epithelial cells. EMBO Rep 4, 88-93 Prof. K. Tasken, University of Oslo, Norway AKAPs in cardiac myocytes Tsunoda SP, Wiesner B, Lorenz D, Rosenthal W, Pohl P (2004) Aquaporin-1, nothing but a water channel. J Biol Dr. M. Zaccolo, University of Padova, Italy Chem 279, 11364-11367 Modulation of Ca2+ fluxes in cardiac myocytes

Tamma G, Wiesner B, Furkert J, Oksche A, Schaefer M, Professor J. D. Scott, Howard Hughes Medical Institute, Valenti G, Rosenthal W, Klussmann E (2003) The prosta- Vollum Institute, Oregon Health & Sciences University, glandin E2 analogue sulprostone antagonizes vasopres- Portland, Oregon, USA sin-induced antidiuresis through activation of Rho. J Cell Peptide disruptors of AKAP-PKA interactions Sci 116, 3285-3294 A Professor F. W. Herberg,UniversityofKassel reabsorption Rho-dependent signallingintheAVP-mediated water P Role ofphosphodiesterasesinrenalprincipalcells Prof. M.Houslay, UniversityofGlasgow, Scotland,UK rof. G.Valenti, UniversityofBari,Italy nalyses ofAKAP-PKAinteractions

45 Cellular Signalling/ Molecular Genetics CELLULAR IMAGING Light-microscopy cannot differentiate whether the recep- Group Leader: Dr. Burkhard Wiesner tor is located at the intracellular side of the membrane or in the plasma membrane of the cell. ENHANCEMENT OF THE CELL SURFACE EXPRESSION BY INCREASING CONFOR- For human NDI-causing mutants (hL62P, h∆LAR 62-64, MATIONAL STABILITY OF VASOPRESSIN hH80R, hW164R, hS167T, hS167L, hC319Y, hP322S) and V2 RECEPTORS several in vitro mutants (hD136A, hD368K/S371X, hF328A) transiently expressed in HEK cells, also predominant ER This facility offers a range of conventional and advanced retention was observed. Interestingly, cell surface expres- light and electron microscopic methods as well as electro- sion of h∆LAR 62-64, hD136A, hS167T, hP322S, and hF328A physiology to all interested research groups in the insti- was only restored by SR121463B. In the case of the tute. The group is available for all types of collaboration mutants hL62P, hH80R, hW164R, hS167L, and hD368K/ including advice in preparation techniques, and the pro- S371X none of the two antagonists restored cell surface cessing of common research projects. expression. Only for the mutant hC319Y, both antagonists

promoted cell surface expression as found for the mV2R.

About 50% of nephrogenic diabetes insipidus (NDI)-caus- The data show that ER-retained mutant V2Rs differ in their ing mutations in the vasopressin V2 receptor (V2R) gene sensitivity to antagonist-promoted cell surface expression. code for single amino acid replacements (missense muta- It is likely that these differences can be attributed to the tions). In most cases the encoded mutant V2Rs are misfol- extent of the folding defect and/or an alteration of the bin- ded and retained in the ER. Improvement of this situation ding pocket. Further studies are required to analyze the is to be achieved by the application of specific substan- general applicability of antagonists in the treatment of NDI. ces (e.g. pharmacochaperones). Such cell permeable antagonists should restore cell surface expression. So we Further projects involved: studied the effects of SR121463B and SR49059 (V2R and A-kinase anchoring proteins and the exocytotic recruit-

V1R-specific antagonists) on ER-retained V2Rs transiently ment of aquaporin-2 (D. Lorenz, B. Wiesner, M. Ringling, B. expressed in HEK293 cells. Such investigations can be Oczko in cooperation with Enno Klussmann, Anchored accomplished generally by receptor binding studies Signalling) (radioactive proof of the number of receptors of the cell Application of novel caged compounds (B. Wiesner, J. surface). If, however, the assigned substance (pharmaco- Eichhorst, B. Oczko in cooperation with Volker Hagen, Syn- chaperone) blocks the binding domain for the ligand, this thetic Organic Biochemistry) method is not applicable. In this situation, the employment of confocal microscopy is very helpful. So the receptor Water flux through water channels (D. Lorenz, B. Wiesner, can be located with the help of green fluorescent protein B. Oczko in cooperation with Peter Pohl, Biophysics) (GFP) microscopically. Optical staining of the cell membra- Cellular uptake of peptides (B. Wiesner, B. Oczko in coope- ne by further coloring (trypan blue) makes a simple alloca- ration with J. Oehlke, Peptide Lipid Interaction/Peptide tion possible between intracellular and membrane locali- Transport; and M. Beyermann, Peptide Synthesis) zation of the receptor (see Fig. 1). Constitutive internalization of the human V2 Vasopressin receptor (A. Schmidt, B. Wiesner in cooperation with R. In the case of the wild-type murine V R (mV R), which is 2 2 Hermosilla, Charité group) mainly localized in the ER in untreated controls, both antagonists promoted a time- and dose- dependent restora- DNA binding controls inactivation and nuclear accumulation of the cell surface expression. SR49059-mediated tion of the transcription factor Stat1 (B. Wiesner, B. Oczko restoration of cell surface expression of the mV2R was in cooperation with U. Vinkemeier, Cellular Signal Proces- accompanied by a dramatic increase in [3H]AVP binding sing) sites. Enhancement of the cell surface expression by increasing Figure 2 shows, that with the microscopic investigation the conformational stability of G protein coupled receptors rearrangement (from the ER to the cell membrane) is fast (B. Wiesner, J. Eichhorst, B. Oczko in cooperation with run off. This phenomenon is justified by the optical resolu- A. Oksche, Charité group; and S. Wüller, RWTH Aachen tion of light microscopy. The binding studies show the Medical School). functional receptor in the plasma membrane of the cell. c: 13h).T [ increase inthenormalizedfluorescence intensitiesisshowninblack.Inparallel,membranepreparationswereanalyzed forspecificbindingof siently expressingthemV Antagonist-mediated restorationofcell surfaceexpressionanalyzedbyquantitativelaserscanningmicroscopyandbinding analysis.Cellstran- FIGURE 2 HEK293 cellstransientlyexpr FIGURE 1 3 H]AVP. Thecurverepresentingtheincreaseinspecifically bound[

Trypan blue mV2R.GFP op panel:mV 2 R.GFP fusionpr 2 R.GFP were treated for up to 16 h with the specific vasopressin receptor antagonist. The fitted curve representing the R.GFP weretreatedforupto16hwiththe specificvasopressinreceptorantagonist.Thefittedcurverepresenting essing themV A otein. Bottompanel:Plasmamembranestainedwithtrypanblue. 2 R.GFP wer e treatedwiththevasopressinreceptorantagonistSR49059forupto13h(A:oh;B:7h; 3 H]AVP isshowningray. CC

47 Cellular Signalling/ Molecular Genetics Group members Wüller S, Wiesner B, Löffler A, Furkert J, Krause G, Her- mosilla R, Schaefer M, Schülein R, Rosenthal W, Oksche Dr. Dorothea Lorenz** A (2004) Pharmacochaperones post-translationally enhan- Antje Schmidt (Student)* ce cell surface expression by increasing conformational Brunhilde Oczko (Technical assistance) stability of wild-type and mutant vasopressin V2 receptors. Jenny Eichhorst (Technical assistance)* J Biol Chem 279, 47254-47263 Martina Ringling (Technical assistance)

Collaborations External funding Prof. Ricardo Hermosilla Deutsche Forschungsgemeinschaft Pathology of signal transduction „Degradations-Mechanismen des humanen Vasopressin- Charité-Universitätsmedizin Berlin V2-Rezeptors und einiger von Patienten mit X chromoso- maler nephrogener Diabetis insipidus isolierten V2- PD Dr. Alexander Oksche Rezeptormutanten“ (He 4486/1-1 und 1-2) G protein-coupled receptors: Signal transduction and Ricardo Hermosilla (Charité - University Medicine Berlin), regulation of cell surface expression Burkhard Wiesner Charité-Universitätsmedizin Berlin

Selected publications (FMP authors in bold) Geissler D, Kresse W, Wiesner B, Bendig J, Kettenmann H, Hagen V (2003) DMACM-Caged Adenosine Nucleotides: Ultrafast Phototriggers for ATP, ADP, and AMP Activated by Long-Wavelength Irradiation. ChemBioChem 4, 162-170

Hagen V, Frings S, Wiesner B, Helm S, Kaupp UB, Bendig J (2003) [7 (Dialkylamino)-coumarin 4 yl]methyl-caged compounds as ultrafast and effective long-wavelength phototriggers of 8 Bromo-substituted cyclic nucleotides. ChemBioChem 4, 434-442

Meyer T, Marg A, Lemke P, Wiesner B, Vinkemeier U (2003) DNA binding controls inactivation and nuclear accumulation of the transcription factor Stat1. Genes Dev 17, 1992-2005

Tamma G, Wiesner B, Furkert J, Hahm D, Oksche A, Schaefer M, Valenti G, Rosenthal W, Klussmann E (2003) The prostaglandin E2 analogue sulprostone antagonizes vasopressin-induced antidiuresis through activation of Rho. J Cell Sci 116, 3285-3294

Tsunoda S, Wiesner B, Lorenz D, Rosenthal W, Pohl P (2004) Aquaporin 1, nothing but a water channel. J Biol Chem 279, 11364-11367

Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F, Schmitt R, Vossebein L, Tamma G, Beyermann M, Krause E, Herberg FW, Valenti G, Bachmann S, Rosenthal W, Klussmann E (2004) Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin 2 shuttle in renal principal cells. J Biol Chem 279, 26654-26665

* part of period reported ** part-time with CDstudiesandmolecular modelling indicatedthat Binding studiesbySPRandpeptide mappingcombined The occludin-ZO-1interactionwas studiedinmoredetail. Kolbe). E. Krause,MassSpectrometry)andMDC(K.Gast;M. groups oftheFMP(G.Krause,StructuralBioinformatics; of TJassembly(Figure).Thisworkwassupportedby worked out.Thisprinciplemayserveasacommonfeature ple oftransmembraneTJproteinsviatheZO-1hasbeen ted ZO-1alsoformsadimer. Ageneraldimerization princi- the transmembraneTJproteins,membrane-associa- corroborated byourfindingthattherecruitingproteinof feature oftransmembraneTJproteins.Thispotentialis TJ, wehypothesizethathomodimerizationisastructural junctional adhesionmoleculeJAMisknowntodimerizein its secondextracellularloop.Sincethetransmembrane self-associate viaitscoiledcoil-domainandclaudin-5 second extracellularloopisadimer. Thus,occludinmay domain wasidentified.Inclaudin-5,wedetectedthatthe din, dimerizationofthecytosolicC-terminalcoiledcoil- occludin andofclaudin-5inonecellmembrane.Forocclu- association). Thisdemonstratesself-associationbothof fluorescence resonanceenergytransfer(intracellular calized inthecellmembraneofsameandshowed Differently taggedTJstrand-formingclaudin-5alsocolo- coprecipitated withitself(intracellularassociation). cell membranecontactsofthesameandcouldbe brane TJproteinoccludinwasfoundcolocalizingwithin ing thetightjunctions(TJ).Theregulatoryandtransmem- transmembrane andmembraneassociatedproteinsform- A mainfocuswastheself-associationmechanismof which arenon-permeablethroughtheBBB. ved administrationofsuchantitumororantiepilepticdrugs open theBBBforpharmacokineticapplication,e.g.impro- epilepsy etc.Anotheraspectistodevelopapproaches of cerebraldiseases,suchasstroke,inflammation, n blood-brain barrier(BBB)proteinsforthedevelopmentof molecular aspectsofstructure,functionandregulation stress inthebrain.Theaimofthesestudiesistoexplore normal andpathologicalconditions,suchasoxidative i This groupinvestigatessignaltransductionpathways OF CELL-CELLCONTACT PROTEINS STRUCTURE, FUNCTION,ANDREGULATION Group Leader:Dr. IngolfBlasig M ncluding cell-cellandprotein-proteininteractionsunder ew pharmacologicalapproachestoimprovetreatment OLECULAR CELLPHYSIOLOGY b molecular mechanismofformingintermolecularhelical occludin werealsoidentified.Inconclusion,acommon ween thebasichelicesofZO-1andacidiconesin Leipzig). thelium (collaborationM.Schroeter, MaxPlanck Institute, taining BBBpropertiesinthebarrierformingbrainendo- approaches targeted,amongothers,toastrocytesmain- zophrenic eventswerereducedbypharmacological found foropeningoftheBBB.BBBdisturbancesandschi- orders withdepressionwerestudiedandindications dis- mood Thus, that thesedisturbancescanbetreated. tal findingsthatoxidativestressmayinjuretheBBBand Clinical studieswerecontinuedtosupportourexperimen- boration C.Humpel,Innsbruck/Austria). BCEC which,hence,canmodulateBBBfunctions(colla- expression ofNGFreceptors.Thisisanewpathwayin ration ofthecells,nervegrowthfactor(NGF)releaseand showed thatexposuretointerleukin-1ßresultsinprolife- response tooxidativestress.Continuativeinvestigations results suggestedthatcytokinesareinvolvedinthe wa, Canada)aswellformembraneproteins.Earlier E. Krause,Dept.PeptideChemistry;D.Stanimirovic/Otta- * partofperiodreported Anna Y. Andreeva(Doctoralstudent)* Dr. Darkhan Utepbergenov* Dr. Christine Rückert* Dr. JörgPiontek Dr. ReinerF. Haseloff Group members t ZO-1. AsimilarassociationwasfoundbetweenZO-1and bundle withthreehelicesontheSH3-hinge-GuKunitof occludin’s coiled-coildomaininteractsasathree-helix dant proteinsusingtheRotofor aimed atimprovingtheconditionsfortargetinglowabun- protein phosphorylation).Moreover, investigations were ways byanalyzingpost-translationalmodifications(e.g., introduced whichallowtheelucidationofsignallingpath- selected proteins.Novelproteomictechniqueshavebeen regulated afterhypoxia,activitydatawereacquiredfor In additiontoidentifyingproteinsthatareup-ordown- xia onculturesofbraincapillaryendothelialcells(BCEC). Further studieswereaccomplishedontheeffectsofhypo- (J. Schneider-Mergener) Berlin. F matics; M.Beyermann,PeptideChemistry),andwiththe boration withintheFMP(G.Krause,StructuralBioinfor- association ofZO-1atadherensandtightjunctions.Colla- identified asageneralmolecularprincipleorganizingthe he adherensjunctionprotein ree University(O.Huber)andtheHumboldt undles betweenZO-1and α α - © catenin andoccludinwas - technology (collaboration catenin. Ionbindingsbet-

49 Cellular Signalling/ Molecular Genetics Claudin-5 dimer JAM dimer Occludin dimer

extra cellular

cell membrane

PDZ domains

SH3 domain

ZO -1 di GuK domain mer

FIGURE 1 Scheme of the dimerization concept showing that transmembrane TJ proteins, as well as ZO-1 may dimerize. Claudin-5 dimerizes via its second extracellular loop (2.ECL; solid double arrow) and occludin via its coiled coil-domain (CC; interacting cylinders). JAMs are known to dimerize via its extracellular domain (asterisk). ZO-1 dimerizes via its SH3-GuK unit. The positively charged (+) helices of ZO-1 (H1, H2 in GuK; CC1 between SH3 and GuK) bind to negatively charged (-) CC-domain of occludin (two dotted arrows). PDZ-domain 3 of ZO-1 can associate to JAMs, PDZ-1 to claudins. N, N-terminus; yellow circles, disulfide bridge in claudin-5 first and occludin last loop.

Manjot Singh Bal (Doctoral student) Deutsche Forschungsgemeinschaft Dörte Lorberg (Doctoral student)* „Wechselwirkungen von Blut-Hirnschranken-Proteinen“ Kerstin Mikoteit (Doctoral student)* (BL 308/6-1, 6-2, 6-3) Sebastian L. Müller (Doctoral student) Ingolf Blasig, Gerd Krause Juliane Walter (Doctoral student)* Deutsche Forschungsgemeinschaft Lars Winkler (Doctoral student) „Lokalisation und Phophorylierungsmuster von Occludin“ Inga Roswadowski* (Student) (GK 238/3, Teilprojekt im Graduiertenkolleg „Schadenme- Jenny Kirsch (Student)* chanismus im Nervensystem – Einsatz von bildgebenden Markus Heine (Student)* Verfahren“ Birgit Lassowski (Student)* Ingolf Blasig Christian Niehage (Trainee)* Katrin Schulz (Student)* Bundesministerium für Bildung und Forschung Ariane Schuster (Student)* „Molecular Fingerprinting of the Blood-Brain Barrier in Constanze Wolf (Student)* Hypoxia – Targeting Brain Vessels to Treat Stroke“, NRC Nikolaj Zuleger (Student)* Reiner Haseloff Barbara Eilemann (Technical assistance) Deutscher Akademischer Austauschdienst Gislinde Hartmann (Technical assistance) „Molecular pharmacology of the HISS-resistin system” (PPP-Ungarn 324/ssch) External funding Ingolf Blasig Deutsche Forschungsgemeinschaft Deutscher Akademischer Austauschdienst „Extrazelluläre Loops von Blut-Hirnschranken-Proteinen“ „Natural polyphenols in the cardiovascular system” (BL 308/7-1) (323/bis Slowakei) Ingolf Blasig Ingolf Blasig

* part of period reported cit schizophrenia.SchizophreniaRes62,231-236 sed duringearlytreatmentwithantipsychoticsandindefi- press brain capillaryendothelialcells.JNeuro-Oncology, in caspase-3 inducescelldeathinmalignantC6gliomaand Mol CellNeurobiol25,25-39 barrier functionsinbraincapillaryendothelialcellsvitro. search oftheastrocyticfactor(s)modulatingblood-brain Stephan Christen,UniversitätBern, Switzerland Christian Humpel,UniversitätInnsbruck, Austria Maria Balda,UniversityCollegeLondon, UK Hans-Christian Bauer, UniversitätSalzburg,Austria Ottawa, Canada Danica Stanimirovic,InstituteofBiologicalSciences,NRC, Medizin Berlin-Buch,Germany Hannelore Haase,Max-Delbrück-CenterfürMolekulare Marina Bigl,UniversitätLeipzig,Germany lare MedizinBerlin-Buch,Germany Wolf-Hagen Schunck,Max-Delbrück-CenterfürMoleku- Hartwig Wolburg, UniversitätTübingen Collaborations Schmidt A,UtepbergenovDI,MuellerSL,BeyermannM Haseloff RF, Blasig IE Müller SL S Anna Andreeva „Phosphorylierung vonOccludin”(Stipendium) Schering-Stiftung G. Schreibelt Marie CurieGrant(HPMT-CT-2001-00399) European Community Moser KV, ReindlM, tion. CellMolLifeSci61,1354-1365 protein 1-potentialmechanismoftightjunctionregula- din bindstotheSH3-hinge-GuKunitofzonulaoccludens Zassler B, Chem, inpress s Huber O, G, DiefenbacherA, Schroeter ML,Abdul-KhaliqH,FrühaufS,HöhneR,Schick tion. BrainRes1017,53-60 express NGFreceptorsandsecreteafterinflamma- capillary endothelialcellsproliferateinresponsetoNGF, Schneider-Mergener J, tein occludinandtheadherensjunctionprotein hare acommoninteractionmechanismwithZO-1.JBiol elected publications Blasig IE,KrauseG , PortwichM, Blasig IE Blasig IE , HumpelC(2005)Proteindeliveryof Blasig IE , BauerH-C,H(2005).In Krause G,BlasigIE Schmidt A,UtepbergenovDI ( (2003) Serum S100B isincrea- FMP authorsinbold) (2005) Thetightjunctionpro- , HumpelC(2004)Brain (2004) Occlu- β -catenin , ,

51 Cellular Signalling/ Molecular Genetics BIOCHEMICAL NEUROBIOLOGY ACE2, an enzyme closely related to ACE: in addition, to its Group Leader: Dr. Wolf-Eberhard Siems peptidolytic activity, ACE2 was identified as the essential receptor of the SARS virus, and represents the entry por- UNEXPECTED FUNCTIONS OF PEPTIDOLY- tal of the body for the virus. TIC ENZYMES Kohlstedt et al. (2002) described receptor-like functions The Biochemical Neurobiology Group deals with the bio- also for ACE. They proved e.g. that the reaction of ACE with chemical, pharmaceutical and molecular aspects of pep- some, but not all substrates or inhibitors resulted in spe- tidases. The current research is focused on angiotensin- cific phosphorylation at its own cytoplasmatic site. converting enzyme (ACE), neutral endopeptidases (NEP) Another unexpected finding: the two enzymes, especially and some related enzymes. We evaluate their biochemi- the NEP, play an important role in degrading the Alzhei- cal and functional relations to various disorders in mer’s disease (AD) peptide ß-amyloid (Aß). This knowl- humans, like alcohol addiction, obesity, neuronal disor- edge is expected to lead to new basic approaches for ders, problems in male fertility and heart diseases. understanding and treatment of this disease.

In the Biochemical Neurobiology Group additional func- Peptidases play a frequently underestimated role in main- tions of these peptidases have been discovered during the taining and controlling essential functions in the body. last years. Anthony Turner, one of the Nestors of research on NEP, wrote: “Peptidases and peptidolysis play vital roles in cel- ACE and voluntary alcohol consumption lular processes from fertilization – to death”. A look at the Our experiments of the last years had demonstrated that ACE confirms this sentence: This enzyme plays a key role Ang II, the main product of ACE, directly influences volun- not only for regulation of blood pressure, but also at the tary consumption of alcohol. Open questions concern the beginning and at the end of life: it is essential for mamma- role of central and peripheral Ang II, the involved recep- lian fertilization (male ACE-knock out mice are infertile in tor(s) and the downstream signaling. spite of normal sperm and libido) as well as for apoptosis, in which angiotensin II (Ang II, the main product of ACE) is We characterized the role of central Ang II in alcohol in- essentially involved. take by using transgenic rats [TGR(ASrAOGEN)680], which In recent years, the use of molecular techniques and of express an antisense RNA against angiotensinogen and genetically modified animals in peptidase research resul- consequently have sharply reduced Ang II levels exclu- ted in tremendous progress and innovative perspectives. sively in the central nervous system. These rats consumed In 2003 A. Turner commented the newly found functions of markedly less alcohol in comparison to their wild-type ACE and NEP as follows: “... (they) provide new avenues controls. Moreover, Spirapril, an ACE inhibitor, which pas- for the treatment of some of the major human diseases of ses the blood-brain barrier, did not alter voluntary alcohol the aging population of the Western world: cardiovascu- consumption in the TGR(ASrAOGEN)680, but it significant- lar disease, cancer and dementia”. ly reduced alcohol intake in wild-type rats. Studies in different types of knockout mice have proved that the effect of ACE, NEP and related enzymes are transmembranal Ang II on alcohol consumption is mediated by the angio- metallopeptidases, which are prevalent in many tissues tensin receptor AT1, whereas the AT2 receptor and the and cleave a broad spectrum of endogenous substrates. bradykinin B2 receptor are not involved. With reference to They consist of a short cytoplasmatic domain, a single signal transduction we found that the TGR(ASrAOGEN)680 transmembranal part and a very big extracellular part, with the very low central Ang II showed a markedly redu- which includes the catalytic domain(s), each of them con- ced dopamine concentration in their ventral tegmental taining one zinc ion. area (VTA), confirming a role of dopaminergic transmission in Ang II-controlled alcohol preference. Our results indi- ACE and NEP play an important role in circulation, and cate that a distinct drug-mediated control of the central inhibitors of the two enzymes, especially ACE inhibitors, renin-angiotensin system (RAS) could be a new principle are widely used for treatment of hypertension and for for therapy of alcohol disease. cardioprotection. Because of their broad spectrum of other substrates, these enzymes are also significantly NEP-deficient mice – a model of human obesity involved in many other pathophysiological processes. In long-lasting experiments on the functions of NEP, we In recent years, a lot of novel, in part surprising results observed that elderly NEP-deficient mice (NEP-/-) had a were published on ACE and NEP. One example concerns considerably higher body weight than wild-types. In con- in wild-typemembranes. interesting resultistheupregulationofnon-NEPactivity(yellowarrows)inNEP-deficientmembranes,sothatdegradationfasterthan A largepercentageoftheANP-degradationandtotalBNP-degradingactivity(greenarrows)cannotbeascribedtoNEP(blackarrow).An C FIGURE 1 Charité undFreeUniversityofBerlin. tions aresummarizedinajointpatent applicationofFMP, results andderiveddiagnostic therapeuticapplica- model ofthetypical,polyfactorial humanobesity. These systems. Therefore,thesemicemaybeabetteranimal cern arelativelygreatnumberofsubstratesandreceptor sing obesephenotypes,thealterationsinNEP-/-micecon- In contrasttoothergeneticallymodifiedanimalsexpres- model arestillbeinginvestigatedatpresent. analysis ofoldNEP-/-micewithregardtoanewobesity NEP onthesepeptidesandamoredetailedbiochemical NEP (e.g.galanin,ghrelin,orexin).Thecataboliceffectof tain aminoacidsequencesthatshouldbehydrolyzedby ral otherpeptideswitheffectsonfoodingestionalsocon- like NPY, seve- MCH andsomeoftheopioids.Furthermore, among themorexigenes(peptidesstimulatingfoodintake), NEP isknowntohydrolyzeagreatnumberofhormones, logical consequencesarepresentlybeinginvestigated. basis ofNEP-relatedobesityinmiceandthepathophysio- exclusively duetoanaccumulationoffat.Themolecular dies showedthatthehigherbodyweightinNEP-/-miceis became mostapparentinthesecondhalfoflife.NMR-stu- with typicalhumanobesity, thedifferencesinbodyweight trast tootheranimalmodelsofobesity, butinaccordance atabolism ofmurineANPandBNPbylungmembraneswild-typeNEP-knockoutmice:NEPisinvolvedinthecleavageonly.

Recovery of ANP (%) 1 20 40 6 8 00 0 0 b wild-type 10 b wild-type +Candoxatrilat 20 Incubation time(min) 30 40 Mouse ANP b 50 NEP -/- 60 b NEP -/-+Candoxatrilat

NEP old homozygousNEP-knockoutmice (>2years)displayed However, we foundinbehavioralexperimentsthatthevery ACE degradedAßbymeansofan endopeptidolyticattack. enzymes areinvolvedinAßcatabolism.Surprisingly, also degradation studiesindicatedthatfurthermembrane the generallackofAD-likeprocessesinrodentbrains.Our in AßdegradationbyNEPcannotbetheonlyreasonfor However, thedifferences degradation rates(mAß>hAß). peptides ofhumansandrodentshadindeeddifferent membrane preparationsofNEP-/-andNEP+/+mice.Aß Aß-partial sequencesbyusingpurifiedenzymeaswell the NEP-induceddegradationofhumanandmurineAßor in Therefore, furtherexperimentsfocusedondifferences didnotrevealanyAßdepositions. Heidelberg/Berlin) against murineAß-peptide(cooperationwithMulthaup, iments withbrainslicesandmonoclonalantibodies But inevenveryoldNEP-/-animalsimmunostainingexper- its impactinthepathogenesisandtherapyofAD. vivo studiesandisnowbeingintensivelydiscussedasto was recentlyconfirmedbyseveralinvitroaswell the amyloidß-peptide(Aß).ThisimportantfunctionofNEP NEP belongstotheenzymeswhichareablecatabolize NEP deficiencyinmice:learningexperiments

Recovery of BNP (%) 1 8 6 20 40 00 0 0 b 10 heat-inactivated 20 Incubation time(min) 30 wild-type 40 Mouse BNP 50 60

53 Cellular Signalling/ Molecular Genetics A FIGURE 2 A: Three-dimensional structure of the catalytic centre of NEP. B: Hypothesis of the NEP – NP interaction.

B catalytic site

cavity

two large tails: spatial clashes

• NP moves into the cavity of NEP • Complementary recognition sites are supporting the orientation of NP within the cavity • Large N - and C - terminal tails (simultaneously) hinder the orientation towards the catalytic site

an even better performance in learning tests, e.g. in a Mor- forms was characterized by Western blots and enzymatic ris water maze, compared to their wild-type littermates. studies with domain selective substrates (Hip-His-Leu, This result was confirmed in long-term potentiation (LTP) Z-Phe-His-Leu, Ac-SDKP), and the clones each with the experiments in two brain regions (hippocampus and amyg- highest enzyme activities were selected. dala). In contrast, there were no differences between the two genotypes in younger animals (~9 months). The mole- These different domain-selective ACE forms are now used cular background of this effect is still unclear. These to investigate experiments demonstrate the Janus head-like action of (i) the receptor-like function of cell-bound ACE, NEP in brain function: it improves the brain function by (ii) the unusual endopeptidolytic activity of ACE on Aß and degradation of the plaque-forming Aß-peptides, and it Aß partial sequences, apparently degrades other peptides which improve learn- (iii) the role of angiotensin (1-7) as substrate and inhibitor. ing processes. Involved peptides are still unknown. Gluca- gon-like Peptide-1 (GLP-1) could play a role. This peptide Peptidolytic degradation of natriuretic peptides strengthens cognitive functions, and we found a greater Natriuretic peptides (NP), like atrial- (ANP), B-type- (BNP) stability of this peptide in membrane preparations of and C-type natriuretic peptide (CNP), are cyclic peptide NEP-/-- mice. hormones with relevance to cardiovascular, endocrine and renal homeostasis. All three peptides contain an intact Domain-selective forms of ACE 17 amino acid disulfide-linked loop which is essential for To analyze the newly discovered ACE-functions and the their biological activity. Two different mechanisms are dis- unexpected interactions with substrates and inhibitors in cussed as being responsible for the inactivation of the NP: more detail, we transfected CHO cells with the following murine constructs: a) wild-type ACE (both domains are (i) binding to specific receptors (C-type-receptor) with intact), b) C-terminal ACE, c) N-terminal ACE, d) inactivat- subsequent internalization and degradation, ed ACE (no intact domain). The specificity of these ACE- (ii) degradation by extracellular peptidases. ** part-time * partofperiodreported Bettina Kahlich(Technical assistance) Kristin Pankow(Doctoralstudent)* Xiaoou Sun(Doctoralstudent) Matthias Becker(Doctoralstudent)* Dr. WinfriedKrause** Group members peutic use. forpotentialthera- resistant NP-analoguesareinteresting is notasubstrateforNEP. Moreover, modifiedpeptidase- loop regions.ThisshouldhelptodetectwhyhumanBNP (b) replacedaminoacidsinthehighlyconservedinner- derivates from humanBNPwith(a)shorter terminal tailsor To provethismodel,weplanstudieswithNEPandNP ween thecatalyticcenterofNEPandNP(Figure2). enabled afirstmodelforthemolecularinteractionsbet- human BNPwastotallyresistantinourtest.Theseresults on thesizeofN-andC-terminaltailsNP. Only nant NEP. Theobserveddegradationratewasdependent These degradationstudieswereperformedwithrecombi- modifications. different specieswithvariousstructure structure ofNPontheircatabolismbycomparing In afirststep,weprovedtheinfluenceofchemical molecular basisofthedifferentdegradationNPbyNEP. In asecondpartofthisprojectwewanttofindoutthe tic treatments. tective actionofNPsandpossiblyenablenewtherapeu- would protecttheNP. Thiswouldpotentiatethecardiopro- mode ofaction.Aspecificinhibitionthesepeptidases stillunknownNP-degradingenzyme(s)andits this/these The nextaimoftheprojectwillbecharacterization tion thaninwild-typemembranes.(Figure1) membranes resultinginanunequivocallyfasterdegrada- up-regulation ofthecatabolicactivityinNEP-deficient n of theANP-degradingandtotalBNP-degradingcan- only involvedinthecleavageofANP. Alargepercentage but incontrasttothegenerallypublishedopinion,NEPwas r observed anunexpectedresult:BothNPpeptideswere ferent organsfromNEP-deficientandwild-typemice.We catabolism ofmouseANPandBNPbymembranesdif- In HPLC-monitoreddegradationstudiesweanalyzedthe at theCys-Phebond. which initiallycatabolizesNPbycleavagewithintheloop The NEPisgenerallyacceptedtobethemainenzyme apidly degradedbymouselungandkidneymembranes, ot beascribedtoNEP. Aninteresting sideresultwasthe Walther T, StepanH, Receptor. FASEB J19:1474-1481 Angiotensin IIControlsAlcoholConsumptionviaitsAT1 retic peptide.Heart91,385-387 vival inChagasdiseasemoreeffectivelythanatrialnatriu- HP, Walther T(2005)Brainnatriureticpeptidepredictssur- Reibitz F Gembardt F S Wolf-Eberhard Siems Strathmann AG W ASTA Medica Wolf-Eberhard Siems an transgenenundknockout-Tieren“ (SI483/3-1/2) „ Deutsche Forschungsgemeinschaft External funding Sergej Danilov, University ofChicago,USA Gisela Grecksch,UniversityofMagdeburg Doris Albrecht,Humboldt-UniversitätzuBerlin Michael Bader, MDCBerlin Thomas Walther, EMCRotterdam, TheNetherlands Collaborations Siems WE,MaulB,WiesnerBeckerM Siems WE Maul B, Walther T, StepanH, and brainnatriureticpeptide.BiolChem385,179-184 tidase activityrevealsindependentcatabolismofatrial Andrologia 35,44-54 spermatozoa andtheimpactofpeptidolyticenzymes. Gembardt F, 111, 452-455 ance ofBNPtoneutralendopeptidaseinthefetus.BJOG for enhancedneutralendopeptidaseactivityandresist- to theirN-terminalfragmentsinfetalcirculation:evidence Schultheiss HP, Rothe L, Silvia-Barra J,PenaJL, Heringer-Walther S,MoreiraMC,Wessel N,SalibaJL, peptidase activityinrodents.Peptides26,1270-1277 Organ specificdistributionofACE2mRNAandcorrelating Neuropeptidasen undAlkoholkonsum–Untersuchungen elected publications olf-Eberhard Siems Winkler A , SchultheissH-P, Walther T (2004) Biochemicalanalysisofneutralendopep- , Sterner-Kock A,ImbodenH,SpalteholzM, Alenina N,BaderM, Siems WE (2003) Effectsofkininsonmammalian , KrauseW, PankowK,BeckerM, Pankow K,BeckerM Pankow K,GembardtF Becker M,SiemsWE (2004) RelationofANPandBNP ( Siems WE FMP authorsinbold) Siems WE , Walther T(2005) , SchultheissHP, (2005) Central , Schultheiss , Walther T, , FaberR,

55 Cellular Signalling/ Molecular Genetics BIOPHYSICS membrane osmotic gradient. The protein was claimed to Group Leader: PD Dr. Peter Pohl have a secondary function as a cyclic-nucleotide gated ion channel. However, upon reconstitution into planar COMBINED TRANSPORT OF WATER AND bilayers, the ion channel exhibited a tenfold lower single IONS THROUGH MEMBRANE CHANNELS channel conductance than in Xenopus oocytes and a hun- dredfold lower open probability (<10-6) of doubtful physio- Life in all its diversity became possible due to the separa- logical significance (Saparov et al., 2001, J.Biol.Chem. tion of living entities from the lifeless and hostile environ- 276:31515). Investigating AQP1 expressed in human ment by means of membranes. It requires preservation of embryonic kidney cells, we now have shown that the selective transmembrane material and information discrepancy is not due to alterations of AQP1 properties exchange. During evolution cells developed, the function upon reconstitution into bilayers but rather due to regula- of which depends on the well-controlled interplay and tory processes of the oocyte expression system that may material exchange between different compartments. The have been misinterpreted as AQP1 ion channel activity. As different transport functions are performed by a sophisti- confirmed by laser scanning reflection microscopy, from cated apparatus of membrane proteins, each of them is 0.8 to 1.4 x 106 AQP1 copies per cell contributed to osmo- fulfilling a very distinct function. Investigation of the latter tic cell swelling. The proper plasma membrane localiza- comprises the main interest of the biophysics group. We tion was confirmed by observing the fluorescence of the aim to explore the molecular mechanisms of water, pro- N-terminal yellow fluorescent protein tag. Whole cell ton and oxygen movement. Additional activities in the area patch-clamp experiments of wild-type or tagged AQP1 of applied biophysics are devoted to selected modifiers of expressing cells revealed that neither cGMP nor cAMP membrane permeability (e.g. ribosome inactivating pro- mediated ion channel activity. The lack of significant CNG teins and block copolymers). In this short report, we will ion channel activity rules out a secondary role of AQP1 focus on water transport. The investigations are carried water channels in cellular signal transduction. out on different levels of organization: starting from proteins reconstituted into planar and vesicular membranes, Anomalously high water mobility in membrane followed by proteins overexpressed in different cell lines ion channels and completed by proteins identified in primarily cultured Along with aquaporins, a variety of other membrane pro- cell monolayers. teins is likely to contribute to transmembrane water movement as well. Dr. Saparov carried out simultaneous measurement of the electrical and hydraulic conductivities of a Water transport by aquaporins bacterial potassium channel. He folded the artificial mem- Water transport is essential to all forms of life. Never- branes from two protein containing lipid monolayers in the theless, the pathways taken by water across a membrane aperture of a teflon film. Water flow was measured by barrier and the mechanism of solute-solvent coupling are imposing an osmotic gradient across these bilayers and still not entirely resolved. Commonly, it is considered that detecting the resulting small changes in solute concentra- water may pass the lipid bilayer by diffusion. The extent to tion close to the membrane surface with scanning ion which the lipid part of membranes contributes to water selective microelectrodes. Dr. Saparov made the surpris- transport varies considerably between different cells. Epi- ing observation that the amount of transported water thelial cells, for example, have to maintain large chemical molecules exceeds the number of transported ions by and osmotic gradients. Consequently, the membrane several orders of magnitude. Water molecules are con- matrix has to be effectively impermeable to ions and most strained to move with K+ ions through the narrow part of other small molecules. Tightening of the lipid barrier requi- the KcsA channel because of the single file nature of res proteinaceous transport routes of water. Proteins spe- transport. In the presence of an osmotic gradient, a water cialized on water transport are called aquaporins. We molecule requires less than 10 picoseconds to cross the have investigated the selectivity of several members of the purified protein reconstituted into planar bilayers. Rinsing aquaporin family both in artificial model membranes and K+ out of the channel, water may be 1000 times faster than in their cellular environment. the fastest experimentally observed K+ ion and 20 times For example, in cooperation with the department “Mole- faster than the one dimensional bulk diffusion of water cular Medicine” (Burkhard Wiesner, Dorothea Lorenz, (Fig. 1). Both the anomalously high water mobility and its Walter Rosenthal) Dr. Tsunoda investigated the transport inhibition observed at high K+ concentrations are consist- mediated by aquaporin-1 (AQP1). It is a membrane chan- ent with the view that liquid-vapor oscillations occur due nel which allows rapid water movement driven by a trans- to geometrical confinements of water in the selectivity Sci. USA. tude. Furtherinvestigationofthe ultrafast diffusionwillbe and inthebulkdifferedbymore thananorderofmagni- result themobilityofwatermolecules insidethechannel profound changesofitsphysico-chemical properties.Asa Confinement ofwaterintoaverynarrowgeometryledto tion towaterhomeostasisinneuronsislikely. channels inthenodesofRanvier, forexample, acontribu- this phenomenon.WithrespecttothehighdensityofK be elucidated,aswellthephysiologicalimportanceof rin family, remainsto whichallrealizesinglefiletransport, Beckstein, O.&Sansom,M.S.P. (2003) Rasaiah, J.C.&Noworyta,J.P. (2001)Nature414,188-190; res bymoleculardynamicssimulations(Hummer, G., so farhaveonlybeendiscoveredinhydrophobicnanopo- the channelleavingbehindanearvacuum(the“vapor”), imbedded inthechannel(the“liquid”)cooperativelyexits filter. Theseoscillations,wherethechainofmolecules process. WhetherthefasttransportmodeoccursinNa sons butaddimportantfeaturestotheoveralltransport the ions.Theyarenotonlyrequiredforelectrostaticrea- molecules aremorethanjustspacerbetween Our results clearlyshowthatinsinglefiletransport, water Ca 2+ , otherK 100, 7063-7068). + channels oreveninmembersoftheaquapo- Proc. Natl.Acad. + + , ** part-time * partofperiodreported Peter Pohl Heisenbergstipendium (Po533/7-1) Deutsche Forschungsgemeinschaft External funding Elena Sokolenko(Guestscientist)* Alexander Lentz(Guestscientist)* Christina deSouza(Guestscientist)* Dr. ArtemAyuyan (Guestscientist)* Dr. Valerij Sokolov(Guestscientist)* Prof. Dr. Yuri Antonenko(Guestscientist)* Matthias Prigge(Student)* Rustam Mollajew Steffen Serowy(Doctoralstudent)* Dr. Valentina Margania** Dr. OxanaO.Krylova Dr. SatoshiTsunoda Dr. SaparM.Saparov Group members sche Forschungsgemeinschaft“Micro-andNanofluidics”. conducted intheframeofpriorityprogramDeut- Chem. 2004,385:921–926). allow differentmoleculestopasseachother(Fig.3fromBiol. which incontrasttotheselectivityfilteriswideenough potassium ion(yellow)hasbeenplacedintothecavity binding sitesareoccupiedbywatermolecules(red).The the filter. Thissituationisillustratedinthefigurewhereall channel (KcsA)occursonlywhenallionsarerinsedoutof Fast osmoticwaterflowthroughthebacterialpotassium FIGURE 1

57 Cellular Signalling/ Molecular Genetics Deutsche Forschungsgemeinschaft Tsunoda SP, Wiesner B, Lorenz D, Rosenthal W, Pohl P “Single file water transport across peptidic nanopores” (2004) Aquaporin-1, Nothing but a Water Channel. J Biol (Po533/11-1) Chem 279, 11364-11367 Peter Pohl Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl Deutsche Forschungsgemeinschaft P, Maric K (2003) Cyclic AMP is sufficient for triggering the „Molekulare Mechanismen des Wassertransports durch exocytic recruitment of aquaporin-2 in renal epithelial Epithelzellmonoschichten“ (Po533/8-1, 8-2) cells. EMBO Rep 4, 88-93 Peter Pohl Serowy S, Saparov SM, Antonenko YN, Kozlovsky W, Deutsche Forschungsgemeinschaft Hagen V, Pohl P (2003) Structural Proton Diffusion along „Wechselwirkung zwischen ebener Bilipidmembran und Lipid Bilayers. Biophys J 84, 1031-1037 Photosensibilisator“ (Po533/4-3) Peter Pohl Collaborations Deutsche Forschungsgemeinschaft „Migration von Protonen an der Oberfläche ebener Bilipid- - Proton migration (DFG Po 533/5-1, 2, 3; 436 RUS113/551): membranen“ (Po533/5-2, 5-3) Prof. Yuri N. Antonenko, Lomonossov University Mos- Peter Pohl cow, Belozersky Laboratory - Photosensibilisator (DFG Po 533/4-3; 436 RUS113/634): Deutsche Forschungsgemeinschaft Dr. Valerij Sokolov, Russian Academy of Science, Frum- Kooperation mit der Lomonossov-Universität Moskau (436 kin Institute of Electrochemistry RUS113/551) - Blockcopolymers and multidrug resistance (VW): Prof. Peter Pohl J. Kressler, Martin-Luther-Universität Halle; Prof. Dr. Deutsche Forschungsgemeinschaft Frey, University of Mainz Kooperation mit dem Frumkin-Institut für Elektrochemie Moskau (436 RUS113/634) Peter Pohl

Volkswagenstiftung ”Control of membrane permeability with novel types of amphiphilic macromolecules” (I/77743) Peter Pohl

Volkswagenstiftung ”Control of membrane permeability with novel types of amphiphilic macromolecules” (I/80074) Peter Pohl

Selected publications (FMP authors in bold)

Pohl P (2004) Combined transport of water and ions through membrane channels. Biol Chem 385, 921-926

Saparov SM, Pohl P (2004) Beyond the diffusion limit: Water flow through the empty bacterial potassium channel. Proc Natl Acad Sci U. S. A 101, 4805-4809

Krylova OO, Pohl P (2004) Ionophoric activity of pluronic block copolymers. Biochemistry 43, 3696-3703

Sun J, Pohl EE, Krylova OO, Krause E, Agapov II, Tonevit- sky AG, Pohl P (2004) Membrane destabilization by ricin. Eur Biophys J 33, 572-579 position ofthemaincellularcompartments oftheimmune and lackofISG15didnotaffectthe developmentandcom- stem cells.ISG15-/-micedisplayno obviousabnormalities, rated micelackingISG15viagene targetinginembryonic To directlyassessthefunctionalroleofISG15,wegene- and vesicularstomatitisvirus(VSV). infection withlymphocyticchoriomeningitisvirus(LCMV) published thatUBP43-/-micearemoreresistantagainst al. 2003,Genes&Development17,455-460).Itwasalso on ofJAK/STAT andinterferonsignaling (MalakhovaOAet ISG15 conjugationplaysanimportantroleintheregulati- induces prolongedSTAT1 phosphorylation andthatthe mented conjugationofISG15intheabsenceUBP43 hypersensitivity toPoly(I:C).Itwasreportedthatanaug- lop aseverephenotypewithbraininjuriesandlethal belongs tothefamilyofdeubiquitinatingenzymes,deve- UBP43 (USP18),theISG15deconjugatingenzymewhich defence, immuneresponses,andpregnancy. Micelackin variety ofbiologicalactivitieswhichencompassantiviral gated todistinctproteinsandhasbeenimplicatedina the familyofubiquitin-likemodifiers.ISG15canbeconju- interferon treatmentandlikeSUMOorNEDD8belongsto ISG15 isoneofthemoststronglyinducedgenesupon molecule andUBPy, adeubiquitinatingenzyme. focused onISG15,aninterferonstimulatedubiquitinlike in thecontextofwholeorganism.Currentresearchis to addressthebiologicalrelevanceandfunctiondirectly In ourgrouptargetedmutagenesisinthemouseisused for drugintervention. and deconjugationpathwaymightserveasnoveltargets Thus distinctcomponentsoftheUbandUBLconjugation interaction ofproteins. tion, receptordegradation,transcriptionalcontroland wide rangeofmolecularmechanismslikeproteinlocaliza- been demonstratedtobeinvolvedintheregulationofa for proteasomaldegradationUbiquitinconjugationhas r embryogenesis, cellcycle,growthcontrol,andimmune controlling apleiotropyofbiologicalprocessesincluding molecules servesasanimportantregulatorymechanism Covalent attachmentofUbiquitinandlike(UBL) USING GENETARGETING INTHEMOUSE Q TIONAL PROTEINMODIFICATION BYUBI- FUNCTIONAL ANALYSIS OFPOSTTRANSLA- Group Leader:Dr. Klaus-PeterKnobeloch C esponse. Beside serving asamarker thattargetsproteins serving Beside esponse. YTOKINE SIGNALING UITIN ANDUBIQUITINLIKEMOLECULES are affectedbythelackofUBPy. Inparallel,wegenera- phenotypic alterations,andmolecular mechanismsthat activated thegeneinadultmice andareanalyzingthe loxP system.Bymatingthemice toMx-crewehavein- allow conditionalinactivationofthegeneusingcre- mice dieearlyinembryogenesiswegeneratedthat approach wehavegeneratedmicelackingUBPy. Asthese nositol-3-kinase andUBPycouldbeisolated.Inaninitial a fusionproductofthep85betasubunitphosphatidyli- tion motif.Frompatientswithamyeloproliferativedisorder domains ofcertaintargetproteinsviaanunusualrecogni- ted uponseruminductionandwasshowntobindSH3 UBPy (USP8)isanubiquitinisopeptidasethatupregula- teins inthecontextofwholeorganism. reports challengingthefunctionofdeubiquitinatingpro- 2002, Trompouki etal2003).However, no sofarthereare kappa Barecontrolledbyubiquitinisopeptidases(Lietal observation thatcellularkeymoleculeslikep53orNF- quitinating enzymeshasbeendemonstratedbythe mes isjustbeginningtoevolve.Theimportanceofdeubi- tin toproteins,theunderstandingofdeubiquitinatingenzy- has beenmadeincharacterizingenzymesthatlinkubiqui- functions ofsometargetproteins.Althoughmuchprogress zation intothelysosomalsystem,aswelltomodify teins fordegradationbytheproteasomeandinternali- Ubiquitination ofproteinsisnowrecognizedtotargetpro- fic proteaseUBPy/USP8 Conditional inactivationoftheubiquitin-speci- c VSV andLCMVwerenotsignificantlyalteredintheabsen- ced antiviralstateandimmuneresponsesdirectedagainst system. IncontrasttoUBP43-/-mice,theinterferon-indu- ISG15 andUBL14. generated micedeficientforUBL14,aswellboth, in vivo,wealsousedalossoffunctionapproachand homology toISG15.To elucidatetheroleofthismolecule named UBL14,thatencodesaproteinwith95%aminoacid We werealsoabletocloneanewmousegene,whichwe lysis. me. Thesedoubleknockoutmicearecurrentlyunderana- functions besideactingasanISG15deconjugatingenzy- s an enhancedISG15conjugation.Thisapproachisalso alterations ofUBP43-/-mice,providingtheyarecausedby for bothmoleculesinanattempttorescuephenotypic to bereassessed.Therefore,wegeneratedmicedeficient p lack ofISG15.ThustheroleISG15inpathology typical STAT1 targetgenesremainedunaffectedbythe STAT1 tyrosine-phosphorylation,aswellexpressionof uitable totestwhetherUBP43possessesadditional henotype ofUBP43-/-andthespecificityUBP43needs e of ISG15. Furthermore, interferon orendotoxininduced e ofISG15.Furthermore,interferon

59 Cellular Signalling/ Molecular Genetics FIGURE 1 FIGURE 2 ISG 15 conjugation cycle Editing functions of deubiquitinating enzymes. Deubiquitinating enzymes may negatively regulate proteolysis or other signaling functions of ubiquitination such as internalization or altered protein function by removing the ubiquitin chain from the target proteins.

ted mice lacking UBPy in specific hematopoietic cell linea- Selected publications (FMP authors in bold) ges like T-cells and granulocytes/macrophages in order to Osiak A, Utermohlen O, Niendorf S, Horak I, Knobeloch KP. determine the functional role of UBPy in these cell linea- (2005) ISG15, an interferon-stimulated ubiquitin-like pro- ges. tein, is not essential for STAT1 signaling and responses against vesicular stomatitis and lymphocytic choriomenin- Group members gitis virus. Sandra Niendorf (Doctoral student) Mol Cell Biol., 6338-6345 Agnes Kisser (Doctoral student) Van Spriel AB, Puls KL, Sofi M, Pouniotis D, Hochrein H, Markus Wietstruck (Technical assistance) Orinska Z, Knobeloch K-P, Plebanski M, Wright MD (2004) Anna Osiak (Doctoral student)* A regulatory role for CD37 in T-Cell Proliferation. J Immu- Liane Boldt (Technical assistance) nol 172, 2953-2961

External funding Rosenbauer F, Wagner K, Zhang P, Knobeloch KP, Iwama A, Tenen DG (2004) pDP4, a novel glycoprotein secreted by Deutsche Forschungsgemeinschaft mature granulocytes, is regulated by transcription factor „Herstellung und Haltung genetisch veränderter Mäuse“ PU.1. Blood 103, 4294-4301 (TP Z3 im Sonderforschungsbereich 366 „Zelluläre Signal- erkennung und Umsetzung“) Schuh K, Cartwright EJ, Jankevics E, Bundschu K, Lieber- Ivan Horak mann J, Williams JC, Armesilla AL, Emerson M, Oceandy D, Knobeloch KP, Neyses L (2004) Plasma membrane Ca2+ Deutsche Forschungsgemeinschaft ATPase 4 is required for sperm motility and male fertility. J „Untersuchungen zur Funktion des Interferon-stimulierten Biol Chem 279, 28220-28226 Genes 15 (ISG15).“ (KN 590/1-1) Klaus-Peter Knobeloch

* part of period reported Universität zuKöln Immunologie undHygiene Dr. OlafUtermöhlen,InstitutfürMedizin.Mikrobiologie, Georg-August-Universität Göttingen PD Dr. Prinz,Abt.Neuropathologie Marco Charité –UniversitätsmedizinBerlin und Hygiene Prof. Dr. med.OliverLiesenfeld,InstitutfürMikrobiologie National U Biology Prof. Dr. RobertKrug,InstituteforCellularandMolecular Washington UniversitySchoolofMedicine H International Collaborations Huebner A,KaindlAM, E in ALADIN,anovelmemberofthenuclearporecomplex. Koehler K(2004)ThetripleAsyndromeisduetomutations ndorcr Res30,891-899 erbert W. Virgin IV, DepartmentofPathology niversity ofTexas atAustin Knobeloch KP , PetzoldH,MannP,

61 Cellular Signalling/ Molecular Genetics MOLECULAR MYELOPOIESIS shown to be specifically expressed by GMP, and the RNA Group Leader: Dr. Dirk Carstanjen expression level was within the group of the five transcription factors the most prominently expressed transcription ICSBP – A CRITICAL TRANSCRIPTION factors in these cells, in concert with prominent proteins FACTOR FOR MYELOPOIESIS as PU.1, and Cebpα. These important findings further confirms the pivotal role of ICSBP for myeloid development The research aim of our group is to elucidate the role of and will now allow to study systematically the analysis of the transcription factor ICSBP (interferon consensus transcription factors regulating myeloid and erythroid cell sequence binding protein) within hematopoietic cell deve- development using genetic approaches e.g., mouse dele- lopment. This hematopoietic specific transcription factor tion-mutants, RNA interference or retroviral overexpres- is a major element regulating transcriptional control of sion. These studies are currently being undertaken. myelopoiesis. Mice deficient in this transcription factor develop a proliferative syndrome characterized by the Dissecting the transcriptional network orche- expansion and accumulation of immature myeloid proge- strated by ICSBP nitor cells within the bone marrow and spleen. Further- We further analyzed the global gene expression profile in more, it has been shown by our group that ICSBP influen- the GMP of the ICSBP mouse deletion mutant. This analy- ces lineage choice of granulocyte-monocyte progenitor sis revealed insight into the developmental program indu- cells (GMP) leading to an imbalance in development and ced by the loss of ICSBP within myeloid progenitor cells. maturation of progenitor cells in favor of granulocytes. We found several major myeloid transcription factors downregulated in the GMP of ICSBP-deleted mice. We are currently studying the contribution of these transcription Genetic profile of myeloid development factors to the myeloproliferative syndrome of these mice This asymmetric lineage choice is at least partially due to using mouse deletion mutants and retroviral overexpres- a reduced response to a macrophage specific cytokine sion in bone marrow derived stem and progenitor cells. (macrophage colony stimulating factor: M-CSF). Our group Furthermore, we are studying potential direct transcriptio- has recently shown that M-CSF signal transduction in nal regulation of ICSBP on genomic target sequences. We macrophages is altered due to altered protease expressi- therefore cloned several murine genomic sequences with on in these cells and enhanced proteasomal degradation known or potential regulatory functions for the genes we leading to shortened signaling. In contrast, the reason for found differentially expressed in GMPs of ICSBP-deleted accumulation of immature progenitor cells and preferen- mice. Luciferase promoter studies are currently being tial production of granulocytes is further obscure. In an undertaken to identify the impact on ICSBP in direct or approach to decipher the function of ICSBP in immature combinatorial gene expression regulation with other pro- progenitor cells as well as to define the expression profi- minent myeloid transcription factors. Furthermore, many le of granulocyte versus erthrocyte precursors, we sorted of the genes overexpressed in ICSBP-deleted mice are GMP and erythrocyte progenitor (EP) cells by flow cyto- genes preferentially expressed in mature granulocytic and metry in collaboration with H.R. Rodewald, Ulm. Global monocytic cells. Interestingly, at the GMP stage, there was gene expression profiling was performed in these highly no clear preferential expression pattern of granulocytic purified cells and analyzed using Affymetrix technology. genes implicating that branching between monocytic and The data sets of this analysis have been published via the granulocytic development occurs beyond the GMP stage GEO database. These data now allow for the first time to and additional extrinsic factors, e.g. cytokine signaling, are study systematically the transcriptional profile of these responsible for the preferential generation of granulocy- functional distinct cell populations. As a first approach, we tes versus macrophages in the ICSBP deleted mouse, a identified the global pattern of transcription factors speci- hypothesis consistent with our previous findings showing fically expressed in either lineage. While this analysis con- abnormal M-CSF signaling in macrophages derived from firmed the exclusive or highly preferential expression of the ICSBP deleted mouse. several transcription factors with pivotal importance for erythroid development (e.g. erythroid Krüppel-like factor, GATA-1, Friend of GATA-1 (FOG1)), we also identified several transcription factors specifically expressed in myeloid progenitors. Several of those are known: Cebpα, Cebpβ, Gfi1, Fli1, PU.1, and Meis1 to name but a few, others have not yet been implicated in myeloid development, e.g. Mef2c, KLF4, and SAT1b. Importantly, ICSBP has been * partofperiodreported Ivan Horak HO 493/12-1und12-2 „Gene inEntwicklungundFunktionvonmyeloidenZellen“ Deutsche Forschungsgemeinschaft I. Horak reich 506„OnkotherapeutischeNukleinsäuren“) scher Targetstrukturen“ (TPG1imSonderforschungsbe- sen: Identifizierungneuerpotentielleronkotherapeuti- „Myeloproliferatives SyndrombeiICSBP-defizientenMäu- Deutsche Forschungsgemeinschaft External funding Melanie Benedict(Technical assistance) Joanna Selfe(Doctoralstudent)* Axel Kallies(Doctoralstudent)* Jessica Königsmann(Doctoralstudent)* Maja Djurica(Doctoralstudent)* Group members c-Kit IL-3R α GMP GMP mann J,CarstanjenD,HorakI M Selected publications Mucha V, JakubickovaL,BiesovaZ,RafajovaM, Zatóvicova M,Tarabkova K,SvastovaE,GibadulinovaA, Terszowski G,Waskow C,ConradtP, LenzeD, Am JPhysiolGastrointestLiver286,G244-G252 leukin-2-deficient mousemodelofcolonicinflammation. Schulzke JD(2004)Mechanismsofdiarrheaintheinter- 1937-1945 the colony-formingunit-erythrocyte (CFU-E).Blood105, spective isolationandglobalgene expressionanalysisof Mankertz J,SeidlerU, Barmeyer C,HarrenM,SchmitzH,Heinzel-PleinesU, ced carbonicanhydraseIX.JImmunolMethods282,117-134 nize differentdomainsoftumourassociatedhypoxia-indu- generated incarbonicanhydraseIX-deficientmicerecog- Pastorek J,PastorekovaS(2003)Monoclonalantibodies Dirk Carstanjen signalvermittlung inhämatopoetischenZellen“(CA306/1-1) „Die RolledesAdaptorproteinsDisabled-2beiZytokin- Deutsche Forschungsgemeinschaft , ParkkilaS,AK,Waheed A,SlyWS, Affymetrix™ technology. and globalgeneexpressionprofilingwasperformedusing locytes. Thispopulationwassortedtopurity, RNAisolated, cells thatgiverisetomonocytes/macrophagesandgranu- against Fc populations canbeidentifiedthroughtheuseofantibodies (MP), andcommonmyeloidprogenitorcells(CMP).Those progenitor cells(GMP),megakaryocyte tor positivecellscontainsocalledgranulocyte-monocyte tor) aswellinterleukin-3receptor. c-kithigh,Il-3recep- stained withantibodiesagainstc-kit(stemcellfactorrecep- cytes. Thoseso-calledlineagenegativecellswerefurther ker forgranulocytes,monocytes,T-, B-cells anderythro- (lower part)weredepletedofmaturecellsexpressingmar- Bone marrowcellsfromICSBPwt(upper)anddeleted FIGURE 1 γ R IandCD41.TheGMPpopulationcontainsonly Horak I (FMP authorsinbold) , WiedenmannB,FrommM, , RodewaldHR(2004)Pro- Ortova Gut Koenigs- Horak I ,

63 Cellular Signalling/ Molecular Genetics MOUSE MODELS and have used these models to study the function of FMIP Group Leader: Dr. Rüdiger Pankow and in particular, its relevance in myeloid development.

BCL-2 ASSOCIATED TRANSCRIPTION Group members FACTOR AND FMS INTERACTING PROTEIN Dr. Rosel Blasig Kerstin Bohne (Technical assistance) Btf (Bcl-2 associated transcription factor) and FMIP (fms Janet Klemm (Technical assistance) interacting protein) are two genes which have been impli- Mina Thakur (Technical assistance)* cated in cellular regulatory processes. Both are intracellular proteins with subcellular distributions in the cytoplasm and the nucleus. We have raised antibodies against Btf and found its expression in a broad spectrum of tissues and organs including those of the hematopoietic system. Its highest expression, however, is found in brain regions of young developing mice. This expression de- clines with age, with Btf being detectable in only a few neural cells in adulthood.

Due to its association with Bcl-2, Btf was thought to be a transcriptional repressor, with an apoptotic function. Since no rigorous evidence for a role of Btf in apoptosis was found, its biological function remains unknown. To generate mice deficient for Btf we inserted a marker cassette containing the β-galactosidase gene into the btf gene. The inserted β-galactosidase gene is under the control of the endogenous btf promoter which allows the detection of btf gene expression. Mice, homozygous for this mutation, lack all wild type btf transcripts and are deficient in Btf protein. These mice display a severe phenotype resulting in premature death. Molecular mechanisms behind the phenotypic changes are being analyzed. To assess the role of Btf in the hematopoietic system we reconstituted lethally irradiated wild type mice with fetal liver or bone marrow cells from Btf deficient mice. These mice reconstituted a Btf-/- hematopoietic system including all resulting cell lineages but do not develop any of the phenotypic signs characteristic of Btf deficient mice. We could therefore rule out the influences of the hematopoietic system and conclude that other factors provide the major cause for the phenotype.

For FMIP, an interacting partner of the activated M-CSF receptor, a possible role in myeloid differentiation was suggested. By means of targeted gene disruption, we generated a mouse model with a null mutation of the fmip gene. Our studies revealed, however, that embryos lacking Fmip die early during embryogenesis. Thus, to allow studies of Fmip in physiological processes in the mouse, we have generated FMIP conditional knockout mice using the cre/loxP system. By intercrossing these mice with Cre- deleter mouse strains we have obtained Fmip floxed mice that allow organ or cell lineage specific deletion of Fmip * part of period reported activity ofreceptor-associated JAKkinases,leadingtothe phorylated state.Stimulationwith cytokinesincreasesthe cytokines theSTAT moleculeexistsinanon-tyrosinephos- in termsofsignaling:beforethe stimulationofcellswith consider thattheSTAT proteinsexistintwodifferentstates analysis oftheirnucleocytoplasmictranslocationhasto exceeding 40kDcanfreelyenterthecellnucleus.The kD forthemonomer),asonlyionsandsmallmoleculesnot dable diffusionbarriertoproteinsthesizeofSTATs (>85 named nuclearporecomplexes(NPC),providesaformi- res. However, passagethroughthenuclear gateways, sion controlledandnotdirectedalongpermanentstructu- genes. MovementofSTATs ineithercompartmentisdiffu- membrane withthepromotersofcytokine-responsive cross thenuclearenvelopetofunctionallylinkcell act astranscriptionfactors.Thus,theseproteinsneedto plasm andcarrythemintothenucleus,wheretheythen kine signalsatintracellularreceptorchainsinthecyto- kines andgrowthfactors.TheSTAT receivecyto- proteins interferon systems,are responsive toawiderangeofcyto- of transcription(STAT) pathways,firstidentifiedinthe The Januskinase(JAK)/signaltransducerandactivator stand theireffectsinthenormalanddiseasedstate. events thataretriggeredbycytokinesinordertounder- cytokine action,weneedtoexploretheintracellular goal. Asthealterationofgenetranscriptionisbasis situations, inhibitionofcytokineactionisatherapeutic cause diseasessuchasrheumatoidathritis.Inthose cer). However, ofcytokines mayalso overexpression (e.g. hepatitis)aswellforsometumorsskincan- is thestandardtherapyforseveralsevereviralinfections the treatmentoftumors.Today, treatmentwithinterferons cal trials,andinterleukin-2wasthefirstdrugapprovedfor Several cell-activatingcytokinesarebeingtestedinclini- cytokines arehighlyrelevantforclinicalapplications. cells againstviralandmicrobialattack.Notsurprisingly, interferons areknowntocausegrowtharrestandprotect control stemcelldevelopmentandgrowth,whilethe l their roleinimmunecelldifferentiation,inflammationand known aretheinterleukinsanderythropoietine(Epo)for roles incelldifferentiationandinnateimmunity. Best Cytokines aresecretedsmallproteinsthatfulfillcrucial k group ofextracellularsignalingmoleculestermedcyto- We arestudyingtheeffectsongenetranscriptionofa KINETIC CONTROLOFGENETRANSCRIPTION Group Leader:Dr. UweVinkemeier C actation. OthercytokinessuchasLIFandOncostatinM ines, morethanahundredofwhicharepresentlyknown. ELLULAR SIGNALPROCESSING process. plasm andhenceconfersdirectionality tothetransport nuclear membranediscriminates cytosolfromnucleo- tric distributionofnucleotideexchangefactorsacrossthe RanGDP/RanGTP gradientthatformsthroughtheasymme- the formationofexportin/cargo-complexes.Thus, RanGTP disruptsimportin/cargocomplexesbutstabilizes complexes isdeterminedbythesmallGTPaseRan,as signals, respectively. Thestabilityofcargo/karyopherin med nuclearlocalization-NLS-orexport-NES- served stretchesofaminoacidsonthecargosurface,ter- the transportfactorsrequirespresenceoflooselycon- export fromthenucleus.Recognitionofcargoproteinsby superfamily ofproteins,whichmediateeitherimportor majority oftransportfactorsbelongstothekaryopherin chaperones duringpassagethroughthepore.Thevast need toassociatewithtransportfactors,whichactas as theremainingproteins(alsocalledcargoproteins) carrier-independent where- nucleocytoplasmictransport, ductive interactionswiththenucleoporinsarecapableof contain regionsthatenablethemtodirectlyengageinpro- cal interactionswithchannelcomponents.Proteinsthat ars tooccurindependentofmetabolicenergyviaidenti- and inbothcasestheactualtranslocationprocessappe- nuclear poreisdiffusioncontrolledforthetwopathways, cytoplasmic translocationarerelated.Dockingtothe Biochemically carrier-free andcarrier-dependent nucleo- exerted bytheFGrepeat-richinteriorofnuclearpore. les needtoovercomethehydrophobicrepulsionthatis elucidated, itiswidelyacceptedthatpermeatingmolecu- details thatallowpassagethroughtheporeremaintobe polar residuesofvaryingnumber. Althoughthemolecular nylalanine-glycine (FG)repeatsthatareinterspersedwith nucleoporins (Nups),manyofwhichcontainmultiplephe- copies ofabout30differentproteinscollectivelycalled 125 MDainmammaliancellsiscomposedofseveral lope. Thisstructurewithanestimatedmolecularmassof nel spanningthedoublemembraneofnuclearenve- i teins throughthenuclearpore.Theporecomplex of translocationareknowntoexistforthepassagepro- Carrier-dependent aswellcarrier-independent modes e This isachievedbydirectlytargetingcognaterecognition required fortheinductionofcytokine-responsivegenes. since onlythedimerisahigh-affinityDNA-bindingprotein p nical phosphotyrosine-SH2domaininteractions.Tyrosine instantly assembleintohomo-orheterodimersviacano- formation oftyrosine-phosphorylatedSTATs, which s amulti-proteinstructurethatcreatesanaqueouschan- lements namedgammaactivatedsites(GAS). hosphorylation isoftendescribedasSTAT activation

65 Cellular Signalling/ Molecular Genetics Our lab discovered that the STATs make use of both of Mandy Kummerow (Technical assistance) these translocation mechanisms. Due to their constant Stephanie Meyer (Technical assistance) carrier-independent and -dependent nucleocytoplasmic cycling the STATs are distributed throughout the cell, all External funding the time. Deviations from an even pancellular distribution Deutsche Forschungsgemeinschaft result from modulated nuclear export. This is caused eit- „Konstitutiver nucleocytoplanmatischer Transport von her by enhancing the translocation rate or by nuclear STAT1“ (VI 218/2) retention due to a co-factor-independent mechanism. The Uwe Vinkemeier Deutsche Forschungsgemeinschaft recognition that the dephosphorylation reaction is under kinetic control of DNA binding provided a strikingly simple Deutsche Forschungsgemeinschaft example of a self-controlling mechanism that integrates „Einfluss der Tyrosin-Dephosphorylierung auf die Zielgen- central elements of cytokine-dependent gene regulation, findung von STAT1“ (VI 218/3) namely receptor monitoring, promoter occupancy, and Uwe Vinkemeier transcription factor activity. It is important to point out that Deutsche Forschungsgemeinschaft nuclear accumulation is not a mechanism sui generis, but „Analyse einer konservativen Protein-Interaktionsdomä- merely reflects the formation of a conformation that is resi- ne“ (VI 218/4) stant to dephosphorylation. For this reason nuclear accu- Uwe Vinkemeier mulation is not suited as a diagnostic marker of transcriptional activity. In contrast, the translocation rate of STAT Bundesministerium für Bildung und Forschung transcription factors critically determines the outcome of „Molekulare Grundlagen der zellulären Signalverarbei- cytokine signaling and hence constitutes a potential spe- tung“ (Sieger im Nachwuchsgruppenwettbewerb „Bio- cificity determinant. The molecular mechanisms that give Future“) rise to flux modulation, either physiologically or in patho- Uwe Vinkemeier logical situations such as microbial infections, and how European Molecular Biology Organization this affects both duration and strength of cytokine signals, “Regulation of Transcription” (EMBO-Young-Investigator as well as the overall sensitivity and latency of the system Award 2002) will be the task of future research. A further important area Uwe Vinkemeier of our research is post-translational modifications, with focus on arginine methylation and sumoylation. Selected publications (FMP authors in bold) In the last years our lab has made a number of exciting and Chen X, Bhandari R, Vinkemeier U, Van Den Akker F, Dar- unexpected discoveries, which revealed that the STATs nell JE Jr, Kuriyan J (2003) A reinterpretation of the dime- are an intriguing model system for studying the intracellu- rization interface of the N-terminal domains of STATs. Pro- lar dynamics of a signal transducer. In addition, it is increa- tein Sci 142, 361-365 singly being appreciated that STAT nucleocytoplasmic Meyer T, Vinkemeier U, Meyer U (2003) Medizinische cycling also constitutes an important area for microbial Implikationen pharmakogenomischer Behandlungsstrate- intervention. Currently, we are generating mice that gien. Ethik in der Medizin 12, 207-209 express STAT mutants. This will allow us to study the effects of defective nucleocytoplasmic shuttling on deve- Meyer T, Vinkemeier U, Meyer U (2003) Evidence-based lopment and disease. These approaches are complemen- medicine – Was geht verloren? Ethik in der Medizin 14, ted by chemical and structural biology to explore the 3-10 possibilities of rational intervention. Meyer T, Marg A, Lemke P, Wiesner B, Vinkemeier U (2003) DNA binding controls inactivation and nuclear Group members accumulation of the transcription factor Stat1. Genes Dev Prof. Dr. mult. Thomas Meyer 17, 1992-2005 Dr. Andreas Begitt Dr. Regis Cartier Dr. Ying Shan** Dr. Andreas Marg* Torsten Meissner (Doctoral student)* Inga Lödige (Doctoral student) Nicola Venta (Doctoral student)*

* part of period reported ** part-time tische Biophysik,Humboldt-UniversitätzuBerlin Thomas HöferandReinhardHeinrich,InstitutfürTheore- Animal Health,Winnipeg,Canada Markus Czub,CanadianScienceCenterforHumanand Institut fürPhysikalischeundTheoretischeChemie Ulrich Kubischeck,Friedrich-Wilhelm-UniversitätBonn, Gino Cingolani,StonyBrookUniversity, USA Institut fürBiochemie Ilian JelesarovandH.R.Bosshard,UniversitätZürich, nobiology Susan John,Kings’s KollegeLondon,DepartmentofImmu- Collaborations Vinkemeier U 4612 of STAT transcriptionfactors.EurJBiochem271,4606- Meyer T, Vinkemeier U Meyer T 587-589 Arginine MethylationofSTAT1: AReassessment.Cell119, Meissner T, KrauseE,LödigeI,Vinkemeier U Meissner T, KrauseE,Vinkemeier U Vinkemeier U Marg A,ShanY, MeyerT, MeissnerT, Brandenburg M, Biol 167,197-201 Design principlesofamolecularsignalingcircuit.JCell cription factors.JBiolChem279,18998-19007 oligomerization, andnuclearaccumulationofstattrans- A singleresiduemodulatestyrosinedephosphorylation, Stat1. JCellBiol165,823-833 n nucleoporins Nup153andNup214CRM1-dependent i leptomycin BblockCRM1-dependentnuclearexportby dentical mechanisms.FEBSLett576,27-30 uclear exportcontrolthesubcellulardistributionoflatent , HendryL, (2004) GettingthemessageacrossSTAT! (2004) Nucleocytoplasmicshuttlingby Begitt A (2004) Nucleocytoplasmicshuttling , JohnS, (2004) Ratjadoneand Vinkemeier U (2004) (2004)

67 Cellular Signalling/ Molecular Genetics

CHEMICAL BIOLOGY SECTION CHEMICAL BIOLOGY per insights at a molecular and atomic level are required in order to unravel the subtle modes of protein-protein Prof. Dr. Michael Bienert, communication. The analysis of interactions between che- Department Head: Peptide Chemistry mical moieties of proteins, and the identification of protein (Secretary: Marianne Dreissigacker) substructures, affected by drug-like compounds, as well as the design and the synthesis of compounds with modi- Hartmut Berger fied recognition characteristics, demand chemical thin- Michael Beyermann king and chemical methodologies. This makes clear that Margitta Dathe chemistry has become a more central science in our Volker Hagen efforts to understand the molecular basis of life. This dra- Eberhard Krause matic trend has led to the creation of the term “Chemical Jens-Peter von Kries Biology”, defining all kinds of chemical research focused Johannes Oehlke on the unravelling of biological phenomena. Naturally, Jörg Rademann there is an overlap with other areas of chemistry, such as “Medicinal Chemistry” and “Bioorganic Chemistry”. The Although in the last decade our knowledge about genome interaction with a powerful Structural Biology and the sequences (genomics) and the occurrence of cellular pro- involvement of biochemical, biophysical and analytical teins (proteomics) has increased dramatically, this pro- methodologies are necessities for the further successful gress is often not sufficient to understand the function and development of the novel science “Chemical Biology”. the significance of biomacromolecules in cellular systems or in organs of different organisms. All proteins evoke their In the context of Chemical Biology, chemical and biologi- function by specific interactions with other proteins or cal research in the FMP are traditionally excellently inter- constituents of membranes and the cytoskeleton, and dee- connected. Research efforts of the Department of Peptide

INTRODUCTION

BEREICH CHEMISCHE BIOLOGIE Ebene sind nötig, um die Feinstruktur der Protein-Protein- Kommunikation zu erfassen. Sowohl die Analyse der Prof. Dr. Michael Bienert, Wechselwirkungen zwischen chemischen Resten an Pro- Abteilungsleiter: Peptidchemie teinen und Identifizierung von Proteinsubstrukturen, die (Sekretariat: Marianne Dreissigacker) durch Wirkstoffe beeinflussbar sind, als auch das Design und die Synthese von Verbindungen mit veränderter Hartmut Berger Erkennungscharakteristik erfordern chemisches Denken Michael Beyermann und chemische Methodologie. Dies macht klar, dass die Margitta Dathe Chemie für unseren Bemühungen, die molekulare Basis Volker Hagen des Lebens zu verstehen, zu einer zentralen Wissenschaft Eberhard Krause geworden ist. Dieser dramatische Trend hat zur Ent- Jens-Peter von Kries stehung des Begriffs „Chemische Biologie“ geführt, der Johannes Oehlke alle Arten chemischer Forschung beschreibt, die auf die Jörg Rademann Untersuchung biologischer Phänomene gerichtet sind. Natürlich gibt es Überlappungen mit anderen Gebieten der Obwohl unser Wissen über Gensequenzen (Genomics) Chemie, wie zum Beispiel der Medizinischen und der Bio- und über das Auftreten zellulärer Proteine (Proteomics) organischen Chemie. Datenaustausch mit einer starken dramatisch angewachsen ist, reicht der Fortschritt oft Strukturbiologie und die Einbeziehung biochemischer, bio- noch nicht aus, um die Funktion und Bedeutung von biolo- physikalischer und analytischer Methoden sind eine Not- gischen Makromolekülen in zellulären Systemen oder in wendigkeit für eine weitere erfolgreiche Entwicklung der den Organen der verschiedenen Organismen zu verste- jungen Wissenschaft Chemische Biologie. hen. Alle Proteine funktionieren, indem sie spezifische Interaktionen mit anderen Proteinen oder mit Bestandtei- Im Kontext der Chemischen Biologie ist die chemische und len der Membranen und des Zytoskeletts eingehen, und biologische Forschung am FMP traditionell exzellent ver- tiefergehende Einsichten auf molekularer und atomarer netzt. Forschungsarbeiten der Abteilung Peptidchemie Chemistry and Biochemistry are focused on the molecular active biomolecule, e. g. a transmitter, is rapidly liberated mechanisms of peptide-protein (Peptide Synthesis Group, by UV light, thus allowing the analysis of fast biological Peptide Biochemistry Group) and peptide-lipid interactions processes in cells. (Peptide-Lipid Interaction Group), leading to a better In 2004, Chemical Biology at the FMP was considerably understanding of the action of biologically active peptides strengthened by the installation of the Medicinal on their respective targets. The study of protein-protein Chemistry Group. A central aim of this group consists in interactions in the cell requires the improvement of the the creation and development of synthetic methods for the cellular uptake of peptides and other low-molecular com- combinatorial organic chemistry and solid phase organic pounds (Peptide-Lipid Interaction Group). The function of synthesis. Focused libraries of potential ligands are gene- proteins is often regulated by distinct chemical, post- rated in order to find small organic molecules which bind translational modification, which can be analyzed by specifically to selected target proteins, thus assisting in newly developed mass spectrometric procedures (Mass the analysis of functions and interactions of the proteins. Spectrometric Group). In addition, the department is in- Naturally, such small molecules could also serve as lead volved in methodological studies in order to improve the structures for the design of drug candidates. For analyzing chemical synthesis of small-sized proteins, as well as to the biological activity of small-molecule libraries, a synthesize fluorescently labeled peptide analogues for Screening Unit is now established, developing concepts localization and interaction studies. for the handling of libraries and providing the technical Very useful tools for the study of intracellular biological equipment for testing hundreds and thousands of chemi- processes, “caged compounds”, are synthesized and cal compounds.

characterized in the Synthetic Organic Biochemistry y g o l o

Group. Caged compounds are photolabile, inactive deriva- i B

tives of biologically active substances, from which the a c i m e h C 1 7 und Biochemie zielen auf die molekularen Mechanismen Transmitter, auf Einwirkung von ultraviolettem Licht hin von Peptide-Protein- (Arbeitsgruppe Peptidsynthese; freisetzen und so die Analyse schneller biologischer Pro- Peptidbiochemie) und Peptid-Lipid-Interaktionen (Arbeits- zesse in Zellen ermöglichen. gruppe Peptid-Lipid Interaktion) und verhelfen zu einem Im Jahr 2004 wurde die Chemische Biologie am FMP durch besseren Verständnis der Wirkung biologisch aktiver Pep- die Einrichtung der Arbeitsgruppe Medizinische Chemie tide auf ihre jeweiligen Zielstrukturen. Die Untersuchung beträchtlich gestärkt. Ein zentrales Ziel der Gruppe ist die von Protein-Protein Interaktionen in der Zelle erfordert Entwicklung und Etablierung von Methoden für die kombi- eine verbesserte Aufnahme der Peptide und anderer klei- natorische organische Chemie und Festphasensynthese. ner Moleküle (Arbeitsgruppe Peptid-Lipid Interaktion). Die Fokussierte Bibliotheken potentieller Liganden werden Funktion von Proteinen ist oftmals durch bestimmte che- aufgestellt, um kleine organische Moleküle zu suchen, die mische posttranslationale Modifikationen reguliert, die spezifisch an ausgewählte Zielproteine binden. So wird die mittels neuartiger massenspektrometrischer Verfahren Analyse der Funktionen und Interaktionen von Proteinen analysiert werden können (Arbeitsgruppe Massenspek- unterstützt. Natürlicherweise können solche kleinen Mole- trometrie). Darüber hinaus ist die Abteilung an methodo- küle auch als Leitstrukturen für das Design von Wirkstof- logischen Studien beteiligt, die die chemische Synthese fen dienen. Um die biologische Aktivität von Bibliotheken kleiner Proteinmoleküle verbessern und fluoreszenz- kleiner Moleküle untersuchen zu können, hat das FMP markierter Peptidanaloga für Lokalisierungs- und Inter- eine Screening Unit etabliert, die Konzepte für das Hand- aktionsstudien gewährleisten sollen. ling von Substanzbibliotheken erarbeitet und technische Äußerst nützliche Werkzeuge für die Untersuchung intra- Voraussetzungen für die Testung Hunderter und Tausen- zellulärer biologischer Prozesse, „caged compounds”, der chemischer Verbindungen schafft. werden in der Arbeitsgruppe Synthetische Organische Biochemie synthetisiert und charakterisiert. Caged compounds sind photolabile, inaktive Derivate biologisch aktiver Substanzen, die aktive Biomoleküle, z. B. einen PEPTIDE SYNTHESIS ted significant CRF2/CRF1 selectivity in terms of affinity Group Leader: Dr. Michael Beyermann (19 and 260 fold, respectively). Replacing the apparent selectivity motif VPIG in truncated Ucn II by TFH, residues DEVELOPMENT OF A MODEL FOR LIGAND- at corresponding positions of non-selective Ucn I gave the RECEPTOR INTERACTION most selective antagonist for CRF2, exhibiting an about 1500-fold higher affinity for CRF2 compared to CRF1. Most Insights into the molecular basis of interaction between remarkably, the CRF2/CRF1 selectivity in terms of antago- peptide ligands and their membrane-embedded receptors, nistic potencies and receptor affinity of those antagonists in particular G-protein-coupled receptors class B, will strongly diverge, although no significant intrinsic activity open a way for development of new drugs for treatment was found. The quotient of the relative selectivity in terms of a number of diseases, since those receptors are very of affinity and potency of a certain antagonist may provi- important targets of peptides regulating many essential de a basis to evaluate its antagonistic efficiency. The an- biological functions. Those receptors are functional only tagonistic efficiency varied between 2.35 and 33.2 for the when embedded in the membrane, which is why it is dif- peptides investigated, showing that looking at receptor ficult to obtain direct structural information by spectros- affinity only in screenings for antagonists as a global meas- copic methods such as X-ray crystallography or NMR ure may be insufficient to reflect their inhibitory potency. methods. Indirect methods, such as structure-activity relationship studies, modifying the ligand or/and receptor Dimerization of corticotropin-releasing factor structure, e.g. by point substitutions, and looking at corre- receptor type 1 is not coupled to ligand binding sponding effects on ligand-receptor binding/activation, (2) may give information about interaction modes between As reported, receptor dimerization of G-Protein-coupled ligand and receptor. The incorporation of light-directed receptors may influence signaling, trafficking and regula- crosslinkers into ligands gives the possibility to crosslink tion in vivo. Up to now, most studies aiming at the possible the ligand bound to receptors. By this approach, contact role of receptor dimerization in receptor activation and points between ligand and receptor can be determined. signal transduction are focused on class 1 GPCRs. We Furthermore, many evidence points to a crucial contribu- have investigated dimerization behavior of CRF1 receptors tion of extracellular receptor domains to ligand binding. tagged with fluorescence labels (CFP/YFP) transiently Therefore, we are investigating ligand binding to extracel- expressed in HEK293 cells. We measured Fluorescence lular receptor domains. From all these studies, which also Resonance Energy Transfer and found that CRF1 receptors require development of efficient chemical and biotechno- form mainly dimers. Upon addition of CRF-related agonists logical methods for preparation of receptor domains and or antagonists, no significant change of the FRET signal constructs as artificial receptors for appropriate structu- was observed, indicating no change in the dimer-mono- re analysis in complex with ligands, we will stepwise mer ratio by ligand binding. deduce an appropriate interaction model, also taking into consideration whether receptors interact with ligands as Photoaffinity cross-linking of the corticotro- mono- or oligomers. pin-releasing factor receptor type 1 using pho- toreactive urocortin analogues (3) CRF receptor antagonists showing different Interaction of peptide ligands to G-protein-coupled recep- efficiency (1) tors of class B, such as glucagon, secretin and corticotro- Corticotropin-releasing factor (CRF), urocortin I (Ucn I), pin-releasing factor (CRF), is characterized by a common sauvagine (Svg), and urotensin (Uts) are non-selective, orientation of two binding domains, in that ligand C-termi- endogenous agonists of the G-protein-coupled receptors nus binds to extracellular receptor N-terminus (high affini- CRF1 and CRF2. Determinants of subtype receptor selecti- ty binding) and receptor juxta-membrane domain binds vity, particularly for such long-chain (~40 aa) peptides, are ligand N-terminus. N-terminal truncation, particularly of widely unknown. First subtype (CRF2) selective agonists residues 6-8 in the case of CRF, leads to antagonists, sug- have only been discovered on basis of genomic DNA infor- gesting that those residues constitute the receptor acti- mation and subsequent cDNA cloning. A strikingly diffe- vating sequence. Here, we identified by photoaffinity rent amino acid motif (VPIG) at the N-terminus of the cross-linking using Bpa-analogues (Bpa: p-benzoyl-L-phe- selective agonist Ucn II led to CRF2 selective agonists, nylalanine) of urocortin (Ucn) interaction domains of the when incorporated into non-selective Ucn I and Svg but receptor (CRF1) with individual amino acids. Specific not Utn. Receptor antagonists generated by N-terminal digestion patterns of ligand-receptor complexes, obtained truncation of CRF2 selective VPIG-Ucn I and Ucn II exhibi- using different cleavage methods and SDS-PAGE for frag- enclosing thoseresidues. in thatpeptideantagonistsbindtoreceptorN-terminus Our findingsareinconflictwithrecentlypublishedmodels specific interactionofaminoacidresidues12,17and22. membrane segment,isfixedtojuxta-membranedomainby vating sequence,whichmightpenetrateintothetrans- receptor loop.Theseresultsshowthatthesuggestedacti- Ucn and arrangement oftheextracellular N-terminal domainofthe C3 free.Thelatterpatternisconsistent withthedisulfide led tothedisulfidepatternC2-C5 andC4-C6,leaving ing C5free,whereastheC-terminal positionoftheHis-tag tag, thedisulfidebondsC2-C3andC4-C6werefound,leav- fide patternswerefound.InthecaseofaN-terminalHis- Depending onthepositionofaHis-tag,twotypesdisul- type 2abearing5cysteines(C2toC6)wasinvestigated. domains ofthecorticotropin-releasingfactorreceptor ulfide bondsofrecombinantextracellularN-terminal The oxidativefolding,particularlythearrangementofdis- de pattern(4) Hexa-histidine tagpositioninfluencesdisulfi- 125 ment separation,haveshownthat

% Intensity 100 % Intensity 100 10 20 30 40 50 60 7 80 9 1 20 3 40 50 6 70 8 90 I-Y 0 0 0 0 0 0 0 0 0 3010 3020 300 2800 2300 1800 1300 2214,0 800 1951,2 1688,4 1425,6 1162,8 900,0 0 -Bpa 9 941,36 39,39 ( ( b) a) 125 12 I-Y -Ucn bindtothesecond,whilst 1040,41 0 -Bpa 1139,47 1141,44 Voyager Spec#1=>BC=>NFO.7=>SM35[BP=2042.2,264] Spec #1=>BC=>NFO.7=>SM11[BP=666.5,13430] 22 -Ucn bindatthefirstextracellular 1240,47 1 339,52 1942,18 2020,60 1440,53 125 Mass (m/z) Mass (m/z) I-Y- 2003,66 1539,60 1 2 2058,56 -Bpa 042,69 125 1640,58 I-Y0-Bpa 0 -Ucn and 1739,64 17 1840,67 - recent textbooks.TheextentofundesiredN peptide synthesisand,thus,arenotevenmentionedin nized asleadingtoserioussidereactionsinFmoc-based all protectinggroups.N,O-acylshiftshavenotbeenrecog- acid (TFA) usedinFmocchemistryforthefinalremovalof prisingly, forthemoderatelystrongacidtrifluoroacetic acids.Wetreatment withstrong observedsuch shifts,sur- peptides orproteinshavepreviouslybeenobservedupon N-to-O acylmigrationsatserineorthreonineresiduesin tool: N An unexpectedsidereactiongetsabeneficial were farmoreefficientthanthe syntheses ofthosewith- in whichtheesterunitappears at appropriatepositions ducts. Moreover, wefoundthatsynthesesofsequences TFA maygiverisetolarge amountsofdepsipeptideby-pro- depends onthepeptidesequenceandevenincaseof bond C4-C6forligandbinding. N-terminal receptordomains,particularlythedisulfide indicating theimportanceofC-terminalportion cant differencesinbindingaffinitiestoselectedligands, two differentlydisulfide-bridgeddomainsshownosignifi- in whichC1ispairedwithC3.However, bindingdataofthe CRF receptortype1,whichhassixcysteines(C1toC6)and 1939,71 2040,73 ↔ O-acyl shifts(5) 2670,7 264,1 depsi isomer(b)(M[H] dard Fmoc-chemistry(a)andastheall- (crude products)synthesizedusingstan- MALDI-MS spectraofH-(Val-Thr) FIGURE 1 + calc : 2019.18) → O shifts 10 -NH 2

73 Chemical Biology out the ester unit. A final ammonium hydroxide-induced Deutsche Forschungsgemeinschaft O→N-shift yielded the authentic peptides in much better „Struktur, Stabilität und Spezifikationen von nichtkataly- yields. This new method opens a way for efficient synthe- tischen Proteindomänen und deren Verwendung als sis of difficult sequences, thus facilitating investigations Werkzeuge für das Design einer stabilen minimalen ß-Falt- on the role of individual amino acid residues on structural blattstruktur und das Verständnis von pathologischen Pro- stability of the WW domain FBP-28. Compared with other zessen“ (TP 2/2-1 in der Forschergruppe 299 „Optimierte methods, such as pseudoproline methodology, an impor- molekulare Bibliotheken zum Studium biologischer Erken- tant advantage of the depsipeptide method results from nungsprozesse“) the fact that the conformation-disrupting modification Hartmut Oschkinat, Michael Bienert introduced by the depsipeptide unit is still present during chromatographic purification of the deblocked peptide. Selected publications (FMP authors in bold) The suppression of conformation-induced association Klose J, Fechner K, Beyermann M, Krause E, Wendt N, leads to an increased solubility and narrower peaks for Bienert M, Rudolph R, Rothemund S (2005) Impact of depsi isomers, thus allowing a much more efficient purifi- N-terminal domains for corticotropin-releasing factor cation. (CRF) receptor-ligand interactions. Biochem, in press

Group members Klose J, Wendt N, Kubald S, Krause E, Fechner K, Beyer- mann M, Bienert M, Rudolph R, Rothemund S (2004) Hexa- Dr. Jana Klose (4) histidin tag position influences disulfide structure but not DC Angelika Ehrlich (SPOT library synthesis)* binding behavior of in vitro folded N-terminal domain of rat Doreen Wietfeld (Doctoral student) (1)* corticotropin-releasing factor receptor type 2a. Protein Sci Sandra Tremmel (Doctoral student) (5) 13, 2470-2475 Oliver Krätke (Doctoral student) (2,3) Stephan Pritz (Doctoral student)* Carpino LA, Krause E, Sferdean CD, Schümann M, Fabian Irene Coin (Doctoral student) (5)* H, Bienert M, Beyermann M (2004) Synthesis of ”Difficult” Annerose Klose (Technical assistance peptide chemistry) Peptide Sequences: Application of a Depsipeptide Tech- Dagmar Krause (Technical assistance peptide purification, nique to the Jung-Redemann 10- and 26-mers and the analysis) Amyloid Peptide A‚(1-42). Tetrahedron Lett 45, 7519-7523 Barbara Pisarz (Technical assistance Biacore)** Eggelkraut-Gottanka R, Klose A, Beck-Sickinger AG, Bey- Bernhard Schmikale (Technical assistance peptide syn- ermann M (2003) Peptide (alpha)thioester formation using thesis)** standard Fmoc-chemistry. Tetrahedron Lett 44, 3551-3554

External funding Kaupp UB, Solzin J, Hildebrand E, Brown JE, Helbig A, Hagen V, Beyermann M, Pampaloni F, Weyand I (2003) The Deutsche Forschungsgemeinschaft signal flow and motor response controlling chemotaxis of Projekt im Schwerpunktprogramm „Molekulare Sinnes- sea urchin sperm. Nature Cell Biol 5, 109-117 physiologie“ (SPP 1025) Michael Beyermann Wietfeld D, Fechner K, Heinrich N, Seifert R, Bienert M, Beyermann M (2003) Development of CRF receptor-2 Deutsche Forschungsgemeinschaft selective peptide ligands. Biopolymers 71, 376 „Untersuchungen von CRF- und CRF-Rezeptor-Mutanten zur Entwicklung eines Modells für die Ligandenerkennung Collaborations von G-Protein-gekoppelten Rezeptoren der Familie 2“ (TP A6 im Sonderforschungsbereich 449 „Struktur und Funk- In a number of projects we have collaborated either with tion membranständiger Rezeptoren“) groups within and/or outside the FMP, particularly with Michael Bienert, Michael Beyermann, Walter Rosenthal L.A. Carpino (University of Massachusetts/USA) on development of a new method for synthesis of difficult peptides, Deutsche Forschungsgemeinschaft with A.G. Beck-Sickinger (University of Leipzig) on prepa- „Organisation und Funktion eines Transduktionskomple- ration and use of peptide thiol esters for peptide and pro- xes in Sehstäbchen“ (BE 1434/3-3) tein synthesis, and with U.B. Kaupp (FZ Jülich)/V. Hagen Michael Beyermann (FMP) on studies of chemotaxis using appropriately caged peptides. Of particular impact is the joint work with the Peptide Biochemistry Group. * part of period reported ** part-time acids (PNAs)ascargomolecules bypursuingtheuptake being studied,usingcovalently boundpeptidenucleic abilityofCPPsare structural requirementsforthedelivery delivery tothebrain(M.Dathe). Asafurthertopic,the the developmentofpeptide-liposomecomplexesfordrug nistic insightsintotransmembranetransportroutesandon interactions ofCPPswithlipidmembranestogetmecha- research effortsinthiscontextarefocusedonstudying docytotic aswellendocytoticmechanisms.The complex-bound drugsacrosscellmembranesbynonen- tural requirementsforpeptidestodelivercovalentlyor The maingoalofourgroupistheelucidationstruc- very. membrane provideanopportunityforsite-specificdeli- addressing well-definedreceptorstructuresatthecell Peptides whichcontainrecognitionmotifssuitablefor gested thatthelipidbilayerofcellmembranesistargeted. lization andenergy-independentuptake,ithasbeensug- tation processesinvolvingtemporarymembranedestabi- Since manyofthepeptidespromotemembrane-reorien- rable propertiessuchaspoorsolubilityandlowstability. transport oflargeamountsdrugmoleculeswithunfavo- philic compoundsprovidetheadditionaladvantageof which canbeloadedwithhydrophobicaswellhydro- coupling topotentialdrugcontainerssuchasliposomes drawbacks ofthecommondeliverysystems.Peptide into andacrossavarietyofcells,therebyavoidingthe proteins andnucleicacidsledtohighlyefficientdelivery such peptideswithbioactivepolymersuptothesizeof litating compounds.Covalentlinkageorcomplexationof on domains(PTDs)havebeenintroducedasuptake-faci- transducti- as cell-penetratingpeptides(CPP)orprotein during thelastdecadesmallpeptidecarriersdesignated lop safe,efficientapplicationforms.Asanalternative, ced indiagnosticsandtherapyifwearenotabletodeve- cal risks.Thus,promisingnewdrugswillnotbeintrodu- specificity, poorbioavailability, toxicityandimmunologi- or viruscomponentsarehamperedbylowefficiency, poor u low permeabilityofthecellmembrane.Commonlyused limited bytheirinefficientcellularuptakecausedthe able drugsofe.g.proteinaceousornucleicacidoriginis The applicationofmanypotential,biotechnologicallyavail- UPTAKE-MEDIATING PEPTIDES P O Group Leaders:Dr. MargittaDatheandPDDr. Johannes TRANSPORT P ptake-promoting carriersystemsbasedoncationiclipids ehlke ROPERTIES OFCELLULAR EPTIDE LIPIDINTERACTION/PEPTIDE peptides amphipathic, conjugate ofthePNAwithcell-penetrating ge andsize(Table). Astheleadweusedanamide-bound properties,char- des showingdifferentstructureforming nin FQreceptorafterdisulfidebridgingwithvariouspepti- PNA directedagainstthemRNAofnociceptin/orpha- peptides, weinvestigatedthecellularuptakeofa12-mer requirements fortheshuttlingabilityofcell-penetrating In ordertocontributeanelucidationofthestructural Bienert, andJohannesOehlke Y positively andnegativelycharged Cellular uptakeofPNAafterconjugationwith as wellintoHEKcellswasfound forconjugatesbearing after energydepletionanenhanced uptakeintoCHOcells rements fortheshuttlingabilityof peptides.Surprisingly, contradicting speculationsabout specificstructuralrequi- ence theextentofuptakeintoHEK-andCHOcells(Fig.1), of thedisulfidebridgedconjugatedidnotseriouslyinflu- nally. Evenstrongstructuralalterationsinthepeptidepart tration inthiscaseonlyapproachedthatprovidedexter- which stillremainunclear, theintracellularPNA concen- disulfide bridgedKLA-PNA-conjugate.But,forreasons extensive cellularuptakewasfoundalsofortheanalogous activated cellsorting(FACS) supportedtheseresults.An cal laserscanningmicroscopy(CLSM)andfluorescence mined ratioofabout10µlcellvolume/mgprotein).Confo- to theexternalconcentrationandanexperimentallydeter- and itsamideboundKLAconjugate,respectively(related cell interiorofabouttwoandtenfoldforthenakedPNA cardiomyocytes byCE-LIFrevealedenrichmentswithinthe Assessment ofthecellularuptakeintoCHOcellsand thus avoidingbiasoftheresultsbysurfaceadsorption. reducing environmentofthecellinteriorwaspossible, PNA generatedbycleavageofthedisulfidebondin separate quantificationoftheconjugateandnaked fluorescence detection(CE-LIF).Usingthisapproacha of capillaryelectrophoresiscombinedwithlaser-induced the nakedPNA.Thecellularuptakewasstudiedbymeans tenfold highercellularuptakeandbiologicalactivitythan (KLA). We haveshownpreviouslythatKLAexhibits upto amphipathic modelpeptideKLALKLALKALKAALKLA-NH new classofpeptidicantibiotics(M.Dathe). These studiesaimatcontributingtothedevelopmentofa selectively killbacteriabydestroyingtheircellmembrane. c nally, weareinterestedinelucidatingthestructuralprin- effects ofthePNAsinvarioussystems(J.Oehlke).Additio- into differentcelltypesincomparisonwiththebiological vonne Wolf, AngelikaEhrlich,BurkhardWiesner, Michael iples andthemechanismofactionpeptideswhich β -sheet formingandunstructured ∝ ∝ -helical α -helical 2

75 Chemical Biology Compound Sequence Structural properties

Peptide nucleic acid Fluos-GGA GCA GGA AAG-Cys

KLA Dansyl-G-C-KLALK LALKA LKAAL KLA-NH2 α-helical, amphipathic KGL Dansyl-G-C-KGLKL KGGLG LLGKL KLG- NH2 unstructured ELA Dansyl-G-C-ELALE LALEA LEAAL ELA-NH2 α-helical, amphipathic RLA Dansyl-G-C-RLALR LALRA LRAAL RLA-NH2 α-helical, amphipathic Penetratin Dansyl-G-C-RQIKI WFQNR RMKWK K-NH2 poor α-helical amphipathic VT5 Dansyl-G-C-DPKGDPKGVTVTVTVTVTGKGDPKPG- NH2 β-sheet, amphipathic

TABLE 1 Components of disulfide-bridged PNA-peptide conjugates.

an acidic helical amphipathic and a ß-sheet forming pep- rity of the lipid matrix and formation of transient and local tide, respectively (Fig. 1). It can be inferred from the latter lipid structures. findings that the uptake under normal conditions can be counteracted differently by active transport systems in Brain targeting by apolipoprotein E-peptide- dependence on both the peptide and the cell type, thus liposome complexes providing a potential basis for achieving cell selective I. Sauer, O. Liesenfeld3, H. Scharnagel4 , K. Weisgraber5, cargo delivery. M. Bienert, M. Dathe

The treatment of central nervous system diseases is a par- Peptide transport across lipid bilayers ticular challenge. Different from blood vessels of other S. Keller, E. Bárány-Wallje1, S. Serovy2, M. Bienert, M. organs which are equipped with small pores, the tightly Dathe connected brain capillary endothelial cells forming the Conflicting reports on the translocation ability of the cell- blood-brain barrier (BBB) prevent paracellular transport. penetrating peptides through pure lipid bilayers have Furthermore, most potential drugs and modern molecular stimulated biophysical studies on the interaction of pene- tools are neither able to diffuse across the cell layer nor tratin, a peptide capable of transporting large hydrophilic are accessible to the specialized receptor-mediated trans- cargos into the cell with model lipid membranes to com- port systems. An approach to overcome unfavorable prop- pare its bilayer behavior with the ability to enter cells. The erties provides for drug incorporation into carriers. Lipo- results, which provided no evidence for peptide trans- somes have emerged as a very potential drug container. location through pure lipid bilayers, support suggestions that the highly cationic peptide enters living cells by an 3) Institute for Infectious Medicine, Charité Campus Benjamin Frank- endocytotic pathway rather then by disturbing the integ- lin, Berlin 4) Dept. of Clinical Chemistry, University Hospital Freiburg im Breisgau 1) Dept. of Biochemistry and Biophysics, Stockholm University 5) Gladstone Institute of Cardiovascular Disease, San Francisco 2) Junior Research Group Biophysics 6) NMR-Spectroscopy Group, FMP preparations renderingvesicles leakyandpeptides peptides couldpromotethedestabilization ofliposomal anchoring domains.Sincecoating vesiclesurfaceswith transmembrane helicesandfatty acidchainsservedas adsorption totheliposomalsurface.Amphipathicand that enablesbindingtotheLDLreceptorrelieson somes withanapoEpeptideandtoinducethestructure vector sequence.Acost-effectiveapproachtocoatlipo- binding domainofapolipoproteinE(apoE)wasusedas dimer peptide(141-150)2derivedfromtheLDLreceptor- cies. Apeptideencompassingthehighlycationictandem expressed oncapillaryendothelialcellsofdifferentspe- to liposomalcarriers.Thereceptorwasfoundbehighly cells, andtodevelopstrategiesoftheirefficientcoupling tein receptor(LDLreceptor)onbraincapillaryendothelial peptidic vectorswhichrecognizethelowdensitylipopro- It wasourobjective(M.Datheandcoworkers)todevelop molecules couldbetransported. capillary endothelialcells,thousandsofincorporateddrug pound thatrecognizesmembraneconstituentsofbrain Equipped withanappropriatetransport-mediatingcom- and thehydrophobicshellwithnon-polarcompounds. self-assembled fromlipidscanbeloadedwithpolardrugs The solvent-containinginteriorofthenanoscopicparticles on with2deoxy-glucose/Na-azide25/10mM).Eachbarrepresentsthemeanofthreesamples+SEM. with variouspeptides(table)for60minat37°Cwithout(normal)andenergydepletion(afterpreincubati- Q FIGURE 1 uantities ofPNA-CysintheextractsHEK-andCHO-cellsexposedbeforeto0.2µMPNAaloneordisulfidebridged played adecisiveroleintheuptake process. matrix componentheparansulfate proteoglycan(HSPG) tuents suggestedthattheubiquitouslyexpressedcell of thecationizedliposomeswithanionicmembraneconsti- route. Studiestoinfluenceunspecificchargeinteraction dominating roleofanLDLreceptor-independent transport with up-anddown-regulatedreceptorlevelpointedtothe affinity totheLDLreceptorandinternalizationintocells dependent endocytoticprocess(Figure1).Lowpeptide into endothelialcellsofbrainmicrovesselsbyanenergy- The peptide-coatedliposomeswereefficientlyinternalized somes providestablecomplexes. of thecationicapoEpeptideontostericallystabilizedlipo- capillary endothelialcells.Furthermore,covalentcoupling purposes ofastabledrugcontainerandtargetingtobrain peptide provedtobethemostpromisingcombine Complexes ofliposomeswithadipalmitoyl-modifiedapoE the vesicles,andtheirtoxiceffectwereevaluated. cal conformation,theirabilitytoconservetheintegrityof face, thepotentialtoassumeareceptor-recognizing heli- membranes, theirstableanchorageattheliposomalsur- released incirculationcouldcauseperturbationofcell

77 Chemical Biology FIGURE 2 Confocal laser scanning microscopic picture of mouse brain capillary endothelial cells exposed to fluorescence-labeled and apoE peptide-covered liposomes at T = 37 °C for t = 30 min. Uptake is documented by the spotted fluorescence within the cytoplasm.

The efficient uptake via HSPG-involving endocytosis may Deutsche Forschungsgemeinschaft provide a promising strategy for facilitated drug delivery “Passage of the blood-brain barrier using surface-modi- across the blood brain barrier. Furthermore, the observed fied nanosuspensions and apolipoprotein-E peptide- internalization emphasized the potential of cationic pepti- covered carrier systems” (DA 324/5-1) des to ferry complex drug carriers into cells, and the Margitta Dathe adsorptive coupling strategy offers great adaptability to a Deutsche Forschungsgemeinschaft broad spectrum of ligands destined to diverse biological “Hirntargeting mittels oberflächenmodifizierter Nano- targets. suspensionen und Apolipoprotein E-Peptid beladener Trägersysteme“ (Teilprojekt 7B der Forschergruppe 463 Group members “Innovative Arzneistoffe und Trägersysteme – Integrative Ines Sauer (Doctoral student) Optimierung zur Behandlung entzündlicher und hyperpro- Sandro Keller (Doctoral student)* liferativer Erkrankungen“) Yvonne Wolf (Doctoral student)* Margitta Dathe Axel Wessolowki (Doctoral student)* European Community Heike Nikolenko (Technical assistance) “Investigation of structural prerequisites for cell-selective Gabriela Vogelreiter (Technical assistance) drug uptake mediated by peptide vectors” (Subproject of QLK3-CT-2002-01989 “Target specific delivery systems for External funding gene therapy based on cell penetrating peptides”) Deutsche Forschungsgemeinschaft Johannes Oehlke, M. Bienert “Linear and cyclic hexapeptides: interaction with membra- European Community nes and modulation of selective cell-lytic processes” (DA “Interaction of cell-penetrating with artificial lipid mem- 324/4-1, 4-2) branes and peptide translocation through lipid layers” Margitta Dathe (Subproject of QLK3-CT-2002-01989 “Target specific deli-

* part of period reported Hällbrink M, synthetic modelpeptide.EurJBiochem271,3043-3049 activity ofPNAafterconjugationwithacell-penetrating Enhancement ofintracellularconcentrationandbiological Biochim BiophysActa1667,222-228 peptide-to-cell-ratio ratherthanon peptideconcentration. Uptake ofcell-penetratingpeptidesisdependentonthe Oehlke J Wessolowski A,BienertM,DatheM chemistry 43,9140-9150 arginine- andtryptophan-containinghexapeptides.Bio- tion increasestheantimicrobialactivityandselectivityof Dathe M,NikolenkoH,KloseJ,BienertM Selected publications Ines Sauer Molecules“, (Budapest2003),Conferencegrant Strategies forTargeting ofEpitopes,DrugsandReporter Organizing committeeoftheCongress“CellularTransport Axel Wessolowski Karolinska Institut,Stockholm,Schweden) (DAAD-Stipendium: 2-monatigerStudienaufenthaltam branen undModulationselektiverzelllytischerProzesse“ “Lineare undcyclischeHexapeptide:InteraktionmitMem- Deutscher AkademischerAustauschdienst Ines Sauer J. DavidGladstoneInstitute,SanFrancisco,USA (DAAD-Stipendium: 4-monatigerStudienaufenthaltam the brain” nes: modulationofareceptor-mediated into drugtransport “ Deutscher AkademischerAustauschdienst Yvonne Wolf der UniversitätMontpellier, Frankreich) ( toren“ Peptidvek- einen zellselektivenWirkstofftransportdurch “Untersuchungen zustrukturellenVoraussetzungen für Deutscher AkademischerAustauschdienst Margitta Dathe,MichaelBienert peptides”) very systemsforgenetherapybasedoncellpenetrating ger H,BienertM cyclization. JPeptRes64,159-169 effect ofaromaticclusters,D-aminoacidsubstitutionand activity of arginine- and tryptophan-richthe hexapeptides: DAAD-Stipendium –3-monatigerStudienaufenthaltan Interaction ofamphipathicApoE-peptideswithmembra- , Wallukat G, Oehlke J,PapsdorfG,BienertM (2004) Wolf Y, EhrlichA,WiesnerB,Ber- (FMP authorsinbold) (2004) Antimicrobial (2004) Cycliza- (2004) lar Disease,SanFrancisco P sity HospitalFreiburgimBreisgau Dr. HubertScharnagel,Dept.ofClinicalChemistry, Univer- Franklin, Berlin I Prof. OliverLiesenfeld,Dept.ofMedicalMicrobiologyand Collaborations r uptake ofstericallystabilizedliposomesintobraincapilla- (2005) AnapolipoproteinE-derivedpeptidemediates Sauer I mmunology ofInfection,CharitéCampusBenhamin y endothelialcells.Biochemistry44,2021-2029 rof. KarlWeisgraber, GladstoneInstituteofCardiovascu- , DunayIR,Weisgraber K, Bienert M,DatheM

79 Chemical Biology PEPTIDE BIOCHEMISTRY Studying the characteristics of the coupling of CRFR1 to

Group Leader: Dr. Hartmut Berger Gs, Gi, and Gq in more detail, we developed the following model for the activation of the signaling by CRFR1 in HEK CORTICOTROPIN-RELEASING FACTOR cells. A peptide agonist ligand binds to a high-affinity, low-

RECEPTOR TYPE 1 (CRFR1): REGULATION capacity receptor binding site activating Gs-protein and OF THE COUPLING TO DIFFERENT G-PRO- adenylate cyclase with high ligand potency. At higher con- TEINS AND THE TWO-DOMAIN RECEPTOR centration the ligand additionally binds to one or more low- MODEL affinity, high-capacity sites of which the main part remains

non-coupled, however a small number couples to Gi-pro- Corticotropin-releasing factor (CRF) receptor type 1 teins, attenuating the ligand-stimulated production of (CRFR1) is a G-protein-coupled receptor (GPCR) of the cAMP. The activation of the G-proteins is accomplished by class B, secretin receptor family, which is activated by increasing the affinity of the nucleotide-binding site in the several peptide ligands and which is thought to be the G chain for GTP, the affinity of GTP S to G being stronger α γ i principal physiological mediator of stress responses. For increased as compared to Gs. Very high ligand concentra- the activation of the receptor by peptide ligands, a two- tions also stimulate IP3 accumulation through activation domain model has been established by several authors: of Gq and, in addition, through a pertussis toxin-dependent the N-terminal domain of the receptor (N-domain) is re- way, obviously by activation of phospholipase C by the G βγ quired for ligand binding, whereas the juxtamembrane chain released from Gi when Gi is activated. Stimulation of region, consisting of transmembrane regions and inter- the receptor by ligands desensitizes Gs and Gq activation, vening loops (J-domain), is involved in the activation of the but not Gi, so that the inositol phosphate pathway is about signaling steps by the ligand, bitethered between N and J. half-desensitized. Furthermore, whereas peptide antagonists bind predomi- From our findings it seems clear that the CRFR1 can be nantly to the N-domain with high affinities, non-peptide added to the growing list of GPCRs that simultaneously antagonists bind only to the J-domain. We have provided couple to unrelated G-proteins, possibly through different evidence that the CRFR1 expressed in HEK (HEK-CRFR1) active receptor states and/or different affinities of the cells couples to G - and G -proteins with different charac- s i G-proteins to one or more such states. To get more insight teristics. The two-domain binding model and our model on into these possibilities, we studied the influence of pepti- G-protein coupling regard different aspects of the activa- de and non-peptide antagonist on the G-protein coupling, tion of the CRFR1. Therefore, it is important to investigate on the basis of the two-domain model for CRFR1. The pep- whether they can be fitted into one combined model. tide antagonist α-helical CRF(9-41), assumed to bind predominantly to the N-domain, antagonized G as well as G For this reason, we studied the regulation of G-protein s i coupling competitively with equal potencies, suggesting coupling of the CRFR1 in HEK-CRFR1 cell membranes and that similar ligand binding to N is responsible for G as well the influence of peptide and non-peptide antagonists on s as G activation. However, the non-peptide antalarmin, the coupling of the receptor, using the GTP 35S binding i γ assumed to bind exclusively to the J-domain, antagonized assay (Hartmut Berger, Nadja Heinrich, Monika Georgi, Gs activation competitively but the G response non-com- Doreen Wietfeld*, Jens Furkert**). Corresponding to i petitively (Fig. 2). This should mean that nonpeptide ant- biphasic binding of the ligand to the CRFR1, G - and G -pro- s i agonists antagonize G and G coupling of CRFR1 by com- tein coupling of the receptor were seen in the biphasic s i petitive and allosteric mechanisms, respectively, and that ligand-evoked stimulation of GTP 35S binding to HEK- γ different conformation ensembles of two possibly over- CRFR1 membranes (Fig. 1). G -activation could be separa- s lapping J domains of the receptor are responsible for the ted by inactivation of G with pertussis toxin, G activation i i coupling to G and G . by pre-stimulation of the cells with a ligand, which only s i desensitized Gs activation (Fig. 1). The specificity of Gs and Taken together, it is concluded that CRFR1 signals through

Gi activation was confirmed by immunoprecipitation of Gs-, Gi-, and Gq-proteins. The concentrations of the stimu- GTP 35S-bound G subunits. By this means and a substan- lating ligand and GTP and, furthermore, selective desensi- γ α tial accumulation of inositol phosphates (IP3) in cells, it tization may be part of a regulatory mechanism determin- was found that the receptor also activates Gq-proteins. ing the actual ratio of the coupling of CRFR1 to different

G-proteins. Gs and Gi coupling of the receptor can be described within the framework of the two-domain receptor * Peptide Synthesis Group, **Cellular Biology/Molecular Medicine Group GTP as sauvagine-stimulatedbindingof cells. G-proteincouplingwasmeasured CRF receptortype1expressedinHEK the cellsafterinactivationofG G FIGURE 1 m gine-stimulated bindingofGTP receptor type1asmeasuredbysauva- (G sitization ofGscouplingbysauvagine tussis toxin(Gsactivityleft,A)ordesen- respectively, G nism bythenon-peptideantalarminof, Competitive andnoncompetitiveantago- FIGURE 2 expressing thereceptor. s embranes obtainedfromHEKcells i - activity left,B). andG γ 35 S tomembranesobtainedfrom i - protein couplingofCRF s and G i coupling ofthe i by per- γ 3 5 S to

81 Chemical Biology model in that they are accomplished by different active conformations of the J-domain.

Our group was also engaged in receptor assays for studying structure-activity relationships of CRF peptides (Klaus Fechner, Gabriela Vogelreiter, results are given by the Pep- tide Synthesis Group).

Group members

Dr. Nadja Heinrich** Dr. Klaus Fechner* Monika Georgi (Technical assistance) Gabriela Vogelreiter (Technical assistance)**

Selected Publications (FMP authors in bold)

Wietfeld D, Heinrich N, Furkert J, Fechner K, Beyermann M, Bienert M, Berger H (2004) Regulation of the coupling to different G proteins of rat corticotropin-releasing factor receptor type 1 in human embryonic kidney 293 cells. J Biol Chem 279, 38386-38394

Oehlke J, Wallukat G, Wolf Y, Ehrlich A, Wiesner B, Ber- ger H, Bienert M (2004) Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide. Eur J Bio- chem 271, 3043-3049

* part of period reported ** part-time its receptor. strategiesseemtohaveanenor- Proteomic tive cellularresponsesinitiatedafter thebindingofEPOto the inductionofproliferative,antiapoptotic ordifferentia- subdomains ordistinctsignaling molecules/pathwaysto relate thefunctionofsingletyrosineresidues/receptor distinct signalingproteins.However, itisstill difficultto of tyrosine residuesthatarecriticalfortherecruitment forms oftheEPOreceptor(EPOR)haveidentifiedsingle lation afterreceptordimerization.Studiesusingmutated associated enzymewhichisundergoingtransphosphory- activity oftheJanusfamilykinase2(JAK2),areceptor- lation ofreceptortyrosineresiduesismainlyduetothe proteins tothephosphorylatedreceptor. Thephosphory- ment ofcytosolicsrchomology2(SH2)domain-containing cells, initiatesmultiplesignalingpathwaysbytherecruit- receptor, whichisexpressedonlateerythroidprogenitor The interactionbetweenerythropoietin(EPO)andits ons usingdifferentproteomicstrategies Examination oferythropoietinreceptorfuncti- the FMP. in collaborationwithgroups from bothwithinandoutside work andthemethodologicalstudiesarebeingcarriedout sed signalingproteins,respectively. Boththeproteomics high sensitiveidentificationandquantitationoflow-expres- for theMS-basedanalysisofphosphoproteinsand isotope-labeling, improvedmethodsarebeingdeveloped dies. Usingchemicalderivatizationstrategiesandstable proteins,and the group is performingstu- methodological order togaininsightintotheionizationbehaviorofpeptides important post-translationalmodificationsofproteins.In functional stimulationandtheelucidationoffunctionally specific proteinsofmolecularnetworksincellsfollowing ral proteomprojectswhichincludetheidentificationof seve- to contributed cular weightproteins.TheMSgroup well assequenceinformationofpeptidesandhighmole- vide veryaccurateandsensitivemassmeasurementsas (MS/MS), stateoftheartmassspectrometryisabletopro- 2002) andnewpowerfulfragmentationtechniques a niques. Basedonthemethodologicaladvancesinpeptide signaling proteins require highlysensitiveanalyticaltech- complex proteinmixturesaswelloflowabundance lular biology. Theidentificationandquantitationofvery a Recent resultsemphasizetheroleofmassspectrometry ANALYSIS MASS SPECTROMETRY-BASED PROTEIN Group Leader:Dr. EberhardKrause M s an essential tool for proteom studiesinthefieldofcel- nd proteinionization(awardedwiththeNobelPrizein ASS SPECTROMETRY weight proteins,remainsfarfrom routine.Thelowabun- site ofphosphorylation,especially forhighmolecular analyze phosphorylatedproteins, thedeterminationof mass spectrometryhasbecome apreferredmethodto proliferation, energystoring,andapoptosis.Although transduction processes,forexample,celldifferentiation, modification ofproteinswhichisinvolvedinmanysignal ne, andtyrosinewithaphosphategroupistheubiquitous Esterification ofthehydroxylfunctionsthreonine,seri- of post-translationalmodificationsproteins Chemical derivatizationstrategiesforanalysis chemistry andMolecularBiology, UniversityofRostock). with T. BittorfandS.Körbel,InstituteforMedicalBio- sive analysisofcomplexcellularresponses(cooperation molecules andoffersthepotentialforamorecomprehen- required forthedetectionoflowexpressedsignaling 18O-labeling, andLC-MS/MSprovidesthesensitivity combination ofone-dimensionalgelseparation,in-gel our experimentsthataproteomicstrategybasedon dent proteinscouldbeachieved.We canconclude from and relativequantificationofmorethan200EPOR-depen- the EPORsignaling.Theresultsshowthatidentification used forquantitativeanalysisofgel-separatedproteins In addition,astableisotopelabelingmethod(Fig.2)was reported tobeinvolvedinEPORinducedregulatoryevents. been not have which modification/regulation of G-proteins GTP bindingproteinsaswellinvolvedinthe the covalentubiquitinattachment.We identified several tegies repressingcytokineinducedsignalsinanalogyto rylation ofSUMO-1hasbeensuggestedtobepartstra- linked toEPO-inducedsignallingsofar. Thus,thephospho- pathways, proteinscouldbeidentifiedwhichhavenotbeen cellular signaling.Besidesproteins of already established allowed ustoidentifyproteinswhichareinvolvedinintra- proteins andLC-tandemmassspectrometry(MS/MS) alternative strategyusing1-Dgelseparationofphospho- signaling proteinscannotbedetectedbythistechnique.An identification byMALDI-MSrevealedthatlowexpressed b However, theanalysisusingclassicalapproachcom- stateofproteins. phosphorylation changes inthetyrosine identical cellularbackground(Fig.1),wefocusedonrapid l tion of structure-function relationships inthe EPORsigna- We proteomicstrategiestotheinvestiga- different applied regulatory moleculesareexpressedatratherlowlevels. m cation. Amajorchallengeincreatingphosphoproteome ce ofproteins,butalsointheirpost-translationalmodifi- mous potentialintheanalysisofchangesabundan- ing complex. Using a set of EPOR mutants expressed in an in ing complex.UsingasetofEPORmutantsexpressed ining two-dimensionalgelelectrophoresis(2-DE)and aps, however, remainsthefactthatphosphorylated

83 Chemical Biology FIGURE 1 Schematic representation of wild-type and mutated EGF/EPO receptors. All receptors consist of the human EGFR ligand-binding domain and the transmembrane and cytoplasmatic domains of the murine EPOR.

FIGURE 2 Scheme for the identification and relative quantitation of EPOR-dependent phosphoproteins by enzymatic isotope labeling. * partofperiodreported Thomas Bittorf(UniversityofRostock), E.Krause 599/2) giger SignaledurchfunktionelleProteomanalyse“ (BI „Simultane UntersuchungErythropoietinrezeptor-abhän- Deutsche Forschungsgemeinschaft External funding Stephanie Lamer(Technical assistance)* Kareen Tenz (Technical assistance)* Heidi Lerch(Technical assistance) Clementine Klemm(Doctoralstudent) Dr. MichaelSchümann Group members Meissner andU.Vinkemeier, FMP). inhibitor methylthioadenosine(incooperationwithT. which accumulatehighlevelsofthemethyltransferase mechanism ofreducedinterferonsensitivitytumorcells tion havetobeexploredunderstandthemolecular extent. Inconclusion,alternativeexplanationstomethyla- in position31ofStat1isnotmethylatedtoasignificant tandem MSmeasurementsclearlyrevealedthatarginine Arg31 methylationintheDNAbindingofStat1.MSand vide evidencecontradictingrecentresultsontheroleof In addition,massspectrometricanalysiswasabletopro- normally suppressedincomplexpeptidemixtures. covering thephosphoserineposition727ofStat1,whichis allowed directMSdetectionofthepeptidesequence necessary fortranscriptionalactivity. Thederivatization vation, whereasserinephosphorylationatposition727is ral post-translationalmodificationsarerequiredforacti- important roleintheregulationofgeneexpression.Seve- cytokine-responsive transcriptionfactorswhichplayan (signal transducersandactivatorsoftranscription)are to determinethephosphorylationofStat1.Statproteins lysis ofproteinphosphorylation.Thismethodwasapplied ionization efficienciescanbeusedforsensitiveMSana- 2 phenylethanethiol(PET)wedemonstratethatenhanced r reaction conditionsforbeta-elimination/additionprocedu- regard toenhancedionizationefficiency, andoptimized metry. SeveralN-andS-nucleophileswerestudiedwith kably enhancedsignalintensitiesinMALDImassspectro- n the phosphatemoietyofphosphoserineorphosphothreo- elimination/Michael additionreactionwasusedtoreplace tion ofspecificphosphopeptides.Inourgroup,thebeta- t ionization efficiencyofphosphopeptidescomparedwith sequence coveragebyspecificproteolysis,andthelow dance ofphosphoproteins,thedifficultyobtainingfull heir non-phosphorylatedanalogsmaypreventthedetec- es weredeveloped.Basedonin-gelderivatizationwith ine peptidesbyagroupwhichshouldgiverisetoremar- loid peptideAbeta(1-42).Tetrahedron Lett45,7519-7523 que totheJung-Redemann10-and26-mersamy- peptide sequences:applicationofadepsipeptidetechni- Spicher K, Czupalla C,CuloM,MüllerEC,BrockReuschHP, N-terminal glycineresidue.EMBOJ22,826-832 Commun MassSpectrom18,863-868 sted laserdesorption/ionizationmassspectrometry. Rapid side chainstosignalintensitiesofpeptidesinmatrix-assi- Kraus M,BienertKrauseE Selected publications Side reactioninpeptidesynthesis MA, USA Louis A.Carpino,UniversityofMassachusetts, Amherst, Modifications ofG-proteins University MedicineBerlin Christiane Kleuss,InstituteforPharmacology, Charité– Proteom analysisforriskassessmentofchemicals Ursula Gundert-Remy, AxelOberemm,BfRBerlin Phosphoproteomics ofEPOreceptorsignaling Thomas Bittorf,UniversityofRostock Collaborations Meissner T, KrauseE,LödigeI,Vinkemeier U Klemm C,SchröderS Baumgart S,LindnerY, KühneR Kleuss C, ne methylationofSTAT1: areassessment.Cell119,587-589 H, H, Carpino LA, Spectrom 17,222-228 zation andelectrosprayionization.RapidCommunMass spectrometry usingmatrix-assistedlaserdesorption/ioni- studies onAlzheimer’s amyloid-betapeptidesbymass C phoinositide 3-kinaseisoformsbetaandgamma.JBiol characterization oftheautophosphorylationsitesphos- Massenspektrometrie) EberhardKrause „Identifizierung hepatozellulärerBiomarker“(0312618/UA Bundesministerium fürBildungundForschung Krause E trometry. RapidCommunMassSpectrom18,2697-2705 cy inmatrix-assistedlaserdesorption/ionizationmassspec- with S-andN-nucleophilesforenhancedionizationefficien- hem 278,11536-11545 Krause E Bienert M,BeyermannM (2004) Derivatizationofphosphorylatedpeptides Krause E Krause E (2004) Thecontributionsofspecificaminoacid Krause E (2003) Galphasispalmitoylatedatthe , NürnbergB(2003)Identificationand , SferdeanCD, , GlückmannM, (FMP authorsinbold) (2004) Synthesisof“difficult“ (2003) Hydrogenexchange , OberemmA,Wenschuh Schümann M Beyermann M, (2004) Argini- , Fabian

85 Chemical Biology SYNTHETIC ORGANIC BIOCHEMISTRY {7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl Group Leader: Dr. Volker Hagen (BCMACM), [6,7-bis(ethoxycarbonylmethoxy)coumarin-4-yl]methyl CAGED COMPOUNDS (BECMCM), [7-(diethylamino)-α-methylcoumarin-4-yl]methyl Controlled temporal and spatial release of biomolecules (DEAMCM), from photolabile precursors, called caged compounds, α-carboxy-4,5-dimethoxy-2-nitrobenzyl (CDMNB), have become increasingly interesting in the chemical and and 4-carboxymethoxy-2-hydroxycinnamoyl (CMHC) biological communities. When caged, a biomolecule is moieties as novel caging groups. Nearly all these caging rendered biologically inactive by derivatization with a groups bear anionic substituents which confer high photolabile protecting or caging group. As a result, it can aqueous solubility. Furthermore, the coumarinylmethyl be applied to cells under steady state conditions without caging groups absorb at wavelengths >350 nm and there- evoking biological responses. Flash photolysis using UV fore allow uncaging at long-wavelength irradiation. light cleaves the modifying group and rapidly generates the biologically active molecule. Because photolysis of Using the novel coumarinylmethyl protecting groups and caged compounds generates effectors in situ, much faster our earlier introduced caging groups, we prepared impro- and more spatially uniform, concentration jumps can be ved caged versions of cyclic nucleoside monophosphates triggered than with other techniques. There are no diffu- (cNMPs). Their usefulness was demonstrated in physiolo- sion problems and no spatial inhomogeneity problems gical studies of different types of cyclic nucleotide-gated with addition of substrates. Desensitization is minimized. ion channels of olfactory sensory neurons (B. Wiesner, Caging and uncaging of biomolecules are widely used for FMP; A. Menini, Trieste; K. Benndorf, Jena) and of sperm studies of mechanisms and the kinetics of cellular proces- (U. B. Kaupp, Jülich). The most useful caged cNMPs are ses. Other applications of photoremovable protecting the BCMACM, BCMCM and BECMCM-caged derivatives. groups which are currently being explored are time resol- We could show that photocleavage of these coumarinyl- ved X-ray studies, protein folding, gene expression con- methyl-caged compounds was possible with two-photon trol, and also combinatorial chemistry and photolitho- excitation as well (collaboration with B. Wiesner, FMP) graphy. and that the liberation of the cNMPs occur in the nanose- cond time scale. A fluorescence spectroscopic method for Caged compounds must meet specific requirements. They the calculation of the rate constants of the steps of the should undergo fast and highly efficient photochemical photolysis pathways was developed (collaboration with J. reactions and display high molar absorptivities at wave- Bendig, Berlin and R. Schmidt, Frankfurt/Main). The high lengths >300 nm, or better >350 nm. Furthermore, they soluble BCMACM-caged compounds allow unpreceden- should be sufficiently soluble in aqueous solutions, stable ted large instantaneous steps of the cNMP concentration. towards solvolysis, and biologically inert. Sometimes Using membrane-permeable BECMCM-caged cGMP and membrane-permeability is required. Commonly used cAMP, it was demonstrated for the first time that cGMP caged compounds do not meet these requirements, the- promotes the influx of Ca2+ into sperm of sea urchin or star- refore the design of novel caging groups and the develop- fish and it succeeds in combination with caged resact the ment of novel caged biomolecules are necessary. Further- identification of the motor response in free swimming more, a comparatively small number of caged compounds sperm (Kaupp, Jülich). exists at present and the variety of applications is such that it is desirable to have a range of compounds with dif- The CDMNB group was used for caging and uncaging of ferent characteristics to choose from. the vanilloid receptor agonist capsaicin (collaboration with S. Frings, Heidelberg). Caged capsaicin is a novel tool for kinetic examinations of TRPV1 channels in somatosenso- Our group deals with the development of novel caging ry neurons. First applications of CDMNB-caged capsaicin groups and the synthesis, characterization and application to noniceptors from rat dorsal root ganglia confirmed that of caged compounds. In the past few years, we developed it can be used as a phototrigger for the activation of noni- [5,7-bis(carboxymethoxy)coumarin-4-yl]methyl ceptors in physiological studies. (5,7-BCMCM), The group was also concerned with the development of [7,8-bis(carboxymethoxy)coumarin-4-yl]methyl caged protons. Protons interact with all proteins, which (7,8-BCMCM), modulate their structure and their catalytic properties. Therefore protons trigger events in protein folding/unfold- lipid bilayers(P. Pohl,S.Keller, FMP). tons wasdemonstratedinstudiesoftheH three-dimensional resolution.Theutilityofthecagedpro- (collaboration withB.Wiesner, FMP)thatshould allowtrue also sensitivityofthecompoundtotwo-photonphotolysis high solubilityallowstoinducelargepHjumps.We found ble inwater. Thecombinationofhighphotosensitivity and tum yield.Furthermore,DMACMsulfateisveryeasilysolu- properties, ahighextinctioncoefficientandquan- constant ofatleast5x10 ton sodiumDMACMsulfateexhibitsaphotoreleaserate rinylmethyl (DMACM)caginggroup.Thenovelcagedpro- variants ofournewlyintroduced7-(dimethylamino)couma- thesized andcharacterizedcagedprotonsbasedon described, buttheyhavealotofdisadvantages.We syn- rapid pHjumps.Anumberofcagedprotonshavebeen photoactivatable protonprecursorsforthegenerationof Kinetic studiesofalltheseprocessesmaybeaidedby ing andplayacrucialroleincellularsignaltransduction. 8 s -1 , long-wavelengthabsorption + migration along * partofperiodreported Hagen V, Beyermann M Selected publications Volker Hagen,U.BenjaminKaupp(ResearchCenterJülich) CT98-0034) „Cyclic nucleotidesforthestudyofcellularevents“(Bio4- European Community Volker Hagen chemie undbiologischeAnwendungen“(HA2694/1-2) „Neue ‚caged’cyclischeNucleotide–Synthese,Photo- Deutsche Forschungsgemeinschaft External funding Brigitte Dekowski(Technical assistance) Nico Kotzur(Student)* Ralf Lechler(Doctoralstudent) Daniel Geißler(Doctoralstudent)* Group members Kaupp UB,SolzinJ,HildebrandE,BrownJE,HelbigA, sea urchinsperm.NatureCellBiol 5,109-117 signal flowandmotorresponse controling chemotaxisof , PampaloniF, Weyand I(2003) The t (F =pH-sensitivefluorescenceindica- flash photolysisofphototriggersforH L FIGURE 1 or). arge jumpsinpHcanbeachievedupon (FMP authorsinbold) +

87 Chemical Biology Geißler D, Kresse W, Wiesner B, Bendig J, Kettenmann H, Caged cNMPs as tools for studies of gating kinetics of Hagen V (2003) DMACM-caged adenosine nucleotides: CNG channels Ultrafast phototriggers for ATP, ADP and AMP activated by K. Benndorf, Friedrich Schiller University Jena long-wavelength irradiation. ChemBioChem 4,162-170 Studies of the function of CNG channels on isolated olfac- Serowy S, Saparov SM, Antonenko YN, Kozlovsky W, tory sensory neurons using caged cNMPs Hagen V, Pohl P (2003) Structural proton diffusion along A. Menini, International School for Advanced Studies, lipid bilayers. Biophys J 84, 1031-1037 Trieste, Italy Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl P, Maric K (2003) Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells. EMBO Rep 4, 88-93

Hagen V, Frings S, Wiesner B, Helm S, Kaupp UB, Bendig J (2003) (7-Dialkylaminocoumarin-4-yl)methyl-caged compounds as ultrafast and effective long wavelength phototriggers of 8-bromo-substituted cyclic nucleotides. Chem- BioChem 4, 434-442

Matsumoto M, Solzin J, Helbig A, Hagen V, Ueno S, Kawase O, Maruyama Y, Ogiso M, Godde M, Minakata H, Kaupp UB, Hoshi M, Weyand I (2003) A sperm-activating peptide controls a cGMP-signaling pathway in starfish sperm. Dev Biol 260, 314-324

Collaborations

Photochemistry of caged compounds J. Bendig and P. Wessig, Institute of Chemistry, Humboldt University Berlin

Time-resolved fluorescence spectroscopy R. Schmidt, Institute for Physical and Theoretical Chemi- stry, Johann Wolfgang GoetheUniversity, Frankfurt / Main

Studies of cellular signaling using caged compounds and caged chemoattractants U. B. Kaupp, Institute for Biological Information Process- ing, Research Center Jülich

Caged vanilloid receptor agonists as tools for kinetic examinations of TRPV1 channels S. Frings, Department of Molecular Physiology, University of Heidelberg

Analysis of receptor systems in CNS using caged compounds H. Kettenmann, Max Delbrück Center for Molecular Medi- cine, Berlin-Buch

Controling of gene expression by using caged compounds S. Cambridge, Max Planck Institute of Neurobiology, Munich planning oflibrarycompositionand hitevaluation. modelling groupandtheScreening Unitforthecoherent organized amedicinalchemistry paneltogetherwiththe cular modellingtoolsisessential. WithintheFMPwehave libraries, theinputofcomputationalchemistryandmole- For theplanningofbothsyntheticandgenericscreen subsequent chemicalrefinement. an initialscreencanbetakenasthestartingpointfora with lowormediumaffinity. Theprimaryhitsidentifiedin will beonthedetectionoffragment-basedinteractions In order to limit this library toamanageablesize, thefocus library availableorwithlittlestructuralinformationknown. tion iscreatedtotargetproteinswithouthavingafocused gned generic(base)screenlibrary. Thiscompoundcollec- ligand libraries,weareengagedintheset-upofadesi- Beyond ourownsyntheticeffortsdirectedatfocused or in-housescreens. red forsubsequentbioassayinginexternalcollaborations one proteinoragroupoftargetproteins.Theyareprepa- sed librariesarecomposedofpotentialligandstargeting screening librariesaredesignedandconstructed.Focu- On thebasisofdevelopedsyntheticmethods,focused parallel withmediumthroughput. developed. Thesynthesesinmostcasesareoperated ic chemistryandsolidphasesynthesisareapplied chemical libraries.Forthispurposecombinatorialorgan- synthetic andanalyticalmethodologytofurnishdesigned Therefore, thestartingpointofourworkiscreation tic methodsincludingproductisolationandanalysis. vided. Librarygenerationposesnovelchallengestosynthe- cules, denominatedaschemicallibraries,havetobepro- initial compounds,rationallydesignedcollectionsofmole- biological activitiesandthesubsequentoptimizationof synthesis methods.However, fortheidentificationofnew cal synthesis.Theycanbetargetedbyclassicalorganic Small moleculeligandsaregenerallyaccessiblebychemi- p specific proteins.Inaddition,proteinligandsarethe proteins ortheassignmentofanobservedfunctionsto over, ofnovel theyhavebeenefficientinthediscovery elucidate andmodulatethefunctionsofproteins;more- e Small moleculesarepowerfulbiologicaltoolsthatcanbe MOLECULES CHEMICAL BIOLOGYWITHSMALL Rademann Group Leader:Prof.Jörg M otential startingpointfordrugdevelopmentefforts. mployed asbioactiveproteinligands.Theyareusedto EDICINAL CHEMISTRY LPS, theserhamnolipidsdonot activatethecytokine similar waytobacteriallipopolysaccharides (LPS).Unlike pounds whichstimulatetheinnate immunesystemina interesting inthattheyarelow molecularweightcom- rhamnolipids hasbeenprepared.Thesemoleculesare anchor. Inthenextstep,alibraryofimmuno-stimulating yielding oligosaccharidesattachedtoahydrophobic concept hasbeenemployedforrepetitiveglycosylations phase.The carrier solution orattachedtothehydrophobic phase switching,eachreactionstepcanbecarriedoutin (HASP) -synthesis”.Thisconceptallowsforaflexible nated as“hydrophobicallyassistedswitchingphase synthetic strategyforlibraryproductionthatwasdenomi- For thepreparationofglycolipidswehavesetupanovel logical activitythroughmultivalentbinding. ligands lesspronetoproteolysisandincreasetheirbio- cial macromoleculesisexpectedtorenderthepeptide on. Themultivalentanddendriticstructureoftheseartifi- activity ofsuchdendriticconstructsisunderinvestigati- an endocytoticpathway. Currentlythespecificbiological ciently deliverpeptideligandsacrosscellmembranesvia mimetics. Thesecomplexconstructswereshowntoeffi- copies ofsmallmolecules,e.g.peptidechainsorpeptido- to 60kDa)wasfeasible,carryingacontrollednumberof facile preparationofhighmolecularweightdendrimers(up sible cross-linkingofbranchedpolyethyleneimine,the dendritic, multivalentpeptidestocelltargets.Via arever- has beenextendedtowardsthesynthesisanddeliveryof commercially availablecarriers.Theultraresinconcept higher yieldofproductspergramresincomparedto economical insolidphasesynthesisastheyallowamuch cycles andpolymerreagents.Thenewcarriersarevery developed andusedinthesynthesisofpeptides,hetero- were product, veryhighloadedresins(“ultraresins”) tion. Basedonpolyethyleneimines,amulti-tonindustrial carriers andpolymerreagentsforfocusedlibraryproduc- the lasttwoyearswasdevelopmentofnovelpolymer In thespecificprojects,centralpartofourworkover users. of librariestogetherwithscreeningresultstoexternal accompanied byadatabasetocommunicatethecontents accessible forscreening.Thelibraryconceptwillbe s collections inwhichsyntheticchemistsprimarilyfromout- screen libraryoflimitedsize,andspecializedcompound ing librarythatistoconsistoffocusedlibraries,ageneric n “ChemBioNet” andrepresenttheFMPonboardofthis logy, weco-initiatedthenationalcollaborationnetwork To strengthen FMP’s activitiesintheareaofchemicalbio- ide theFMPcandeposittheircompoundstomakethem etwork. TheFMPhasofferedtosetupacentralscreen-

89 Chemical Biology Synthetic methods Modelliung/ & analysis Structural biology

(Focussed) libraries

Screening concepts

FIGURE 1

excretion via the Toll-like receptors (Tlr-II and -IV) by a A future keystone of our research will be the development hitherto unknown mechanism. of novel screening concepts. In this area we have initiated a project on primary screen techniques together with The main focus of the synthetic efforts was in the area of the Screening Unit. Several collaborative projects within protease inhibitors. Stabilized phosphoranes were found the FMP and on-Campus have been started as well. to be an efficient tool for polymer-supported C-acylation reactions. With this concept, it was possible to construct Group members the central element of protease inhibitors, the isosteric core, directly on the solid support. As a consequence, all Dr. Samer Al-Gharabli substituents of peptide isoster inhibitors now can be intro- Dr. Ludmila Perepellichenko* duced and varied flexibly. This variability is a new oppor- Dr. Michael Barth tunity especially in respect to the central side chain, the Dr. Syed Tasadaque Ali Shah* P1-site, and thus could be employed to investigate the Dr. Steffen Weik effect of P1-site variations on specificity and selectivity of Jörg Bauer (Doctoral student) protease inhibitors. In a model study, conducted with the Adeeb El-Dashan (Doctoral student) plasmepsin II, the malaria-linked aspartyl protease of plas- Viviane Uryga-Polowy (Doctoral student)* modium falciparum, we discovered that P1-site mutations Franziska Meier (Student)* enhanced the affinity of norstatine inhibitors by the factor of 60 compared to the natural, i.e. substrate-derived side chain. Further protease projects yielded reversible inhibitors of the SARS-main protease (in cooperation with R. Hilgenfeld, Lübeck) and of industrial targets (patent filed with Boehringer Ingelheim Pharma in May 2004).

* part of period reported J CombChem6,340-349 mance insolidphasesynthesisofveryhigh-loadedresins. the mobility, thechemicalaccessibility, andtheperfor- investigation ofthechemicalcomposition,swelling, the cross-linkingofpolyethyleneimines.Comparative Press SanDiego,366-390 Enzymology 369,BuninBA,MoralesG(eds),Academic novel toolsindiversity-orientedsynthesis.In:Methods activated reactantsandreactiveintermediates:Powerful 2491-2494 Versatile C-C-CouplingReactions.AngewChemIntEd42, Acylanion Equivalent.LinkerReagentsforSmoothand Barth M,RademannJ Weik S,RademannJ Tetrahedron Lett44,5019-5023 te derivatives(including5pagessupplementalmaterial). employed intheparallelsynthesisoflabeledcarbohydra- (TAL) amines –highlyorthogonalconversionsofprimary Bauer J,RademannJ Rademann J Selected publications Jörg Rademann Merck Biosciences Jörg Rademann Boehringer IngelheimPharma Jörg Rademann „Reaktionskaskaden“ (Landesforschungsschwerpunkt) Land Baden-Württemberg Jörg Rademann ses“ Teilprojekt imGraduateCollege„ChemistryinInterpha- D Jörg Rademann (Ra895-3) „Diversitätsorientierte SynthesevonRhamnolipiden“ D Jörg Rademann sowie derenbiochemischeEvaluierung“(Ra895-2) dung inparallelenSynthesenvonProteaseinhibitoren „ Deutsche Forschungsgemeinschaft External funding Reaktive IntermediateinpolymerenGelen,ihreAnwen- eutsche Forschungsgemeinschaft eutsche Forschungsgemeinschaft (2003) Advancedpolymerreagentsbasedon (2003) APhosphoraneasSupported (2004) Tailoringon based Ultraresins (2003) Trimellitic anhydridelinker (FMP authorsinbold) A the combinatorialdecorationofmultivalentscaffolds. cross-linking ofhyperbranchedpolymers:Astrategyfor C very through thechemicalmodificationofproteins.Angew sium inPrint60,8703-8709 E mer-Supported ReductionsandOxidationswithIncreased Polymer ReagentsbasedonPolycationicUltragels.Poly- T. Mayer, MPIMartinsried U. Zähringer, K.Brandenburg,H.Heine,FZBorstel R. Hilgenfeld,UniversityofLübeck R. Brock,UniversityofTübingen G. Klebe,UniversityofMarburg Collaborations Bauer J,RademannJ Barth M Rademann J Barth M ride Preparation.JAmChemSoc127,7296-7297 and solution-phasereactionsemployedforoligosaccha- phase synthesis:theflexiblecombinationofsolid-phase fficiency. Tetrahedron –CombinatorialChemistrySympo- hem IntEd43,4554-4556 ngew ChemIntEd44,1560-1563 , FischerR,Brock , AliShahT, (2004) Organicproteinchemistry:drugdisco- Rademann J (2005) Hydrophobicallyassisted Rademann J (2004) HighLoading (2005) Reversible

91 Chemical Biology SCREENING UNIT tion of many functions, if the overexpressed domain con- Group Leader: Dr. Jens Peter von Kries tains multiple binding sites for other factors. Contribution of individual interactions cannot be determined from these SCREENING FOR BIOACTIVE SMALL experiments. Although some protein functions have been MOLECULES IN AN ACADEMIC SET UP - identified in the past, only a small part of the complex inter- CHEMICAL BIOLOGY INITIATIVE actions and functions of the proteins encoded in the human genome is known as of yet. Beside their medical potential as drugs which cure diseases, bioactive small molecules may also serve as power- Small molecules: new tools for analysis ful tools for analysis of complex biological systems. Syste- The functional characterization of the proteins encoded in matic screening of large compound libraries for bioactive the humane genome and those of a growing number of small molecules which serve as molecular „switches“ in model organisms present a big challenge for the biosci- biological networks may become a key technology in ences. Chemistry may become a driving force for solution, Chemical Biology. Therefore, the “Forschungsinstitut für as has been shown by recent publications presenting inhi- Molekulare Pharmakologie“ provides a Screening Unit for bitors of protein interactions and of enzymatic activities of academic research groups which has been functional proteins. Molecular “switches“ may be generated by che- since autumn 2004. Furthermore, the institute supplies the mical synthesis which modulate the activity of proteins or central compound collection of the German Initiative for genes. The combination of biological and chemical Chemical Biology: ChemBioNet. methods may allow the investigation of the function of proteins which have, until now, defied functional analysis. Biological networks Small bioactive molecules often bind in hydrophobic pock- About 30,000 genes determine the development and stabi- ets of the surface of proteins. This hydrophobic character lity of a human organism. They code for proteins of the also favors crossing of cellular membranes. As small protein synthesis machinery, ion channels, receptors, molecules extend on only small regions of a protein sur- kinases, adaptor- or scaffolding proteins of cellular com- face, they potentially inhibit only individual and not all munication pathways, cell adhesion molecules or proteins functions of a protein. Therefore small molecules offer an of the immune system. Their correct function is maintai- ideal tool for identification of individual contribution of ned by complex interactions including enzymatic modifi- interactions towards function of multiprotein complexes, cations, like phosphorylation or proteolytic processing. For i.e. in biological signaling networks. Furthermore, selec- instance, adaptor proteins may serve as scaffolds after tive interference with protein binding or enzyme function modification by phosphorylation which then in turn recruit allows the comparison with the consequence of loss of enzymes and their corresponding substrates for further specific functions in human diseases. Moreover, the data signal transduction. collection and analyses of interactions of small molecules Comparative genomics has shown that evolution does not with proteins provides insights into substrate recognition solely correspond to a growing number of genes, but cor- mechanisms and will allow more effective de novo design responds to an increase in the complex combination of of agonists or antagonists in the future. regulatory modules in multifunctional proteins or genes. Perturbation of these complex interaction networks, i.e. by Small molecules and structural biology mutation, viral infection or environmental influences, may Small molecules, which resemble natural substrates of result in diseases like cancer, cardiac disease or diabetes. enzymes (like ATP), but cannot be processed, fix dynamic structures of enzymes for x-ray analysis of crystals. They Classical methods for analysis allow the analysis of the active state of an enzyme. This is Many proteins have been positioned into pathways using an important feature as many enzymes in cancer become classical genetic methods, which are based on loss of permanent active after mutation. Therefore, this fixed function by mutation and on complementation studies to structure provides an ideal starting point for the design of define for example the position of a given protein in a path- inhibitors. Alternatively, this information has been used to way. Biochemical in vitro assays measure enzyme activ- design an enzyme-specific cosubstrate (ATP) which is only ities or characterize binding domains of proteins. Methods recognized by a specifically mutated enzyme for labeling in modern molecular biology comprise overexpression of its specific downstream target proteins. In general, experiments of wild-type or mutant proteins in cell cultu- small molecules may help to stabilize protein structures, re. Many of those strategies result in deletion of all func- which cannot be structurally analyzed in their absence. tions of a given protein (antisense) or result in perturba- function ofthehumangenome. molecules presentanidealtoolforanalysisofthecomplex targeting ofindividualinteractions.Insummarysmall even discriminatebetweenfamilymembersandallowthe ce. Therefore,aftermodificationsmallmoleculesmay hot spotsshowsignificantvariationinaminoacidsequen- ly interfereinthesamemanner. Inprotein families these the proteins,smallmolecules,whichbindthere,potential- regions (pointmutants)abrogatethecomplexformationof As substitutionsofsingleaminoacidresiduesinthese surface. Thisregionisdefinedasahotspotofinteraction. contact pointsclusterinasmallregionoftheinteraction of manyinteractionsurfacesdemonstratethatessential tely blockedbysmallmolecules.Butmutagenesisstudies extensive interactionsurfaces,whichcannotbecomple- cause forproblemstargetingproteininteractionsarethe because ithasbeenshowntobeextremelydifficult.One Inhibition ofproteininteractionsisnotafavoredproject, ities fordrugdevelopment. Pharmaceutical companiesprefertoinhibitenzymeactiv- Small moleculesandproteinfamilies inless Screening robotssupporttoscreenthe20.000compoundsofFMPlibrary thanonereach FIGURE 1 institutions inclosecontactwith industrialpartners(for bining theindividualscreeninginitiatives ofmanyresearch BioNet, iscurrentlyorganizingan academicnetworkcom- The GermanInitiativeforChemical Biology, namedChem- ties forChemicalBiology ChemBioNet: chemistryandscreeningfacili- catalytic sites. docking ofvirtuallibrariesintopocketshotspotsor faces willbeanalyzedandusedforcomputer-aided pounds tobeorderedforeachproject.Theproteinsur- (3). Useofvirtualdrugscreeningfortheselectioncom- library characterizesthehotspotsforinteractions mutated peptidesisoffered.Thescreeningofthispeptide derived fromtheminimalbindingdomains,containingper- (2) Paralleltothis,aspotsynthesisofpeptidelibraries readers tosupportmoststandardscreeningtechniques. robot andautomaticdispensers,washersdifferent Screening Unitiswellequippedwithaliquidhandling or fluorescencetechniques.Thescreeninglabofthe (1) Screeningwithmoderate“HTS”usingstandardELISA Three mainstrategiesofscreeningaresupported: Concept oftheScreeningUnit

93 Chemical Biology detailed information see: www.chembionet.de) for support Current academic projects: Mycobacterium of academic screening projects. This screening network tuberculosis enzyme inhibitor screens will also build up a central shared compound library at the Infection with Mycobacterium tuberculosis, the causative FMP and a database of combined bioactivity profiles and bacterium of human tuberculosis, results predominantly in chemical data of compounds (see www.chembionet.de an asymptomatic persistent infection, often referred to as page “Datenbank”). The network will be supported by che- latency. Infected individuals are at risk over their lifetime mistry for the modification of molecules and by the exper- to convert their asymptomatic infection into what is called tise of the screening centers for the setup of robust reactivation tuberculosis, a disease state both potentially screening assays. The research projects will identify and fatal and highly contagious. The long persistence of the use bioactive compounds for functional analysis of bio- bacteria in the host during asymptomatic infection in the logical networks in development or in disease situations face of a robust host immune response poses fundamen- like cancer, cardiac disease, autoimmune reactions or tal biological questions. The bacteria could be in a nonre- others. Beside the established role of small molecules for plicative, metabolically and transcriptionally inactive and identification and characterization of protein functions, dormant state. Recent molecular genetic approaches have these compounds may serve for the development of new yielded Mycobacterium proteins and lipids important for drugs for interference with human diseases. virulence and persistence. Hence, it can be argued that the bacteria manipulate the host immune response to Competitor or partner of pharmaceutical com- ensure their persistence. panies? It is estimated that nearly 2 billion people currently suffer Screening in an academic setup has the advantage that from latent Mycobacterium tuberculosis infection. Al- no obvious disease relevance is a prerequisite for usage of though the key front-line antituberculosis drugs are effec- small molecules for interference and characterization of tive in treating individuals with acute tuberculosis, these biological functions. A specific inhibitor of an individual drugs are ineffective in eliminating M. tuberculosis during protein function is a valuable tool, even when it “only” pro- the persistent stages of latent infection. Consequently, vides a molecular switch for characterization of a functi- therapeutics that directly target persistent bacilli are on, without any perspective for serving as a new drug. This urgently needed. independence from commercial aspects allows free collection of chemical and biological data of bioactive mole- The “Structural Proteomics Consortium” selected proteins cules. These data are not limited to a few types of enzy- from Mycobacterium tuberculosis on the basis of gene mes as they include a much broader panel of targets. ablation experiments (MPI für Infektionsbiologie, Berlin) Therefore this database may provide important clues resulting in reduced infectiveness. The structures of towards the design of new classes of bioactive molecules. selected proteins are solved at DESY in Hamburg (EMBL and MPG research groups) and screened for small mole- Another advantage of the independence from commerci- cule inhibitors at the Screening Unit in Berlin and by Com- al aspects is the chance to identify and characterize the binature (NMR-screening, Berlin). mode of action of potential drugs years before the disease relevance becomes established. At this special point Two primary screens using the 3-isopropylmalate dehydro- the interests of pharmaceutical companies and academic genase (IPMDH, enzyme for biosynthesis of branched Screening Units could partially overlap, since academic chain amino acids) and sterol 14?-demethylase (CYP51) institutions do not have the resources for successful drug resulted in identification of inhibitors with IC50-values in development, and pharmaceutical companies do not want the micromolar range (Rajesh et al. 2005). Two structural to focus on protein interactions as targets. Pharmaceuti- classes of inhibitors for CYP51 resemble already published cal companies could get rights of privileged usage of compounds as estriol (IC50: 100 µM) or 4-phenylimidazole patents and sponsor the academic units with know how or (IC50: 1 mM) demonstrating the reliability of this screen be. money for library enlargement. In summary, this partner- One of the CYP51 antagonists inhibits growth of tuberculo- ship would benefit people suffering from a disease for sis bacteria in human macrophages (Stefan HE Kaufmann, which no drug is available yet. MPI-IB, Berlin). Therefore this compound may be used for development of new drugs against tuberculosis infections. The novel compound classes identified will be cocrystal- lized to analyze substrate recognition mechanisms in the catalytic site of the enzyme. The IPMDH inhibitors have been identified in a dual approach. First, a compound libra- screened andinhibitorsofIPMDHidentified(IC F ** part-time * partofperiodreported W. Birchmeier, MDC,Berlin Hans Bartunik,MPG,Hamburg Manfred S.Weiss, EMBL,Hamburg Matthias Willmanns,EMBL,Hamburg Collaborations Christoph Erdmann(Technical assistance)* DC AngelikaEhrlich** Group members affinity ofallinhibitorsidentified(IC enzyme assay. Onecompounddemonstratedthehighest number ofhydrogenbondsinthesiteandtested s and defaultlibrary screening high- settings.From30virtual tic siteoftheenzymeusingcomputerprogramGOLD ry ofabout37,000compoundswasdockedintothecataly- mik Konsortium”). (0312992J, “MycobacteriumtuberculosisStrukturproteo- The ScreeningUnitissupportedbyagrantoftheBMBF pharmaceutical companies. of drugsagainsttuberculosisinfectionsinpartnershipwith compounds couldpotentiallybeofuseinthedevelopment sis toescapefromimmunedefence.Besidethisresult,the the biologicalmechanismsofMycobacteriumtuberculo- identification ofsmallmoleculeswhichenabletoidentify ness oftreatedbacteria.Theseprojectsmayresultinthe for analysisinmodelsystemswhichmeasureinfective- p asaninputfororderingrelatedcom- structures serve respective enzymeshavebeenalreadystarted.Their µM). Cocrystallizationexperimentsofinhibitorswith core compounds,10wereselectedduetotheproposed MP smallmoleculelibraryof20,000compoundswas ound structurestoidentifyinhibitorswithhigheraffinity 5 0 : 35the Second, µM). 50 : 75-250

95 Chemical Biology

SCIENTIFIC AND TECHNICAL SERVICES MICRODIALYSIS enzyme (IDE; insulysin) and angiotensin-converting enzy- Group Leader: Dr. Regina M. Richter me (ACE) have recently been identified to degrade extracellular Aβ, the proteolytic pathways are less well under- DIRECT MEASUREMENT OF IN VIVO stood. In particular, in vivo studies are rare and differ PROCESSING OF BRAIN NEUROPEPTIDES evidently from in vitro reports (for review Hersh, Curr USING MICRODIALYSIS Pharm Des 9, 449, 2003). Reverse microdialysis in combination with advanced mass spectrometric techniques is used Elevated cerebral levels of a variety of neuropeptides are to study the clearance of these neuropetides in the brain believed to be risk factors for the development of diseases of rodents (Figure 1). that are related to the stress syndrome, cardiovascular Using these methods, we have recently demonstrated the dysfunction and, ultimately to neurodegenerative proces- pathways of in vivo processing of several A species by ses such as Alzheimer’s disease (AD). Specifically, exces- β NEP using specific protease inhibitors. Current studies sive accumulation and deposition of -amyloid peptides β focus on the interplay of structural parameters and protea- (A ) form senile plaques in AD brains which are conside- β se activity as well as on the contribution of other candida- red to be one hallmark of the disease. Emerging evidence te proteases to the regulation of brain A levels using rele- suggests that A accumulation is not in all AD cases asso- β β vant protease knockout and overexpressing mouse lines. ciated with increased A production. Impaired clearance β Another project explores whether astrocytes could have a of A is, therefore, considered to be a critical factor in the β direct role in A degradation either by A -induced activa- development of AD pathology. However, the mechanisms β β tion or chemotactic migration to A deposits (Wyss-Coray of the in vivo clearance of these critical neuropeptides are β et al., Nature Med 9: 453-456, 2003). Astrocyte motility was still not understood, and elimination of A from tissues and β demonstrated to be controlled by the ryanodine type 3 plasma by activation of degrading proteases appears to receptor (RyR3) (Matyash et al., FASEB J 16: 84-86, 2002). have considerable therapeutic potential. Thus, the major These findings encouraged us to test the hypothesis that focus of research in our laboratory is to understand the extracelluar A degradation is reduced in RyR3 knockout proteolytic mechanism of in vivo A degradation. β β mice associated with impaired astrocyte motility (in collaboration with H. Kettenmann, MDC, Berlin). Finally, studies are For all investigated peptides we identified primary cleava- underway directed at understanding the principles of exten- ge sites and involved proteases by using specific protea- sive amyloid deposition in cerebral vessels leading to cere- se inhibitors. The impact of structural parameters on A β bral amyloid angiopathy. While chronically reduced micro- clearance was explored in greater detail by using double circulation is believed to impair the clearance of circulating D-amino acid replacement sets and N-C inverted peptides. A , the reported vasoconstrictor action of circulating A In addition, other factors such as astrocytes which may β β (Niwa et al., Am. J. Physiol 281, H2417, 2001) is still contro- have a direct role in A degradation have been studied. To β versial. In order to address these questions, we have initia- address specific questions of peptide processing, we ted collaborative work with M.J. Mulvany, University of include transgenic and gene-targeted mouse lines that ei- Aarhus, Denmark, to explore the direct contractile effects ther overexpress or carry deletions of genes of interest. of A in microvessels, as well as the possibility that the Our experimental tools comprise the use of reverse micro- β resulting hypoperfusion results in accumulation of A . dialysis both to introduce neuropeptides or inhibitors into β the brain of awake rodents, as well as to collect metabo- A second area of research concerns the generation of bio- lic fragments. In collaborative work, advanced mass spec- active angiotensin peptides from angiotensin I. In particu- trometric techniques are applied to monitor the clearance lar, angiotensin (1-7) [Ang-(1-7)] is thought to mimic and of these neuropeptides close to real-time in dialysates of oppose the multiple actions of angiotensin II. Both the role a few microliters volume. Further efforts are directed of substrate availability and the metabolic pathways yield- towards quantitative analysis of the peptide fragments. ing to the formation of Ang-(1-7) remain unclear. Using protease knockout mouse models and protease inhibitors, Work report: Major objectives of the laboratory we have elucidated the major pathways of Ang-(1-7) for- Normally soluble β-amyloid peptides (Aβ), generated by mation including the involved proteases in vivo. proteolytic cleavage of the β-amyloid precursor protein (APP), is a mainly 40-42 amino acid species which may Main objective 1. Impact of structural parameters on Aβ extend to Aβ residue 43/46 in neuritic plaques (Zhao et al., processing JBC 49, 50647, 2004). Although multiple proteases such as Little is known about the impact of structural parameters neutral endopeptidase (NEP; neprilysin), insulin-degrading on the catabolic pathways of Aβ peptides. Therefore, we % Intensity % Intensity 100 100 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 9. 59425. 10232. 4701.0 3920.6 3140.2 2359.8 1579.4 799.0 9. 59425. 10232. 4701.0 3920.6 3140.2 2359.8 1579.4 799.0 0 0 NEP knockoutmouse Mass (m/z) Mass (m/z) Wildtype mouse 5.3E+4 4.6E+4 charge ratio. given inarbitaryunits;m/z=mass-to- low flowrate.Relativesignalintensityis the hippocampusofanimalsatavery (bottom). Thepeptidewasinfusedinto wildtype (upper)andNEPknockoutmice mouse. Cerebral microdialysisinafreelymoving FIGURE 1 A Comparison ofMALDImassspectra FIGURE 2 β (1-40) peptidefragmentsobtainedfrom

99 Scientific and Technical Services tested the hypothesis that both the sequence of amino rous mass spectra displayed an additional cleavage site at acids and the secondary structure may play a substantial position 34/35 (Leu-Met) attributed to a matrix metallopro- role in the processing of Aβ. Thus, two N-C inverted Aβ teinase-9 (MMP-9) which is believed to prevent plaque chains, (42-1) and (40-1), and a double D-amino acid repla- formation (Backstrom et al., J Neurosci 16:7910, 1996). cement set of A were involved in the paradigm to study β To study the particular role of IDE in A degradation, we their proteolytic cleavage pattern. The two inverted A β β extended our studies to awake NEP knockout versus wild- species serve as inactive controls because of lacking neu- type mice. The in vivo profile of hippocampal A proces- rotoxic properties in cell cultures (Simmons et al., Mol β sing revealed cleavage patterns which match well to in Pharmacol 45, 373, 1994). In contrast to regular A , the β vitro findings from Mukherjee and colleagues (J Neurosci inverted A species were cleaved to only a few short C- β 20:8745-8749, 2000) in so far as short N-terminal fragments terminal fragments at the peptide bond His27-His28, Leu24- (1-11 to 1-16) dominated in mass spectra while the C-ter- His25 and Phe22-Val23 in the (40-1) chain (Richter & Kraus, minal region remained intact (Figure 2) (Richter et al., Soc Soc Neurosci Abstr # 409.3, 2003). The cleavage site at His- Neurosci Abstr # 220.6, 2004). These results provide strong His is considered to be a preferred target of IDE, whereas evidence that the major proteases NEP and IDE contribu- cleavage sites at Phe-Phe and in the N-terminal region of te to the clearance of A by cleavage of differing peptide the N-C inverted molecule are thought to be caused by β bonds in distinct regions within the peptide chain. NEP. The lack of cleavage sites caused by NEP suggests that only IDE but not NEP was involved in the degradation Group members of Aβ under these experimental conditions. Antje Muschter (Technical assistance)* ** Systematic double D-amino acid replacement of adjacent Christian Wolff (Technical assistance)** amino acids within the Aβ(1-42) molecule was used to study the impact of the secondary structure on A clearance. In β External funding contrast to former reports, more recent studies revealed that double D-amino acid substitution efficiently inhibited Schering AG-Ri1; Regina Richter the peptide degradation primarily around the replaced amino acids. In aCSF, a complete protection of the molecu- Selected publications (FMP authors in bold) le against enzymatic degradation indicating a folding of the Richter RM, Kraus M (2003). Profile and Inhibition of the in molecule was not found. These results are strongly suppor- vivo Degradation of Alzheimer’s -Amyloid Peptides in Rat ted by findings obtained with A dissolved in buffer and β β Brain. Soc Neurosci Abstr # 409.3 subsequently analyzed with CD and ITC methods (S. Keller, unpublished data). These results suggest that the Richter RM, Heyne A, Schuchardt S (2004). Degradation of proteolytic clearance of Aβ peptides in vivo depends highly Amyloid β-Peptide in the Hippocampus of a NEP knockout on both the primary and secondary structure of the mole- Mouse Model. Soc Neurosci Abstr # 220.6 cule and differs significantly from that ex vivo. Collaborations Main objective 2. Proteases involved in Aβ clearance First, we explored the two principal regulators of Aβ clea- Hersh, L.B. (Dept. Molec. and Cellular Biochemistry, Uni- rance: NEP and IDE. The postulated primary cleavage site versity of Kentucky, Lexington, KY, USA) at positions 33/44 (Gly-Leu) and the N-terminal directed Degradation of β-amyloid peptides in transgenic and ladder-like degradation in rat hippocampus were success- knockout mouse models fully blocked by the NEP inhibitors phosphoramidon and Kettenmann, H. (Dept. Cellular Neuroscience, MDC for thiorphan, suggesting a major role of this particular pro- Molecular Medicine, Berlin, Germany) tease in A clearance. However, compared to the litera- β Clearance of -amyloid peptides in the brain of RyR3- ture our in vivo findings indicate additional metabolic path- β knockout mice ways of Aβ. For example, the intermediate fragment (10-37), identified as the major product of Aβ(1-42) clea- Mulvany, M.J. (Dept. Pharmacology, Aarhus University, vage by NEP (Iwata et al., Nature Med 6, 143, 2000), was Aarhus, Denmark) not detected in our close to real-time experiments. More- Cerebral hypoperfusion and beta-amyloid plaque formation over, separate metabolic studies revealed a very rapid Schuchardt, S. (PLANTON GmbH), Kiel degradation of the fragment which does not correspond to MALDI-TOF and MALDI-MS/MS analysis of the in vivo the A (10-37) accumulation in catabolic studies on radio- β metabolism of neuropeptides labeled Aβ reported by Iwata and collegues. Also, nume- * part of period reported ** part-time Barbara Mohs(Technical assistance)** Group members from congenitalnephrogenicdiabetesinsipidus(NDI). receptor genesofpatients(andtheirfamilies)suffering work onthemolecularanalysisofvasopressinV2 In additiontotheservicefunction,groupcontinues occasional samplesfromothergroups. (Vinkemeier)Group CellularSignalProcessing 30%,with submits about60%ofthesamples,JuniorResearch Medicine Department ofSignalTransduction/Molecular At presentabout100samplesareprocessedweekly. The 310). automated sequencersareavailable(ABI377andABI It comprisesascientistandtechnician(part-time);two sequencing forthemolecularresearchgroupsofFMP. The DNASequencingServiceGroupconducts Group Leader:Dr. ErhardKlauschenz DNA SEQUENCING ** part-time Renate Peters Academic library Petra Göritz Eva Lojek Dr. ReginaM.Richter Animal housing Uwe Hackel† Central glasswarewashingfacility Dr. Hans-UlrichHeyne Safety Officer Stefanie Wendt Helmut Blick Holger Panzer Michael Uschner Jürgen Mevert Scientific workshop ADDITIONAL SERVICES

101 Scientific and Technical Services Visit of the Federal President on Berlin-Buch Campus 2004 Besuch des Bundespräsidenten auf dem Campus Berlin-Buch 2004

Hans-Olaf Henkel, President of the Leibniz Association, during the inauguration of the 900 Mhz spectrometer Hans-Olaf Henkel, Präsident der Leibniz-Gemeinschaft, zur Einweihung des 900 MHz-Spectrometers

PUBLIC RELATIONS AND THE MEDIA In April 2004 the FMP, together with ten other scientific Dr. Björn Maul institutions from Berlin and the German Human Genome Project (DHGP), celebrated the fiftieth anniversary of the The Forschungsinstitut für Molekulare Pharmakologie discovery of the DNA helix structure with an exhibition in (FMP) uses public funds to conduct the major part of its the Berlin Museum of Natural History (Naturkundemu- research into the basis of the effects of substances on the seum) that received striking attention. About 40,000 people organism. In order to give the taxpayer, who provides the paid a visit to the exhibition, which was the only one of its funds, some insight into how these resources are utilized, kind in all of Germany. the FMP presents its work to the general public in an easi- The FMP constantly strives to inspire enthusiasm for ly understood form. scientific research in young people of school age. Scien- In 2003 and 2004 the FMP successfully participated in tists regularly visit schools, presenting easily understood various public exhibitions and presentations. Scientists lectures to promote discussion with pupils and teaching from the Institute used exhibits of interest to the layperson staff. In addition, the FMP once a year awards a practical to introduce their fields of research at the Science Fair of course in the Life Science Learning Lab of the Berlin-Buch the Free University Berlin, at the “Müncher Wissen- Campus to a winner of one of the country-wide competiti- schaftstage”, and at the presentation "Window on Sci- ons in "Youth in Research.“ In 2004, the college student ence" held in the arcades at Potsdamer Platz. Sylke Höhne from Chemnitz won the prize and was hosted by the FMP in Berlin. The FMP also participated in the exhibition "Swimming lab (MS Chemie)" held on a ship in summer of the Year of Che- In each of the two reporting years, the FMP took the mistry, 2003. The ship, which was initiated by the science- opportunity to present itself to the general public during to-public platform “Wissenschaft im Dialog”, moored in 25 the Berlin Long Night of the Sciences. At this event, more cities along the Rhine River and attracted more than 40,000 than 1000 visitors came to isolate their own DNA from their visitors. als 40,000Menschenbesucht. lief 25StädteentlangdesRheins anundwurdevonmehr senschaft imDialog”initiierte,schwimmende Ausstellung Sommer desJahresderChemie2003. Diesevonder„Wis- Das FMPbeteiligtesichamChemieschiff(MSChemie)im Wissenschaftstagen vor. in denPotsdamer-Platz-Arkaden undaufdenMünchner lin, aufderPräsentation„SchaufensterWissenschaft“ Exponaten aufderScienceFairFreienUniversitätBer- ihre ForschungsgebieteanhandvonfürLaieneingängigen und Präsentationen.WissenschaftlerdesInstitutsstellten erfolgreich anverschiedenenöffentlichenAusstellungen In denJahren2003und2004beteiligtesichdasFMP Öffentlichkeit vor. seine ArbeitinleichtverständlicherFormderbreiten wofür dieseMittelverwendetwerden,stelltdasFMP Steuerzahler, derdieGelderaufbringt,Einblickzugeben, deröffentlichenHand.Umdem Organismus mitGeldern über dieGrundlagenderWirkungvonStoffenaufden (FMP) betreibtdenüberwiegendenTeil seinerForschung Das ForschungsinstitutfürMolekularePharmakologie Dr. BjörnMaul PRESSE- UNDÖFFENTLICHKEITSARBEIT vortrag S W chaufenster derW indow onscience,PotsdamerPlatzinBerlin:Openinglecture i ssenschaft amPotsdamerPlatz2003:Eröffnungs- o Exhibition “50thanniversar Ausstellung „50JahreDoppelhelix“imNaturkundemuseum machkurs „MeineDNA“zuabsolvieren unddieeigene Veranstaltung andasFMP, insbesondereumdenMit- ren. Insgesamtkamenmehrals1000 Menschenzudieser Wissenschaften einerbreitenÖffentlichkeit zupräsentie- Gelegenheit wahr, sichinderBerlinerLangen Nachtder In jedemderbeidenBerichtsjahrenahmdasFMPdie Gast desFMP. Höhne ausChemnitzdenPreisundwareineWoche lang bewerb derAktion.In2004erhieltdieAbiturientinSylke des CampusalsPreisfüreinenausgewähltenLandeswett- einmal imJahreinenPraktikumskursGläsernenLabor unterstützt denWettbewerb „Jugendforscht“.Esvergibt gen derDiskussionmitSchülernundLehrern.DasInstitut len undstellensichdortmitleichtverständlichenVorträ- begeistern. RegelmäßigbesuchenWissenschaftlerSchu- im SchulalterfürdienaturwissenschaftlicheForschungzu Das FMPversuchtkonsequent,jungeMenschenbereits lung, dieeinzigeihrerArtinDeutschlandwar. museum. Etwa50,000MenschenbesichtigtendieAustel- mit einerSonderausstellungimBerlinerNaturkunde- (DHGP) den50.JahrestagderEntdeckungDNA-Helix Brandenburg unddemDeutschenHumangenom-Projekt teren wissenschaftlichenEinrichtungenausBerlinund Im April2004begingdasFMPzusammenmitzehnwei- f NaturalHistor y y oftheDNAhelixstructure”inmuseum

103 Scientific and Technical Services “My DNA“ – The Long Night of the Sciences 2003 at the FMP Exhibition “Swimming lab“ 2003 „Meine DNA“ – Lange Nacht der Wissenschaften 2003 am FMP Ausstellung auf der MS Chemie 2003

saliva. This special offer was first made in 2002 and beca- the Berlin Buch Campus. The public relations consultant me a real attraction over the years. supported telecasts like “nano” (3sat), “Quarks & Co.” (West German Broadcast WDR), and “ZDF-Expeditionen” The FMP greeted delegations from Germany and abroad (ZDF) as an adviser and by providing broadcasting and introduced the participants – government delegations, material. journalists, students and school pupils – to its work and that of the entire Campus. In collaboration with BBB Cam- The FMP gave independent presentations at the Parlia- pus Management GmbH, the directorate consultants brief- mentary Evenings of the Leibniz Association in Brussels in ed the numerous visitors on content and organization. 2003 and the Forschungsverbund Berlin e.V. (Berlin With the cooperation of Institute scientists, short lectures, Research Association) in Berlin in 2004. The Institute tours of the laboratories and discussions were available. brought the research of its scientists to the attention of the international biotech community with a presentation at the To give the media the opportunity to report significant international convention BIO 2004. events at the FMP, press releases were researched, writ- ten and issued. The public relations consultant as well as Personnel scientists gave interviews, e.g. for Deutschlandfunk, the Hessian and Berlin broadcasters HR and RBB, as well as Ulrike Lauterjung (Assistant) Radio Eins, Radio Multi-Kulti, Korea TV, and the audio:link Internet radio station. The FMP has been reported in the major Berlin daily newspapers. The national as well as the regional press have published reports on FMP scientists and their research. FMP consultants and scientists have made several contributions to brochures, various periodi- cals, e.g. for the Leibniz Association, the Berlin Research Association, the German Human Genome Project and for und regionalenPresseberichtet, soauchinArtikelnwich- schaftler undihreForschungwurde inderüberregionalen net-Radiostation. ÜberdasFMP unddieFMP-Wissen- Eins, RadioMulti-Kulti,KoreaTVunddieaudio:linkInter- (HR), denRundfunkinBerlin-Brandenburg(RBB),Radio B.fürdenDeutschlandfunk,HessischenRundfunk z. für ÖffentlichkeitsarbeitvermittelteundgabInterviews, DerReferent gen recherchiert,verfasstundveröffentlicht. ge EreignisseamFMPzugeben,wurdenPressemitteilun- Um denMedienAnlasszurBerichterstattungüberwichti- Labore undGesprächeangeboten. des HauseswurdenkurzeVorträge, Führungendurchdie organisatorisch vor. ZusammenmitdenWissenschaftlern nagement GmbH,diezahlreichenBesucheinhaltlichund bereitete, oftinZusammenarbeitmitderBBBCampusma- Arbeit desgesamtenCampusbekannt.DasDirektorat Journalisten, StudentenundSchüler–mitseinerder und machtedieTeilnehmer –Regierungsdelegationen, Das FMPempfingDelegationenausdemIn-undAusland hat sichzueinerechtenAttraktionentwickelt. dere AngebotwurdeimJahr2002vomFMPinitiiertund DNA auseinerSpeichelprobezuisolieren.Diesesbeson- P Parliamentar arlamentarischer AbenddesForschungsverbundsBerlin2004 y EveningoftheForschungsverbundBerlin2004 S Science Fair2004 2004 aufmerksam. ner WissenschaftlermiteinerPräsentationaufderBIO internationale Biotech-CommunityaufdieForschungsei- V. inBerlin2004.DasInstitutmachtedie bunds Berline. Gemeinschaft inBrüssel2003unddesForschungsver- nen andenParlamentarischenAbendenderLeibniz- Das FMPbeteiligtesichmiteigenständigenPräsentatio- Material. Expeditionen” (ZDF)alsBeraterundmitaudiovisuellem „Quarks &Co.”(Westdeutscher Rundfunk)und „ZDF- schiedene Fernsehpoduktionen,wie„nano”(3sat), Der ReferentfürÖffentlichkeitsarbeitunterstütztever- Berlin-Buch. des DeutschenHumangenomprojektsundCampus V., niz-Gemeinschaft, desForschungsverbundsBerline. B.derLeib- für Broschüren undverschiedenePeriodika,z. renten undWissenschaftlerdesFMPverfasstenBeiträge tiger Tageszeitungen derBerlinerPresselandschaft.Refe- cience Fair2004

105 Scientific and Technical Services ADMINISTRATION The cost and performance accounting system (CPA) with Head: Thomas Ellermann its two hierarchical levels, which was implemented in 2002 and has been operative since 2003, was the prerequisite In addition to the Administrative Director, the FMP admini- for the introduction of the program budget. In the project strative staff comprises six employees (three full-time and group established by the Berlin Research Association three part-time). After the positive conclusion of the last (Forschungsverbund Berlin e.V., FVB) the FMP assumed in training cycle, the FMP has decided to take on a new 2004 the role of pilot institute for all institutes of the FVB. office communication trainee. Following the directives of the Joint Federal-State Com- mission on “minimum requirements on program budgets”, A main focus of activity of the administration during the a standard procedure for the fiscal part of the program reporting period was the preparation for the conversion budget could be developed for the institutes of the FVB. from the cameralistic system of accounting to a cost and Initially, with the data from the CPA from the year 2004, a performance accounting system (CPA) for the institute. program budget for the budget year 2006 was prepared. This CPA system will first be applied to the 2006 budget Parallel to that, a financial plan was created in coordina- year. tion with the State of Berlin as funding allocator. The Joint Federal-State Commission for Education Plan- At the request of FMP scientists, an electronic ordering ning and Research Promotion demands that the instituti- system was introduced. A prerequisite for that was the ons of the Leibniz Association introduce program budgets successful implementation of SAP version 4.6. Research at the latest for the budget year 2006. The basis for this is staff members can now submit their purchase requests in the decision of the heads of the federal and state govern- digital form via the intranet of the institute. After verifica- ments from autumn 1997 on “Securing and Quality of tion of the entered data, data relevant to the transaction is Research”.

VERWALTUNG Die im FMP 2002 implementierte und seit 2003 voll produk- Leiter: Thomas Ellermann tive Kosten- und Leistungsrechnung (KLR) mit ihren zwei Hierarchieebenen war die Voraussetzung für die Einfüh- In der Verwaltung des FMP sind neben dem Verwaltungs- rung des Programmbudgets. In der vom Forschungsver- leiter sechs Mitarbeiterinnen beschäftigt (drei Vollzeit und bund Berlin e. V. eingerichteten Projektgruppe hatte das drei Teilzeit) beschäftigt. Nach dem positiven Abschluss FMP 2004 die Rolle des Musterinstituts für alle Einrichtun- des letzten Ausbildungszyklus hat sich das FMP außerdem gen des FVB übernommen. Orientiert an den Vorgaben der entschlossen, erneut eine Bürokommunikations-Kauffrau BLK zu den „Mindestanforderungen an Programmbud- auszubilden. gets“ konnte ein Standardverfahren für den finanzwirt- schaftlichen Teil des Programmbudgets für die Institute Ein Tätigkeitsschwerpunkt der Verwaltung im Berichtszeit- des FVB erarbeitet werden. Zunächst wurde mit Daten der raum war die Vorbereitung zur Umstellung von der Fehlbe- KLR vom Jahr 2004 ein Programmbudget für das Haus- darfsfinanzierung zu einer leistungs- und ergebnisorien- haltsjahr 2006 aufgestellt. Parallel wurde in Abstimmung tierten Finanzierung des Instituts. Die leistungs- und mit dem Zuwendungsgeber Land Berlin ein Wirtschafts- ergebnisorientierte Finanzierung ist erstmalig auf das plan erstellt. Haushaltsjahr 2006 anzuwenden. Auf Wunsch der Wissenschaftler des FMP wurde die elek- Die Bund-Länder-Kommission für Bildungsplanung und tronische Bestellübermittlung eingeführt. Voraussetzung Forschungsförderung (BLK) fordert für die Einrichtungen dafür war die erfolgreiche Implementierung der SAP-Ver- der Leibniz Gemeinschaft die Einführung eines Programm- sion 4.6. Wissenschaftlich Beschäftigte haben jetzt die budgets spätestens zum Haushaltsjahr 2006. Grundlage ist Möglichkeit, ihre Beschaffungsanträge in digitaler Form der Beschluss der Regierungschefs des Bundes und der im Intranet des Instituts zu hinterlegen. Softwareseitig Länder vom Herbst 1997 zur „Sicherung und Qualität der können buchungsrelevante Daten nach Prüfung über eine Forschung“. Schnittstelle in eine „Bestellanforderung“ (BANF) des SAP-Systems übernommen werden. Der Buchungsvor- mit 200m sen. Indem2003neuerrichteteLabor-Gebäude „NMRII“ begonnen undzumTeil imBerichtszeitraumabgeschlos- für MolekulareMedizinseit2002dreiBaumaßnahmen Das FMPhatgemeinsammitdemMax-Delbrück-Center tung vonVerbrauchsmitteln istsoamFMPnichtnötig. Wissenschaftler ausgeliefert.EineaufwendigeLagerhal- innerhalb von24StundennachderBestellungdurchden mittel werden,abhängigvomLieferanten,nichtselten lassen dasInstitutinderRegeltagesgenau.Verbrauchs- Chemikalienbezeichnung, zuvermeiden.Bestellungenver- zeitaufwendige Doppelerfassungen,beispielsweiseder Zugriff Dritterabgekoppelt.Zielistesfehlerträchtigeund gang derVerwaltung istbeidiesemVerfahren immer vom opened with200m covered bythereport.In2003newlabbuildingNMRII projects andhasinpartcompletedthemtheperiod Molecular Medicine,theFMPhasbegunthreebuilding S for costlywarehousingofexpendablematerialattheFMP. tist, dependingonthesupplier. Therefore, there isnoneed often deliveredwithin24hoursoftheorderbyscien- w Orders usuallyleavetheinstituteonsamedaythey ble entries,e.g.inconnectionwithchemicaldesignations. The aimistoavoiderror-prone andtime-consumingdou- t tion’s of transactionworkflowisseparatedfromtherest theadministra- thisprocedure, With se requestiscreated. transferred intoSAPviaaninterface,andpurcha- sich starkerhöhen. Anforderungen. DerMittelaufwand fürTierhaltung wird nächsten JahrenstelltdieVerwaltung desFMPvor große lichen UmgangsmitdiesenneuenRessourceninden samt 385m Haus“ Ende2004kanndasFMPneueTierlabore mitinsge- MDC erstelltenTierlaborgebäudes „Erwin-Negelein- Abschluss derBaumaßnahmedesgemeinsammitdem MHz-NMR-Spektrometer inBetriebgenommen.Nach 385 m 2004, theFMPhasnewanimallaboratorieswithatotalof laboratory buildingErwinNegeleinHouseattheendof After completion,togetherwiththeMDC,ofanimal 2004 the900MHzNMRspectrometerstartedoperation. he organizationandcannotbeaccessedbythirdparties. ince 2002,togetherwiththeMaxDelbrückCenterfor ere enteredusingtheintranet.Expendablematerialis 2 at itsdisposal.Organizinganeconomicallysound 2 vom FMPgenutzterFlächewurde2004das900 2 nutzen. DieOrganisationeineswirtschaft- 2 floor spaceusedbytheFMP, and in from the FMP will use the 1.094 m from theFMPwilluse1.094 open in2006.Fiveworkgroupswithatotalof40employees The newMedicinalGenomicsResearchBuildingisdueto for laboratoryanimalcarewillgreatlyincrease. sents greatchallengesfortheFMPadministration.Costs usage ofthesenewresourcesinthecomingyearspre- b to theinstitute.Innewlaboratorybuildingslabsare Josephine Passow(Trainee)* Dana Hausbeck(Trainee)* Gabriele Schumacher(Secretary) Kerstin Brauße(GeneralAdministration)** Claudia Messing(GeneralAdministration)** Christel Otto(GeneralAdministration) B Silvia Mauks(PersonnelManager) Personnel ** part-time * partofperiodreported chen mitaufwendigentechnischenAusstattungen. nutzen. IndenneuenLaborgebäudenentstehenLaborflä- dem InstitutzuzurechnendenFlächenanteilvon1.094m mit insgesamt40MitarbeiternausdemFMPwerdenden aussichtlich in2006bezogenwerden.FünfArbeitsgruppen Der Neubau„MedizinischeGenomforschung“wirdvor- eing fittedoutwithextensivetechnicalequipment. irgit Sperling (General Administration)* ** irgit Sperling(GeneralAdministration)* 2 of floorspaceallotted 2

107 Scientific and Technical Services COMPUTER SERVICES (e-mail, www (Intranet), ftp-Server). The services www (for Head: Thomas Jahn FMP externally) and e-mail (MX-Service) are provided through cooperation with the BBB. The DFN Association is The EDP Service Group is centrally located to support the the Internet provider for the FMP. The Berlin Research Asso- scientific work of the Research Groups and the admini- ciation (FV Berlin) operates SAP-R/3 server architecture for strative infrastructure (Administration, Library, etc.) and is administrative tasks, used on the client side in the FMP. PC responsible for the following areas: work stations with corresponding peripherals for graphics • Provision of communication channels and resources and image processing are available in the library and in a • Operation and extension of network components graphics room. • Central data storage, backup and interchange, print ser- An EDP Commission advises on investments and develop- vices mental perspectives in the IT field. • Standard equipment for PCs (hardware and software co-ordination) The next tasks of the Group will be implementing measures • Technical support in preparing publications and presen- to increase security, server consolidation and the evalua- tations tion of future Thin Client Applications to reduce administrative overheads. The backbone of all communication channels forms an efficient network infrastructure consisting of flexible, Personnel structured network connections and an active network (modular layer 2/3 switches). Ingrid Hermann (Software Administration) Hans-Werner Pisarz (Service Engineer) All EDP services are based on a heterogenous server (Net- Alexander Heyne (Student)** Ware 6 Cluster, LINUX and Windows 2k Server). The usual Björn Schümann (Student)* ** Internet services are made available by the EDP Group

COMPUTER-SERVICE Durch die Arbeitsgruppe EDV werden die üblichen Inter- Leiter: Thomas Jahn netdienste angeboten (eMail, www (Intranet), ftp-Server). Durch Kooperation mit der BBB werden die Services www Zur Unterstützung der wissenschaftlichen Arbeit der For- (für FMP extern) und eMail (MX-Service) bereitgestellt. schungsgruppen und der administrativen Infrastruktur Der Internetprovider des FMP ist der DFN Verein. Der FV (Verwaltung, Bibliothek u. a.) ist eine Arbeitsgruppe EDV Berlin betreibt eine SAP-R/3-Serverarchitektur für Verwal- zentral angesiedelt, die für folgende Bereiche verantwort- tungsaufgaben die clientseitig im FMP genutzt wird. lich ist: In der Bibliothek und in einem Grafikraum stehen PC- • Bereitstellung von Kommunikationswegen und -res- Arbeitsplätze mit entsprechender Peripherie für die Gra- sourcen fik- und Bildverarbeitung zur Verfügung. • Betrieb und Ausbau aller Netzwekkomponenten • zentrale Datenspeicherung, -sicherung und -austausch, Eine EDV-Kommission berät über Investitionen und per- Printservices spektivische Entwicklungen im IT-Bereich. • Grundausstattung PC’s (Hard- und Softwarekoordinie- Für die nächste Zukunft bereitet die Gruppe Maßnahmen rung) zur Erhöhung der Security vor, plant Schritte zur Server- • technische Unterstützung bei Publikationserstellung konsilidierung und evaluiert künftige Thin-Client-Applika- und -präsentationen tionen zur Senkung des administrativen Aufwandes. Das Rückgrat für alle Kommunikationswege bildet eine leistungsfähige Netzinfrastuktur, bestehend aus einer flexiblen strukturierten Netzwerkverkabelung und einem aktiven Netzwerk (modulare Layer 2/3-Switches).

Basis für alle EDV-Dienstleistungen ist ein heterogener Serverpool (NetWare 6 Cluster, LINUX und Windows 2k Server). * part of period reported ** part-time APPENDIX PEER REVIEWED ARTICLES 2003 Zimmermann J, Kühne R, Volkmer-Engert R, Jarchau T, ORIGINALARBEITEN 2003 Walter U, Oschkinat H, Ball LJ (2003) Design of N-substituted Peptomer Ligands for EVH1 Domains. J Biol Chem SECTION STRUCTURAL BIOLOGY 278, 36810-36818

Protein Structure Solution NMR Castellani F, van Rossum BJ, Diehl A, Rehbein K, Hupfer M, Rübe B, Schmieder P (2003) Origin and diage- Oschkinat H (2003) Determination of solid-state NMR nesis of polyphosphate in lake sediments: A 31P-NMR structures of proteins by means of three-dimensional 15N- study. Limnol Oceanogr 49, 1-10 13C-13C dipolar correlation spectroscopy and chemical shifts analysis. Biochemistry 42, 11476-11483 Otte L, Wiedemann U, Schlegel B, Pires JR, Beyermann M, Schmieder P, Krause G, Volkmer-Engert R, Schneider- Chevelkov V, van Rossum BJ, Castellani F, Rehbein K, Mergener J, Oschkinat H (2003) WW domain sequence Diehl A, Hoh M, Steuernagel S, Engelke F, Oschkinat H, activity relationships identified using ligand recognition Reif B (2003) 1H detection in MAS solid-state NMR spec- propensities of 42 WW domains. Protein Sci 12, 491-500 troscopy of biomacromolecules employing pulsed field gradients for residue solvent suppression. J Am Chem Soc Structural Bioinformatics 125, 7788-7789 Otte L, Wiedemann U, Schlegel B, Pires JR, Beyermann Labudde D, Leitner D, Krüger M, Oschkinat H (2003) Pre- M, Schmieder P, Krause G, Volkmer-Engert R, Schneider- diction algorithm for amino acid type with its secondary Mergener J, Oschkinat H (2003) WW domain sequence structure in proteins (PLATONS) using chemical shifts. J activity relationships identified using ligand recognition Biomol NMR 25, 41-53 propensities of 42 WW domains. Protein Sci 12, 491-500 Ladizhansky V, Jaroniec CP, Diehl A, Oschkinat H, Griffin RG (2003) Measurement of multiple psi torsion angles in Molecular Modelling uniformly 13C, 15N-labeled alpha-spectrin SH3 domain Freund C, Kühne R, Park S, Thiemke K, Reinherz EL, using 3D 15N-13C-13C-15N MAS dipolar-chemical shift Wagner G (2003) Structural investigations of a GYF domain correlation spectroscopy. J Am Chem Soc 125, 6827-6833 covalently linked to a proline-rich peptide. J Biomol NMR Pires JR, Hong X, Brockmann C, Volkmer-Engert R, 27, 143-149 Schneider-Mergener J, Oschkinat H, Erdmann R (2003) Karges B, Karges W, Mine M, Ludwig L, Kühne R, Milgrom The ScPex13p SH3 domain exposes two distinct binding E, de Roux N (2003) Mutation Ala171Thr stabilizes the sites for Pex5p and Pex14p. J Mol Biol 326, 1427-1435 gonadotropin releasing hormone receptor in its inactive Reif B, van Rossum B, Castellani F, Rehbein K, Diehl A, conformation, causing familial hypogonadotropic hypogo- Oschkinat H (2003) Characterization of 1H-1H distances in nadism. J Clin Endocrinol Metab 88, 1873-1879 a uniformly 2H, 15N-labeled SH3 domain by MAS solid- Zimmermann J, Kühne R, Volkmer-Engert R, Jarchau T, state NMR spectroscopy. Walter U, Oschkinat H, Ball LJ (2003) Design of N-substi- J Am Chem Soc 125, 1488-1489 tuted Peptomer Ligands for EVH1 Domains. J Biol Chem Strauss H (2003) A device for facilitating the use of the 278, 36810-36818 French Press. Analytical Biochemistry 321, 276-277 Solid State NMR Toepert F, Knaute T, Guffler S, Pires JR, Matzdorf T, Oschkinat H, Schneider-Mergener J (2003) Combining Chevelkov V, van Rossum BJ, Castellani F, Rehbein K, SPOT Synthesis and Nature Peptide Ligation to Create Diehl A, Hoh M, Steuernagel S, Engelke F, Oschkinat H, Large Arrays of WW Domains. Angew Chem Int Ed 42, Reif B (2003) 1H detection in MAS solid-state NMR spec- 1136-1140 troscopy of biomacromolecules employing pulsed field gradients for residue solvent suppression. J Am Chem Soc Van Rossum B, Castellani F, Pauli J, Rehbein K, Hollander 125, 7788-7789 J, de Groot H, Oschkinat H (2003) Assignment of amide proton signals by combined evaluation of HN, NN and Narayanan S, Bösl B, Walter S, Reif B (2003) Importance of HNCA MAS-NMR correlation spectra. J Biomol NMR 25, low oligomeric weight species for prion propagation in the 217-223 yeast prion system Sup35/Hsp104. Proc Natl Acad Sci U.S.A. 100, 9286-9291

FMP-authors in bold. FMP-authors inbold. antigens. JBiolChem278,22998-23007 surface expressionoftumor-associated O-linkedglycan B, RehmA(2003)TheGolgiproteinRCAS1controlscell state NMRspectroscopy. JAmChemSoc125,1488-1489 a Tamma G, 1525 lation andinteractionwithRhoGDI.JCellSci116,1519- phosphory- RhoA associated withRhoAinhibitionthrough W 1525 lation andinteractionwithRhoGDI.JCellSci116,1519- phosphory- RhoA associated withRhoAinhibitionthrough W Tamma G, Anchored Signalling Engelsberg A, Protein Trafficking MaricK Pohl P, Lorenz D,KrylovA,HahmHagenV, RosenthalW, Tamma G, Cellular Signalling MOLECULAR GENETICS SECTION CELLULARSIGNALLING/ Freund C,KühneR P Oschkinat H R Reif B Rho. JCellSci116,3285-94 vasopressin-induced antidiuresisthroughactivationof The prostagladinE2analoguesulprotoneantagonizes thelial cells.EMBORep4,88-94 ing theexocyticrecruitmentofaquaporin-2inrenalephi- 2 covalently linkedtoaproline-richpeptide.JBiomolNMR Wagner G(2003)StructuralinvestigationsofaGYFdomain spectroscopy inrotatingsolids.JMagnReson160,78-83 Schaefer M,Valenti G, 7, 143-149 eif B,vanRossumCastellaniF, RehbeinK,DiehlA, rotein Engineering uniformly 2H,15N-labeledSH3domainbyMASsolid- , , Valenti G(2003)cAMPinducedAQP2translocationis Valenti G(2003)cAMPinducedAQP2translocationis , Griffin RG(2003)1Hdetected1H,15Ncorrelation Klussmann E Klussmann E Wiesner B,FurkertJ,HahmD,OkscheA (2003) Characterizationof1H-1Hdistancesin Hermosilla R (2003) CyclicAMPissufficientfortrigger- , Park S, Rosenthal W, KlussmannE , , Procino G,SveltoM, Procino G,SveltoM, , Karsen U, Thiemke K Schülein R , Reinherz EL, Rosenthal Rosenthal , Dorken (2003) , 1 porin-2 inhypothyroidrats.AmJPhysiolRenal284, (2003) Renalexpressionofsodiumtransportersandaqua- Sci 116,1261-1268 urocortin usingconfocallaserscanningmicroscopy. JCell after stimulationwithcorticotropin-releasingfactorand cellular calciummeasurementsofsinglehumanskincells by Long-Wavelength Irradiation.ChemBiochem4,162-170 Ultrafast PhototriggersforATP, ADP, andAMPActivated H Hagen V Hagen V Geissler D Cellular Imaging P lators ofAQP2expressioninrenalprincipalcells.AmJ (2003) Osmolalityandsolutecompositionarestrongregu- Storm R,KlussmannE,GeelhaarA,RosenthalW, MaricK 17, 1992-2005 accumulation ofthetranscriptionfactor. Stat1 GenesDev (2003) DNAbindingcontrolsinactivationandnuclear Meyer T, MargA,LemkeP, WiesnerB,Vinkemeier U Wiesner B Siems WE,MaulB,WiesnerBeckerM Kamp G,BüsselmannJonesN, ChemBiochem 4,434-442 phototriggers of8-Bromo-substitutedcyclicnucleotides. compounds asultrafastandeffectivelong-wavelength J Andrologia 35,44-54 spermatozoa andtheimpactofpeptidolyticenzymes. (Sus scrofa)spermatozoa.Reproduction126,517-525 J Rothe L, lestern inZellen.AngewChem114,3775-3777 aktivierbaren [Bis(carboxymethoxy)-cumarin-4-yl]methy- der FreisetzungvoncyclischenNukleotidenausphoto- UB (2003)FluoreszenzspektroskopischeQuantifizierung Schmitt R, 097-104 hysiol RenalPhysiol284,189-98 , (2003) EnergymetabolismandintracellularpHinboar H (2003) [7-(Dialkylamino)coumarin-4-yl]methyl-caged agen V , , Winkler A Frings S, Frings S,BendigJ, , , Klussmann E ( Kresse W, 2003) DMACM-CagedAdenosineNucleotides: Roloff B, Wiesner B (2003) Effectsofkininsonmammalian Fechner K Wiesner B , Kahl T, EllisonDH,BachmannS Lorenz D,WiesnerB , Helm S,KauppUB,Bendig , Slominski A(2003)Intra- , Wiesner B Bendig J,Kettenmann , , Lauterwein Walther T, , Kaupp

111 Appendix Molecular Cell Physiology Meyer T, Vinkemeier U, Meyer U (2003) Evidence-based medicine - Was geht verloren? Ethik in der Medizin 14, Wallukat G, Podlovski S, Nissen ER, Morwinski R, Csonka 3-10 C, Tosaki A, Blasig IE (2003) Functional and structural characterization of anti-beta1-adrenoceptor auto-antibodies Meyer T, Marg A, Lemke P, Wiesner B, Vinkemeier U of spontaneously hypertensive rats. Mol Cell Biochem 251, (2003) DNA binding controls inactivation and nuclear 67-75 accumulation of the transcription factor Stat1. Genes Dev 17, 1992-2005 Schroeter ML, Abdul-Khaliq H, Fruhauf S, Hohne R, Schick G, Diefenbacher A, Blasig IE (2003) Serum S100B is increased during early treatment with antipsychotics and in defi- SECTION CHEMICAL BIOLOGY cit schizophrenia. Schizophrenia Res 62, 231-236

Peptide Synthesis Biochemical Neurobiology Carpino LA, Ionescu D, El-Faham A, Beyermann M, Henk- Siems WE, Maul B, Wiesner B, Becker M, Walther T, lein P, Hanay C, Wenschuh H, Bienert M (2003) Complex Rothe L, Winkler A (2003) Effects of kinins on mammalian polyfluoride additives in Fmoc-amino acid fluoride coup- spermatozoa and the impact of peptidolytic enzymes. ling processes. Enhanced reactivity and avoidance of Andrologia 35, 44-54 stereomutation. Org Lett 5, 975-977

Biophysics Carpino LA, Imazumi H, El Faham A, Fernando JF, Zhang C, Lee Y, Foxman BM, Henklein P, Hanay C, Mugge C, Urbánková E, Voltchenko A, Pohl P, Jezek P, Pohl EE (2003) Wenschuh H, Klose J, Beyermann M, Bienert M (2003) Transport kinetics of uncoupling proteins, Analysis of The uronium/guanidinium peptide coupling reagents: UCP1 reconstituted in planar lipid bilayers. J Biol Chem Finally the true uronium salts. Biopolymers 71, 349 278, 32497-32500 Otte L, Wiedemann U, Schlegel B, Pires JR, Beyermann Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl M, Schmieder P, Krause G, Volkmer-Engert R, Schneider- P, Maric K (2003) Cyclic AMP is sufficient for triggering the Mergener J, Oschkinat H (2003) WW domain sequence exocytic recruitment of aquaporin-2 in renal ephithelial activity relationships identified using ligand recognition cells. EMBO Rep 4, 88-94 propensities of 42 WW domains. Protein Sci 12, 491-500

Molecular Genetics Von Eggelkraut-Gottanka R, Klose A, Beck-Sickinger AG, Beyermann M (2003) Peptide (alpha)thioester formation Wieczorek G, Steinhoff C, Schulz R, Scheller M, Vingron using standard Fmoc-chemistry. Tetrahedron Lett 44, M, Ropers HH, Nuber UA (2003) Gene expression profile of 3551-3554 mouse bone marrow stromal cells determined by cDNA microarray analysis. Cell Tissue Res 311, 227-237 Wissmann R, Bildl W, Oliver D, Beyermann M, Kalbitzer HR, Bentrop D, Fakler B (2003) Solution structure and function Barmeyer C, Horak I, Zeitz M, Fromm M, Schultzke JD of the “Tandem inactivation domain” of the neuronal (2003) The interleukin-2-deficient mouse model. Patho- A-type potassium channel Kv1.4. J Biol Chem 278, 16142- biology 70, 139-142 16150

Cellular Signal Processing Kaupp UB, Solzin J, Hildebrandt E, Brown JE, Helbig A, Hagen V, Beyermann M, Pampaloni F, Weyand I (2003) The Chen X, Bhandari R, Vinkemeier U, Van Den Akker F, signal flow and motor response controlling chemotaxis of Darnell JE Jr, Kuriyan J (2003) A reinterpretation of the sea urchin sperm. Nat Cell Biol 5, 109-117 dimerization interface of the N-terminal domains of STATs. Protein Sci 142, 361-365

Meyer T, Vinkemeier U, Meyer U (2003) Medizinische Implikationen pharmakogenomischer Behandlungsstrate- gien. Ethik in der Medizin 12, 207-209

FMP-authors in bold. FMP-authors inbold. ChemBiochem 4,434-442 phototriggers of8-Bromo-substituted cyclicnucleotides. compounds asultrafastandeffectivelong-wavelength J Beattie KA,ResslerJ,WiegandC, Chem 278,11536-11545 phoinositide 3-KinaseIsoformsBetaandGamma.JBiol Characterization oftheAutophosphorylationSitesPhos- estern inZellen.AngewChem114,3775-3777 aktivierbaren [Bis(carboxymethoxy)-cumarin-4-yl]methyl- der FreisetzungvoncyclischenNukleotidenausphoto- UB (2003)FluoreszenzspektroskopischeQuantifizierung Kleuss C, Spec 17,222-228 zation andelectrosprayionization.RapidCommunMass spectrometry usingmatrix-assistedlaserdesorption/ioni- studies onAlzheimer'samyloid-betapeptidesbymass Kraus M, 284-288 of Nodularin-RinFlounderLivers.EnvironToxicol Chem18, (2003) MassSpectrometricDetectionandQuantification S Czupalla C,CuloM,MüllerEC,BrockReuschHP, 696 mass spectrometry. RapidCommunMassSpec17,690- I Karlsson K,SipiaV, brine shrimpArtemiasalina.AquatToxicol 62,219-226 and metabolismoftwomicrocystinsnodularininthe Steinberg CEW, PflugmacherS(2003)Comparativeeffects by Long-Wavelength Irradiation.ChemBiochem4,162-170 Ultrafast PhototriggersforATP, ADP, andAMPActivated Czupalla C,NürnbergB, te Leishmaniadonovani.Proteomics3,1811-1829 induced changesoftheproteomeinprotozoanparasi- Hagen V Hagen V Geissler D,KresseW, Synthetic OrganicBiochemistry C Bente M,HarderS,WiesgiglHeukeshovenJ,Gelhaus Mass Spectrometry N-terminal glycine.EMBOJ22,826-832 H, phosphoinositide 3-kinaseautophosphorylationsitesby picher K, , Hagen V K (2003) [7-(Dialkylamino)coumarin-4-yl]methyl-caged rause E , , Krause E Frings S, Frings S,BendigJ, Bienert M,KrauseE K (2003) DMACM-CagedAdenosineNucleotides: , rause E Clos J,BruchhausI(2003)Developmentally Wiesner B (2003) G-alpha-sispalmitoylatedatthe Krause E , Nürnberg B(2003)Identificationand Wiesner B Krause E , Lorenz D,WiesnerB , Meriluoto J,PflugmacherS (2003) Hydrogenexchange Helm S,KauppUB,Bendig , (2003) Analysisofclass Bendig J,Kettenmann Krause E , Codd GA, , Kaupp Matsumoto M,SolzinJ,HelbigA, s signal flowandmotorresponsecontrollingchemotaxisof sperm. DevBiol260,314-324 p Kaupp UB,HoshiM,Weyand I(2003)Asperm-activating Kawase O,MaruyamaY, OgisoM,GoddeMinakataH, Kaupp UB,SolzinJ,HildebrandtE,BrownJE,HelbigA, tives. Tetrahedon Lett44,5019-5023 ed intheparallelsynthesisoflabeledcarbohydratederiva- highly orthogonalconversionsofprimaryaminesemploy- Weik S, mol 369,366-390 novel toolsindiversity-orientedsynthesis.MethodEnzy- activated reactantsandreactiveintermediates:powerful Rademann J Medicinal Chemistry cells. EMBORep4,88-94 e P, MaricK Lorenz D,KrylovA,HahmHagenV, RosenthalW, Pohl Hagen V, BeyermannM Rademann J 2494 satile C-Ccouplingreactions.AngewChemIntEd42,2491- acylanion equivalent:linkerreagentsforsmoothandver- ea urchinsperm.NatCellBiol5,109-117 xocytic recruitmentofaquaporin-2inrenalephithelial eptide controlsacGMP-signalingpathwayinstarfish Rademann J (2003) CyclicAMPissufficientfortriggeringthe (2003) Advancedpolymerreagentsbasedon (2003) Trimellitic anhydridelinker(TAL) – (2003) Aphosphoraneassupported , Pampaloni F, Weyand I(2003)The Hagen V , Ueno S,

113 Appendix PEER REVIEWED ARTICLES 2004 Soukenik M, Diehl A, Leidert M, Sievert V, Büssow K, ORIGINALARBEITEN 2004 Leitner D, Labudde D, Ball LJ, Lechner A, Nägler DK, Oschkinat H (2004) The SEP domain of p47 acts as a rever- SECTION STRUCTURAL BIOLOGY sible competitive inhibitor of cathepsin L. FEBS Lett 576, 358-362 Protein Structure Schleinkofer K, Wiedemann U, Otte L, Wang T, Krause G, Brockmann C, Leitner D, Labudde D, Diehl A, Sievert V, Oschkinat H (2004) Comparative Structural and Energetic Büssow K, Kühne R, Oschkinat H (2004) The solution struc- Analysis of WW Domain-peptide. J Mol Biol 344, 865-881 ture of the SODD BAG-domain and a model of the SODD- BAG/HAP70 complex reveal additional SODD subfamily- Waldmann H, Karaguni IM, Carpintero M, Gourzoulidou E, specific electrostatic interactions. FEBS Lett 558, 101-106 Herrm C, Brockmann C, Oschkinat H, Muller O (2004) Sulin- dac-Derived Ras Pathway Inhibitors Target the Ras-Ra Brockmann C, Diehl A, Rehbein K, Strauss H, Schmieder Interaction and Downstream Effectors in the Ras Pathway. P, Korn B, Kuhne R, Oschkinat H (2004) The oxozidid sub- Angew Chem Int Ed 43, 454-458 unit b8 from human complex I adopts a thioredoxin fold. Structure 12, 1645-1654 Wiedemann U, Boisguerin P, Leben R, Leitner D, Krause G, Mölling K, Volkmer-Engert R, Oschkinat H (2004) Quanti- Gaiser OJ, Oschkinat H, Heinemann U, Ball LJ (2004) (1), fication of PDZ Domain Specificity, Prediction of Ligand (13) C and resonance assignments of C-terminal BRCT Affinity and Rational Design of Super-Binding Peptides. J domain from human BRCA1. J Biol NMR 30, 221-222 Mol Biol 343, 703-718 Gaiser OJ, Ball LJ, Schmieder P, Leitner D, Strauss H, Zierler-Gould KM, Pope B, Weeds AG, Ball LJ (2004) Letter Wahl M, Kühne R, Oschkinat H, Heinemann U (2004) Solu- to the Editor: backbone and sidechain 1H, 13 C and 15N tion structure, backbone dynamics, and association beha- resonance assignments of human cofilin. J Biol NMR 29, vior of the C-terminal BRCT domain from the breast can- 429-430 cer-associated BRCA1. Biochemistry 43, 15983-15995 Zimmermann J, Jarchau T, Walter U, Oschkinat H, Ball LJ Kahmann JD, Wecking DA, Putter V, Lowenhaupt K, Kim (2004) 1H, 13C and 15N resonance assignment of the YG, Schmieder P, Oschkinat H, Rich A, Schade M (2004) human Spred2 EVH1 domain. J Biol NMR 29, 435-436 The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its redu- Wüller S, Wiesner B, Löffler A, Furkert J, Krause G, ced affinity to Z-DNA in vitro. Proc Natl Acad Sci U.S.A. Hermosilla R, Schaefer M, Schülein R, Rosenthal W, 101, 2712-2717 Oksche A (2004) Pharmacochaperones post-translationally enhance cell surface expression by increasing confor- Krabben L, van Rossum BJ, Castellani F, Bocharov E, mational stability of wild-type and mutant vasopressin V2 Schulga AA, Arseniev AS, Weise C, Hucho F, Oschkinat H receptor. J Biol Chem 279, 47254-47263 (2004) Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid-state Solution NMR NMR approach. FEBS Lett 564, 319-324 Brockmann C, Diehl A, Rehbein K, Strauss H, Schmieder Mueller U, Bussow K, Diehl A, Bartl FJ, Niesen FH, P, Korn B, Kuhne R, Oschkinat H (2004) The oxozidid sub- Nyarsik L, Heinemann U (2004) Rapid purification and cry- unit B8 from human complex I adopts a thioredoxin fold. stal structure analysis of a small protein carrying two ter- Structure 12, 1645-1654 minal affinity tags. J Struct Funct Genomics 4, 217-225 Gaiser OJ, Ball LJ, Schmieder P, Leitner D, Strauss H, Pahlke D, Leitner D, Wiedemann U, Labudde D (2004) Wahl M, Kühne R, Oschkinat H, Heinemann U (2004) Solu- COPS - Cis/trans peptide bond conformation prediction of tion structure, backbone dynamics, and association beha- amino acids on the basis of secondary structure informa- vior of the C-terminal BRCT domain from the breast can- tions. Bioinformatics 10, 27-28 cer-associated BRCA1. Biochemistry 43, 15983-15995 Pope BJ, Zierler-Gould KM, Kuhne R, Weeds AG, Ball LJ Kahmann JD, Wecking DA, Putter V, Lowenhaupt K, Kim (2004) Solution structure of human cofilin: rationalising the YG, Schmieder P, Oschkinat H, Rich A, Schade M (2004) pH sensitivity of actin binding. J Biol Chem 279, 4840-4848 The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro. Proc Natl Acad Sci U.S.A. 101, 2712-2717 FMP-authors in bold. K tor. Endocrinol46,477-485 in theSecondIntracellularLoopofThyrotropin Recep- ral DeterminantsforG-ProteinActivationandSelectivity Simon B,LetunicK,Bussow G Mol Biol343,703-718 Affinity andRationalDesignofSuper-Binding Peptides.J tification ofPDZDomainSpecificity, ofLigand Prediction Oksche A interface. JBiolChem279,51590-51600 receptor actingasintramolecularsignalling ectodomain FMP-authors inbold. Wüller S,WiesnerB,LöfflerA,FurkertJ,KrauseG Wiedemann U,BoisguerinP Müller SL Krause G Kleinau G with PAR1. RegulatoryPeptides,inpress – Henklein P N Structural Bioinformatics Scheich C, receptor. JBiolChem279,47254-47263 mational stabilityofwild-typeandmutantvasopressinV2 ly enhancecellsurfaceexpressionbyincreasingconfor- Schleinkofer K, Cell MolLifeSci61,1354-365 protein 1:potentialmechanismoftightjunctionregulation. din bindstotheSH3-hinge-GuKunitofzonulaoccludens Huber O, logy 4,4 E fication onfoldedhumanproteindomainsexpressedin Hermosilla R,SchaeferM, Oschkinat H Schneider-Mergener J, Schmidt A, Chem 280,3747-3756 catenin shareacommoninteractionmechanism.JBiol tein occludinandtheadherensjunctionproteinalpha- Analysis ofWWDomain-peptide.JMolBiol344,865-881 . aufmann R,SchulzeB, , eumann S, The PAR3 Neo-N-terminalpeptideTFRGAPinteracts coli suitableforstructuralanalysis.BMCStructuralBio- Mölling K,Volkmer-Engert R, Blasig IE,KrauseG (2004) IdentificationofanovelepitopeintheTSH , (2004) Pharmacochaperonespost-translational- , Jaeschke H,NeumannS, Utepbergenov DI,MuellerSL,BeyermannM (2004) Proteinase-activatedreceptors(PARs) Leitner D Portwich M, K (2004) ComparativeStructuralandEnergetic rause G Wiedemann U , , Claus M,PaschkeR(2004)Structu- Sievert V, K Krause G,BlasigIE rause G Schmidt A (2004) Thetightjunctionpro- , Schülein R Leben R, , Diehl A Otte L,Wang T, Oschkinat H , Leidert M,SchlegelB Mayr LM,SetmacherU, Laettig J , Utepbergenov DI Leitner D,Krause (2004) Fastidenti- , Rosenthal W, (2004) Occlu- (2004) Quan- , Paschke R, Krause G, , , , , Büssow K, case. ProcNatlAcadSciU.S.A.101,7258-7263 homology3(SH3) formation inglobularproteins:theSrc L mal functionofCD2BP2.JBiolChem 279,28292-28297 sequences fortheGYFdomainreveal apossiblesplicoso- specific electrostaticinteractions.FEBSLett558,101-106 BAG/HAP70 complexrevealadditionalSODDsubfamily- ture oftheSODDBAG-domainandamodelSODD- Commun MassSpec18,863-868 massspectrometry. Rapid sted laserdesorption/ionization side chainstosignalintensitiesofpeptidesinmatrix-assi- binding topeptideamyloidfibrils.JBiolNMR29,525-530 couplings inpeptideinhibitorsweaklyalignedbytransient H Wahl M,KühneR,OschkinatH Gaiser OJ,BallLJ,SchmiederP, LeitnerD,StraussH, P Brockmann C,DiehlA,RehbeinK,StraussH,Schmieder B Baumgart S,LindnerY, KuhneR Molecular Modelling Kofler M,HeuerK,ZechT, FreundC Heuer K,KoflerM Protein Engineering Reif B Chevelkov V Chen Z, Solid StateNMR pH sensitivityofactinbinding.JBiolChem279,4840-4848 (2004) Solutionstructureofhumancofilin:rationalisingthe adapter proteinADAP. 12,603-610 Structure Structure ofahelicallyextendedSH3domaintheTcell Pope BJ,Zierler-Gould KM, cer-associated BRCA1.Biochemistry43,15983-15995 vior oftheC-terminalBRCTdomainfrombreastcan- tion structure,backbonedynamics,andassociationbeha- Structure 12,1645-1654 fromhumancomplexIadoptsathioredoxinfold. unit B8 Ventura S,ZurdoJ, Magn Reson172,56-62 13C homonuclearscalardecouplingduringacquisition.J enhancement inMASsolid-stateNMRbyapplicationof , , rockmann C,LeitnerD,LabuddeDiehlA (2004) Shortaminoacidstretchescanmediateamyloid Krause E Korn B, , Chiti F, Giannoni E,DobsonCM,Aviles FX,Serrano Reif B Kuhne R,OschkinatH Kühne R,OschkinatH ( 2004) Thecontributionsofspecificaminoacid , Chen Z,BermelW, (2004) Measurementsofresidualdipolar , Langdon G, Narayanan S Kuhne R Thiemke K,FreundC , Heinemann U(2004)Solu- , , (2004) Thesolutionstruc- (2004) Theoxozididsub- Reif B Oberemm A, Parreno M,ManguesR, , Weeds AG, (2004) Recognition (2004) Resolution , Wenschuh Sievert V, Ball LJ (2004)

115 Appendix SECTION CELLULAR SIGNALLING/MOLE- Protein Trafficking CULAR GENETICS Droese J, Mokros T, Hermosilla R, Schuelein R, Lipp M, Cellular Signalling Hopken UE, Rehm A (2004) HCMV-encoded chemokine receptor US28 employs multiple routes for internalization. Gregan B, Schäfer M, Rosenthal W, Oksche A (2004) Fluo- Biochem Biophys Res Comm 322, 42-49 rescence resonance energy transfer analysis reveals the existence of endothelin A and endothelin B receptor Hermosilla R, Oueslati M, Donalies U, Schönenberger E, homodimers. J Cardiovasc Pharmacol 44, S30-33 Krause E, Oksche A, Rosenthal W, Schuelein R (2004) Disease-causing V2 Vasopressin Receptors are Retained Gregan B, Jürgensen J, Papsdorf G, Furkert J, Schaefer in Different Compartments of the Early Secretory Pathway. M, Beyermann M, Rosenthal W, Oksche A (2004) Ligand- Traffic 12, 993-1005 dependent differences in the internalization and intracellular trafficking of endothelin A and endothelin B receptor Neuschäfer-Rube F, Rehwald M, Hermosilla R, Schülein R, heterodimers. J Biol Chem 279, 27679-27687 Rönnstrand L, Püschel G (2004) Identification of different Ser/Thr residues in the C-terminal domain of the human Hällbrink M, Oehlke J, Papsdorf G, Bienert M (2004) Up- EP4 reseptor for agonist-induced phosphorylation, beta- take of cell-penetrating peptides is dependent on the pep- arrestin interaction and sequestration Biochemistry 379, tide-to-cell-ratio rather than on peptide concentration. 573-585 Biochim Biophys Acta 1667, 222-228 Wüller S, Wiesner B, Löffler A, Furkert J, Krause G, Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F, Hermosilla R, Schaefer M, Schülein R, Rosenthal W, Schmitt R, Vossebein L, Tamma G, Beyermann M, Krause Oksche A (2004) Pharmacochaperones post-translational- E, Herberg FW, Valenti G, Bachmann S, Rosenthal W, ly enhance cell surface expression by increasing confor- Klussmann E (2004) Identification of a novel A-kinase mational stability of wild-type and mutant vasopressin V2 anchoring protein 18 isoform and evidence for its role in receptor. J Biol Chem 279, 47254-47263 the vasopressin-induced aquaporin-2 shuttle in renal principal cells. J Biol Chem 279, 26654-26665 Anchored Signalling Hermosilla R, Oueslati M, Donalies U, Schönenberger E, Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F, Krause E, Oksche A, Rosenthal W, Schuelein R (2004) Schmitt R, Vossebein L, Tamma G, Beyermann M, Krause Disease-causing V2 Vasopressin Receptors are Retained E, Herberg FW, Valenti G, Bachmann S, Rosenthal W, in Different Compartments of the Early Secretory Pathway. Klussmann E (2004) Identification of a novel A-kinase Traffic 12, 993-1005 anchoring protein 18 isoform and evidence for its role in Tsunoda AP, Wiesner B, Lorenz D, Rosenthal W, Pohl P the vasopressin-induced aquaporin-2 shuttle in renal prin- (2004) Aquaporin-1, Nothing but a water channel. J Biol cipal cells. J Biol Chem 279, 26654-26665 Chem 279, 11364-11367 Cellular Imaging Wietfeld D, Heinrich N, Furkert J, Fechner K, Beyermann M, Bienert M, Berger H (2004) Regulation of the Coupling Geissler D, Antonenko YN, Schmidt R, Keller S, Krylova to Different G-Proteins of Rat Corticotropin-Releasing Fac- OO, Wiesner B, Bendig J, Pohl P, Hagen V (2004) (Couma- tor Receptor Type 1 (rCRFR1) in HEK293 Cells. J Biol Chem rin-4-yl)methyl Esters as Highly Efficient, Ultrafast Photot- 279, 38386-38394 riggers for Protons and Their Application to Acidifying Membrane Surfaces. Angew Chem Int Ed 44, 1195-1198 Wüller S, Wiesner B, Löffler A, Furkert J, Krause G, Hermosilla R, Schaefer M, Schülein R, Rosenthal W, Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F, Oksche A (2004) Pharmacochaperones post-translational- Schmitt R, Vossebein L, Tamma G, Beyermann M, Krause ly enhance cell surface expression by increasing confor- E, Herberg FW, Valenti G, Bachmann S, Rosenthal W, mational stability of wild-type and mutant vasopressin V2 Klussmann E (2004) Identification of a novel A-kinase receptor. J Biol Chem 279, 47254-47263 anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells. J Biol Chem 279, 26654-26665

FMP-authors in bold. Moser KV, ReindlM, Neurosi, inpress function inbraincapillaryendothelialcellsvitro.MolCell of theastrocyticfactor(s)modulatingblood-brainbarrier chem 271,3043-3049 with acell-penetratingsyntheticmodelpeptide.EurJBio- c brain capillaryendothelialcells.JNeuro-Oncol71,127-134 caspase-3 inducescelldeathinmalignantC6gliomaand Oksche A B FMP-authors inbold. Müller SL Haseloff RF, Blasig IE Molecular CellPhysiology Wüller S,WiesnerB,LöfflerA,FurkertJ,KrauseG Chem 279,11364-11367 ( Tsunoda AP, WiesnerB,LorenzD,RosenthalW, PohlP Oehlke J Mathas S,LietzA,AnagnostopoulosI,HummelF, ger H,BienertM Zassler B, Cell MolLifeSci61,1354-365 protein 1:potentialmechanismoftightjunctionregulation. din bindstotheSH3-hinge-GuKunitofzonulaoccludens Huber O, receptor. JBiolChem279,47254-47263 mational stabilityofwild-typeandmutantvasopressinV2 ly enhancecellsurfaceexpressionbyincreasingconfor- Schneider-Mergener J, Schmidt A, Chem 280,3747-3756 catenin shareacommoninteractionmechanism.JBiol tein occludinandtheadherensjunctionproteinalpha- on. BrainRes1017,53-60 express NGFreceptorsandsecreteafterinflammati- capillary endothelialcellsproliferateinresponsetoNGF, Hermosilla R,SchaeferM, receptor-induced apoptosis.JExpMed199,1041-1052 r HP, K,SteinH,DorkenB(2004)C-FLIPmediates Bommert 2004) Aquaporin-1,Nothingbutawaterchannel.JBiol esistance ofHodgkin/Reed-Sternbergcellstodeath entration andbiologicalactivityofPNAafterconjugation , Janz M,JundtF, HirschB,Johrens-LederK,Vornlocher , Blasig IE,KrauseG (2004) Pharmacochaperonespost-translational- Wallukat G, , Blasig IE Utepbergenov DI,MuellerSL,BeyermannM Portwich M, (2004) Enhancementofintracellularcon- , , Humpel C(2004)Proteindeliveryof Bauer HC,H(2004)Insearch Wolf Y, EhrlichA,WiesnerB,Ber- Blasig IE Krause G,BlasigIE Schmidt A (2004) Thetightjunctionpro- Schülein R , Humpel C(2004)Brain , Utepbergenov DI , Rosenthal W, (2004) Occlu- Wiesner , , , Immunol Methods282,117-134 associated hypoxia-inducedcarbonic anhydraseIX.J domainsCA IX-deficientmicerecognizedifferent oftumor- Pastorekova S(2004)Monoclonal antibodiesgeneratedin Tonevitzky AG, nel. ProcNatlAcadSciU.S.A.101,4805-4809 Water flowthroughttheemptybacterialpotassium chan- Block Copolymers.Biochemistry43,3696-3703 Krylova O,PohlP OO, WiesnerB,BendigJ,PohlP, HagenV Geissler D,AntonenkoYN,SchmidtR,KellerS,Krylova Biophysics Siems WE Walter T, StepanH, Biochemical Neurobiology Mankertz J,SeidlerU, Barmeyer C,HarrenM,SchmitzH,Heinzel-PleinesU, Molecular Genetics Chem 279,11364-11367 (2004) Aquaporin-1,Nothingbutawaterchannel.JBiol Tsunoda AP, WiesnerB,LorenzD,RosenthalW, PohlP Sun J Saparov SM,PohlP and brainnatriureticpeptide.BiolChem385,179-184 tidase activityrevealsindependentcatabolismofatrial mann J, Terszowski G,Waskow C,ConradtP, LenzeD,Koenigs- Am JPhysiol286,244-252 leukin-2-deficient mousemodelofcolonicinflammation. Schulzke JD(2004)Mechanismsofdiarrheaintheinter- Walter T, StepanH, 111, 452-455 stance ofBNPtoneutralendopeptidaseinthefetus.BJOG for enhancedneutralendopeotidaseactivityandresi- to theirN-terminalfragmentsinfetalcirculation:evidence ricin. EurBiophysJ33,572-579 Parkkila S,AK,Waheed A, Mucha V, JakubickovaL,BiesovaZ,Ortova-GutM, Zatovicova M,Tarabkova K,SvastovaE,GibadulinovaA, 1937-1945 the colony-formingurit-erythrocyte(CFU-E).Blood105, spective Isolationandglobalgeneexpressionanalysisof Membrane Surfaces.AngewChemIntEd44,1195-1198 triggers forProtonsandTheirApplicationtoAcidifying rin-4-yl)methyl EstersasHighlyEfficient,UltrafastPhoto- S chultheiss HP, , Carstanjen D,HorakI Pohl EE, (2004) Biochemicalanalysisofneutralendopep- Pohl P S iems WE (2004) IonophoricActivityofPluronic Krylova OO,KrauseE Pankow K,BeckerM (2004) Membranedestabilizationby Pankow K,GembardF Horak I,WiedenmannB (2004) Beyondthediffusionlimit: ( 2004) RelationofANPandBNP , Rodewald HR(2004)Pro- Horak I , Schultheiss HP, (2004) (Couma- , , Pastorek J, Agapov II, , , Fromm M, Faber R,

117 Appendix Cytokine Signalling SECTION CHEMICAL BIOLOGY

Rosenbauer F, Wagner K, Zhang P, Knobeloch KP, Iwama Peptide Synthesis A, Tenen DG (2004) pDP4, a novel glycoprotein secreted by Cansarek P, Beyermann M, Koch KW (2004) Thermodyna- mature granulocytes, is regulated by transcription factor mics of apocalmodulin and nitric oxide synthase II peptide PU.1. Blood 103, 4294-4301 interaction. FEBS Lett 577, 465-468 Schuh K, Cartwright EJ, Jankevics E, Bundschu K, Lieber- Carpino LA, Krause E, Sferdean CD, Bienert M, Beyermann mann J, Williams JC, Armesilla AL, Emerson M, Oceandy M (2004) Dramatically enhanced N to O acyl migration D, Knobeloch KP, Neyses L (2004) Plasma membrane Ca2+ during the trifluoracetic acid-based deprotection step in ATPase 4 is required for sperm motility and male fertility J solid phase peptide synthesis. Tetrahedon Lett 46, 1361- Biol Chem 279, 28220-28226 1364 Van Spriel AB, Puls KL, Sofi M, Pouniotis D, Hochrein H, Carpino LA, Krause E, Sferdean CD, Schümann M, Fabian Orinska Z, Knobeloch KP, Plebanski M, Wright MD (2004) H, Bienert M, Beyermann M (2004) Synthesis of “difficult“ A regulatory role for CD37 in T cell proliferation. J Immunol peptide sequences:application of a depsipeptide techni- 172, 2953-2961 que to the Jung Redemann 10- and 26-mers and the amyloid peptide Abeta (1-42). Tetrahedron Lett 45, 7519-7523 Molecular Myelopoiesis Gregan B, Jürgensen J, Papsdorf G, Furkert J, Schaefer Heymann GA, Carstanjen D, Kiesewetter H, Salama A M, Beyermann M, Rosenthal W, Oksche A (2004) Ligand- (2004) Polymorphism of the a4-subunit of VLA-4 integrin dependent differences in the internalization and intracel- and bone marrow transplantation. Haematologica 89, lular trafficking of endothelin A and endothelin B receptor 882-884 heterodimers. J Biol Chem 279, 27679-27687 Terszowski G, Waskow C, Conradt P, Lenze D, Koenigs- Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F, mann J, Carstanjen D, Horak I, Rodewald HR (2004) Pro- Schmitt R, Vossebein L, Tamma G, Beyermann M, Krause spective Isolation and global gene expression analysis of E, Herberg FW, Valenti G, Bachmann S, Rosenthal W, the colony-forming urit-erythrocyte (CFU-E). Blood 105, Klussmann E (2004) Identification of a novel A-kinase 1937-1945 anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal prin- Cellular Signal Processing cipal cells. J Biol Chem 279, 26654-26665 Marg A, Shan Y, Meyer T, Meissner T, Brandenburg M, Klemm C, Schöder S, Glückmann M, Beyermann M, Krau- Vinkemeier U (2004) Nucleocytoplasmic shuttling by se E (2004) Derivatization of phorphorylated peptides with nucleoporins Nup153 and Nup214 and CRM1-dependent S- and N-nucleophiles for enhanced ionization efficiency nuclear export control the subcellular distribution of latent in matrix-assisted laser desorption/ionization mass spec- Stat1. J Cell Biol 165, 823-833 trometry. Rapid Commun Mass Spectrom 18, 2697-2705 Meissner T, Krause E, Vinkemeier U (2004) Ratjadone and Klose J, Wendt N, Kubald S, Krause E, Fechner K, leptomycin B block CRM1-dependent nuclear export by Beyermann M, Bienert M, Rudolph R, Rothemund S (2004) identical mechanisms. FEBS Lett 576, 27-30 Hexa-histidin tag position influences disulfide structure Meissner T, Krause E, Lödige I, Vinkemeier U (2004) but not binding behaviour of in vitro folded N-terminal Arginine Methylation of STAT1: A Reassessment. Cell 119, domain of rat corticotropin-releasing factor receptor type 587-589 2a. Protein Sci 13, 2470-2475

Meyer T, Hendry L, Begitt A, John S, Vinkemeier U (2004) Schmidt A, Utepbergenov DI, Mueller SL, Beyermann M, A single residue modulates tyrosine dephosphorylation, Schneider-Mergener J, Krause G, Blasig IE (2004) Occlu- oligomerization, and nuclear accumulation of stat tran- din binds to the SH3-hinge-GuK unit of zonula occludens scription factors. J Biol Chem 279, 18998-19007 protein 1: potential mechanism of tight junction regulation. Cell Mol Life Sci 61, 1354-365

FMP-authors in bold. des attheair/waterinterface.BiophysJ86,3750-3758 tion absorptionspectroscopyofamphipathicmodelpepti- Commun MassSpec18,863-868 sted laserdesorption/ionizationmass spectrometry. Rapid side chainstosignalintensitiesofpeptidesinmatrix-assi- Berger H,BienertM chem 271,3043-3049 with acell-penetratingsyntheticmodelpeptide.EurJBio- centration andbiologicalactivityofPNAafterconjugation H, KrauseE FMP-authors inbold. Carpino LA,KrauseE Baumgart S,LindnerY, KuhneR Mass Spectrometry M, BienertBergerH Wietfeld D,HeinrichN,FurkertJ,FechnerK,Beyermann Oehlke J Peptide Biochemistry cyclization. JPeptRes64,159-69 effects ofaromaticclusters,D-aminoacidsubstitutionand activity ofArginine-&Tryptophan-rich hexapeptides:the Wessolowski A,BienertM,DatheD Oehlke J H chemistry 43,9140-9150 of tryptophanandargininecontaininghexapeptides.Bio- zation increasestheantimicrobialactivityandselectivity D Transport Peptide LipidInteraction/Peptide M, BienertBergerH Wietfeld D,HeinrichN,FurkertJ,FechnerK,Beyermann ger H,BienertM Kerth A,Erbe Biochim BiophysActa1667,222-228 peptide-to-cell-ratio ratherthanonpeptideconcentration. Uptake ofcell-penetratingpeptidesisdependentonthe 279, 38386-38394 tor ReceptorType 1(rCRFR1)inHEK293Cells.JBiol Chem Fac- to DifferentG-ProteinsofRatCorticotropin-Releasing 279, 38386-38394 t Fac- to DifferentG-ProteinsofRatCorticotropin-Releasing J gation withacell-penetratingsyntheticmodelpeptide.Eur concentration andbiologicalactivityofPNAafterconju- H, BienertM,BeyermannM or ReceptorType 1(rCRFR1)inHEK293Cells.JBiolChem ällbrink M,OehlkeJ,PapsdorfG,BienertM athe Biochem 271,3043-3049 M, NikolenkoH,KloseJ,BienertM , , Wallukat G, Wallukat G, (2004) Thecontributionsofspecificaminoacid Dathe M (2004) Enhancementofintracellularcon- , (2004) Enhancementofintracellular Sferdean CD, Wolf Y, EhrlichA,WiesnerB,Ber- (2004) RegulationoftheCoupling (2004) RegulationoftheCoupling Wolf Y, EhrlichA,WiesnerB, , Blume A(2004)Infraredreflec- (2004) Synthesisof“difficult“ , Oberemm A, Schümann M,Fabian (2004) Antimicrobial ( 2004) Cycli- Wenschuh ( 2004) trometry. RapidCommunMassSpectrom18,2697-2705 in matrix-assistedlaserdesorption/ionizationmassspec- S- andN-nucleophilesforenhancedionizationefficiency Peptides. RussJBioChem2,101-110 Use ofhydrogenPeroxideforClosingDisulfideBridgesin Biophys J33,572-579 ky AG,PohlP(2004)Membranedestabilizationbyricin.Eur K Klemm C Henn V, EdemirB,StefanE,WiesnerLorenzD Sun J Meissner T, KrauseE,Vinkemeier U Meissner T, KrauseE,LödigeI,Vinkemeier U mann M,BienertM Klose J,Wendt N Schmitt R,Vossebein L,Tamma G, EV, Sidorova MV, MolokoedovAS,Az’mukoAA,Kydryavtseva mun 25,1251-1256 peptides andsyntheticpolymers.MacromolRapidCom- approach toblockcopolymersofsequence-definedpoly- polymerization withpolypeptide-initiators:ageneral 587-589 Arginine methylationofSTAT1: areassessment. Cell119, Traffic 12,993-1005 in DifferentCompartmentsoftheEarlySecretoryPathway. Disease-causing V2Vasopressin ReceptorsareRetained loid peptideAbeta(1-42).Tetrahedron Lett45,7519-7523 que totheJungRedemann10-and26-mersamy- peptide sequences:applicationofadepsipeptidetechni- Sci 13,2470-2475 Protein rat corticotropin-releasingfactorreceptortype2a. binding behaviourofinvitrofoldedN-terminaldomain histidin tagpositioninfluencesdisulfidestructurebutnot se E Hermosilla R, cipal cells.JBiolChem279,26654-26665 the vasopressin-inducedaquaporin-2shuttleinrenalprin- a Rettig H, identical mechanisms.FEBSLett576,27-30 leptomycin BblockCRM1-dependentnuclearexportby Klussmann E E nchoring protein 18 isoform andevidenceforitsrolein , rause E,OkscheA,RosenthalW, SchueleinR Herberg FW, Valenti G,BachmannS, Krause E (2004) Derivatization of phorphorylated peptides with peptides (2004) Derivatizationofphorphorylated , Pohl EE, , Krause E Schöder S,GlückmannM, , Oueslati M,DonaliesU,SchönenbergerE, Ovchinnikov MV, BespalovaZD(2004)The (2004) IdentificationofanovelA-kinase Krylova OO,KrauseE , , Börner HG(2004)Atomtransferradical Kubald S, , Rudolph R,RothemundS(2004)Hexa- Krause E,FechnerK,Beyer- Beyermann M,Krause (2004) Ratjadoneand Beyermann M,Krau- , Agapov II,Tonevitz- Rosenthal W, , Theilig F, (2004) ( 2004)

119 Appendix Synthetic Organic Biochemistry

Geissler D, Antonenko YN, Schmidt R, Keller S, Krylova OO, Wiesner B, Bendig J, Pohl P, Hagen V (2004) (Couma- rin-4-yl)methyl Esters as Highly Efficient, Ultrafast Photo- triggers for Protons and Their Application to Acidifying Membrane Surfaces. Angew Chem Int Ed 44, 1195-1198

Medicinal Chemistry

Barth M, Rademann J (2004) Tailoring Ultraresins based on the cross-linking of polyethylene imines. Comperative investigation of the chemical composition, the swelling, the mobility, the chemical accessibility, and the performance in solid phase synthesis of very high loaded resins. J Comb Chem 6, 340-349

Barth M, Fischer R, Brock R, Rademann J (2004) Reversi- ble cross-linking of hyperbranched polymers: A strategy for the combinatorial decoration of multivalent scaffolds. Angew Chem Int Ed 44, 1560-1563

Rademann J (2004) High Loading Polymer Reagents based on Polycationic Ultragels. Polymer-Supported Reductions and Oxidations with Increased Efficiency. Tetrahedon Lett 60, 8703-8709

Rademann J (2004) Organic Protein Chemistry: Drug Dis- covery through the Chemical Modification of Proteins. Angew Chem Int Ed 116, 4654-4656

Smerdka J, Rademann J, Jung G (2004) Polymer-bound alkyltriazenes for mild racemization-free esterification of amino acid and peptide derivatives. J Pept Sci 10, 603-611

Sorg G, Thern B, Mader O, Rademann J, Jung G (2004) Pro- gress in the preparation of peptide aldehydes via polymer supported IBX oxidation and scavenging by threonyl resin. J Pept Sci 11, 142-152

Number of original articles published in peer reviewed journals ordered by impact factor Anzahl der Originalarbeiten geordnet nach Impact-Faktor

Impact factor 1999 2000 2001 2002 2003 2004 < 3 14 17 24 16 23 28 3 - 4.5 13 8 7 11 6 16 4.5 - 7 9 8 10 12 12 22 > 7 16 10 12 19 13 14 total 52 43 53 58 54 80

FMP-authors in bold. Funktionen imKörper. Pharmazie29-31 KLA-derived modelpeptides.JMolRecognit17,1-10 on thecellularuptakeofsubstanceP-andlysine-rich, Molecular MarkersofBrainDamage21,141-150 rimental settingsofcardiacsurgery. NeurolNeurosci/ n Lange PE(2003)Releasepatternsofastrocyticandneuro- through membranechannels.BiolChem385,921-926 FMP-authors inbold. Vinkemeier U Schülein R Rademann, J Pohl P Oehme, P Oehme P Oehlke J,LorenzD,WiesnerB,BienertM 4612 of STAT transcriptionfactors.EurJBiochem271,4606- Meyer T, Vinkemeier U 2852-2869 interactions insignaltransduction.AngewChemIntEd44, adaptor domains:Animportantclassofprotein-protein ( Ball LJ,KühneR Crausaz M,CelioSchroeterML, ler M,Troitsch D,Wehsack A,BoettcherW, SchwallerB, A ÜBERSICHTSARBEITEN 2003/2004 R Biol 167,197-201 Design principlesofamolecularsignalingcircuit.JCell Medizin undGesellschaft51,109-119 Sucht. MeineBegegnungenmitderSuchtforschung. 45-91 gical implications.RevPhysiolBiochemPharmacol151, transport ofmembraneproteins:clinicalandpharmacolo- Angew ChemIntEd43,4554-4556 covery throughthechemicalmodificationofproteins. 2003) Recognitionofprolinerichmotifs(PRMs)bysmall al biochemicalmarkersinserumduringandafterexpe- bdul-Kaliq H,SchubertS,Stoltenburg-DidingerG,Hueb- EVIEWS 2003/2004 (2004) Combinedtransportofwaterandions , Schmidt J,HinkelU(2004)Vitamin Kundseine (2004) TheoretischeundklinischeAspekteder (2004) Theearlystagesoftheintracellular (2004) Organic protein chemistry: drug dis- (2004) Organicproteinchemistry:drug (2004) Gettingthemessageacross,STAT. , Schneider-Mergener J, (2004) Nucleocytoplasmicshuttling Blasig IE (2004) Studies Oschkinat H , Hetzer R, Academic Press2003,SanDiego,USA disease (Eds.:SharmaHS,Westmann J),ElsevierScience/ In: Blood-SpinalCordandbrainbarriersinhealth resonance spectroscopy-effectofSIN-1 barrier proteinsZO-1andoccludinusingsurfaceplasmon Direct demonstrationoftheassociationblood-brain lag 2003,Heidelberg,Germany (Eds.: RosenthalW, S)766-770,SpringerVer- Offermanns In: EncyclopedicReferenceofMolecularPharmacology Proteomics Krause E ence Inc2003,Amsterdam,TheNetherlands In: Onlinepharmacologyreferencedatabase,ElsevierSci- Protein kinaseA Klussmann E 4 rier -Biologyandresearchprotocols(Ed.:NagS)89,465- In: MethodsofMolecularMedicine:Theblood-brainbar- cation bymassspectrometry t Proteomics ofthebrainendothelium:Separationpro- Haseloff RF, KrauseE,BlasigIE 2003 B 2003/2004 C lag 2003,Heidelberg,Germany W, Rosenthal (Eds: S)758-762,SpringerVer- Offermanns In: EncyclopedicReferenceofMolecular Pharmacology Protein Trafficking andQuality Control Schülein R,RosenthalW Schmidt A,UtepbergenovDI,KrauseG,BlasigIE lag 2003,Heidelberg,Germany (Eds.: RosenthalW, S)339-345,SpringerVer- Offermanns In: EncyclopedicReferenceofMolecularPharmacology Endothelins Oksche A lag 2003,Heidelberg,Germany (Eds.: RosenthalW, S)527-530,SpringerVer- Offermanns In: EncyclopedicReferenceofMolecularPharmacology, JAK/STAT Pathway Meyer T, Vinkemeier U 2003, Heidelberg,Germany (Eds.: RosenthalW, S)603-609,SpringerVerlag Offermann In: EncyclopedicReferenceofMolecularPharmacology Molecular Modelling Krause G eins bytwo-dimensionalgelelectrophoresisandidentifi- 77, HumanaPress2003,Totowa, NJ,USA ONTRIBUTIONS INMONOGRAPHS EITRÄGE ZUSAMMELWERKEN 2003/2004 (2003)

121 Appendix 2004 Rademann J Combinatorial Chemistry - Concepts and Methods for Bauer HC, Bauer H, Haseloff RF, Blasig IE Tasks of Molecular Optimization The role of glia in the formation and function of the blood- In: Encyclopedic Reference of Molecular Pharmacology brain barrier (Eds.: Offermanns S, Rosenthal W) 257-261, Springer Ver- In: Neuroglia 2nd Edition (Eds.: Ramson B, Kettenmann H) lag 2004, Heidelberg, Germany 325-333, Oxford University Press 2004, Oxford, UK Rademann J Freund C Solid phase synthesis (SPS) and polymer-assisted solution The Gyf Domain phase (PASP) synthesis In: Modular Protein Domains (Eds.: Cesareni G, Gimona M, In: Highlights in Bioorganic Chemistry (Eds.: Wennemers Sudol M, Yaffe M) 107, Wiley-VHC 2004, Weinheim, Germa- H, Schmuck C) 290-293, Wiley-VCH 2004, Weinheim, Ger- ny many Hagen V Rademann J Uncaging and photoconversion/activation Novel polymer- and linker reagents employed for the pre- In: Encyclopedic Reference of Genomics and Proteomics paration of protease inhibitor libraries in Molecular Medicine (Eds.: Ganten D, Ruckpaul K) in In: Highlights in Bioorganic Chemistry (Eds.: Wennemers press, Springer Verlag 2004, Heidelberg, Germany H, Schmuck C) 277-290, Wiley-VCH 2004, Weinheim, Ger- Kuehne R, Krause G, Rosenthal W many Entdeckungsstrategien in der Wirkstoffforschung Rademann J In: Handbuch der Psychopharmakologie (Eds: Holsboer F, Inhibition of Proteases Gruender G, Benkert O) in press, Springer Verlag 2004, Hei- In: Highlights in Bioorganic Chemistry (Eds.: Wennemers delberg, Germany H, Schmuck C) 293-295, Wiley-VCH 2004, Weinheim, Ger- Klussmann E many Protein Kinase A Rosenthal W, Seyberth H In: xPharm 1.0 (Eds.: Enna SJ, Bylund DB), Elsevier 2004, Besonderheiten der Arzneimitteltherapie im Kindesalter Amsterdam, The Netherlands In: Pharmakotherapie/Klinische Pharmakologie, 12th Krause E Edition (Eds.: Lemmer B, Brune K) 507-516, Springer Verlag Mass Spectrometry: MS/MS 2004, Heidelberg and Berlin, Germany In: Encyclopedic Reference of Genomics and Proteomics Schmidt A, Utepbergenov DI, Krause G, Blasig IE in Molecular Medicine (Eds.: Ganten D, Ruckpaul K) in Direct demonstration of association between the blood- press, Springer Verlag 2004, Heidelberg, Germany barrier proteins ZO-1 and occludin using surface plasmon Oehme, P resonance spectroscopy - Effect of SIN-1 Zwischen Wissenschaft und Politik In: Blood-Spinal Cord Barriers in Health and Disease (Ed.: In: Sitzungsbericht der Leibniz-Sozietät (Ed.: Steiger KP) Sharma HS) 11-17, Elsevier Verlag/Academic Press 2004, 67, Trafo-Verlag 2004, Berlin, Germany Heidelberg, Germany

Oehme P, Göres E, Rosenthal W, Ganten D Zimmermann J Pharmakologische Institutionen Berlin-Buch und Berlin- EVH1/WH1 domains Friedrichsfelde In: Modular Protein Domains (Eds.: Cesareni G, Gimona M, In: Geschichte und Wirken der pharmakologischen, kli- Sudol M, Yaffe M) 73-102. Wiley-VHC 2004, Weinheim, nisch-pharmakologischen und toxikologischen Institute Germany (Ed.: Philippou A) 698-711, Berenkamp Verlag Innsbruck 2004, Austria

Oksche A, Pohl P, Krause G, Rosenthal W MONOGRAPHS 2003/2004 Molecular biology of diabetes insipidus MONOGRAPHIEN 2003/2004 In: Encyclopedia of Molecular Cell Biology and Molecular Medicine (Ed.: Meyers RA) 301-324, Wiley-VCH 2004, Offermanns S (Eds.), Rosenthal W Weinheim, Germany Encyclopedic Reference of Molecular Pharmacology Springer Verlag 2004, Heidelberg, Germany

FMP-authors in bold. Springer Verlag, Heidelberg,Germany Handbook ofExperimentalPharmacologySeries Rosenthal, Walter Member 2001-2004 Springer Verlag, Heidelberg Journal ofMolecularMedicine Rosenthal, Walter Member since2002 American ChemicalSociety Journal ofCombinatorialChemistry Rademann, Jörg Kluwer AcademicPublishers,Netherlands Journal ofStructuralandFunctionalGenomics Oschkinat, Hartmut Member since2004 W Glia Blasig, Ingolf Member since2003 T Journal ofReceptorsandSignalTransduction Bienert, Michael BOARDS 2003/2004 M 2003/2004 M aylor andFrancis,Philadelphia iley-Liss, NewYork EMBERSHIPS INEDITORIALBOARDS ITGLIEDSCHAFTEN INEDITORIAL 10th AkaboriConference.Awaji, Japan ming Peptides:TheFBP28WWDomain Design, SynthesisandCharacterizationofBeta-Sheet-For- penetrating peptides.Stockholm,Sweden specific deliverysystemsforgenetherapybasedoncell- Opening lecture-EU-ProjectQLK3-CT-2002-01989: Target ve CompoundsintoCells tide-Derived CarriersfortheDeliveryofBiologicallyActi- Cellular UptakeofPeptides:DesignandSynthesisPep- Workshop. Budapest,Hungary Reporter Molecules–Celltarget Transport StrategiesforTargeting ofEpitopes,Drugsand Cellular UptakeofModelAmphipathicPeptides, 6th GermanPeptideSymposium.Berlin,Germany f the firstextracellulardomainofratcorticotropin-releasing In vitrofolding,disulfidepattern,andcharacterizationof Bienert M Bienert M Beyermann M B EINGELADENE VORTRÄGE2003/2004 I Blasig IE Blasig IE Blasig IE Blasig IE Bienert M N Peptidase-Workshop, The EMCRotterdam.Rotterdam, obesity? NEP-deficient mice-anewmodelforthelateonsethuman Hahn-Meitner-Institut. Berlin, Germany Struktur, FunktionundDichtheitderBlut-Hirnschranke Barriers. Szeged,Ungarn 6th SymposiumSignalTransduction oftheBlood-Brain occludin andoligomerizationofZO-1 Hinge regionoftheSH3-GuKunitZO-1isregulatedby CVB 2003.Amarillo,USA ZO-1 andrecruitmentofoccludin Interaction oftightjunctionproteins-Oligomerization USA Gordon ResearchConference:BarriersoftheBrain.Tilton, Phosphorylation andProtein-ProteinInteractions actor receptor NVITED TALKS 2003/2004 ecker M etherlands (2004) (2003) (2003) (2003) ( (2003) (2003) (2004) 2004) (2003)

123 Appendix Carstanjen D (2003) Knobeloch KP (2003) Die Rolle des Interferon induzierten Transkriptionsfaktors, Zytokin-Rezeptoren und Zytokin-abhängige Signalwege: ICSBP, in der Reifung und Funktion von Zellen des myelo- the role of Interferon Consensus Sequence Binding Pro- poetischen Systems tein in hematopoiesis Charitè Universitätsmedizin, Campus Benjamin Franklin. International Hannover Workshop on Cytokine Receptors Berlin, Germany and Cytokine Signaling. Hannover, Germany

Dathe M (2004) Krause E (2004) Apolipoprotein E-derived peptides and drug delivery to the Massenspektrometrie in der Proteomforschung brain Bundesanstalt für Materialforschung. Berlin-Adlershof, 7th Symposium Signal Transduction of the Blood Brain Germany Barriers. Potsdam, Germany Krause G (2004) Freund F (2003) Gemeinsamkeiten von Struktur und Funktion des Tight Molecular recognition of proline-rich sequences by the Junction Proteins Occludin und des Adherens Junction GYF domain Proteins alpha-Catenin Max-Planck-Institut für Biophysikalische Chemie. Göttin- Institut für Chemie und Pathobiochemie der Freien Univer- gen, Germany sität Berlin, Germany

Freund F (2004) Krause G (2004) Struktur-Funktionsbeziehungen wichtiger T-Cell-Proteine The tight junction protein occludin and the adherens BMBF Bundesministerium für Bildung und Forschung. junction protein alpha-catenin share a common interacti- Berlin, Germany on mechanism with ZO-1 7th International Symposium Signal Transduction in the Freund F (2004) blood-brain barriers. Potsdam, Germany Proline-rich sequence recognition by GYF domains Universität des Saarlandes. Saarbrücken, Germany Krause G (2004) Identifizierung von Wechselwirkungsepitopen zwischen Freund F (2004) Zellkontaktproteinen der tight junctions Molecular Recognition Workshop Neue Peptidtechnologien. Max Brünger Zen- Max-Planck-Institut für Biochemie. München, Germany trum, Universität Leipzig, Germany Freund F (2004) Krause G (2004) Proline-rich sequence recognition by GYF domains Structural determinants for the activation of glycoprotein Johann Wolfgang Goethe-Universität Frankfurt, Germany hormon receptors Haseloff RE (2003) Institut für Reproduktionsmedizin, Universität Münster, Alterations in the protein expression of rat brain capillary Germany endothelial cells induced by oxidative stress von Kries JP (2004) 6th Symposium Signal Transduction of the Blood-Brain Screening for beta-Catenin Antagonists Barriers. Szeged, Ungarn Max-Planck-Institute of Molecular Cell Biology and Gene- Keller S (2004) tics Dresden, Germany Can Cell-Penetrating Peptides Cross Lipid Membranes? von Kries JP (2004) Institut für Physikalische Chemie. Martin-Luther-Universi- Screening in an academic setup tät Halle, Germany EMBL Hamburg, Germany Klussmann E (2004) Leitner D (2004) Anchored cAMP signalling in the vasopressin-induced PASTE und PAPST for biomolecules aquaporin-2 shuttle in renal principal cells Spanish NMR User’s meeting. Madrid, Spain The Biotechnology Centre of Oslo. University of Oslo, Nor- way Maul B (2003) Neuropeptidasen und Alkoholsucht Zentralinstitut für Seelische Gesundheit. Mannheim, Ger- many ference. Savannah,USA 44th ENCExperimentalNuclearMagneticResonanceCon- the investigationofmembraneproteins alpha-spectrin SH3domainandthepotentialofNMRfor The solid-stateMAS-NMRstructureofthe62-residue Recognition andTransduction. Berlin,Germany 8th InternationalDahlemSymposiumonCellularSignal NMR-Spectroscopy ofmembraneproteins Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oehme P Oehme P Berlin, Germany W nen auspharmakologischerSicht Toleranz als essentiellerSchutzmechanismus-Reflektio- H Zentrum fürHuman-undGesundheitswissenschaften,FB Theoretische undklinischeAspektederSucht Euroconference onStructuralGenomics.Obernai,France lar Biology m asviewedbyNMR.Structuresof From GenetoStructure EMBL Heidelberg,Germany Solid-state NMR Protein-protein interactioninvestigated bysolutionand tic Resonance.Chamonix-Mont,France. The 3rdAlpineConferenceonSolid-StateNuclearMagne- the investigationofmembraneproteins alpha-spectrin SH3domainandthepotentialofNMRfor The solid-stateMAS-NMRstructureofthe62-residue ny 4th ColloquiumonTransport. Rauischholzhausen,Germa- NMR-Spectroscopy ofmembraneproteins Denver, USA 45th RockyMountainConferenceonAnalyticalChemistry. tion tothexspectrinSH3domain proteins by3DmagicanglespinningNMRanditsapplica- A logy. Vienna, Austria 5th International ConferenceonMolecularStructuralBio- NMR-Spectroscopy ofmembraneproteins umanmedizin derFreienUniversitätBerlin,Germany embrane proteinsbysolid-stateNMR.NMRinMolecu- concept forstructuredeterminationofsmallmembrane issenschaftliche KonferenzderLeibniz-Sozietäte.V. (2004) (2004) (2003). (2004) (2003) (2003) (2003) (2003) (2003) (2003) Max-Delbrück-Center Berlin-Buch,Germany cology Molecular Profiles-Target searchinmolecularpharma- U stigated bySolutionandSolid-StateNMR Inve- Protein-Protein InteractionsandMembraneProteins Ventura, USA Structural genomics,membraneproteinsandautomation Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Oschkinat H Technische UniversitätMünchen,Germany state NMR Protein-protein interactionsviewedbysolutionandsolid- P state NMR Protein-protein interactionsviewedbysolutionandsolid- B ning MASNMR bymagicStructure determinationofproteins anglespin- Ascona, Schweiz NMR approachestostructuresof membraneproteins Martin-Luther-Universität Halle-Wittenberg,Germany ning MASNMR bymagicStructure determinationofproteins anglespin- Charité-Universitätsmedizin Berlin,Germany ferenz Zelluläre Protein-InteraktionundpharmakologischeInter- New Jersey, USA solid-state NMR Structures ofmembraneproteinsbysolutionansolidand New York, USA NMR analysisofproteinmodules Biozentrum derUniversitätBasel,Switzerland teins bysolutionandsolid-stateNMR Viewing protein-proteininteractions andmembranepro- aris, France arossa Valley, Australia niversity ofFlorence,Italy (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004) (2004)

125 Appendix Oschkinat H (2004) Pohl P (2004) An apoE-derived peptide mediates uptake of PEG-liposo- Fluid transport through membrane channels and epitheli- mes onto brain capillary endothelial cells al cells Bad-Herrenalb, Germany Johannes Keppler Universität, Linzer Winter Workshop. Linz, Austria Oschkinat H (2004) NMR-Spectroscopy of membrane proteins Pohl P (2004) 5th International Conference on Molecular Structural Bio- Fluid transport through membrane channels and epitheli- logy. Vienna, Austria al cells: Calcium and Transport Processes Universität des Saarlandes in Homburg, Germany Oschkinat H (2004) Quality control of the vasopressin V2 receptor in the ER Rademann J (2004) and ER/Golgi intermediate compartment Polymer-unterstützte C-Acylierungen gegen Malaria 4th Action Meeting on new Drugs and Treatment. Berlin, Novabiochem Seminar, Technische Universität Berlin, Germany Germany

Oschkinat H (2004) Rademann J (2004) Aufbau der Technologieplattform NMR-Messtechnik für P1-Variation in Peptidisosteren: Molekulare Diversität die Proteomforschung durch die Kombination von C- and N-Acylierungen Braunschweig, Germany Max-Bergmann Kreis. Munster, France

Oschkinat H (2004) Rademann J (2004) Magic-angel-spinning solid-state NMR of proteins Molekulare Werkzeuge für die chemische Industrie IFIA-BioNMR. Karlsruhe, Germany Bioclub der Freien Universität Berlin. Berlin, Germany

Oschkinat H (2004) Rademann J (2004) Structure determination of proteins by magic angle spin- P1-Site Diversity in Peptide Isoster Libraries via Smooth ning MAS NMR CC-Couplings on Linker Reagents. University of Osaka, Japan ACS National Meeting. Philadelphia, USA

Oschkinat H (2004) Rademann J (2004) Design, Synthesis and Characterization of ß-Sheet-For- New linkers, resins and reagents - some problems of poly- ming peptides: The FBP 28 WW Domain mer-supported synthesis and attemps to solve them. Awaji, Japan ABC Technologies Symposium. Basel, Switzerland

Oschkinat H (2004) Rademann J (2004) Protein structure determination by magic-angle spinning Molekulare Werkzeuge für die Chemische Biologie solid state NMR (Antrittsvorlesung) Lille, France Freie Universität Berlin, Germany

Oschkinat H (2004) Rademann J (2004) Protein Structure Determination by Solid State MAS NMR New roads to Chemical Diversity: From P1-Site Mutants in Frauenchiemsee, Germany Malaria Inhibitors to Combinatorial Synthesis of Complex Modified Dendrimers Pankow K (2004) Quaid-iAzzam University. Islamabad, Pakistan Influence of the natriuretic peptide structure on degradation by NEP Rademann J (2004) Peptidase-Workshop, EMC Rotterdam, The Netherlands Diversitäts-orientierte Synthese von Glycolipiden durch hydrophob unterstützte Synthese von Glycopeptiden Pohl P (2004) Forschungszentrum Borstel, Germany Fluid transport through membrane channels and epithelial cells Rademann J (2004) Tagung der Gesellschaft für Biochemie und Molekularbio- Reversibly cross-linked dendrimers - A strategy for the logie. Münster, Germany facile synthesis and combinatorial variation of complex, multivalent protein-mimics 7th International Symposium on Biomolecular Chemistry - ISBOC-7. Sheffield, United Kingdom Il Ciocco,Italy Multidimensional NMRinStructuralBiology Forschungszentrum Karlsruhe,Germany Protons inMASSolid-StateNMR Baltimore, USA racterized bySolutionStateNMR Molecular InteractionsbetweenSup35andHsp104cha- France biomolecular NMRandneutronscattering.Grenoble, EMBO-ILL workshoponDeuteriumlabelingtechniqesfor peptides andproteins Use ofProtonsinMASSolid-StateNMRperdeuterated Principles toPathologicalImplications”.Jena,Germany IMB-Symposium „ProteinFoldingandAggregation–From state NMRexperiments m Aggregation behaviourofmisfoldingpeptidesandproteins nai, France E Sup35 andthemolecularchaperoneHsp104 Molecular Interactionsbetweentheyeastprionprotein Germany. Berlin,Germany New ConceptsinMolecularMedicine fromFinlandand BioTechnica -Workshop: InternationalBiotechnology - Drug Design Bioprofile –wohinführtderWeg? Von der Heilpflanzezum Biomedical researchandbiotechnologyinBerlin-Buch: Recognition andTransduction. Berlin,Germany VIIIth InternationalDahlemSymposiumonCellularSignal Cyclic AMP-triggeredexocytosis Denver, Colorado,USA Use ofperdeuterationinMASSolidStateNMR USA Asilomar ConferenceCenter, PacificGrove,California, peptides andproteins Use ofProtonsinMASSolid-StateNMRperdeuterated Rosenthal W Rosenthal W Reif B Reif B Reif B Reif B Reif B R Reif B Reif B uresco ConferenceonNMRinMolecularBiology. Ober- eif B odulated bymolecularchaperones:solutionandsolid- (2004) (2004) (2004) ( (2003) (2003) (2004) (2004) 2003) (2003) (2003) Peptidase-Workshop, EMC-Rotterdam,TheNetherlands ACE forms Development andPropertiesofdomain-selectivemurine Herbsttagung desHahn-Meitner-Instituts Berlin,Germany phytochromes Towards thestructureofchromophoreinbacterial Germany krinologie, DiabetologieundErnährung.Berlin/Potsdam, Eröffnung desambulantenHochschulzentrumsfürEndo- S Mechanismen derAntidiurese Germany V Berlin-Brandenburg Leuchttürme derBerlinerWissenschaft.Biotechnologiein Hahn-Meitner-Institut Berlin,Germany NMR-Spektroskopie Aspekte derStrukturbestimmungvonProteinenmittels tugal European DialysisandTransplant Association.Lisbon,Por- Annual congressoftheEuropeanRenalAssociation– mutation andrescuestrategiesofmutantreceptors The humanV2vasopressinreceptor:gene,theprotein, Schmieder P R Rosenthal W Rosenthal W Sun X Peptidase-Workshop, EMC-Rotterdam,TheNetherlands ACE andNEP-relationstoalcoholpreference Siems, WE Schülein R Schülein R Schülein R Schmieder P Germany Symposium onNeuro-ImmuneInteractioninPain.Berlin, along thesecretorypathway Intracellular transportofGprotein-coupledreceptors Symposium onInflammationandPain.Berlin,Germany corticotropin-releasing factorreceptors Functional significanceofthecleavablesignalpeptides 5th GlobalNDIConference.Phoenix,USA receptor mutantsintheearlysecretorypathway Compartmentalization ofNDI-casingvasopressinV2 eranstaltung derInitiative„AnMorgendenken“.Berlin, osenthal W ymposium FortschrittundPraxisderEndokrinologiezur (2004) (2004) (2004) (2004) (2004) ( (2003) (2003) (2004) 2004)

127 Appendix EXTERNAL FUNDING 2003/2004 DRITTMITTEL

7000 6867

6000

5000

4473

4000

3163 2989 3000

2105 2178 2000 1583

1000

1998 1999 2000 2001 2002 2003 2004

Others 11 3 6 3 5 37 28

Foundations 216 174 112 2 231 158 116

Industry 92 53 29 51 134 197 110

EU 43 74 217 122 710 414 1984

Berlin 52 54 51 0 0 0 0

BMBF 514 696 773 5422 1045 974 886

DFG 656 1050 992 1267 1073 1383 1349

Distribution of annual expenditure shown for the sources of income since 1998 Verteilung der jährlichen Ausgaben über die Drittmittelgeber seit 1998 (total expenses per year in italics) (kursiv: Gesamtausgaben pro Jahr) Expenses duringtheperiodr Drittmittelausgaben imBerichtszeitraum: V T T P P 1000 1500 2000 1000 1 1500 250 500 250 5 750 00 0 0 1349 1 F MFBri UIndustr EU Berlin BMBF DFG Others Foundations Industry EU Berlin BMBF DFG 383 eported: contributionofthesources ofincome erteilung überdieDrittmittelgeber 886 974 0 0 2003 2004 1984 414 110 197 y onain Others Foundations 116 158 28 37

129 External Funding PARTICIPATION IN RESEARCH NETWORKS Sonderforschungsbereich 366: Signalerkennung und – 2003/2004 Umsetzung BETEILIGUNG AN NETZWERKEN UND Teilprojekt A11: Verbreitung und Bedeutung N-terminaler VERBUNDPROJEKTEN 2003/2004 Signalpeptide bei G-Protein-gekoppelten Rezeptoren Ralf Schülein, Walter Rosenthal Sonderforschungsbereiche der Deutschen Laufzeit: 01.97-12.05 Forschungsgemeinschaft Sonderforschungsbereich 594: Molekulare Maschinen in Sonderforschungsbereich 449: Struktur und Funktion Proteinfaltung und Proteintransport membranständiger Rezeptoren Teilprojekt A3: Biochemische und NMR-Strukturuntersu- Teilprojekt A3: Struktur und Funktion von Transportsigna- chungen an Sup35p im Komplex mit Hsp104, Hsp40 und len des Vasopressin V2-Rezeptors Hsp70 Ralf Schülein, Walter Rosenthal Bernd Reif Laufzeit: 01.99-12.04 Laufzeit 05.01-04.04

Sonderforschungsbereich 449: Struktur und Funktion Forschergruppen der Deutschen membranständiger Rezeptoren Forschungsgemeinschaft Teilprojekt B1: Bestimmung der Raumstrukturen von Rezeptor-gebundenen Agonisten und Antagonisten mittels Forschergruppe 299: Optimierte molekulare Bibliotheken Festkörper-NMR-Spektroskopie zum Studium biologischer Erkennungsprozesse Hartmut Oschkinat Teilprojekt 2/2-1: Struktur, Stabilität und Spezifikation von Laufzeit: 01.02-12.04 nichtkatalytischen Proteindomänen und deren Verwen- dung als Werkzeuge für das Design einer stabilen minima- Sonderforschungsbereich 449: Struktur und Funktion len ß-Faltblattstruktur und das Verständnis von pathologi- membranständiger Rezeptoren schen Prozessen Teilprojekt A6: Untersuchungen von CRF- und CRF-Rezep- Hartmut Oschkinat, Michael Bienert tor-Mutanten zur Entwicklung eines Modells für die Laufzeit: 06.01-12.05 Liganderkennung von G-Protein-gekoppelten Rezeptoren der Familie 2 Forschergruppe 299: Optimierte molekulare Bibliotheken Michael Bienert, Michael Beyermann, Walter Rosenthal zum Studium biologischer Erkennungsprozesse Laufzeit: 01.99-12.04 Teilprojekt 2/2-2: Theoriegestützte NMR-spektroskopische Analyse von Protein-Ligand-Wechselwirkungen unter Ver- Sonderforschungsbereich 498: Protein-Kofaktor-Wech- wendung von Peptid-Bibliotheken selwirkungen in biologischen Prozessen Hartmut Oschkinat Teilprojekt C1: Schlüsselreaktionen der biologischen Was- Laufzeit: 06.01-12.05 serstoffaktivierung am Beispiel der [NiFe]-Hydrogenasen Hartmut Oschkinat, Bärbel Friedrich (HU) Forschergruppe 299: Optimierte molekulare Bibliotheken Laufzeit: 01.03-12.05 zum Studium biologischer Erkennungsprozesse Teilprojekt 7/2-1: Studium der Ligand-Erkennung von Sonderforschungsbereich 498: Protein-Kofaktor-Wech- CRH-Rezeptoren mit Peptid- und nichtpeptidischen Biblio- selwirkungen in biologischen Prozessen theken Teilprojekt B6: NMR-spektroskopische Untersuchungen Michael Bienert, Jens Schneider-Mergener von lichtinduzierten Strukturveränderungen in Protein- Laufzeit: 06.99-12.05 Chromophorkomplexen Peter Schmieder Forschergruppe 463: Innovative Arzneistoffe und Träger- Laufzeit: 01.03-12.05 systeme – Integrative Optimierung zur Behandlung ent- zündlicher und hyperproliferativer Erkrankungen Sonderforschungsbereich 366: Signalerkennung und – Teilprojekt 7B: Hirntargeting mittels oberflächenmodifi- Umsetzung zierter Nanosuspensionen und Apolipoprotein E-Peptid Teilprojekt Z3: Herstellung und Haltung genetisch verän- beladener Trägersysteme derter Mäuse Margitta Dathe Elvira Rohde, Ivan Horak Laufzeit: 11.01-12.07 Laufzeit: 01.09-12.05 hohem DurchsatzfürmedizinischrelevanteProteine hohem DurchsatzfürmedizinischrelevanteProteine Laufzeit: 10.99-09.07 Jörg Rademann Laufzeit: 04.03-03.07 A Laufzeit: 10.98-09.04 Hartmut Oschkinat,DirkLabudde,IliaPoliakov Laufzeit: 01.03-12.05 Ingolf Blasig satz vonbildgebendenVerfahren Verbundprojekt 0312890G: Verbundprojekt 0312992J: Bildung undForschung Verbundprojekte desBundesministeriumsfür Teilprojekt 10: 01GG9812: Proteinstrukturfabrik Teilprojekt 9: 01GG9812: Proteinstrukturfabrik Bildung undForschung Leitprojekte desBundesministeriumsfür GK 441: GK 865: G G gemeinschaft Graduiertenkollegs derDeutschenForschungs- Laufzeit: 04.04-06.06 Hartmut Oschkinat,JensvonKries zur SuchevonWirkstoffen bacterium tuberculosisGenomundihrerLigandkomplexe molekulare StrukturenvonZielproteinenausdemMyco- Laufzeit: 10.98-09.04 Hartmut Oschkinat,PeterSchmieder, DietmarLeitner Laufzeit: 04.03-03.06 Hartmut Oschkinat brangebundener Proteine Laufzeit: 10.99-09.05 Alexander Oksche,Walter Rosenthal K K lexander Oksche,Walter Rosenthal 238/3: 276: Chemie inInterphasen Vaskuläre Regulationsmechanismen S ignalerkennung und-umsetzung S chadensmechanismen imNervensystem–Ein- NMR-Spectroscopy NMR-structure determination Proteomweite Analysemem- Proteomische Methodenfür Strukturanalyse mit Strukturanalyse mit L Walter Rosenthal ders peutic strategyofurinaryincontinenceandvoitingdisor- action vasopressin–V2-receptoragonistsasanewthera- Laufzeit: 12.00-30.11.03 Hartmut Oschkinat interfere withcancer-related phenotypes s EU-Projekt QLK3-CT-2002-01989: EU-Projekt QLK3-CT-2002-02149: E EU-Projekt QLK3-2000-00924: EU-Projekte Laufzeit: 11.02-20.05 Enno Klussmann,Walter Rosenthal ling –Implicationsfortreatmentofhumandisease Laufzeit: 03.03-02.06 Michael Bienert,JohannesOehlke,MargittaDathe tides systems forgenetherapybasedoncell-penetratingpep- aufzeit: 09.01-08.04 caffolded repertoire libraries to profile cancercellsandcaffolded repertoirelibrariestoprofile U-Projekt QLRT-2000-00987: A Exploiting syntheticSH2- ntidiuretics usingshort- Anchored cAMPsignal- Target specificdelivery

131 Appendix COOPERATIONS WITH CONTRACT - Max-Planck-Gesellschaft zur Förderung der Wissen- 2003/2004 schaften e. V., München, Germany VERTRAGLICHE KOOPERATIONEN 2003/2004 - Max-Planck-Institut für Infektionsbiologie, Berlin, Ger- many Vereinbarung über die Zusammenarbeit zwischen der - Technische Universität München, Wissenschaftszen- Freien Universität Berlin und dem Forschungsverbund trum Weihenstefan, Freising, Germany Berlin e. V. für das FMP - Combinature Biopharm AG, Berlin, Germany - Freie Universität Berlin - Marresearch GmbH, Norderstedt, Germany - Biomax Informatics AG, Martinsried, Germany Vereinbarung über die Zusammenarbeit zwischen der Charité – Universitätsmedizin Berlin und dem For- Forschungs- und Entwicklungsvereinbarung schungsverbund Berlin e. V. für das FMP - IKOSATEC GmbH, Garching, Germany - Charité – Universitätsmedizin Berlin - Combinature Biopharm AG, Berlin-Buch, Germany

Kooperationsvereinbarung: Gemeinsamer Neubau und Kooperationsvereinbarung: Strukturanalyse mit hohem Nutzung des Genomzentrums Druck für medizinisch relevante Proteine - Max-Delbrück-Centrum für Molekulare Medizin, Berlin, - Proteinstrukturfabrik: Projekt des Bundesministeriums Germany für Bildung und Forschung, Germany

Kooperationsverbarung: Gemeinsame Nutzung des Her- Kooperationsvereinbarung zur Erbringung von Verwer- mann-von-Helmholtz-Hauses tungsleistungen - Max-Delbrück-Centrum für Molekulare Medizin, Berlin, - Ascenion GmbH, München, Germany Germany Kooperationsvereinbarung über die Fortsetzung der Kooperationsvereinbarung für das Verbundprojekt „Pro- Öffentlichkeitsarbeit auf dem Forschungscampus Berlin- teomweite Analyse membrangebundener Proteine (Pro- Buch Amp) - BBB Management GmbH, Berlin, Germany - Johann Wolfgang Goethe-Universität Frankfurt, Ger- many Kooperationsvereinbarung: (Forschungs- und Entwick- - GSF-Forschungszentrum für Umwelt und Gesundheit lungsvereinbarung) Identifizierung von 2-D-separierten GmbH, Germany Proteinen über In-Gel-Verdau, Massenspektrometrie und - Max-Planck-Gesellschaft zur Förderung der Wissen- Datenbankanalyse schaften e. V., Germany - Bundesinstitut für Risikobewertung, Berlin, Germany - Bundesinstitut für Verbraucherschutz und Veterinärme- Kooperationsvereinbarung: Ausbau und Intensivierung dizin, Germany der wissenschaftlichen Zusammenarbeit - Deutsches Primatenzentrum GmbH, Göttingen, Germany Kooperationsvereinbarung: Massenspektrometrische - Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Charakterisierung von Fluoreszenz-markierten Glyko- und Germany Phosphopeptiden - Heinrich-Pette-Institut für experimentelle Virologie und - Biosyntan GmbH, Berlin-Buch, Germany Immunologie, Hamburg, Germany - Forschungszentrum Borstel, Zentrum für Medizin und Forschungs- und Entwicklungsvereinbarung: Develop- Biowissenschaften, Borstel, Germany ment of isotope labelled media for protein expression in - Institut für Molekulare Biotechnologie e.V., Jena bacteria, yeast, insect cells and higher organisms - Cambridge Isotope Laboratories, Massachusetts, USA Kooperationsvereinbarung: Automatisierte Methoden für molekulare Strukturen von biologischen Makromolekü- Nutzungs- und Dienstleistungsvereinbarung: Gemein- len aus dem Mycobakterium-tuberculosis-Genom und same Nutzung von Forschungsgeräten und technologi- ihrer Ligandkomplexe zur Suche von Wirkstoffen mit schen Einrichtungen Hochdurchsatz Chiptechnologien (Strukturgenomprojekt) - Charité-Universitätsmedizin Berlin, Germany - European Molecular Biology Laboratory (EMBL), Ham- burg und Heidelberg, Germany Combinature BiopharmAG,Berlin,Germany Infrastrukturnutzungsvereinbarung TheUniversityWisconsin, Madison,USA - Kooperationsvereinbarung UniversityofOxford,UK - NMR Kooperationsvereinbarung: Structuredeterminationby ScheringAG,Berlin,Germany - Prof. Dr. H.Oschkinat Kooperationsvereinbarung fürAuftragsmessungen fürdasDeutsche Humangenom- Ressourcenzentrum - PD Kooperationsvereinbarung: NutzungderPrimärdatenbank PFSbiotechAG,Berlin,Germany - Infrastrukturnutzungsvereinbarung NationalResearchCouncilofCanada,Montreal - sels toTreat Stroke the Blood-BrainBarrierinHypoxia-Targeting BrainVes- Kooperationsvereinbarung: MolecularFingerprintingof Max-Delbrück-CentrumfürMolekulareMedizin,Berlin - Collaboration AgreementmitderAGOschkinat - Derivaten undAnaloga Kooperationsvereinbarung: Transfer vonNaturstoffen, FreieUniversitätBerlin,Germany - R Kooperationsvereinbarung überdieZusammenarbeitim - senschaftsnetz G.WiN gungsstrecken alsZugangsleitungenzumGigabit-Wis- Kooperationsvereinbarung zurNutzungvonÜbertra- ahmen vonProjekten projekt, Germany many V. ,Ger- Hans-Knöll-Institut fürNaturstoff-Forschunge. V., Berlin,Germany zes e. Verein zurFörderungeinesDeutschenForschungsnet- Dr. IEBlasig InstitutfürPflanzengenetikundKulturpflanzenfor- - Saale, InstitutfürPflanzenbiochemie(IPB),Hallea.d. - - IfN,Magdeburg,Ger- Leibniz-InstitutfürNeurobiologie - Heinrich-Pette-InstitutfürExperimentelleVirologie und - - Hans-Knöll-Institut für Naturstoff-Forschung (HKI), Hans-Knöll-InstitutfürNaturstoff-Forschung - Borstel(FZB)–ZentrumfürMedi- Forschungszentrum - - (DDZ)anderHeinrich- DeutschesDiabetesZentrum - Kooperationsvereinbarung China-Konsortium schung (IPK),Gatersleben,Germany Germany Jena, Germany Leibniz-Institut fürMolekulareBiotechnologie(IMB), many Immunologie (HPI),Germany J zin undBiowissenschaften,Borstel,Germany Heine-Universität, Düsseldorf,Germany Deutsches PrimatenzentrumGmbH(DPZ),Göttingen ena, Germany

133 Appendix MEETINGS, WORKSHOPS, SYMPOSIA 2003/2004 WISSENSCHAFTLICHE VERANSTALTUNGEN

2003 2004

6th Symposium Signal Transduction in the Blood-Brain 34. Jahrestagung der Deutschen Gesellschaft für Immu- Barriers nologie Blasig IE, Haseloff RF Horak I Szeged/Ungarn Berlin, Henry Ford Building, Germany September 2003 September 2004

Berlin Magnetic Resonance Seminar and High-Field NMR Festsymposium zur Einweihung des 900-MHz-Spektrome- Facility Users Meeting ters am FMP Oschkinat H Oschkinat H, Steuer A, Maul B Forschungsinstitut für Molekulare Pharmakologie, Berlin- Max-Delbrück-Communications Center, Berlin-Buch, Ger- Buch, Germany many December 2003 July 2004 4th Progress report meeting, EU-Project: Anti-diuresis using short-acting vasopressin-V2-receptor agonists as a 7th International Symposium on Signal Transduction in the new therapeutic strategy of urinary incontinence and voi- Blood-Brain Barriers ding disorders Blasig IE, Haseloff RF Klussmann E, Rosenthal W, Lauterjung U Potsdam, Germany Magnus-Haus, Berlin, Germany September 2004 June 2003 EMBO Practical Course "Multidimensional NMR in Struc- 1st Progress report meeting, EU-Project: Anchored cAMP tural Biology II” signalling / RTD grant - Implication for treatment of human Oschkinat H (Co-Organisator) diseases Ciocco, Italy Klussmann E, Rosenthal W, Lauterjung U September 2004 Magnus-Haus, Berlin, Germany June 2003 25. Tagung des Max-Bergmann-Kreises 6. Deutsches Peptidsymposium (mit internationaler Betei- Bienert M ligung) Munster, France Bienert M, Dreissigacker M, Dathe M October 2004 Humboldt-Universität zu Berlin, Germany March 2003

8th International Dahlem Symposium on Cellular Signal Recognition and Transduction, Berlin Rosenthal W Charite - Universitätsmedizin Berlin, Germany June 2003 SchatzmeisterimVerbund biowissenschaftlicher und - LeiterdesInstitutsfürPharmakologie,Charité-Univer- - KuratoriumsmitgliedderBerlin-Brandenburgischen - BeiratsdesHans- MitglieddesWissenschaftlichen - WissenschaftlicherSekretär desMax-Bergmann-Krei- - Michael Bienert MitglieddesBeiratsin derTransgenic CoreFacilityder - derArbeitsgruppeTierhaltung Sprecher derGemein- - Praxisdes OmbudsmannfürGuteWissenschaftliche - DirektorderForschungseinrichtung Wissenschaftlicher - Ivan Horak PSFBioAG,Berlin MitglieddesAufsichtsrats derFirma - Hartmut Oschkinat Stellvertretender SprecherderSektionCLebenswis- - MitgliedimKuratoriumdesDeutschenInstitutsfür - W GREMIENARBEIT 2003/2004 W alter Rosenthal biomedizinischer Gesellschaften(vbbm) sitätsmedizin Berlin F Knöll-Insituts, Jena ses RCC GenbiotecGmbH,CampusBerlin-Buch 3(3)UniMedG samen Kommissionnach§ Forschungsverbundes Berline.V. für ExperimentelleMedizin,FUBerlin s Ernährungsforschung, Potsdam-Rehbrücke ORK INPANELS 2003/2004 ortbildungsakademie (BBFA) enschaften derLeibnizGemeinschaft Blasig, Ingolf PeptRes - JMolRecognit - JAmChemSoc - JPeptSci - AminoAcids - JMassSpec - Tetrahedron Lett - - OrgLett - Journals Humboldt-UniversitätzuBerlin - - Research Institutions DeutscheForschungsgemeinschaft - Organisations B GUTACHTERTÄTIGKEIT 2003/2004 R BiochimBiophysicaActa - EurJBiochem - JPepRes - Journals TheIsraelScienceFoundation - Organizations Dathe, Margitta Bonemarrowtransplantation - Journals Carstanjen, Dirk NeurochemicalResearch - JNeurochem - JPharmPharmacol - JNeurosci - Glia - Neurochemistry - Journals Charité-UniversitätsmedizinBerlin - UniversitätPotsdam - Philipps-UniversitätMagdeburg - Research Institutions AssoziazoneItalianaperlaRicercaCancro - JubiläumsfondÖsterreichischeNationalbank - ResearchGrantCouncilHongKong - DeutscheForschungsgemeinschaft - Organisations ienert, Michael EVIEW ACTIVITIES2003/2004 Drug DesignReview-Online Universität Halle

135 Appendix - Comb Chem Klussmann, Enno - Biophys J Organisations - J Biol Chem - Health Research Board, Ireland - New J Chem Journals - Biochem J - JCI - Regul Peptides - Glia - Org Biomol Chem - Biol of the Cell - Biol Cell Freund, Christian - Neuroscience Letters Organisations - J Cell Sci - University of Oxford - Kidney Int Journals - J Biol Chem - Regul Peptides - Eur J Cell Biol

Hagen, Volker Krause, Eberhard Research Institutions Journals - Bayrische-Maximilians Universität Würzburg - J Anal Chem - J Pept Sci Journals - J Mass Spec - Angew Chemie - Biochemistry - ChemBioChem - J Am Chem Soc Krause, Gerd - Blood Journals - Eur J Med Chem - QSAR Journal - J Mol Struct - J Comput Aid Mol Des

Haseloff, Rainer Meyer, Thomas Journals Research Institutions - J Photochemistry and Photobiology B-Biology - Georg-August-Universität Göttingen - Free Radicals Research Journals Horak, Ivan - Circulation Organisations - Deutsche Forschungsgemeinschaft Oehlke, Johannes - Deutsche Krebshilfe Journals - Boehringer Ingelheim Fonds - Biochim Biophys Acta - Eur J Biochem Research Institutions - Yorkshire Cancer Research, UK Oschkinat, Hartmut - Universität zu Lübeck Organisations - Taussig Cancer Center, The Cleveland Clinic Foundation - Deutsche Forschungsgemeinschaft Journals - Österreichische Akademie der Wissenschaften - J Biol Chem - Schering AG - Immunity - Swiss Federal Institute of Technology Zürich - Eur J Immunol - Studienstiftung Bonn - J Mol Cell Biol - Schweizerischer Nationalfond zur Förderung von - Blood Wissenschaftlicher Forschung - Nucleic Acids Research - FWF Der Wissenschaftsfond - Journal Leukemia Psychopharmacology - JCardiovascPharma - EurJPharmacol - Vasc Pharmacol - Journals Richter, Regina ProcNatlAcadSciU.S.A. - TIPS - BioMedCentral - JBioelectrochemicalSociety - EurJBiochem - BiophysJ - Biochemistry - Journals Humboldt-UniversitätzuBerlin - Christian-Albrechts-UniversitätzuKiel - Research Institutions Wellcome Trust - Organisations Pohl, Peter GLIA - Journals Piontek, Jörg ProcNatlAcadSciU.S.A. - Biochemistry - Science - NatStructBiol - JAmChemSoc - - JMagnReson - JMolBiol - Journals - Charité-UniversitätsmedizinBerlin - EcoleNormaleSupérieuredeLyon - Humboldt-UniversitätzuBerlin - - Ludwig-Maximilians-UniversitätMünchen - FreieUniversitätBerlin - Research Institutions J Eidgenössische Technische HochschuleZürich Bayerische-Maximilians UniversitätWürzburg Biol NMR KidneyInt - TheLancet - FEBSLett - EurBiophysJ - EurJBiochem - EJB - EurJofCellBiol - EncyclopediaofBiologicalChemistry - HormoneResearch - Pharmacol&Toxicol - Biologyofthecell - HumanMolecularGenetics - NephrologyDialysisTransplantation - NeuroscienceLetters - Journals FachhochschuleLausitz - OregonHealthandScienceUniversityPortland - UniversitätdesSaarlandes - Christian-Albrechts-UniversitätzuKiel - BayerischeMaximilians-UniversitätMünchen - UniversitéMontpellier - InstitutfürZoo-undWildtierforschung - Eberhard-Karls-UniversitätTübingen - EidgenössischeTechnische HochschuleZürich - Philipps-UniversitätMarburg - - FreieUniversitätBerlin - Research Institutions Dellùniversita`EDellaRicera MinisterioDell`Istruzione, - - ÖsterreichischerWissenschaftsfond - BoehringerIngelheim - FakultedeScienceChile - - DeutscheForschungsgemeinschaft - Organisations Rosenthal, Walter Biotechniques - FEBSLett - Journals Schülein, Ralf Chemistry(WileyVCH) - JMagnReson - ChemBioChem - Journals Studienstiftungdesdeutschen Volkes - Organizations Schmieder, Peter Technische UniversitätBerlin/DRFZ Alexander vonHumboldtStiftung Ärztekammer Berlin

137 Appendix Siems, Wolf-Eberhard ACADEMIC TEACHING 2003/2004 Journals LEHRE 2003/2004 - J Mol Med Beyermann, Michael Utebergenov, Darkhan Biophysik-Praktikum zum Biacore-Gerät Journals Freie Universität Berlin - J Neurochem Bienert, Michael Vinkemeier, Uwe Vorlesung Proteine und Peptide Organizations Humboldt-Universität zu Berlin - European Molecular Biology Organizations Vorlesung zum Biophysikpraktikum (Biacore-Gerät) - Boehringer Ingelheim Fonds Freie Universität Berlin - Österreichische Akademie der Wissenschaften - DAAD Blasig, Ingolf Journals Vorlesung Funktionelle Biochemie - Biochim Biophys Acta Universität Potsdam - Biochem Biophys Res Comm Mastercourse „Medical Neurosciense“. Reguläre Vorle- - Dev Cell sung, Seminare und Praktikum - EMBO J Universität Potsdam - Eur J Biochem - FEBS Left Mastercourse „Medical Neurosciense“. Fakultative Vor- - J Cell Biol lesung und Seminar - Mol Cell Biol Universität Potsdam - Nucleic Acids Res - Proc Natl Acad Sci U.S.A. Dathe, Margitta Biophysik für Studenten der Biochemie: CD-Spektrosko- pie Freie Universität Berlin

Freund, Christian Vorlesung Protein Engineering Freie Universität Berlin

Vorlesung Molekulare Immunologie Freie Universität Berlin

Horak, Ivan Vorlesung Knock-Out-Mäuse Freie Universität Berlin

Keller, Sandro Vorlesung Biophysik für Studenten der Biochemie: Iso- therm Titration Calorimetry Freie Universität Berlin

Klussmann, Enno Kursus der Allgemeinen Pharmakologie und Toxikologie Charite-Universitätsmedizin Berlin

Vorlesung Molekulare Pharmakologie und zelluläre Signal- transduktion: A-Kinase-Ankerproteine Charite-Universitätsmedizin Berlin Freie UniversitätBerlin Vorlesung BiologischeNMR-Spektroskopie Oschkinat, Hartmut Universität Göttingen gischer Erkrankungen Vorlesung PathologieundKlinikinternistischerchirur- Universität Göttingen Praktikum InnereMedizin Universität Göttingen Vorlesung InnereMedizinfürZahmediziner Meyer, Thomas Humboldt-Universität zuBerlin Messungen) Kursus MikroskopischeTechniken (IntrazelluläreCa2+- Lorenz, Dorothea Charite-Universitätsmedizin Berlin zelluläre Signaltransduktion Psychopharmakologie imPraktikumMolekulareund Charite-Universitätsmedizin Berlin Spezialkurs Sucht Krause, W informatics Technical UniversityofAppliedSciences,Berlin,FBBio- Vorlesung GrundlagenMolecularModelling Technical UniversityofAppliedSciences,Berlin Vorlesung GrundlagenMolecularModelling Krause, Gerd Charite-Universitätsmedizin Berlin teomics) transduktion –MassenspektrometrieProteinanalytik(Pro- Vorlesung MolekularePharmakologieundzelluläreSignal- K Charite-Universitätsmedizin Berlin für Humanmediziner Kursus derAllgemeinenPharmakologieundToxikologie Charite-Universitätsmedizin Berlin Pharmazeuten transduktion fürBiochemiker, Biologen,Medizinerund Vorlesung MolekularePharmakologieundzelluläreSignal- Charite-Universitätsmedizin Berlin transduktion: Affinitätspräzipationstechniken Vorlesung MolekularePharmakologieundzelluläreSignal- rause, Eberhard Johannes-Kepler UniversitätLinz Übungen zurPhysik Johannes-Kepler UniversitätLinz Vorlesung BiophysikI Johannes-Kepler UniversitätLinz Vorlesung BiophysikIII Humboldt-Universität zuBerlin Vorlesung Biomechanik Humboldt-Universität zuBerlin Vorlesung ExperimentelleBiophysik H Vorlesung Mikroskopie Pohl, Peter Charite-Universitätsmedizin Berlin Neuropharmakologie Vorlesung AnwendungderMikrodialysetechnikin Richter Regina Freie UniversitätBerlin Vorlesung BioorganicandNaturalProductChemistry Freie UniversitätBerlin Synthese Vorlesung AktuelleMethodenderBioorganischen Universität Tübingen Vorlesung ChemiefürBiologen Quaid-i-Azzam University, Islamabad Vorlesung CombinatorialChemistry Freie UniversitätBerlin Vorlesung Stereochemistry Universität Tübingen Vorlesung Heterocyclen Rademann, Jörg Freie UniversitätBerlin s Vorlesung GrundlagenderbiologischenNMR-Spektro- Freie UniversitätBerlin biologischen NMR-Spektroskopie V Freie UniversitätBerlin Vorlesung BiophysikalischeMethoden kopie orlesung GrundlagenundneueTechniken der umboldt-Universität zuBerlin

139 Appendix Rosenthal, Walter Vorlesung Molekulare Pharmakologie und zelluläre Signal- Vorlesung Gentherapie I transduktion Charite-Universitätsmedizin Berlin Charité-Universitätsmedizin Berlin

Vorlesung Weitere alternative Therapieformen Vinkemeier, Uwe Charite-Universitätsmedizin Vorlesung Aktuelle Themen der zellulären Siganltrans- Vorlesung Pharmakokinetik duktion Charite-Universitätsmedizin Berlin Freie Universität Berlin

Vorlesung Arzneimittel-Metabolismus/Pharmakogenetik Vorlesung Mechanismen der molekularen Signalverarbei- Charite-Universitätsmedizin Berlin tung Freie Universität Berlin Vorlesung Gentherapie II (Mechanismen) Charite-Universitätsmedizin Berlin Praktikum Mechanismen der Signalverarbeitung Freie Universität Berlin Vorlesung Stammzellen Charite-Universitätsmedizin Berlin Wiesner, Burkhard Vorlesung Homöopathie Laser Scanning Mikroskopie: Möglichkeiten und Grenzen Charite-Universitätsmedizin Berlin optischer Methoden zur Untersuchung subzellulärer Pro- zesse Vorlesung Placebo Freie Universität Berlin Charite-Universitätsmedizin Berlin Konfokale Mikroskopie (Molekulare Pharmakologie und Vorlesung Antivirale Therapie, Antigenese, Gentherapie zelluläre Signaltransduktion) Charite-Unviersitätsmedizin Berlin FU/FMP

Schmieder, Peter Molekulare Pharmakologie und zelluläre Signaltransduk- Vorlesung Molekulare Pharmakologie und zelluläre Signal- tion transduktion FU/FMP Freie Universität Berlin Mikroskopische Techniken (FRAP, FREt, Reflexionsmes- Vorlesung Multidimensionale NMR-Spektroskopie-Grund- sungen) lagen und Anwendungen in der Strukturaufklärung HU/FMP Technische Universität Berlin Laser Scanning Mikroskopie: Möglichkeiten und Grenzen Vorlesung Molekulare Pharmakologie und zelluläre Signal- optischer Methoden zur Untersuchung subzellulärer Pro- transduktion zesse Technische Universität Berlin Freie Universität Berlin

Grundlagen und Anwendungen der Mehrdimensionalen NMR-Spektroskopie Technische Universität Berlin

Schülein, Ralf Kursus der Allgemeinen Pharmakologie und Toxikologie Charite-Universitätsmedizin Berlin

Vorlesung Molekulare Pharmakologie und zelluläre Signal- transduktion für Biochemiker, Biologen, Mediziner und Pharmazeuten Chartie-Universitätsmedizin Berlin

Kursus der allgemeinen Pharmakologie Charité-Universitätsmedizin Berlin CALLS FOR APPOINTMENTS 2003/2004 POSTDOCTORAL LECTURE QUALIFICATIONS RUFE 2003/2004 2003/2004 HABILITATIONEN 2003/2004 Hermosilla, Ricardo (2003) Juniorprofessur für Pathologie der Signaltransduktion Oksche, Alexander (2003) Molekulare Grundlagen ange- am Institut für Pharmakologie der Charite-Universitäts- borener und erworbener polyurischer Störungen medizin Berlin, Germany Charité-Universitätsmedizin Berlin

Pohl, Peter (2004) Schülein, Ralf (2003) The early secretory pathway of mem- Lehrstuhl für Biophysik brane proteins: clinical and pharmacological implications Technisch-Naturwissenschaftliche Fakultät der Johannes- Charité-Universitätsmedizin Berlin Kepler-Universität Linz

Pires, Ricardo (2002) Associated Professor for Biochemistry and Structural Bio- logy Instituto De Ciencas Biomédicas, Universidade Federal do Rio de Janeiro, Brasil x i d n e p p A 1 4 1 GRADUATIONS 2003/2004 Thielen, Anja (2004) Identifizierung transportrelevanter PROMOTIONEN 2003/2004 Aminosäurereste im proximalen C-Terminus des humanen Vasopressin-V2-Rezeptors Alken, Martina (2004) Functional significance of N-termi- Freie Universität Berlin nal signal peptides of G protein-coupled receptors Storm Robert (2004) Extracellular osmolality and solute Freie Universität Berlin composition participate in the expressional regulation of Andreeva, Anna (2004) Protein kinase C isoform anta- Aquaporin-2 in renal inner medullary collecting duct cells. gonism controls occludin phosphorylisation and tight Evidence for an involvement of the TonE/TonEBP pathway junction assembly Freie Universität Berlin Freie Universität Berlin Wessolowski, Axel (2004) Amphipathische Hexapeptide - Barth, Michael (2004) Entwicklung neuer, hochbeladener Interaktion mit Membranen Trägermaterialien für die organische Festphasensynthese Freie Universität Berlin auf Basis von vernetztem Polyethylenimin – Anwendung im Zimmermann, Jürgen (2004) Struktur und Funktion von Bereich der Peptidsynthese, für Polymerreagenzien und EVH1-Domänen zur Synthese von peptidfunktionalisierten Dendrimeren Freie Universität Berlin Eberhard Karls Universität Tübingen Zühlke, Kerstin (2003) Strukturelle und funktionelle Bedeu- Begitt, Andreas (2004) Nukleocytoplasmatischer Transport tung der konservativen Disulfidbrücke des Vasopressin- und Geninduktion durch den Transkriptionsfaktor STAT1 V2-Rezeptors Freie Universität Berlin Freie Universität Berlin Boisguerin, Prisca (2004) Characterization of PDZ Domain/ Ligand Specifity Freie Universität Berlin

Castellani, Federica (2004) Structure determination of immobilized proteins by solid-state NMR spectroscopy Freie Universität Berlin

Eckhardt, Torsten (2004) Design, Synthese, Photochemie und biologische Anwendung von ausgewählten „caged“ cyclischen Adenosin-3, 5´-monophosphaten Humboldt-Universität zu Berlin

Edemir, Bayram (2004) Klonierung und Charakterisierung einer neuen AKAP18-Isoform, AKAP18δ, und ihre mögliche Beteiligung an der AVP-vermittelten AQP2-Translokation Freie Universität Berlin

Meyer, Thomas (2004) Nukleäre Akkumulation und Ziel- generkennung von STAT1-Transkriptionsfaktoren Freie Universität Berlin

Osiak, Anna (2004) Die Rolle der ubiquitinähnlichen Gene UBL 14 und ISG15 in vivo Freie Universität Berlin

Sauer, Ines (2004) Apolopoprotein E - abgeleitete Peptide als Vektoren zur Überwindung der Blut-Hirn-Schranke Freie Universität Berlin

Serowy, Steffen (2004) Migration von Protonen entlang der Oberfläche ebener Bilipidmembranen Humboldt-Universität zu Berlin DIPLOMA THESES 2003/2004 Kubald, Sybille DIPLOMARBEITEN 2003/2004 Extrazelluläre Domäne des Rezeptors für den Cortico- tropin-Releasing-Faktor: Klonierung in Expression eines N- 2003 Terminus-Schleife-Konstrukts als Bindungsmodell Technische Fachhochschule Berlin Brandenburg, Martin Kerntransportverhalten trunkierter STAT-Porteine Lassowski, Brigitte Technische Fachhochschule Berlin Untersuchungen zur Clonierung und Reinigung von Tight junction-Proteinen Hahn, Janina Universität Potsdam Untersuchungen zu Chromophor-Protein-Interaktionen in ortsspezifischen Mutanten von Phytochrom Cph1 aus Cya- Renner, Armin nobakterium Synechocystis Funktionelle Bedeutung des Signalpeptides des Cortico- Freie Universität Berlin tropin-Releasing-Faktor Rezeptors 2a Julius-Maximilians-Universität Würzburg Heine, Markus Wasserstoffperoxyd-induzierte Veränderungen der Aktivi- Roswadowski, Inga tät und Expression ausgewählter Proteine in Epithelzellen Untersuchungen zur Struktur und Funktion von Tight Freie Universität Berlin Junction-Proteinen Technische Fachhochschule Berlin Paschke, Carmen Erweiterte Peakformanalyse zur Verbesserung der auto- Schröder, Stephan matischen Resonanzzuordnung von NMR-Spektren Derivatisierung phophorylierter Peptide zur Erhöhung der Fachhochschule Lausitz Ionisierungen der Massenspektrometrie x i d

Technische Universität Berlin n Schröder, Stephan e p p

Derivatisierung phosphorylierter Peptide zur Erhöhung der Zech,Tobias A Signalintensität in der Massenspektrometrie Functional studies of the adapter protein CD2BP2 Technische Universität Berlin Freie Universität Berlin 3 4 Thurisch, Boris 1 Strategien zur gezielten Expressions-Inhibition des muri- nen FMIP-Gens Technische Fachhochschule Berlin

Wendt, Norbert Strukturuntersuchungen der Disulfidstruktur an N-termi- nalen CRF-Rezeptoren Technische Universität Berlin

2004

Cubic, Ivona Charakterisierung von Proteinen mittels Kapillar-HPLC und Massenspektrometrie Technische Fachhochschule Berlin

El-Dashan, Adeeb Milde C-Acylierungen von polymergebundenen Carbox- ylatphosphoranen für Anwendungen in der Medizinischen Chemie Eberhard-Karls-Universität Tübingen

Kotzur, Nico Synthese und Photochemie von photolabilen cAMP-Deri- vaten Humboldt-Universität zu Berlin INTERNSHIPS 2003/2004 Cubic, Ivona PRAKTIKANTEN 2003/2004 01.09.2003–29.02.2004 Technische Fachhochschule – Biochemie Ballmer, Boris Supervision: Dr. E. Krause 10.11.2003–05.12.2003 Curth, Sebastian Freie Universität Berlin – Biochemie 14.06.2004–10.09.2004 Supervision: Dr. Berger Technische Fachhochschule Berlin – Biotechnologie Behling, Katja Supervision: Dr. Donalies 01.06.2004–25.06.2004 Elß, Franka Freie Universität Berlin – Biochemie 04.10.2004–25.03.2005 Supervision: Dr.Berger Fachhochschule Magdeburg – Pharmakologie Behnken, Swantje Supervision: Dr. E. Krause 16.08.2004–08.10.2004 Erel, Funda Universität Potsdam – Biochemie 01.02.2003–31.03.2003 Supervision : Dr. Donalies Vorpraktikum Biotechnologie Bibow, Stefan Supervision: Dr. Klußmann 01.05.2004–31.07.2004 Ernst, Oliver Humboldt-Universität zu Berlin – Biophysik 20.10.2003–19.04.2004 Supervision: Prof. Reif Technische Universität Berlin – Biotechnologie Bieberstein, Andrea Supervision: Dr. Donalies 06.10.2003–20.02.2004 Femmer, Christian Fachhochschule Lausitz – Biotechnologie 01.08.2004–15.10.2004 Supervision: Dr. Donalies Technische Universität Berlin – Biotechnologie Blick, Kristin Supervision: Dr. Klose 12.05.2003–20.06.2003 Gartmann, Marco Freie Universität Berlin – Biochemie 20.10.2003–31.12.2003 Supervision: Dr. Berger Universität Potsdam – Biochemie Böthe, Matthias Supervision: Dr. Haseloff 08.12.2003-30.01.2004 Gasser, Carlos Freie Universität Berlin – Biochemie 14.05.2004–16.07.2004 Supervision: S. Keller Freie Universität Berlin – Biochemie Böthe, Matthias Supervision: Dr. Pohl 01.07.2004–11.08.2004 Giebel, Sebastian Freie Universität Berlin – Biochemie 01.06.2004-16.07.2004 Supervision: Dr. Dathe Freie Universität Berlin – Biochemie Brückner, Kathrin Supervision: S. Keller 07.04.2003–09.05.2003 Groß, Anika Technische Universität Dresden – Chemie 02.02.2004–12.03.2004 Supervision: Dr. Siems Freie Universität Belin – Pharmazie Christian, Frank Supervision: Dr. Schmieder 15.09.2003–24.10.2003 Güngör, Volkan Freie Universität Berlin – Biochemie 01.02.2004–30.04.2004 Supervision: Dr. Klussmann Berufsanfänger Supervision: H.-J. Mevert Guse, Katrin Kamitz, Anne 19.04.2004-04.06.2004 08.01.2004–02.04.2004 Freie Universität Berlin – Biochemie Abiturientin Supervision: S. Keller Supervision: Dr. Vinkemeier

Han, Soeng-Ji Kieseritzky, Gernot 19.07.2004-03.09.2004 01.03.2004–09.04.2004 Freie Universität Berlin – Biochemie Freie Universität Berlin – Biochemie Supervision: S. Keller Supervision: Dr. Pohl

Hamidzadek, Nadja Kirsch, Jenny 27.01.2003–27.03.2003 13.04.2004–30.04.2005 Weiterbildung (Arbeitsamt) – Biochemie Universität Potsdam – Biochem. Supervision: Dr. Labudde Supervision: Dr. Blasig

Heinze, Mathias Kotzur, Nico 01.02.2004–31.03.20004 01.03.2004–31.12.2004 Humboldt-Universität zu Berlin Humboldt-Universität Berlin – Chemie Supervision: T. Jahn Supervision: Dr. Hagen

Heuberger, Julian Kraemer, Florian 08.11.2004–14.12.2004 01.08.2004–24.09.2004 Freie Universität Berlin – Biochemie Universität Potsdam – Biochemie Supervision: Dr. Dathe Supervision: Prof. Horak x i d n

Hübner, Florian Kron, Anja e p p

01.11.2004-18.02.2005 01.09.2003–30.01.2004 A Technische Fachhochschule Berlin Fachhochschule – Biotechnologie Supervision: Dr. Pohl Supervision: Dr. Leitner 5 4 Hühn, Stephan Kronsbein, Helena 1 29.03.2004-07.05.2004 10.03.2003–28.03.2003 Freie Universität Berlin – Biochemie Technische Universität München – Biochemie Supervision: S. Keller Supervision: Dr. Schmieder

Jahnke, Nadine Krüger, Kerstin 01.06.2004-16.07.2004 15.04.2003–14.10.2003 Freie Universität Berlin – Biochemie Rharmazie-Praktikantin Supervision: S. Keller Supervision: Dr. Siems

Jehle, Stefan Krüger, Magnus 11.08.2003–24.10.2003 01.11.2004–30.04.2005 Freie Universität Berlin – Biochemie Freie Universität Berlin – Pharmazie Supervision: Prof. Oschkinat Supervision: Dr. Beyermann

Kalmbach, Norman Kuhn, Ramona 06.01.2003–31.01.2003 04.08.2003–26.09.2003 Technische Fachhochschule Berlin – Biotechnologie Technische Universität – Berlin – Umweltschutz Supervision: Dr. Vinkemeier Supervision: Dr. Wiesner

Kaltofen, Sabine Lassowski, Birgit 21.07.2003–10.10.2003 01.01.2003–31.03.2003 Fachhochschule Zittau Universität Potsdam – Biotechnologie Supervision: Dr. Krabben Supervision: Dr. Blasig Leddin, Mathias Overath, Thorsten 01.08.2003–31.01.2004 01.09.2003–26.10.2003 Freie Universität Berlin – Bio Technische Universität Berlin – Biotechnologie Supervision: Dr. Knobeloch Supervision: Dr. Siems

Lisewski, Ulrike Overath, Thorsten 14.04.2004–18.05.2004 23.02.2004–14.05.2004 Freie Universität Berlin – Biochemie Technische Universität – Biotechnologie Supervision: Dr. Pohl Supervision: Dr. Siems

Marter, Katrin Petrovska, Silvija 05.05.2004–31.10.2004 15.11.2004–10.12.2004 Freie Universität Berlin – Biologie Skopje-Mazedonien Supervision: Dr. Haseloff Supervision: Dr. Siems

Mehmedi, Burhan Pietske, Matthias 24.11.2003–26.12.2003 19.07.2004-03.09.2004 Freie Universität Berlin – Biochemie Universität Potsdam – Biochemie Supervision: Dr. Siems Supervision: S. Keller

Mitschke, Doreen Prigge, Matthias 02.06.2003–11.07.2003 24.09.2003–07.11.2003 Freie Universität Berlin – Biochemie Freie Universität Berlin – Biochemie Supervision: Dr. Klußmann Supervision: Dr. Pohl

Müller, Jeanette Reibitz, Franziska 19.04.2004-04.06.2004 01.02.2004–31.07.2004 Freie Universität Berlin – Biochemie Pharmazeutin Supervision: S. Keller Supervision: Dr. Siems

Narayanan, Raghav Roswadowski, Inga 17.05.2004–07.08.2004 01.06.2004–30.09.2004 Neu Delhi Technische Fachhochschule Berlin – Biotech. Supervision: Dr. Freund Supervision: Dr. Blasig

Neumann, Christine Santamaria, Katja 23.02.2004–02.04.2004 19.02.2003–28.02.2003 Universität Marburg – Biologie Freie Universität Berlin Supervision: Dr. Vinkemeier Supervision: Dr. Klussmann

Neumann, Marleen Schäfer, Zasia 03.02.2003–08.03.2003 06.09.2004–08.10.2004 Technische Fachhochschule Berlin – Biotechnologie Universität Potsdam – Biochemie Supervision: Dr. Wiesner Supervision: Dr. Carstanjen

Niehage, Christian Schlede, Stephanie 01.08.2004–31.10.2004 04.08.2003–29.08.2003 Freie Univerisät Berlin – Biochemie Universität Potsdam – Biotechnologie Supervision: Dr. Blasig Supervision: Dr. Siems

Oehlke, Elisabeth Schmidt, Marco 12.07.2004–30.08.2004 08.12.2003–31.01.2004 Freie Universität Berlin – Chemie Freie Universität Berlin – Biochemie Supervision: Dr. Hagen Supervision: Dr. Hagen Supervision: Dr. Blasig FU Berlin–Biochemie 07.01.2003–15.02.2003 Strohschein, Susan Supersision: Dr. Knobeloch Fachhochschule Lausitz–Biotechnologie 01.09.2003–30.01.2004 Stenzel, Denise Supervision: Dr. Siems Freie UniversitätBerlin–Biochemie 25.05.2004–06.07.2004 Stengel, Florian Supervision: Dr. Hagen Humboldt-Universität zuBerlin–Chemie 17.02.2003–19.03.2003 Socher, Elke Supervision: Dr. Siems Freie UniversitätBerlin–Biochemie 03.03.2003–28.03.2003 Seifert, Christoph Supervision: Dr. Blasig Universität Potsdam–Biochemie 01.07.2004–30.04.2005 Schuster, Ariane Supervision: Dr. Beyermann Technische FachhochschuleBerlin.–Chemietechnologie 15.09.2004–28.02.2005 Schumann, Franziska Supervision: Dr. Diehl Freie UniversitätBerlin–Biologie 01.06.2004–31.07.2004 Schumacher, Anne S Humboldt-Universität zuBerlin–Biologie 04.08.2003–12.09.2003 Schumacher, Anne S Charitè-Universitätsmedizin Berlin 01.12.2004–28.02.2005 Schulz, Katrin S Technische UniversitätBerlin–Biotechnologie 24.02.2003–14.03.2003 Schröder, Kati upervision: Dr. Krabben upervision: Dr. Blasig upervision: M.Alken Supervision: Dr. Siems Technische FachhochschuleBerlin–Biochemie 26.07.2004–26.08.2004 Winkler, Franziska Supervision: Dr. Krabben Freie UniversitätBerlin:Biotechnologie 29.03.2004–13.08.2004 Wellmann, Anke S Freie UniversitätBerlin–Biochemie 08.03.2004–07.05.2004 Vorwinkel, Jakob S Technische HochschuleBerlin–Biotechnologie 19.10.2004–11.01.2005 Trippens, Jessica S Italien –Biotechnologie 07.06.2004–27.08.2004 Teodorczyk, Marcin Supervision: Dr. Dathe Hochschule Anhalt–PharmazeutischeTechnologie 25.10.2004–18.03.2005 Zimmerling, Katrin Supervision: Dr. Vinkemeier Universität Regensburg–Biochemie 25.08.2003–26.09.2003 Zschörnig, Barbara Supervision: Dr. Blasig Fachhochschule Lausitz–Biotechnologie 01.09.2003–30.01.2004 Zuleger, Nikolaj Supervision: Dr. Wiesner Universität Potsdam–Biotechnologie 01.01.2003–30.06.2003 Wolkenhauer, Jan Supervision: Dr. Blasig Universität Potsdam–Biologie 13.04.2004–12.07.2004 Wolf, Constanze upervision: Dr. Pohl upervision: Dr. Carstanjen upervision: Dr. Klussmann

147 Appendix GUEST SCIENTISTS 2003/2004 2004 GASTWISSENSCHAFTLER 2003/2004 Alexandrov, Alexej 01.11.2003–15.03.2004 2003 Kazan University, Russia Antonenko, Juri Antonenko, Juri 30.01.2003–27.02.2003 30.04.2004–30.05.2004 01.11.2003–30.11.2003 01.10.2004–31.10.2004 Belozerski Insitut Moscow, Russia Belozerski Institute, University of Moscow, Russia Alexandrov, Alexej Andreeva, Anna 15.03.2003–15.09.2003 01.04.2004–31.05.2004 01.11.2003–31.05.2004 Russia, Scholarship holder Kazan University, Russia Arbuzova, Anna Ayuyan, Artem 19.04.2004–28.05.2004 13.08.2003–30.11.2003 University of New York in Stony Brook, USA Academy of the Sciences, Moscow, Russia Barany-Wallje, Elsa Balla, Zsolt 08.09.2003–29.02.2004 01.09.2003–14.09.2003 University of Stockholm, Sweden University of Debrecen, Hungary Coin, Irene Barany-Wallje, Elsa 01.10.2004–31.10.2004 08.09.2003–29.02.2004 Italy, Scholarship holder University of Stockholm, Sweden Khanfar, Monther Lents, Alexander 08.07.2004–19.09.2004 24.07.2003–28.09.2003 University of Zarga, Hashemite, Jordanien Uni Academy of the Sciences Moscow, Russia Kumari, Neha Pechanova, Olga 01.05.2004–31.12.2004 25.06.2003–20.07.2003 India, Scholarship holder Academy of the Sciences Slovakia, Bratislava Lents, Alexander Paulis, Ludovit 11.08.2004–11.10.2004 17.11.2003–21.11.2003 Frumkin Institute for Electrochemistry, University of Mos- Academy of the Sciences Slovakia, Bratislava cow, Russia Pires, Jose R. Magalhaes de Souza, Crista 01.11.2003–31.01.2004 01.01.2004–30.11.2004 University of Rio de Janeiro, Brasil Oswald Criz Institute, Rio de Janeiro, Brasilia Woineshet, Zenebe Pires, Richardo 25.06.2003–20.07.2003 01.11.2003–31.01.2004 Academy of the Sciences Bratislava, Slovakia 01.12.2004–28.02.2005 Sokolov, Valerij University of Rio de Janeiro, Brasil 20.06.2003–18.07.2003 Schreibelt, Gerty 01.10.2003– 31.10.2003 03.05.2004–25.09.2004 Academy of the Sciences Moscow, Russia VU Medical centre Amsterdam, The Netherlands Universität ofLinz,Austria 1 Sommer, Klaus cow, Russia Frumkin InstituteforElectrochemistry, UniversityofMos- 1 Sokolenko, Elena cow, Russia Frumkin InstituteforElectrochemistry, UniversityofMos- 1 18.10.2004 11.03.2004–11.04.2004 Sokolov, Valerij 5.11.2004–28.01.2005 1.08.2004–11.10.2004 7.11.2004 25.11.2003 teins, Structure, FunctionandRegulationofTight JunctionPro- gie, Berlin-Buch,Germany) Host: SiemsWE 24.11.2003 me usingmonoclonalantibodies Structure-function studiesofangiotensin-convertingenzy- cago, USA) 11.03.2003 domänen Struktur- undBindungsstudienextracellulärerRezeptor- Pharmakologie, Berlin-Buch,Germany) Beyermann, Michael Berger, Hartmut Beitz, Eric B KOLLOQUIEN UNDSEMINAREAMFMP2003 L Danilov, SergeiM Carstanjen, Dirk Blechert, Siegfried Blasig IE Host: OschkinatH 17.07.2005 tion Metathese undWirkstoffchemie–einestarkeKombina- Universität Berlin,Germany) 14.01.2003 GS/GI couplingoftheCRFreceptorstype1inHEKcells makologie, Berlin-Buch,Germany) 09.12.2003 poetischen Systems ICSBP inderReifungundFunktionvonZellendesmyelo- Die RolledesInterferoninduziertenTranskriptionsfaktors makologie, Berlin-Buch,Germany) Host: KlussmannE 0 Aquaporin mediatedwaterpermeabilityintheinnerear sität Tübingen,Germany) H 26.02.2003 Tight junctionproteins Austria) 7.10.2003 ECTURES AT THEFMP2003 ost: BlasigIE auer, Hans (Forschungsinstitut fürMolekularePharmakolo- (Institut fürPharmazeutischeChemie,Univer- ( Institut fürMolekularbiologie,Salzburg, (Forschungsinstitut fürMolekularePhar- (Forschungsinstitut fürMolekularePhar- (Anestesiology ResearchCenter, Chi- (Institut fürChemiederTechnischen (Forschungsinstitut fürMolekulare

149 Appendix Dathe, Margitta (Forschungsinstitut für Molekulare Phar- Hagen, Volker (Forschungsinstitut für Molekulare Pharma- makologie, Berlin-Buch, Germany) kologie, Berlin-Buch, Germany) ApoE-Peptid-Lipidkomplexe: Trägermodelle für eine Wirk- Caged compounds: Design and applications stoffaufnahme ins ZNS 13.05.2003 14.10.2003 Hauri, Hans-Peter (Universität Basel, Switzerland) Dieckmann, Torsten (University of California) Protein traffic early in the secretory pathways: dynamics Mechanism of action of Rab GTPases in intracellular vesi- and signals cular protein transport 23.09.2003 02.07.2003 Host: Hermosilla R Host: Oschkinat H Heinz, Dirk (Department of Structural Biology, Braun- Diehl, Anne (Forschungsinstitut für Molekulare Pharma- schweig, Germany) kologie, Berlin-Buch, Germany) Bacterial invasion at atomic resolution Protein production for structure determination by NMR 16.09.2003 and X-ray Host: Rosenthal W 25.02.2003 Heise, Bernd (Institut für Experimentelle Physik der Uni- Duffy, Heather S. (Albert-Einstein College of Medicine, versität Ulm, Germany) New York, USA) Structural investigations on peptaibols via liquid and solid pH dependent inter- and intramolecular interactions on state NMR connexin43: Regulation of a junctional complex 18.03.2003 02.09.2003 Host: Oschkinat H Host: Blasig IE Helm, Volkhard (MPI Frankfurt, Germany) Folkers, Gerd (Eidgenössische Technische Hochschule Studying protein-protein interactions in silico Zürich, Switzerland) 11.02.2003 Designing the Locks and Creating new Keys: Molecular Host: Freund C Design Methodology for Genetic Switches Hennemann, Hanjo (Center of advanced european studies 06.05.2003 and research, Bonn, Germany) Host: Bienert M Ras recruitment system: analysis of protein function and Freund, Christian (Forschungsinstitut für Molekulare Phar- protein networks and its high throughput application makologie, Berlin-Buch, Germany) 21.10.2003 GYF and AH3 domain mediated interactions in eukayotic Host: Rosenthal W signaling Hermosilla, Ricardo (Forschungsinstitut für Molekulare 10.06.2003 Pharmakologie, Berlin-Buch, Germany) Glockshuber, Rudi (Eidgenössische Technische Hoch- Intracellular degradation pathways of wild-type and trans- schule Zürich, Switzerland) port-defective mutant vasopressin V2 receptors Catalysis of disulfide bond formation in Escherichia coli 09.07.2003 21.01.2003 Hougardy, Stefan (Institut für Informatik der Humboldt-Uni- Host: Bienert M versität zu Berlin, Germany) Goody, Roger (Max-Planck-Institut für Molekulare Physio- Three-Dimensional similarity of Small Molecules logie, Dortmund, Germany) 13.11.2003 Mechanisms of actions of Rab GTPases in intracellular Host: Vinkemeier U vesicular protein transport Johnson, Nils (Forschungszentrum Karlsruhe, Germany) 01.07.2003 Analysis of protein network in living cells Host: Oschkinat H 02.12.2003 Gottschalk, Kay (Weizmann Institute of Science, Israel) Host: Vinkemeier U Computational Approaches to Transmembrane Protein Structure 27.11.2003 Host: Reif B Host: OschkinatH 23.09.2003 Structural GenomicsoftheEukaryoteArabidopsisthaliana 30.09.2003 NMR amMembranprotein makologie, Berlin-Buch,Germany) Host: OschkinatH 10.01.2003 Techniques formodernsolid-stateNMR Cambridge, UK) 08.04.2003 tages andlimitsofstructuralbioinformatics relationships:Advan- Predictions ofstructure-function kologie, Berlin-Buch,Germany) 11.02.2003 in theAVP-indused shuttlingofaquaporin Protein kinaseAanchoringproteinsandRhoareinvolved m Markley, JohnL. Lang, Christine Ladizhansky, Vladimir Kühne, Ronald Krause, Gerd Krause, Eberhard K Klussmann, Enno Jordt, Sven 22.04.2003 trometry Structural analysisofpeptidesandproteinsbymassspec- makologie, Berlin-Buch,Germany) 24.06.2003 cular dynamicsandmodel-basedliganddesign Luteinizing HormoneReleasingReceptor:Mole- makologie, Berlin-Buch,Germany) Host: SchadeM 30.04.2003 as hosts genomicsusingyeast Production ofproteinsforstructural Technischen UniversitätBerlin,Germany) Host: RosenthalW 16.12.2003 R The PainGate–FunctionalDomainsintheCapsaicin cology, UCSF, USA) rabben, Ludwig eceptor akologie, Berlin-Buch,Germany) (Department ofCellular&MolecularPharma- (Forschungsinstitut fürMolekularePharma- (Forschungsinstitut fürMolekularePhar- (Institut fürMikrobiologieundGenetikder (University ofWisconsin-Madison,USA) ( (Forschungsinstitut fürMolekularePhar- (Forschungsinstitut fürMolekularePhar- Forschungsinstitut fürMolekularePhar- (Francis BitterMagnetLaboratory, Host: RosenthalW 20.03.2003 and variousothersignalingproteins C-TAK1 protein KSR isaregulatoroftheMAPKscaffold USA) Host: DatheM 12.06.2005 Krankheit Apolipoprotein E,LipidstoffwechselunddieAlzheimersche ny) Host: SiemsWE 13.01.2003 tionelle BedeutungfürdieSpermatogenese E logie, Giessen,Germany) Host: PohlP 2 Proton Transfer alongHydrogen-BondedNetworks Universität Bochum,Germany) Müller, Jürgen M Monsees, Thomas Marx, Dominik Scharnagl, Hubert Reiser, Oliver Palczewski, Krysztof Multhaup, Gerd Host: BienertM 13.02.2003 Inhibitor Arglabin, Farnesyltransferase- einvielversprechender sität Regensburg,Germany) Host: KrauseG 10.04.2003 brate rhodopsin Structure, functionandmembraneorganisationofverte- University ofWashington, USA) Host: ReifB 26.08.2003 dogenesis Implication ofamyloidprecursorproteinligandsinamyloi- Universität Berlin,Germany) Host: PohlP tinic receptorbyphotoaffinitylabeling,12.12.2003 Investigation oftheconformationaltransitionsnico- France) 0.05.2003 xpression vonKininrezeptorenimRattentestis:Einefunk- ourot, Alexandre (Institut fürOrganischeChemiederUniver- (Lehrstuhl fürTheoretischeChemie,Ruhr- (Institut fürChemieundBiochemie,Freie (National CancerInstituteatFrederick, (Universitätsklinikum Freiburg,Germa- (Zentrum fürDermatologieundAndro- ( Laboratoire DeChimieBioorganique, (Department ofOphtalmologythe

151 Appendix Schmalz, Hans-Günther (Universität zu Köln, Germany) Stubbs, Milton T. (Institut für Biotechnologie, Martin- Mediated synthesis of biologycally relevant small mole- Luther-Universität Halle, Germany) cules Understanding protein - ligand interactions: Effects of fle- 27.03.2003 xibility Host: Oschkinat H 18.02.2003 Host: Oschkinat H Schmidt, Gundula (Albert-Ludwigs-Universität Freiburg, Germany) Vinkemeier, Uwe (Forschungsinstitut für Molekulare Phar- Mechanism and function of Rho targeting bacterial toxins makologie, Berlin-Buch, Germany) 19.08.2003 Regulation der subzellulären Verteilung von STAT1 Host: Rosenthal W 27.05.2005

Schmieder, Peter (Forschungsinstitut für Molekulare Phar- Zamora, Salvador Ventura (Departemente de Bioquimica i makologie, Berlin-Buch, Germany) Biologia Molecular, University of Barcelona, Spain) Strukturen von GPCRs mit Lösungs-NMR Short amino acid sequences can trigger protein amyloid 22.07.2003 formation in globular proteins 15.07.2003 Schubert, Mario (University of British Columbia in Vancou- Host: Reif B ver, Canada) Insights into mRNA degradation in E. coli - The S1 domain of Rnase E 19.06.2003 Host: Oschkinat H

Schülein, Ralf (Forschungsinstitut für Molekulare Pharma- kologie, Berlin-Buch, Germany) The early secretory pathway of G protein-coupled receptors: clinical and pharmacological implications 28.01.2003

Serrano, Luis (European Molecular Biology Laboratory, Heidelberg, Germany) Sequence determinants of amyloid formation 20.10.2003 Host: Oschkinat H

Siems, Wolf-Eberhard (Forschungsinstitut für Molekulare Pharmakologie, Berlin-Buch, Germany) NEP und ACE, zwei „alte“ Enzyme mit vielen neuen Chan- cen 16.12.2003

Sinning, Irmgard (Biochemie-Zentrum Heidelberg, Germany) Expression of G-protein coupled receptors in the eye of transgenic Drosophila 04.03.2003 Host: Rosenthal W

Stiles, Brad (Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Freiburg, Germany) Biotoxins: Antrax to Venoms 14.02.2003 Host: Rosenthal W Host: KlussmannE 1 heterotrimeric G-proteins Regulation ofvesicularneurotransmitter transporters by Universitätsmedizin Berlin,Germany) Host: OschkinatH 15.11.2004 schaften undMolekularbiologie BIOMINERALISATION. Von BiologiezuMaterialwissen- Martinsried, Germany) Host: OschkinatH 03.05.2004 NMR onMetalloproteininStructuralGenomics Florence, Italy) 13.04.2004 and MolecularDynamics P makologie, Berlin-Buch,Germany) Host: BienertM 10.02.2004 Critical oligomersinproteinmisfoldingandaggregation ny) Host: KühneR 18.05.2004 Conformation DatabasesforVirtual Screening technik, Berlin,Germany) Host: KrauseG 02.03.2004 tors: SimilaritiesandDifferences Pharmacology ofThyrotropin-ReleasingHormoneRecep- Gershengorn, Marvin Gast, Klaus Cordes, Frank Boelens, Wilbert Banci, Lucia Baeuerlein, Edmund Appelt, Christian A KOLLOQUIEN UNDSEMINAREAMFMP2004 L Host: ReifB 06.04.2004 in cellularstressregulation Involvement ofthesmallheatshockproteinaB-crystallin Biochemistry, TheNetherlands) 7.02.2004 eptide-lipid interaction:structuralinvestigationsbyNMR ECTURES AT THEFMP2004 hnert-Hilger, Gudrun (Max-Delbrück-Center, Berlin-Buch,Germa- (Centro RisonanzeMagnetiche,Universityof (Konrad-Zuse-Zentrum fürInformations- (Forschungsinstitut fürMolekularePhar- (University ofNjimegen,Department (Max-Planck-Institut fürBiochemie, (NIH/NIDDK Bethesda,USA) ( Institut fürAnatomiederCharitè- 07.12.2004 Recognition CodeofGYFdomains FMP, Engineering NWGProtein makologie, Berlin-Buch,Germany) H 07.09.2004 molekulare Vielfalt derTRP-Kanäle Kationenkanäle aufderSuchenachneuenFunktionen:Die C Host: KühneR 07.12.2004 CCR1 receptorantagonistsfromthebenchtoClinic Henning, Mirko Heise, Tilmann Harteneck, Christian Gohla, Antje Kofler, Michael John, Susan Viola Hundsrucker, Christian/Weber, Host: BlasigIE 15.06.2004 The fateofCell-cellcontactsinapoptoticepithelialcells ny) Huber, Otmar Horuk, Richard Host: RosenthalW 30.03.2004 n Regulation ofcytoskeletaldynamicsbyChronophin,a logy, LaJolla,USA) 07.12.2004 Peptide DisruptorsofAKAP-PKAInteractions für MolekularePharmakologie,Berlin-Buch,Germany) Host: Vinkemeier U 16.11.2004 Regulating thetranscriptionalactivityofSTAT5 Host: VinkemeierU 05.10.2004 essential forcellcycleprogression The LaProtein,amultifunctionalRNAbindingprotein Germany) Host: ReifB 02.06.2004 Labeling Methodologyand19FNMRofFluorinatedRNA Skaggs InstituteofChemicalBiology, LaJolla,USA) ovel unconventionalcofilinphosphatase harité UniversitätsmedizinBerlin,Germany) ost: RosenthalW (King's College,London,UK) (Departments ofImmunologyandCellBio- (Charité UniversitätsmedizinBerlin,Germa- (Berlex AG,Richmont,Californien,USA) (Forschungsinstitut fürMolekularePhar- (Department ofMolecularBiology&The (Heinrich-Pette-Institut Hamburg, (Institut fürPharmakologieder (Forschungsinstitut

153 Appendix Kungl, Andreas (Universität Graz, Switzerland) Oelgeschläger, Thomas (Marie Curie Research Institut, Biophysical Investigations of Chemokine-Receptor and Transcription Laboratory, Oxted, UK) Co-Receptor Interactions Core promoter-specific regulation of RNA polymerase II 06.01.2004 transcription Host: Oschkinat H 04.05.2004 Host: Vinkemeier U Labudde, Dirk/Leitner, Dietmar (Forschungsinstitut für Molekulare Pharmakologie, Berlin-Buch, Germany) Overduin, Michael (University of Birmingham, UK) Tools and concepts for automation of protein structure Membrane binding domains: the good, the bad, and the determination by NMR ugly 09.03.2004 14.04.2004 Host: Oschkinat H Langer, Gernot (Schering AG, Berlin, Germany) Early Drug Discovery - Target identification, Assay deve- Pardo, Leonardo (Universidad Autonoma de Barcelona, lopment & High throughput screening 16.11.2004 Spain) Host: Kühne R Bioinformatic Approaches Leading to an Understanding of Livett, Bruce G (University of Melbourne, Australia) the Structure and Activity of G-protein coupled receptors Beauty and the Beast: molecular Prospecting for Novel 30.11.2004 Drugs from the Sea: the discovery, properties and deve- Host: Kühne R lopment of the novel analgesic, conotoxin Vc1.1 (ACV1) Rapp, Wolfgang (Rapp Polymer GmbH, Tübingen, 26.01.2004 Germany) Host: Oehme P Ein universelles Linkerkonzept zur sequenziellen Abspal- Llinas, Miguel (Department of Chemistry of the Carnegie tung von Peptiden und Erzeugung von geschützten Pepti- Mellon University, Pittsburgh, USA) damiden Is it NMR or is it crystallography? CLOUDS: a direct 06.05.2004 method for protein structure elucidation via NMR proton Hosts: Bienert M, Beyermann M densities Reif, Bernd (Forschungsinstitut für Molekulare Pharmako- 15.06.2004 logie, Berlin-Buch, Germany) Host: Oschkinat H Misfolding proteins and molecular chaperones by solution Lorenz, Dorothea (Forschungsinstitut für Molekulare Phar- and solid-state NMR makologie, Berlin-Buch, Germany) 10.11.2004 Electron microscopy in cell biology. Methods and perspec- Rosen, Michael (Southwestern Medical Center Dallas, tives University of Texas, USA) 24.02.2004 Structural and Biochemical Mechanisms of Signal Inte- Luy, Burkhard (Institut für Organische Chemie und Bioche- gration by the Wiskott-Aldrich Syndrome Protein mie II der Technischen Universität München, Germany) 25.03.2004 Structural Investigations on the Pulmonary Surfactant Host: Oschkinat H Associated Lipopeptide SP-C and Novel NMR-Techniques Salditt, Tim (Institut für Röntgenphysik, Universität Göttin- Concerning Small Molecules in Organic Solvents gen, Germany) 29.07.2004 Probing the Structure, and Interactions of Polypeptides in Host: Reif B Lipid Bilayers by X-ray and Neutron Scattering Marg, Andreas (Forschungsinstitut für Molekulare Phar- 12.03.2004 makologie, Berlin-Buch, Germany) Host: Freund C STAT – Abwege Scott, John D. (FRS, Oregon Health & Sciences University, 25.05.2004 Portland, USA) Mayer, Thomas (Max-Planck-Institute of Biochemistry, The Molecular Architecture of Signal Transduction com- Department of Cell Biology, Martinsried, Germany) plexes Small molecules: versatile probes to study mitotic kinesins 05.02.2004 05.10.2005 Host: Klussmann E Host: von Kries J 22.06.2004 from MD-simulations S makologie, Berlin-Buch,Germany) 23.03.2004 terial phytochromeCph1d2 Biophysical investigationsontheN-terminusofcyanobac- makologie, Berlin-Buch,Germany) Host: PohlP 28.10.2004 oncogenic c-H-Ras Inhibitor ofapoptosisproteinsurvivinisupregulatedby 11.05.2004 on ofthewaterchannelaquaporin-2 mediated waterreabsorbtionbyregulatingthelocalizati- Phosphodiesterase pde4isinvolvedinthevasopressin- makologie, Berlin-Buch,Germany) Host: BienertM u Erzwungene Interkalation–WiemanBasenlückenstopft Wüstner, Daniel Hosts: ReifB,OschkinatH rimental ConstraintsfromSolidStateNMR13.10.2004 Structure ofUnfoldedProteinsandAmyloidFibrils:Expe- Bethesda, USA) Tycko, Robert Strauss, Holger Stefan, Eduard Sommer, Klaus Soderhäll, Arvid Seitz, Oliver Seelig, Joachim Host: BienertM 0 Detergents, PeptidesandMicrodomainsinMembranes land) Hosts: RosenthalW, BienertM 01.12.2004 Transport undfunctionofcholesterolinepi many) 4.02.2004 nd MutationeninDNAnachweist19.10.2004 uggested functionoftheantimicrobialcyc-RWpeptide (Humboldt-Universität zuBerlin,Germany) (Laboratory ofChemicalPhysics,NIDDK, (Medizinische UniversitätWien,Austria) (Forschungsinstitut fürMolekularePhar- (Forschungsinstitut fürMolekularePhar- (Max-Delbrück-Center, Ger- Berlin-Buch, (Forschungsinstitut fürMolekularePhar- (Biozentrum UniversitätBasel,Switzer-

155 Appendix TECHNOLOGY TRANSFER 2003/2004 Inventions and patents Erfindungen und Schutzrechte Also in 2003 and 2004, the FMP submitted patent applications for economically promising research results, asses- Hagen V, Bauer PJ sed the prospects for commercial success and, where Als Verknüpfungsreagenzien einsetzbare dimaleinimido- appropriate, initiated commercialization activities. Due to substituierte Dihydroxyalkane und Verfahren zu deren Her- the proximity to biotech companies on the Berlin-Buch stellung campus and in the Berlin-Brandenburg region, excellent DE 195 33 867 C1 (filed 24.04.1997) conditions for the utilization of research results are pro- EP 96938012.0 (filed 02.05.2003) vided. PCT/DE96/01742 US 09/043,263 (filed 15.06.1999) To further optimize commercialization activities, the FMP priority establishing patent application: 13.09.1995 entered a contract agreement with Ascension GmbH in Munich. Since that time Ascension has been assessing Hagen V, Kaupp UB inventions of FMP employees with regard to patents and Neue photolabile 8-substituierte cyclische Nucleotidester, especially to market-relevant aspects. Verfahren zu ihrer Herstellung und Verwendung DE 195 29 025.9 The FMP currently holds eight registered or granted EP 96927617.9-1270 patents on a total of four inventions. Applications for addi- WO 97/05155 tional patents have been submitted. priority establishing patent application: 29.07.1995

TECHNOLOGIETRANSFER 2003/2004

Auch in den Jahren 2003 und 2004 hat das FMP für wirt- schaftlich vielversprechende Forschungsergebnisse Schutzrechte angemeldet, Verwertungschancen geprüft und gegebenenfalls Maßnahmen zur kommerziellen Ver- wertung ergriffen. Durch die räumliche Nähe zu biotech- nologischen Firmen auf dem Campus Berlin-Buch und in der Region Berlin/Brandenburg sind optimale Vorausset- zungen für die Anwendung von Forschungsergebnissen gegeben.

Um die Verwertungsaktivitäten weiter zu optimieren, wurde im August 2004 eine Zusammenarbeit mit der Ascenion GmbH (München) vertraglich vereinbart. Seit- dem bewertet die Ascenion Erfindungen von FMP-Mitar- beitern hinsichtlich patent- und vor allem marktrelevanter Gesichtspunkte.

Das FMP hält derzeit acht eingetragene oder erteilte Schutzrechte auf insgesamt vier Erfindungen. Für weitere Erfindungen sind Anmeldeverfahren zur Erteilung von Schutzrechten eingeleitet.

FMP-inventors in bold. priority establishingpatentapplication: 7.2.2003 10/526.768 US 502327850 JP EP 03750490.9-2405 CA PCT/EP2003/09892 PCT/EP2003/09892 DE 10306085.5 und Verwendung dieser Neue SpleißvarianteeinesProteinkinaseA-Ankerproteins Rosenthal W, KlußmannE,OkscheA priority establishingpatentapplication:17.01.2002 EP 03702426.2-2107 PCT/EP2003/000240 10201791.3 DE Bindungsdomäne ons-Signalpeptiden (dsNLS)abgeleitetausderSTAT-DNA Verwendung dimer-spezifischen von Nuclear-Lokalisati- Meyer T, Vinkemeier U priority establishingapplication:8.12.2000 2002109.5(filed05.04.2001) DE Tablett fürFeuchtkammer(Gebrauchsmuster) Maric K priority establishingpatentapplication:09.03.2000 EP 01916923.4 US 10/221,064 JP 002003527899T2 PCT/DE01/00955 DE einem Bildschirm reale durchTumorbildung undderenDarstellungauf Verfahren zurErmittlungvonVerschiebungen der Hirna- Labudde D priority establishingpatentapplication:11.09.2001 US 10/799,447 PCT/EP02/09959 EP 02777009.8 10144661.6-33(filed14.08.2003) DE nale vonPolypeptiden Vorrichtung undVerfahren zurZuordnungderNMR-Sig- S Labudde D,LeitnerSchubertM,WinterR,OschkinatH, priority establishingpatentapplication:20.04.2000 US 01/03512 J EP 01929464.4-2110 PCT/EP01/03512 DE 10021256A1 w Nucleotiden, Verfahren zuderenHerstellungundihreVer- Neue, PhotolabileCumarinylmethylestervoncyclischen Hagen V P chmieder P endung 2001-578448 100 13360.6(filed22.07.2004) , Kaupp B,Bendig, Wiesner B rights soldin2004 n Neue, peptidomimetische,oralverfügbareLHRHAntago- priority establishingpatentapplication:14.07.2000 US 10/333,082 EP 01954035.0 PCT/EP01/08065 DE 10035867A1 de FusionsproteinesowiederenVerwendung Neue Nucleus-Export-Signalpeptide(NES),sieenthalten- Vinkemeier U Siems W Soderhäll A,KühneR priority establishingpatentapplication:29.06.2004 D und ProteinkinaseA-Ankerprotein Peptide zurInhibitionderInteraktionvonProteinkinaseA Rosenthal W, KlußmannE,HundsruckerC FMP-inventors inbold. priority establishingpatentapplication:17.09.2004 EP 04077629.6 employing geneactivation Hyperactive Statmoleculesandtheiruseinassays Vinkemeier U,MeyerT, MargA priority establishingpatentapplication:20.09.2004 DE 102004046327.1 Transportprozessen Verfahren zurDetektionvonnukleozytoplasmatischen Vinkemeier U,MeyerT priority establishingpatentapplication:10.2.2004 PCT/EP2004/001462 EP 04090039.1 scription factors oligomerization, andnuclearaccumulationofStatTran- A Vinkemeier U,MeyerT priority establishingpatentapplication:12.3.2003 PCT/DE2004/000491 10311984.1-41 DE sationskrankheiten l Verwendung vonNEP-assoziiertenMolekülenzurBehand- ung vonnichtimmunogenen-nichthypertensivenZivili- isten mitTetrahydrocarbazol-Grundkörper 102004031579.5 E single residuemodulatesTyrosine Dephosphorylation, , Walther T, MelzigMF , et al.

157 Appendix STRUCTURE OF THE FORSCHUNGSINSTITUT FÜR MOLEKULARE PHARMAKOLOGIE (FMP)

Forschungsverbund Public Relations Berlin e. V. Björn Maul Director Walter Rosenthal Staff Council Safety Officer Burkhard Wiesner Hans-Ulrich Heyne

Structural Cellular Signalling / Chemical Administration, Biology Molecular Genetics Biology Technical and Scientific Services

Peptide Chemistry Protein Structure Cellular Signalling Molecular Genetics Administration & Biochemistry Hartmut Oschkinat Walter Rosenthal Ivan Horak Thomas Ellermann Michael Bienert

Solution NMR Protein Trafficking Cytokine Signaling Peptide Synthesis Peter Schmieder Ralf Schülein Klaus P. Knobeloch Michael Beyermann Computer Services Thomas Jahn Peptide Lipid Interaction/ Structural Molecular Anchored Signalling Peptide Transport Bioinformatics Myelopoiesis Enno Klußmann Margitta Dathe Gerd Krause Dirk Carstanjen Technical Services Johannes Oehlke Hans-Jürgen Mevert Molecular Cellular Signal Peptide Biochemistry Modelling Cellular Imaging Processing Hartmut Berger Ronald Kühne Burkhard Wiesner Library Uwe Vinkemeier Michael Beyermann Renate Peters Mass Spectrometry Solid State NMR Molecular Cell Eberhard Krause Bernd Reif Physiology Ingolf E. Blasig Animal Housing Regina Richter Synthetic Organic Protein Engineering Biochemical Biochemistry Christian Freund Volker Hagen Neurobiology Microdialysis Wolf-Eberhard Siems Service Regina Richter Medicinal Chemistry Jörg Rademann DNA Sequencing Service Screening Unit Erhard Klauschenz Jens Peter von Kries O Molekulares Modelling RGANIGRAMM Forschungsverbund Protein Engineering B Hartmut Oschkinat P Festkörper-NMR Strukturbiologie Christian Freund urkhar P Lösungs-NMR eter Schmieder Ronald Kühne Bioinformatik Gerd Krause roteinstruktur Strukturelle Betriebsrat elne V. Berlin e. Bernd Reif d Wiesner Wolf-Eberhard Siems Anchored Signalling Burkhard Wiesner Protein-Trafficing Walter Rosenthal Enno Klußmann Zellphysiologie Z Ingolf E.Blasig Neurobiologie Biochemische R Zell-Imaging ell-Signalling Molekulare alf Schülein Zell-Signalling/Molekulare Genetik Zell-Signalling/Molekulare Molekulare Genetik Signalverarbeitung Klaus P. Knobeloch Z Uwe Vinkemeier ytokin-Signalling Walter Rosenthal Dirk Carstanjen Myelopoese Molekulare Ivan Horak Zelluläre Direktor Synthetische Or aktion/Peptidtransport Massenspektrometrie Medizinische Chemie Jens PetervonKries Chemische Biologie M P Johannes Oehlke Peptidbiochemie Eberhard Krause Peptidchemie & ichael Beyermann Michael Bienert eptid-Lipid-Inter- Jör Peptidsynthese Hartmut Berger Screening-Unit Margitta Dathe V Biochemie Biochemie olker Hagen g Rademann ganische Verwaltung, technische und wissenschaftliche Öffentlichkeitsarbeit Mikrodialyseservice Hans-Jürgen Mevert Technischer Service Michael Beyermann Hans-Ulrich Heyne Erhard Klauschenz Thomas Ellermann Computer-Service DNA-Sequenzier- Arbeitssicherheit Regina Richter Regina Richter Renate Peters Thomas Jahn Verwaltung Tierhaltung Björn Maul Bibliothek Dienste service

159 Structure of the FMP INDEX D Dasari, Muralidhar ...... 29 A Dathe, Margitta ...... 70, 75, 76 Al-Gharabli, Samer ...... 90 Dekowski, Brigitte ...... 87 Alken, Martina ...... 40 Diehl, Anne ...... 13 Andreeva, Anna Y...... 49 Djurica, Maja ...... 63 Antonenko, Yuri ...... 57 Donalies, Ute ...... 40 Appelt, Christian ...... 16, 18 Dreissigacker, Marianne ...... 70 Ayuyan, Artem ...... 57 E B Ehrlich, Angelika ...... 74, 75, 95 Ball, Linda ...... 13 Eichhorst, Jenny ...... 48 Balling, Rudolf ...... 2 Eilemann, Barbara ...... 50 Bárány-Wallje, Elsa ...... 76 Eisenmenger, Frank ...... 24, 26 Barth, Michael ...... 90 El-Dashan, Adeeb ...... 90 Bauer, Jörg ...... 90 Ellermann, Thomas ...... 106 Becker, Matthias ...... 55 Erdmann, Christoph ...... 95 Beck-Sickinger, Annette G...... 2 Evers, Heide ...... 14 Begitt, Andreas ...... 66 Benedict, Melanie ...... 63 F Ben-Slimane, Uta ...... 33 Fälber, Katja ...... 13 Berger, Hartmut ...... 70, 80 Fechner, Klaus ...... 82 Beyermann, Michael ...... 70, 72 Fink, Uwe ...... 29 Bienert, Michael ...... 70, 75, 76 Flinders, Jeremy ...... 13 Blasig, Ingolf ...... 49 Fossi, Michele ...... 13 Blasig, Rosel ...... 64 Freund, Christian ...... 31, 32 Blick, Helmut ...... 101 Blum, Christopher ...... 43 G Bohne, Kerstin ...... 64 Geelhaar, Andrea ...... 43 Boisguerin, Prisca ...... 13 Geißler, Daniel ...... 87 Boldt, Liane ...... 60 Georgi, Monika ...... 82 Brauße, Kerstin ...... 107 Gomoll, Michael ...... 43 Bräutigam, Matthias ...... 2 Göritz, Petra ...... 101 Brockmann, Christoph ...... 13, 25, 26 Griesinger, Christian ...... 2 Buschner, Michael ...... 101 H Hackel, Uwe ...... 101 C Hagen, Volker ...... 70, 86 Carstanjen, Dirk ...... 62 Hahn, Janina ...... 16, 18 Cartier, Regis ...... 66 Handel, Lilo ...... 14 Castellani, Federica ...... 13 Hartmann, Gislinde ...... 50 Chen, Zhongjing ...... 29 Haseloff, Reiner F...... 49 Chernogolov, Alex ...... 29 Hausbeck, Dana ...... 107 Chevelkov, Veniamin ...... 29 Heine, Markus ...... 50 Coin, Irene ...... 74 Heinrich, Nadja ...... 82 Heinze, Matthias ...... 31, 33 Henn, Volker ...... 43 Hermann, Ingrid ...... 108 Heuer, Katja ...... 32, 33 Heyne, Alexander ...... 108 Heyne, Hans-Ulrich ...... 101 Hiller, Matthias ...... 13 umrw ad ...... 66 ...... Kummerow, Mandy 24,25 Kühne, Ronald...... 57 Krylova, OxanaO...... 3,70,92 Kries, Jens-Petervon...... 55 Winfried...... Krause, 20 Gerd...... Krause, 70,83 ...... Krause, Eberhard Krause, Dagmar...... 74 ...... 74 Krätke, Oliver 13 ...... Krabben, Ludwig Kotzur, Nico...... 87 ...... 31, 33 Kofler, Michael ...... 63 Königsmann, Jessica 13 ...... Köhler, Christian 59 ...... Knobeloch, Klaus-Peter 42 ...... Klussmann, Enno 74 Klose, Jana...... 74 ...... Klose, Annerose 64 ...... Klemm, Janet Klemm 20,23 ...... Kleinau, Gunnar 101 ...... Klauschenz, Erhard 60 ...... Kisser, Agnes 50 ...... Kirsch, Jenny 36 ...... Kiesling, Alexandra 76,78 ...... Keller, Sandro 55 ...... Kahlich, Bettina 63 ...... Kallies, Axel K 13 ...... Joshi, Mangesh 2 ...... Joost, Hans-Georg 14 ...... Jehle, Stefan 108 ...... Jahn, Thomas J J 43 Hundsrucker,...... Christian H 36 ...... Horak, Ivan 13 ...... Holtmann, Henrik ...... 29 Hologne, Maggy h,Rihr ...... 2 ...... ahn, Reinhard bl tfn ...... 26 ...... übel, Stefan , lmnie...... 85 Clementine ...... uctr nj ...... 100 ...... Muschter, Antje 20,23,50 ...... Müller, SebastianL. 31,33 ...... Motzny, Katrin 57 Mollajew,...... Rustam 101 ...... Mohs, Barbara 50 ...... Mikoteit, Kerstin 40 ...... Michl, Dagmar 66 ...... Meyer, Thomas 66 ...... Meyer, Stephanie Mevert, Jürgen...... 101 107 ...... Messing, Claudia 2 ...... Melchior, Frauke 66 Meissner, Torsten...... 90 ...... Meier, Franziska 43 McSorley,...... Theresa 102, 103 ...... Maul, Björn 57 ...... Margania, Valentina 66 ...... Marg, Andreas 107 ...... Manks, Silvia 14 ...... Mac, Thien-Thi M 43,48 ...... Dorothea Lorenz, 50 ...... Lorberg, Dörte 66 ...... Lödige, Inga 101 ...... Lojek, Eva L 85 ...... Lerch, Heidi 13 ...... Leitner, Dietmar 57 Lentz, Alexander...... 14 ...... Leidert, Martina L 104 ...... Lauterjung, Ulrike 50 ...... Lassowski, Birgit 14 ...... Lange, Vivien L 20,23 Lättig, Jens...... 13 ...... Labudde, Dirk L ioek,Hie ...... 78 ...... Nikolenko, Heike 60 ...... Niendorf, Sandra 50 ...... Niehage, Christian 43 ...... Nedvetsky, Pavel 29 ...... Narayanan, Saravanakumar N mr tpai ...... 85 ...... amer, Stephanie eefl,Oie ...... 76 ...... iesenfeld, Oliver clr af ...... 87 ...... echler, Ralf

161 Index O Schumacher, Gabriele ...... 107 Oczko, Brunhilde ...... 48 Schumann, Björn ...... 108 Oehlke, Johannes ...... 70, 75 Schrey, Anna ...... 24, 26 Oschkinat, Hartmut ...... 10, 12 Schröder, Nikolaj ...... 14 Osiak, Anna ...... 60 Schulz, Katrin ...... 50 Otto, Christel ...... 107 Schuster, Ariane ...... 50, 152 Oueslati, Morad ...... 40 Selfe, Joanna ...... 63 Serowy, Steffen ...... 57, 76 P Shan, Ying ...... 66 Pahlke, Doreen ...... 14 Siems, Wolf-Eberhard ...... 52 Pankow, Kristin ...... 55 Singh Bal, Manjot ...... 50 Pankow, Rüdiger ...... 64 Söderhäll, Arvid ...... 24, 25, 26 Panzer, Holger ...... 101 Sokolenko, Elena ...... 57 Passow, Josephine ...... 107 Sokolov, Valerij ...... 57 Perepellichenko, Ludmila ...... 90 Soukenik, Michael ...... 14 Peters, Renate ...... 101 Souza, Christina de ...... 57 Petschick, Heidemarie ...... 36 Sperling, Birgit ...... 107 Piontek, Jörg ...... 49 Stefan, Eduard ...... 43 Piotukh, Kirill ...... 31, 33 Steuer, Andrea ...... 10 Pires, Ricardo ...... 13 Strauss, Holger ...... 16, 18 Pisarz, Barbara ...... 74 Sun, Xiaoou ...... 55 Pisarz, Hans-Werner ...... 108 Sylvester, Marc ...... 32, 33 Pohl, Peter ...... 57 Poliakov, Ilja ...... 14 T Prigge, Matthias ...... 57 Tasadaque Ali Shah, Syed ...... 90 Pritz, Stephan ...... 74 Tenz, Kareen ...... 85 Thielen, Anja ...... 40 R Thiemke, Katharina ...... 31, 32, 33 Rademann, Jörg ...... 3, 70, 89 Thakur, Mina ...... 64 Reif, Bernd ...... 28 Tremmel, Sandra ...... 74 Rehbein, Kristina ...... 14 Tsunoda, Satoshi ...... 56 Richter, Regina M...... 98, 101 Ringling, Martina ...... 48 U Rosenthal, Walter ...... 6, 36 Uryga-Polowy, Viviane ...... 90 Rossum, Barth van ...... 13 Uschner, Michael ...... 101 Roswadowski, Inga ...... 50 Utepbergenov, Darkhan ...... 49 Rötzschke, Olaf ...... 25 Rückert, Christine ...... 49 V Rutz, Claudia ...... 40 Vargas, Carolyn ...... 14 Venta, Nicola ...... 66 S Vinkemeier, Uwe ...... 65 Santamaria, Katja ...... 43 Vogelreiter, Gabriela ...... 78, 82 Saparov, Sapar M...... 57 Sauer, Ines ...... 76, 78 W Scharnagel, Hubert ...... 76 Waldmann, Herbert ...... 2 Schlegel, Brigitte ...... 16, 18 Walter, Juliane ...... 50 Schmidt, Antje ...... 46 Weber, Viola ...... 43 Schmieder, Peter ...... 16 Weik, Steffen ...... 90 Schmikale, Bernhard ...... 74 Weisgraber, Karl ...... 76 Schneeweiß, Ulrike ...... 33 Wendt, Stefanie ...... 101 Schülein, Ralf ...... 33 Wessolowki, Axel ...... 78 Schümann, Michael ...... 85 Wichard, Jörg ...... 26 uee,Nklj...... 50,145 Zuleger,...... Nikolaj 32,33 ...... Zimmermann, Jürgen Z W 75,78 ...... Wolf, Yvonne 50 Wolf,...... Constanze 50 ...... Winkler, Lars W 74 ...... Wietfeld, Doreen 46,75 ...... Wiesner, Burkhard 14,20,23 ...... Wiedemann, Urs lf hita ...... 100 ...... olff, Christian esrc,Mru ...... 60 ...... ietstruck, Markus

163 Index Forschungsinstitut für Molekulare Pharmakologie (FMP) Campus Berlin-Buch Robert-Rössle-Straße 10 13125 Berlin Tel.: +49-30-9489-2920 Fax: +49-30-9489-2927 www.fmp-berlin.de

Ausfahrt/Exit Schönerlinder Str.

Ausfahrt/Exit Weißensee FMP

A10 Bibliothek C71 Tier- und Laborgebäude A13 Infocenter, Gläsernes Labor C81 Forschungsinstitut für Molekulare Pharmakologie (FMP) A14 Mensa C81.1 NMR-Haus I (FMP) A15 Charles River Deutschland GmbH C81.2 NMR-Haus II (FMP) A8 Torhaus C83 Max-Delbrück-Communactions Center A9 Pförtner C84 Hermann-von-Helmholtz-Haus B46 Robert-Rössle-Klinik C87 Genomforschung B54 Hans-Gummel-Gästehaus D16 Bebig GmbH B55 Oskar-und-Cécile-Vogt-Haus D23 Eckert & Ziegler AG B61 Salvadore-Luria-Haus D72 BioTeZ GmbH B63 Tierhaus D79 Erwin-Negelein-Haus B64 epo GmbH D80 Otto-Warburg-Haus C27 Walter-Friedrich-Haus D82 Karl-Lohmann-Haus C31 Max-Delbrück-Haus D85 Arnold-Graffi-Haus

165 Maps/Lagepläne 4 0 0

2 RESEARCH REPORT I 3 0

0 2003 I 2004 2 T R O P E R H C R A E S E R . V . E N I L R E B D N U B R E V S G N U H C S R O F M I E I G O L O K A M R A H P E R A L U K E L O M R Ü F T U T I T S N I S G N U H C S R O F