Research Report 2010/2011 online - Institute of Biochemistry

Research Report Biochemical Institute, Christian-Albrechts-University KielThe Biochemical InstituteChristian-Albrechts-University ofKielhttp: www.uni-kiel.de/Biochemie2010/2011

Legends to the cover figuresTop: Hematoxylin and eosin (H&E), BrdU, anti–phosho-STAT3, and anti-cyclin D1 stainingof colons from mice challenged for 10 d with DSS. Bars: (H&E) 200 µM; (BrdU, p-STAT3,and cyclin D1) 100 µm. Representative microscopic images of three experiments (H&E andBrdU), two experiments (pSTAT3), and one experiment (cyclin D1) of ADAM17 ex/ex ,ADAM17 WT/ex , and WT controls (five mice per group) are shown.Left down: Embryonic stem cells were differentiated along neuroectodermal differentiationso that neural precursor cells were generated in high numbers as shown by anti-TUBB3/ TuJ1staining in green (day 14). When Notch1 signaling was activated this differentiation wasabolished and instead glia cells were generated as shown by anti-GFAP marker in orange (day16). All cells were counterstained with DAPI (blue).Right down: The structure of acanthaporin, the pathogenic factor of Acanthamoebaculbertsoni.ContactBiochemical InstituteChristian-Albrechts-University of KielOlshausenstr. 40D-24098 KielGermanyphone: +49 (0) 431/880-2211fax: +49 (0) 431/880-2007email: mkanter@biochem.uni-kiel.dehttp: www.uni-kiel.de/Biochemie2

Research Report Biochemical Institute, Christian-Albrechts-University KielBiochemical InstituteChristian-Albrechts-University of KielResearch Report 2010/2011

Research Report Biochemical Institute, Christian-Albrechts-University KielEditorialAfter the Institute of Biochemistry at the Christian-Albrechts-Universität, Kiel has undergonefundamental changes in the years 2000-2002, we are now in a phase of consolidation and expansion.Renovations in the Institute have been completed and all the laboratories and teaching areas of theinstitute are now fully modernized.In the years 2010/2011, members of the Biochemistry Institute were represented in three biomedicalcollaborative research centers (SFB415: 'Specifity and Pathophysiology of Signal TransductionPathways', SFB654: "Plasticity and Sleep") and SFB841 "Liver inflammation: infection, immuneregulation und consequences"). The SFB415 ended in summer 2010, and Stefan Rose-John and PaulSaftig from the Institute of Biochemistry have taken the initiative to coordinate an application for anew SFB with the title 'Proteolysis as a regulatory event in pathophysiology'. This new SFB877 hasbeen approved for funding by the Deutsche Forschungsgemeinschaft and has started in summer 2010.Part of the SFB877 is an Integrated Research Training group for natural science and medical graduatestudents. The first funding period will end in summer 2014. Funding of the SFB877 can be renewedtwice and is therefore envisioned to continue until summer 2022.A new junior professorship of Biochemistry to support the SFB877 has recently been established atthe Institute of Biochemistry and has been filled with Christoph Becker-Pauly.Furthermore, the scientific work of most members of the Institute of Biochemistry is supported byadditional grants from the European Union and the Deutsche Forschungsgemeinschaft, including aresearch training group for graduate students (GRK1459) together with the Universität Hamburg.Since 2008, the Cluster of Excellence ‘Inflammation at Interfaces’ formed by scientists from theUniversität of Kiel, the Universität Lübeck and the Research Center Borstel is funded for five years.Scientists from the Biochemistry Institute are prominently involved in the cluster of excellence andone of the three major research projects of the cluster is coordinated at our Institute. This fundingallowed the Christian-Albrechts-Universität to appoint Jürgen Scheller W2 professor of Biochemistryat our institute. In 2010, Jürgen Scheller accepted the position of a W3 professor of Biochemistry andhead of the Institute of Biochemistry II at the Medical Faculty of the Heinrich-Heine UniversitätDüsseldorf. The position of Jürgen Scheller has recently been filled by a junior professor ofBiochemistry at our institute.The Cluster of Excellence ‘Inflammation at Interfaces’ in 2011 has filed a proposal for renewal foradditional 5 years and will be reviewed in February 2012. Members of the Institute of Biochemistrywill help to represent the Cluster of Excellence to the reviewers. A decision about the renewal isexpected in summer 2012.Thus, our Institute continues to represent a spearhead in biomedical research in the Northern Germanylandscape of science. In this brochure you find an overview of the projects being carried out in theInstitute of Biochemistry in 2010 and 2011, along with internet addresses where you can find moredetailed information.Kiel, January 2012Stefan Rose-John

ContentScientific Staff Members 2010/2011...................................................................................... 11. Research Group Prof. Dr. Stefan Rose-John................................................................... 32. Research Group Prof. Dr. Hilmar Lemke ....................................................................... 93. Research Group Prof. Dr. Joachim Grötzinger ............................................................. 114. Research Group Prof. Dr. Jürgen Scheller .................................................................... 155. Research Group Dr. Athena Chalaris............................................................................ 196. Research Group Dr. Dirk Schmidt-Arras...................................................................... 237. Research Group Prof. Dr. Paul Saftig ........................................................................... 278. Research Group PD Dr. Michael Schwake ................................................................... 399. Research Group Dr. Judith Blanz.................................................................................. 4110. Research Group Dr. Bernd Schröder............................................................................ 4511. Research Group Prof. Dr. Ursula Just .......................................................................... 4912. Research Group Dr. Ralf Schwanbeck......................................................................... 5313. Research Group Prof. Dr. Roland Schauer................................................................... 57Seminars 2010/2011............................................................................................................. 61Publications 2010/2011 ........................................................................................................ 63Accumulated Impact Factors................................................................................................ 696

Research Report Biochemical Institute, Christian-Albrechts-University KielScientific Staff Members 2010/2011Scientists:Prof. Dr. rer. nat. Stefan Rose-John – Executive DirectorProf. Dr. rer. nat. Hilmar LemkeProf. Dr. rer. nat. Joachim GrötzingerProf. Dr. rer. nat. Jürgen SchellerDr. Dirk Schmidt-ArrasDr. Athena ChalarisProf. Dr. rer. nat. Paul Saftig – DirectorPD Dr. rer. nat. Michael SchwakeDr. rer. physiol. Bernd SchröderDr. rer. nat. Judith BlanzProf. Dr. med. Ursula Just – DirectorDr. rer. nat. Ralf SchwanbeckProf. Dr. med. Roland Schauer – EmeritusPost-DocsDr. Ahmad TradDr. Alexander SchneedeDr. Andrea RittgerDr. Björn RabeDr. Christoph GarbersDr. Doreen FlossDr. Inken LorenzenDr. Kozuke YamamotoDr. Meng LinDr. Nathalie JännerDr. Sascha JungDr. Simone MartiniDr. Thomas HöfkenDoctoral studentsAnna ZaslawskiAntje SchüttBjörn SpudyChristian RaabChristin DewitzChristina ZachosChristoph GarbersEva WagenerFriederike ZunkeGerina VollmersJan SommerJana KieperJanna SchneppenheimJeanette Schwarz1

Jessica GewieseJohann GrothJohannes ProxJudith PetersKatja MöllerKristina BernothMadlen MohrMarija StevanovicMatthias MichalekMichelle DanaherMirja OldefestMirka AllerdingMiryam MüllerMohammad ShomaliNina AdamOlga BraunSandra KissingSebastian WetzelSilvio WeberSimone MartiniStefan DüsterhöftSusann HüttlSven MalchowTelly SavallasTimo EffenbergUlrike MayVerena PawlakMD StudentsJohannes KnabbeMaike LüdemannMiriamWagnerNina HedemannStephanie BlumWolfgang Thaiss2

Research Report Biochemical Institute, Christian-Albrechts-University Kiel1. Research Group Prof. Dr. Stefan Rose-JohnA Group Leader: Prof. Dr. Stefan Rose-JohnB Lab Members: Post-Docs:Dr. Athena ChalarisDr. Björn RabeDr. Dirk Schmidt-ArrasDr. Kozuke YamamotoDr. Nathalie JännerDoctoral Students:Antje SchüttChristin DewitzChristoph GarbersEva WagenerJan SuthausJessica GewieseNina AdamOlga BraunSven MalchowTimo EffenbergUlrike MayTechnicians:Melanie BossSilke HornStephanie SchnellCResearch ReportC.1 The soluble interleukin-6 receptor: generation and physiologic functionThe soluble interleukin-6 receptor (IL-6R) in complex with interleukin-6 (IL-6) stimulates cells, which expressthe signaling receptor subunit gp130 but no ligand binding IL-6R. Such cells in the absence of the soluble IL-6Rare unable to respond to IL-6. This process has been named 'trans-signaling'. Trans-signaling has been shown tobe important for inflammation reactions, neuronal survival, hematopoiesis and tumor rejection.We have characterized the metalloproteinase, which is responsible for the release of the soluble IL-6R bybiochemical and genetic means. This protease belongs to the Metalloproteinase with Disintegrin Domain(ADAM) family of metalloproteinases. We perform experiments to better understand the biochemical andstructural prerequisites of limited proteolysis of the IL-6R by members of the ADAM family. We also try tounderstand the regulation of the cleavage reaction. In this respect it is interesting that we could recently show3

that the induction of apoptosis leads to activation of the shedding protease ADAM17 (see below). This seems tobe a general phenomenon, which might play an important role in the regulation of the inflammatory process. Wecould show that neutrophils, which are the first line of defense of the body during infection and inflammation,are a major source of the soluble IL-6R in vivo. Interestingly, induction of apoptosis leads to a selectiveactivation of ADAM17, which in turn is responsible for shedding of the IL-6R.As mentioned above, we could show that the protease ADAM-17 (also called TACE), which is responsible forcleavage of TNFα is also strongly involved in shedding of the IL-6R. Using a novel homologous recombinationstrategy, we have recently generated ADAM17 hypomorphic mice (called ADAM17 ex/ex mice) to analyze theinvolvement of this protease in various shedding processes. Using these ADAM17 ex/ex mice we currently studythe physiologic role of cleavage of proteins of the ligands of the EGF-Receptor and of the Notch protein.The phenotype of the ADAM17 hypomorphic mice clearly shows an involvement of ADAM17 in inflammatoryprocesses. Therefore these mice are an excellent experimental tool to study the overall physiologic role of theADAM17 metalloprotease and its involvement in inflammation and cancer.C.2 Viral Interleukin-6: Strukture, Pathophysiology and Strategies of NeutralisationOn target cell Interleukin-6 (IL-6) binds to a receptor complex consisting of the ligand binding subunitInterleukin-6-receptor (IL-6R) and the signal transducing subunit gp130. The complex of soluble IL-6R and IL-6acts on cells, which do not express IL-6R. Such cells would not be able to respond to IL-6 alone. Such cellscomprise hematopoietic progenitor cells, endothelial cells, smooth muscle cells, T-cells and neural cells.Interestingly, the recently identified viral IL-6 (vIL-6) encoded by Human Herpes Virus 8 (HHV8) binds to andactivates gp130 directly. Such, vIL-6 activates a significant larger spectrum of target cells than human IL-6. Wecharacterized the biochemical and physiological properties of vIL-6. Furthermore, we have generated transgenicmice, which overexpress vIL-6. Using these mice we evaluate the involvement of vIL-6 in human diseases likeCastleman disease, primary effusion lymphoma and multiple myeloma.Using a novel strategy, we used our recently generated recombinant antibodies against vIL-6 to target the vIL-6within cells expressing the protein. The underlying principle of this strategy is to anchor the recombinant vIL-6antibodies within the endoplasmic reticulum (ER) with the help of the canonical ER retention sequence KDEL.Indeed we could demonstrate that vIL-6 can induce signaling from within the cell and that such signaling can becompletely blocked from within the cell.We have performed structure-function analysis to clarify how vIL-6 can bind directly to gp130 whereas humanIL-6 needs the IL-6R in order to bind to and activate gp130. These data clearly show that the so-called site III ofvIL-6 is responsible for this property and that the ability to directly bind to gp130 can be transferred to humanIL-6 by transferring site III.Transgenic mice, which over-express the vIL-6 protein show a phenotype resembling human Castleman disease.These data indicate that vIL-6 is strongly involved in the phathophysiology of the HHV8 virus.C.3 Development of the IL-6 trans-signaling antagonist sgp130FcWe could show in the past years that IL-6 trans-signaling can specifically be inhibited by the sgp130Fc proteinwithout affecting IL-6 signaling via the membrane bound IL-6R. These data established the sgp130Fc proteinnot only as a molecular tool to experimentally distinguish between classic- and trans-signaling. The sgp130Fcprotein can also be used to block the course of inflammatory diseases in animal models of rheumatoid arthritis,peritonitis, inflammatory bowel disease and many animal models of inflammation induced cancer.We have improved the properties of the sgp130Fc protein in terms of protein stability, affinity towards the IL-6/sIL-6R complex and feasibility of production. Furthermore, together with the group of Joachim Grötzinger, weare engaged in a study to solve the three dimensional structure of the sgp130Fc bound to the IL-6/sIL-6Rcomplex.Since the sgp130Fc protein has a considerable therapeutic potential, Stefan Rose-John together with Prof. StefanSchreiber (Director of the Institute of Clinical Molecular Biology at the University Hospital in Kiel) founded abiotechnology company (Conaris Research Institute), which develops the sgp130Fc protein into a drug. InDecember 2008, Conaris Researcha Institute and the company Ferring have completed an exclusive worldwidelicense agreement for the development of sgp130Fc for inflammatory conditions such as IBD and rheumatoidarthritis. Sgp130Fc is undergoing pre-clinical testing prior to moving into Phase I Clinical Trials in 2012.4

Research Report Biochemical Institute, Christian-Albrechts-University KielD Publications 2010/2011Publications 20101. Stumhofer JS, Tait ED, Quinn W, Hosken N, Spudy B, Goenka R, JonesM, Saris CJM, Rose-John S, Elloso MM, Grötzinger J, Cancro M, LevinS, Hunter CA (2010) A role for IL-27p28 as an antagonist of gp130-mediated signaling. Nat Immunol 11: 1119-262. Gewiese-Rabsch J, Drucker C, Malchow S, Scheller J, Rose-John (2010)Role of IL-6 Transsignaling in CCl4 induced liver damage, BBA-MolBasis Disease 1802: 1054-613. Waldner MJ, Wirtz S, Jefremow A, Neufert C, Warntjen M, Becker C,Weigmann B, Vieth M, Rose-John S, Neurath MF (2010)VEGF receptorsignaling links chronic inflammation and tumorigenesis in colitisassociatedcolon cancer via proliferation of epithelial cells. J Exp Med207: 2855-684. Bender M, Hofmann S, Stegner D, Chalaris A, Bösl M, Braun A, SchellerJ, Rose-John S, Nieswandt B (2010) Differentially regulated GPVIectodomain shedding by multiple platelet-expressed proteinases, Blood116: 3347-555. Lam SP, Luk JM, Man K, Cheung CK, Rose-John S, Lo CM (2010)Activation of IL-6-induced gp130/STAT3 Pathway in Mesenchymal StemCells via the Trans-Signaling Mechanism Enhances HepaticDifferentiation, Proliferation and Liver Regeneration. Liver Transplant16: 1195-206. Hammer M, Echtenachter B, Weighardt H, Jozefowski K, Rose-John S,Männel DN, Holzmann B, Lang R (2010) Increased inflammation andlethality of Dusp1-/- mice in polymicrobial peritonitis models,Immunology 131: 395-4047. Suthaus J, Tillmann A, Lorenzen I, Rose-John S and Scheller J (2010)Forced heterodimerization of all gp130-type receptor complexes leads toconstitutive ligand independent signaling activation, and cytokineindependent growth. Mol Biol Cell 21: 2797-28078. Chalaris A, Adam N, Sina C, Rosenstiel P, Lehmann J, Schirmacher P,Hartmann D, Cichy J, Gavrilova O, Schreiber S, Jostock T, Matthews V,Häsler R, Becker C, Neurath MF, Reiß K, Saftig P, Scheller J, Rose-JohnS (2010) Critical Role of the Disintegrin Metalloprotease ADAM17 forIntestinal Inflammation and regeneration in Mice. J Exp Med 207: 1617-16249. Rodriguez N, Dietrich H, Mossbrugger I, Weintz G, Scheller J, HammerM, Quintanilla-Martinez L, Rose-John S, Miethke T, Lang R (2010)Increased inflammation in Dusp1-/- mice impairs resistance toChlamydophila pneumoniae via IL-6 trans-signaling. J Leuk Biol 88:579-8710. McGreal EP, Davies PL, Powell W, Rose-John S, Spiller BO, Doull I,Jones SA and Kotecha S. (2010) Inactivation of IL-6 and soluble IL-6receptor by neutrophil derived serine proteases in cystic fibrosis.Biochimica et Biophysica Acta - Molecular Basis of Disease 1802: 649-65811. Febbraio MA, Rose-John S, Pedersen BK (2010) Is interleukin-6 receptorblockade the Holy Grail for inflammatory diseases? Clin Pharmacol Ther87: 396-8.12. Jones GW, McLoughlin RM, Hammond VJ, Parker CR, Williams JD,Malhotra R, Scheller J, Williams AS, Rose-John, Topley N and Jones SA(2010) Loss of CD4+ T cell IL 6R expression during inflammation for IL6 trans-signaling in local Th17 cell regulation. J Immunol 184: 2130-913. Chalaris A, Gewiese J, Paliga, K, Fleig L, Schneede A, Krieger K, Rose-John S and Scheller J (2010) ADAM17-mediated shedding of the IL6Rinduces cleavage of the membrane stub by g-secretase. Biochimica etBiophysica Acta-Molecular Cell Research 1803: 234-245Impact Factor25.6685.21114.77610.5583.0683.3025.86114.7764.6265.2116.3785.7454.7335

14. Matsumoto S, Hara T, Mitsuyama K, Yamamoto M, Tsuruta O, SataM,Scheller J, Rose-John S, Kado S, Takada T (2010) Essential roles ofIL6 trans-signaling in colonic epithelial cells, induced by the IL6/soluble-IL6-receptor derived from lamina propria macrophages, on thedevelopment of colitis-associated pre-malignant cancer in a murinemodel. J Immunol 184: 1543-5115. Waetzig GH, Chalaris A, Rosenstiel P, Suthaus J, Holland C, Karl, N,Uriarte LN, Till A, Scheller J, Grötzinger J, Schreiber S, Rose-John S,Seegert D (2010) N-linked glycosylation is essential for the stability, butnot the signaling function of the interleukin-6 signal transducer gp130. JBiol Chem 285: 1781-178916. Floss DM, Schallau K, Rose-John S, Conrad U, Scheller J (2010)Elastin-like polypeptides revolutionize recombinant protein expressionand their medical. application. Trends Biotechnol 28: 37-4517. Vahdat AM, Reiners KS, Simhadri VL, Eichenauer DA, Böll B, ChalarisA, Simhadri VR, Wiegmann K, Krell H-W, Rose-John S,Engert A, Pogge von Strandmann E and Hansen HP (2010) TNF-alphaconverting enzyme (TACE/ADAM17)-dependent loss of CD30 inducedby proteasome inhibition through reactive oxygen species. Leukemia 24:51-5718. Drucker C, Gewiese J, Malchow S, Scheller J, and Rose-John S (2010)The impact of Interleukin-6 classic- and trans-signaling on liver damageand regeneration. J Autoimmun 34: 29-37Publications 20111. Garbers C, Thaiss W, Jones GW, Waetzig GH, Lorenzen I, Guilhot F,Lissilaa R, Ferlin WG, Grötzinger J, Jones SA, Rose-John S, Scheller J.(2011) Inhibition of classic signaling is a novel function of soluble GP130which is controlled by the ratio of interleukin 6 and soluble interleukin 6receptor.J Biol Chem. 286, 42959-429702. Atreya R, Zimmer M, Bartsch B, Waldner MJ, Atreya I, H. Neumann H,Hildner K, Hoffman A, Kiesslich R, Rink AD, Rau T, Rose-John S,Kessler H, Schmidt J and Neurath MF (2011) Anti-TNF antibodies targetT-cell apoptosis in inflammatory bowel diseases via TNFR2 and intestinalCD14+ macrophages. Gastroenterol, 14, 2026-2038.3. Fisher DT, Chen Q, Skitzki JJ, Muhitch JB, Zhou L, Appenheimer MM,Vardam TD, Unger E, Passanese J, Wang W-C, Dewhirst MW, Rose-JohnS, Repasky EA, Baumann H, Evans SS (2011) IL-6 trans-signalinglicenses murine and human tumor microvascular gateways for traffickingof cytotoxic T cells. J Clin Invest 121: 2846-38594. Jones SA, Scheller J, Rose-John S (2011) Therapeutic strategies for theclinical blockade of IL-6/gp130 signaling. J Clin Invest 121: 3375-33835. Scheller J, Chalaris A, Garbers C, Rose-John S (2011) ADAM17: amolecular switch to control inflammation and tissue regeneration. TrendsImmunol 32: 380-3876. Garbers C, Jänner N, Chalaris A, Moss ML, Floss DM, Meyer D, Koch-Nolte F, Rose-John S, Scheller J (2011) Species specificity of ADAM10and ADAM17 in IL-6 transsignaling and novel role of ADAM10 ininducible IL-6R shedding. J Biol Chem 286: 14804-148117. Schiechl G, Bauer B, Fuss IJ, Lang SA. Moser C, Rose-John S, NeurathMF, Geissler E, Schlitt HJ, Strober W, Fichtner-Feigl S (2011) Tumorgrowth in murine ulcerative colitis depends on innate signaling of colonicF4/80+ CD11bhigh Gr1 low macrophages. J Clin Invest 121: 1692-17088. Lesina M, Kurkowski MU, Ludes K, Rose-John S, Treiber M, Klöppel G,Yoshimura A, Reindl W, Sipos B, Akira S, Schmid RM, and Algül H(2011) Stat3/Socs3 activation by IL-6 transsignaling promotes progressionof pancreatic intraepithelial neoplasia and development of pancreaticcancer. Cancer Cell 19: 456-4699. Scheller J, Chalaris A, Schmidt-Arras D, Rose-John S (2011) The pro- andanti-inflammatory properties of the Cytokine Interleukin-6. BBA -Molecular Cell Research 1813: 878-8885.7455.3289.6448.9668.136Impact Factor5.32812.03214.15214.1529.5335.32814.15226.9254.7336

Research Report Biochemical Institute, Christian-Albrechts-University Kiel10. Chalaris A, Garbers C, Rabe B, Rose-John S, Scheller J. (2011) Thesoluble Interleukin 6 receptor: Generation and role in inflammation andcancer. Eur J Cell Biol 90: 6-711. Suthaus J, Adam N, Grötzinger J, Scheller J, Rose-John S (2011) ViralInterleukin-6: Structure, pathophysiology and strategies of neutralization.Eur J Cell Biol 90: 495-50412. Lorenzen I, Shang W, Perbandt M, Petoukhov MV, Svergun DI, WaetzigGH, Rose-John S, Hilgenfeld R, Grötzinger J (2011) The structure of theunliganded extracellular domain of the interleukin-6 signal transducergp130 in solution.Eur J Cell Biol 90: 515-52013. Barkhausen T, van Griensven M, Vonberg R-P, Dorsch M, Seegert D,Chalaris A, Scheller J, Rose-John S, Hildebrand F, Krettek C, Tschernig T,Waetzig GH (2011) Selective blockade of IL-6 trans-signaling improvessurvival in a murine polymicrobial sepsis model. Crit Care Med 39: 1407-141314. Loppnow H, Buerke M, Werdan K, Rose-John S (2011) Contribution ofvascular cell-derived cytokines to innate and inflammatory pathways inatherogenesis. J Cell Mol Med 15: 484-50015. Malchow S, Thaiss W, Jänner N, Gewiese-Rabsch J, Garbers C,Yamamoto K, Rose-John S, Scheller J (2011) Essential role of neutrophilmobilization in concanavalin A-induced hepatitis is based on classic IL-6signaling but not on IL-6 trans-signaling. BBA-Mol Basis Disease 1812:290-30116. Lo C-W, Chen MW, Hsiao M, Wang S, Chen C-A, Hsiao S-M,Chang J-S, Lai T-C, Rose-John S, Kuo M-L, Wei L-H (2011) IL-6 transsignalingin formation and progression of malignant ascites in ovariancancer Cancer Progression. Cancer Res 71:424-43417. Greenhill CJ, Rose-John S, Lissilaa R, Ferlin W, Ernst M, Hertzog PJ,Mansell A, and Jenkins BJ (2011) IL-6 trans-signaling modulates TLR4-dependent inflammatory responses via STAT3. J Immunol 186: 1199-120818. Nechemia-Arbely Y, Shriki A, Denz U, Raub J, Pappo O, Rose-John S,Galun E and Axelrod JH (2011) Early Hepatocyte DNA SyntheticResponse Posthepatectomy is Dependent on IL-6 Trans-Signaling andPI3K/AKT Activation. J Hepatol 54: 922-92919. Loppnow H, Zhang L, Buerke M, Lautenschläger M, Chen L, Frister A,Schlitt A, Song N, Hofmann B, Rose-John S, Silber R-E, Müller-WerdanU, Werdan K (2011) Statins potently reduce the cytokine-mediated IL-6release in SMC / MNC cocultures. J Cell Mol Med 15: 994-100420. Conrad U, Plagmann I, Malchow S, Sack M, Floss D, Kruglov AA,Nedospasov S, Rose-John S and Scheller J (2011) ELPylated anti-humanTNF-therapeutic single-domain antibodies for prevention of lethal septicshock. Plant Biotech J 9: 22-313.6303.6303.6306.2544.6085.2118.2345.7459.3344.6084.886Impact factors 2010: 147.732Impact factors 2011: 166.105Total impact factors 2010/2011: 313.837EGrantsE.1 The role of gp130-Trans-Signaling in liver-regeneration and -cancer: therapeutic perspectivesCollaborative Research Centre 841, Project C1, (DFG, Germany)Total granted sum: (2010 – 2013) 367.200,00 €E.2 The function of the gp130-signaling-family for sleep and plasticityCollaborative Research Centre 654, Project C5, (DFG, Germany)Total granted sum: (2009 – 2013) 387.200 €E.3 Analysis of the role of the shedding protease ADAM17 in vivoCollaborative Research Centre 877, Project A1 (DFG, Germany)Total granted sum: (2010 – 2014) 433.760 €7

E.4 BIO-DISC5: Stem-Cell based Treatment of Xerostomia (BMBF, Germany)Total granted sum: (2010 – 2013) 450.000 €E.5 Jürgen Scheller und Stefan Rose-JohnDer lösliche Interleukin-6-Rezeptor: Entstehung und physiologische BedeutungenSonderforschungsbereich 415, Teilprojekt B5, Fördersumme (2007 – 2010) 248.700 €E.6 Stefan Rose-JohnVirales Interleukin-6: Struktur, Pathophysiologie und Strategien zu seiner NeutralisierungSonderforschungsbereich 415, Teilprojekt C6, Fördersumme (2007 – 2010) 268.800 €E.7 Excellenzcluster Inflammation at Interfaces, Integrated Research Network F: gp130 signalling,Fördersumme (2008-2012) 2.749.800 €E.8 Excellenzcluster Inflammation at Interfaces, Miniproposal (2011): 50.000 €8

Research Report Biochemical Institute, Christian-Albrechts-University Kiel2. Research Group Prof. Dr. Hilmar LemkeA Group Leader: Prof. Dr. Hilmar LemkeB Lab Members: Dr. rer. nat. Ahmad TradCResearch ReportC.1 The adaptive immune system of T and B cells forms a completely integrated physiological networkthrough specific = idiotypic interaction of their antigen-receptors, known as the idiotypic network. The mutualinteractions in this network depend on the recognition of non-genetically determined portions of T and B cellantigen-receptors. These Non-Self parts are created by imprecisions during V(D)J recombination and somatichypermutations during thymus-dependent immune responses. After antigen stimulation, second-step idiotypicresponses of T and B cells are induced. The products of such intrinsic activations represent cellular and humoralregulatory information that may be both, suppressive or stimulatory. The humoral information in form of antibodiesis transferred via the maternal, but not the paternal route to the next generation. Therefore, maternal antibodiesrepresenting a good deal of the mother’s immunological experience have to be regarded as transgenerationalmessengers. Maternal idiotypic as well as anti-idiotypic antibodies represent the first environmental influenceon the nascent immune system. Thus, the collective maternal immunological experience to the offspringenables a maternally mediated immunological education before the first encounter with external antigens. Duringsensitive phases, maternal antibodies are involved in the induction of a long-lasting and sometimes life-long determinativeimmunological imprinting. This effect of maternal antibodies will mostly be beneficial, but transferof autoantibodies may be detrimental for the development of the nascent immune system (Lemke et al., in press).In the latter context, it was a remarkable finding that maternally-derived immune or monoclonal IgG antibodies(idiotypes) reactive with suitable model allergens like ovalbumin or bee venom phospholipase A 2 (bvPLA 2 ) areable to induce long-lasting IgE unresponsiveness in the offspring. Our latest investigations have shown that antiidiotypicantibodies to the transgenerational IgE-suppressive IgG are likewise able to induce such IgE suppression.Since the effective anti-idiotype did not mimic epitopes of the allergen (bvPLA 2 ), we conclude that idiotypicinteractions are mechanistically responsible to the transgenerational IgE-suppression by maternal IgG antibodies.These results may offer new ways for a prophylactic and possibly therapeutic treatment of human IgE-mediatedallergies (Tanasa et al., 2010).9

C.2 In this project, we analyze the model immune response to the hapten 2-phnel-oxazolone (phOx) and obtainedsome interesting results which are of general importance.1.) The analysis of the thymus-dependent (TD) anti-phOx response following a pre-immunization with the thymus-independenttype 2 (TI-2) form of the antigen demonstrated that the latter, in contrast to current understanding,induces a special sort of immunological memory which we named network memory. In addition, evidencewas obtained for the first time that receptor revision by VH replacement may occur during immune maturationin genetically non-engineered wildtype mice.2.) Antigen affinity is commonly viewed as the driving force behind the selection for dominant clonotypes thatcan occur during the T cell-dependent processes of class switch recombination (CSR) and immune maturation.To test this view, we analyzed the variable gene repertoires of `natural´ monoclonal antibodies to the hapten 2-phenyloxazolone (phOx) from non-immunized mice as well as those generated after primary, secondary, tertiaryand quaternary immunization with phOx protein carrier-induced thymus-dependent or Ficoll-induced thymusindependent (TI-2) antigen stimulation. In contrast to expectations, the extent of IgM heterogeneity provedsimilar and many IgM from these three populations exhibited similar or even greater affinities than the classicOx1 clonotype that dominates only after CSR among primary and memory IgG. The population of clones thatwere selected during CSR exhibited a reduced VH/VL repertoire that was enriched for variable domains withshorter and more uniform CDR-H3 lengths and almost completely stripped of variable domains encoded by thelarge VH1 family. Thus, contrary to the current paradigm, T cell dependent clonal selection during CSRappeared to select for VH family and CDR-H3 loop content even when the affinity provided by alternativeclones exhibited similar to increased affinity for antigen. (Lange et al., Eur J Immunol, in press)C.3 It is assumed that the VHCDR3 has 2 functions: on the one hand, it is of particular importance for bindingof the epitope and, on the other, it is the primary target for idiotypic regulation, not only in B but also in Tcells with their analogous VßCDR3. However, the significance of VHCDR3 for the regulation of an immune responseis not known. Previous results had indicated that there might be a correlation between immune maturationand VHCDR3-dependent idiotypic network regulation (Lange et al, 1996, Eur J Immunol 26: 2234-42).For a further investigation of this subject, we started to analyze the anti-phOx response in mutant mice whichlack most of the 13 D gene segments of BALB/c mice and express only one of them. Three different strains areavailable: Strain ΔD-DFL contains the normal DFL-D-segment DFL16.1 which preferentially codes for neutralamino acids. Strain ΔD-iD contains a D gene segment with an inverted reading frame (`inverted D´ = iD) codingpreferentially for charged amino acids. Strain ΔD-rf2 contains one D gene segment in the second reading frame(`reading frame´ 2 = rf2) which preferentially codes for hydrophobic amino acids. This project is done in collaborationwith Dr. Michael Zemlin (Pediatric Clinics, University of Marburg, Germany) who participated in thegeneration of these mice in the laboratory of Prof. Dr. Harry Schroeder (Birmingham, USA).The hitherto obtained results in strains ΔD-iD and ΔD-rf2 show already a) that the full repertoire of D gene segmentsis essential for a good immune response and b) that the normally dominant Ox1 idiotype is only of minorimportance in these mice although the typical VHCDR3 sequence DRG can principally, but rarely be generated.Further investigations will show how the immune maturation develops in these mice (Ahmad Trad, DoctoralThesis, Kiel 2009).D Publications 2010/2011Publications 20101. Tanasa RI, Trad A, Lange H, Grötzinger J, Lemke H (2010) Allergen IgEisotype-specificsuppression by maternally derived monoclonal anti-IgGidiotype.Allergy 65: 16-23Publications 20111. Lange H, Hecht O, Zemlin M, Trad A, Tanasa RI, Schroeder HW andLemke H (2011) Immunoglobulin class switching appears to be regulatedby B cell antigen receptor-specific T cell action. Eur J Immunol, in press2. Lemke H, Tanasa RI, Trad A and Lange H (2011) Function of maternalidiotypic and anti-idiotypic antibodies as transgenerational messengers.In: Berencsi, G. (Ed.) Maternal-Fetal Transmission of Human Viruses andtheir Influence on Human Tumorigenesis. Springer Publishing, in pressImpact Factor6.204Impact Factor4.942Impact factors 2010: 6.024Impact factors 2011: 4.942Total impact factors 2010/2011: 10.96610

Research Report Biochemical Institute, Christian-Albrechts-University Kiel3. Research Group Prof. Dr. Joachim GrötzingerA Group Leader: Prof Dr. rer nat Joachim GrötzingerB Lab Members: Post-DocsDr. rer. nat. Ahmad TradDr. rer. nat. Inken LorenzenDr. rer. nat. Sascha JungDoctoral Students:Anna ZaslawskiBjörn SpudyJana KieperMadlen MohrMatthias MichalekMirja OldefestMohammad ShomaliNina HedemannStefan DüsterhöftVerena PawlakDiploma/Bachelor/Master students:Juliane LokauJuliane SchmähLaura RuschkiesMichel RieseTim KerkowTechnicians:Britta HansenJanina Schröder (Trainee Lab. Technician)Jessica SchneiderJonas Hiesener (Trainee Lab. Technician)Sina Viehweg (Trainee Lab. Technician)CResearch ReportC.1 Cytokines and their receptorsInterleukin-27 is a heterodimeric protein, which consists of the two proteins p28 and EBI-3. Whereas p28 isthought to belong to the four-helix bundle family of cytokines, EBI-3 is a soluble cytokine receptor containing11

the typical signatures of this family. The signaltransducers of IL-27 are gp130 and WSX-1, which are activatedby IL-27 upon binding to their extracellular parts. EBI-3 as well as WSX-1 contains a so called cytokine bindingregion, which consists of two domains with the signature of two conserved cysteine bridges in the N-terminaland a conserved WSXWS motive in the C-terminal domain. Like interleukin-6 and interleukin-15, p28 is able tointeract with three different receptor chains. The structure, the location and the affinity of the different receptorbindingepitopes of p28 to their respective receptor chains are not known. The goal of the planned work is toestablish a structure/function relationship for p28 and its receptors EBI-3 and WSX-1. Therefore, we will solvethe structure of p28 and the cytokine binding regions of EBI-3 and WSX-1 by multidimensional heteronuclearNMR spectroscopy. By using one 15 N labeled component and a second unlabeled component we will identify theamino-acid residues involved in the interaction between the two molecules by 15 N- 1 H correlated NMR spectra.The identification of the residues crucial for the ligand/receptor interaction and the three-dimensional structureof these molecules is the prerequisite for the development of therapeutically interesting antagonists and/oragonists.C.2 Antimicrobial proteinsThe primary defence of an organism against pathogens is performed by the physical barrier of the epidermis andthe epithel. The secondary defence is mediated by the epidermal cells, by their secretion of antimicrobialpeptides and proteins, like defensins and pore forming proteins. The molecular mechanisms of their interactionwith the pathogens are not well understood. The enlightening of the three dimensional structure of these proteinsis the basis for understanding their mechanisms of action. In this project the three-dimensional structures of thenewly discovered antimicrobial peptides will be solved by NMR spectroscopy.C.3 A disintegrin and metalloprotease 17 (ADAM17)ADAM17 is a membrane-spanning multi-domain protease. Besides several cell adhesion molecules, like L-selectin and VCAM-1, cytokines and cytokine receptors are important substrates of ADAM17. As an example,the soluble IL-6R as well as the soluble IL-15Rα are generated via shedding of the membrane-bound moleculesby ADAM17. Its extracellular part consists of a pro-, a catalytic-, a disintegrin- and an EGF-like domain.The three dimensional structure of the catalytic domain has been solved, whereas the structure and function ofthe disintegrin and EGF-like domain is unknown. A dominant negative ADAM17 mutant, lacking the proteasedomain, might inhibit the proteolytic cleavage by 1) binding to the ligand and thereby competition with the fulllengthADAM17 or 2) binding to the full-length ADAM17 thereby generating inactive dimers or multimers.For the closest relative of ADAM17, namely ADAM10, it has been shown that the disintegrin/EGF-like domainis involved in binding and specific recognition of their substrates. Co-immunoprecipitation experiments withdifferently tagged full-length ADAM17 showed that the molecule forms dimers/multimers. From these findingswe hypothesize that both possibilities might act in concert.On a cellular level we will define the role of the disintegrin- and EGF-like domain of ADAM17 in its mode ofaction. In parallel we will solve the three-dimensional structure of these domains by multidimensionalheteronuclear NMR-spectroscopy. Furthermore, the structures will be the basis for the determination of theepitopes responsible for the interaction with ADAM17 substrates and or dimerisation.D. Publications 2010/2011Publications 20101. Stumhofer, JS, Tait, ED, Quinn, W, Hosken , N, Spudy, B, Goenka, R,Jones, M, Rose-John, S, Elloso, MM, Grötzinger, J, Cancro, M, Levin,S, Hunter, C.A. 2010. IL-27p28 antagonizes gp130-mediated cytokinesignaling. Nature Immunol, 11, 1119-1126.2. Roeder, T., Stanisak, M., Gelhaus, C., Bruchhaus, I., Grötzinger, J.,Leippe, M. 2010. Caenopores are antimicrobial peptides in thenematode Caenorhabditis elegans instrumental in nutrition andimmunity. Dev Comp Immun 34, 203-209.3. Kuenzel, S., Till, A., Winkler, M., Häsler, R., Lipinski, S., Jung, S.,Grötzinger, J., Fickenscher, H., Schreiber, S., Rosenstiel, P. 2010. Thenucleotide oligomerisation binding domain-like receptor NLRC5 isinvolved in interferon-dependent antiviral immune responses. JImmunol 184, 1990-2000.4. Mysliwy, J., Dingley, A.J., Stanisak, M., Jung, S., Lorenzen, I.Roeder, T., Leippe, M., Grötzinger. J. 2010. Caenopore-5: The three-Impact Factor25.6683.2935.7453.29312

Research Report Biochemical Institute, Christian-Albrechts-University Kieldimensional structure of an antimicrobial protein from Caenorhabditiselegans. Dev Comp Immun 34, 323-330.5. Waetzig, H.G., Chalaris, A., Rosenstiel, P., Suthaus, J., Holland, C.,Karl, N., Uriarte, L.V., Till, A., Scheller, J., Grötzinger, J., Schreiber,S., Rose-John, S., Seegert, D. 2010. N-linked glycosylation is essentialfor the stability, but not the function of the interleukin-6 signaltransducer gp130. J Biol Chem 285, 1781-1789.6. Bens, S., Mohn, A., Yüksel, B., Kulle, A., Michalek, M., Chiarelli, F.,Özbek, M.N., Leuschner, I., Grötzinger, J., Holterhus, P.M., Riepe,F.G. 2010. Congenital Lipoid Adrenal Hyperplasia: Functionalcharacterization of three novel mutations in the STAT Gene. J ClinEndocrinol Metab. 95, 1301-1308.7. Tanasa, R.I., Trad, A., Lange, H., Grötzinger, J., Lemke, H. 2010.Allergen IgE-isotype-specific suppression by maternally derivedmonoclonal anti-IgG-idiotype. Allergy 65, 12-23.Publications 20111. Trad, A., Hedemann, N., Shomali, M., Pawlak, V., Grötzinger, J.,Lorenzen, I. Development of sandwich ELISA for detection andquantification of human and murine a disintegrin andmetalloproteinase17. J. Immunol. Methods 371, 91–96 (2011)2. Parajes, S., Kamrath, C., Rose, I.T., Taylor, A.E., Mooij, C.F., Dhir,V., Grötzinger, J., Arlt, W., Krone, N. A novel entity of clinicallyisolated adrenal insufficiency caused by a partially inactivatingmutation of the gene encoding for P450 side chain cleavage enzyme(CYP11A1). J Clin Endocrinol Metab. 96, 1798-1806, (2011)3. Lorenzen, I., Trad, A., Grötzinger , J. Multimerisation of A disintegrinand metalloprotease protein-17 (ADAM17) is mediated by its EGFlikedomain. Biochem Biophys Res Commun 415, 330-336 (2011)4. Bruhn, O., Cascorbi, I., Grötzinger, J., Jung, S. Antimicrobial peptidesand proteins of the horse - insights into a well armed organism.Veterinary Research 42, 1-22 (2011)5. Lorenzen, I., Shang, W., Perbandt, M., Svergun, D.I., Waetzig, G.H.,Rose-John, S., Hilgenfeld, R., Grötzinger. J. The structure of theunliganded extracellular domain of the interleukin-6 signal transducergp130 in solution. Eur J Cell Biol 90, 515-520 (2011)6. Marischen, L., Wesch, D., Oberg, H.H., Rosenstiel, P., Trad, A.,Shomali, M., Grötzinger, J., Janssen, O., Tchikov, V., Schütze, S.,Kabelitz, D. Functional expression of NOD2 in human peripheralblood γδ T-cells. Scand J Immunol. 74, 126-134 (2011)7. Jung, S., Mysliwy, J., Spudy, B., Lorenzen, I., Gelhaus, C., Podschun,R., Leippe, M., Grötzinger, J. Human β-defensin2 and β-defensin3chimeric peptides reveal the structural basis of the pathogenspecificity of their parent molecules. Antimicrob Agents CH 55, 954-960 (2011).8. Milenkov, M., Thummer, R., Glöer, J., Grötzinger, J., Jung, S.,Schmitz, R.A. Insights into membrane association of Klebsiellapneumoniae NifL under N 2 - fixing conditions by mutational analysis.J Bacteriol 193, 695-705 (2011)9. Thiele, S., de Sanctis, L., Werner R., Jueppner, H., Bastepe, M.,Grötzinger, J., Cumur, Hiort, O. Reclassification ofPseudohypoparathyroidism type I c based on molecular genetic andfunctional in-vivo data from naturally occurring mutations in theGNAS gene. Human Mutation 32, 653-660 (2011)10. Garbers C, Thaiss W, Jones GW, Waetzig GH, Lorenzen I, Guilhot F,Lissilaa R, Ferlin WG, Grötzinger J, Jones SA, Rose-John S, SchellerJ. (2011) Inhibition of classic signaling is a novel function of solubleGP130 which is controlled by the ratio of interleukin 6 and solubleinterleukin 6 receptor.J Biol Chem. 286, 42959-429705.3286.4956.297Impact Factor2.3406.4952.5953.7653.631.9354.6723.7265.9565.32813

Impact factors 2010: 56.119Impact factors 2011: 40.442Total impact factors 2010/2011: 96.561E. GrantsE.1 The structure of Interleukin-27 (p28) and its receptors WSX-1 and EBI-3, DFG SFB 415-B7,Fördersumme (2007 – 2010) 224.400 €.E.2 Excellenzcluster Inflammation at Interfaces, Integrated Research Network G: NOD-like receptors, TP5:NOD2 structure, Fördersumme (2010-2010); 170.000 €.E.3 Excellenzcluster Inflammation at Interfaces, Integrated Research Network F: gp130 signalling, TP1:Structural and molecular desing of novel cytokine antagonists, Fördersumme (2010-2011); 200.000 €.E.4 Excellenzcluster Inflammation at Interfaces, Cluster Stipend: NOD27 – Structure-Function and CellularLocalisation, Fördersumme (2010-2011); 78.600 €.E.5 Structure-function analysis of the extracellular part of ADAM17. DFG SFB 877-A6,Fördersumme (2010-2014) 301.200 €.E.6 Microscopy core facility and antibody production: Analysis of protease structure, cell biology andfunction. (Saftig/Grötzinger) DFG SFB 877-Z3 Fördersumme (2010-2014) 200.000 €.14

Research Report Biochemical Institute, Christian-Albrechts-University Kiel4. Research Group Prof. Dr. Jürgen SchellerA Group Leader: Prof. Dr. Jürgen SchellerB Lab Members: Post-Doc:Dr. Christoph Garbers (since 2011)Dr. Doreen FlossPh.D. Students:Christin DewitzChristoph GarbersEva WagenerJan SommerKatja MöllerOlga BraunTimo EffenbergerMedical doctoral studentsWolfgang ThaissDiploma studentsJan SommerTimo EffenbergerTechnicians:Annett LickertStefanie SchnellCResearch ReportC.1 Role of Interleukin 6 in vitro and in vivoThe immunoregulatory cytokine Interleukin-6 (IL6) acts in a pro- and anti-inflammatory fashion. Synthesized bymyeloid cells, fibroblasts and endothelial cells, IL6 on target cells, binds to the IL6 receptor (IL6R) and signalsvia complex formation with the ubiquitously expressed gp130 receptor. Paradoxically, most cells, which respondto IL6 during inflammatory states do not express the IL6R and are themselves not responsive to the cytokine. Anaturally occurring soluble form of the IL6R renders all cells responsive to IL6. Interleukin 6 (IL6) transsignalinghas emerged as a prominent regulator of immune responses during both innate and acquired immunity.Regulation of IL6 trans-signaling is reliant upon the release of soluble IL6 receptor (sIL6R), which binds IL6 to15

create an agonistic IL6/sIL6R complex capable of activating cell types that would not normally respond to IL6itself.C.2ADAM17:amolecularswitchtocontrolinflammationandtissueregenerationA disintegrin and metalloproteinase 17 (ADAM17), also known as tumor necrosis factor-a converting enzyme(TACE), is a membrane-bound enzyme that cleaves cell surface proteins, such as cytokines (e.g. TNFa),cytokine receptors (e.g. IL-6R and TNF-R), ligands of ErbB (e.g. TGFa and amphiregulin) and adhesion proteins(e.g. L-selectin and ICAM-1). Ectodomain shedding of these molecules can alter their biology and impact onimmune and inflammatory responses and cancer development. Gene targeting of Adam17 is embryonic lethal,highlighting the importance of ectodomain shedding during development. Tissue-specific deletion, orhypomorphic knock-in, of Adam17 demonstrates an in vivo role for ADAM17 in controlling inflammation andtissue regeneration.C.3 Elastin-like polypeptidesElastin-like polypeptides (ELPs) are highly biocompatible and exhibit a potentially highly useful property – thatof a thermally responsive reversible phase transition. These characteristics make ELPs attractive for drugdelivery, appealing as materials for tissue repair or engineering, and improve the efficiency with whichrecombinant proteins can be purified. ELP fusion proteins (referred to as `ELPylation´) inherit the reversiblephase transition property. ELPylation technology has recently been extended to plant cells, and a number ofplant-based expression systems have been evaluated for the production of ELPylated proteins. Here, we discussrecent developments in ELP technology and the substantial potential of ELPs for the deployment of transgenicplants as bioreactors to synthesize both biopharmaceuticals and industrial proteins.D Publications 2010/2011Publications 20101. Waetzig, G. H., Chalaris, A., Rosenstiel, P., Suthaus, J., Holland, C., Karl, N., Uriarte, L.V., Till, A., Scheller, J., Grötzinger, J., Schreiber, S., Rose-John, S., and Seegert, D. (2010)N-linked glycosylation is essential for the stability, but not the function of the interleukin-6signal transducer gp130. J Biol Chem 285, 1781-1789.2. Suthaus, J., Tillmann, A., Lorenzen, I., Bulanova, E., Rose-John, S., and Scheller, J. (2010)Forced homo- and heterodimerization of all gp130-type receptor complexes leads toconstitutive ligand-independent signaling and cytokine-independent growth. Mol Biol Cell21, 2797-2807.3. Rodriguez, N., Dietrich, H., Mossbrugger, I., Weintz, G., Scheller, J., Hammer, M.,Quintanilla-Martinez, L., Rose-John, S., Miethke, T., and Lang, R. (2010) Increasedinflammation and impaired resistance to Chlamydophila pneumoniae infection in Dusp1(-/-) mice: critical role of IL-6. J Leukoc Biol 88, 579-587.4. Matsumoto, S., Hara, T., Mitsuyama, K., Yamamoto, M., Tsuruta, O., Sata, M., Scheller,J., Rose-John, S., Kado, S., and Takada, T. (2010) Essential roles of IL-6 trans-signaling incolonic epithelial cells, induced by the IL-6/soluble-IL-6 receptor derived from laminapropria macrophages, on the development of colitis-associated premalignant cancer in amurine model. J Immunol 184, 1543-1551.5. Larsen, J. V., Hansen, M., Møller, B., Madsen, P., Scheller, J., Nielsen, M., and Petersen,C. M. (2010) Sortilin facilitates signaling of ciliary neurotrophic factor and related helicaltype 1 cytokines targeting the gp130/leukemia inhibitory factor receptor beta heterodimer.Mol Cell Biol 30, 4175-4187.6. Jones, G. W., McLoughlin, R. M., Hammond, V. J., Parker, C. R., Williams, J. D.,Malhotra, R., Scheller, J., Williams, A. S., Rose-John, S., Topley, N., and Jones, S. A.(2010) Loss of CD4 T cell IL-6R expression during inflammation underlines a role for IL-6 trans-signaling in the local maintenance of Th17 cells. J Immunol 184, 2130-2139.7. Giersberg, M., Floss, D., Kiprijanov, S., Conrad, U., and Scheller, J. (2010) Covalentdimerization between a camelidae anti-human TNF single domain antibody and theconstant kappa light chain domain improves neutralizing capacity. Biotechnol Bioeng 106,161-166.8. Gewiese-Rabsch, J., Drucker, C., Malchow, S., Scheller, J., and Rose-John, S. (2010) Roleof IL-6 trans-signaling in CCl₄induced liver damage. Biochim Biophys Acta 1802, 1054-1061.ImpactFactor5.5815.8614.4036.2776.1886.2772.9364.13916

Research Report Biochemical Institute, Christian-Albrechts-University Kiel9. Drucker, C., Gewiese, J., Malchow, S., Scheller, J., and Rose-John, S. (2010) Impact ofinterleukin-6 classic- and trans-signaling on liver damage and regeneration. J Autoimmun34, 29-37.10. Chalaris, A., Adam, N., Sina, C., Rosenstiel, P., Paliga, K., Lehmann, J., Schirmacher, P.,Hartmann, D., Cichy, J., Gavrilowa, O., Schreiber, S., Jostock, T., Matthews, V., Häsler,R., Becker, C., Neurath, M. F., Reiß, K., Scheller, J., and Rose-John, S. (2010) Critical roleof the disintegrin metalloprotease ADAM17 for intestinal inflammation and regenerationin mice. J Exp Med 207, 1617-1624.11. Chalaris, A., Gewiese, J., Paliga, K., Fleig, L., Schneede, A., Krieger, K., Rose-John, S.,and Scheller, J. (2010) ADAM17-mediated shedding of the IL6R induces cleavage of themembrane stub by gamma-secretase. Biochim Biophys Acta 1803, 234-245.12. Bender, M., Hofmann, S., Stegner, D., Chalaris, A., Bösl, M., Braun, A., Scheller, J., Rose-John, S., and Nieswandt, B. (2010) Differentially regulated GPVI ectodomain shedding bymultiple platelet-expressed proteinases. Blood 116, 3347-3355.Publications 20111. Suthaus, J., Adam, N., Grötzinger, J., Scheller, J., and Rose-John, S. (2011) ViralInterleukin-6: Structure, pathophysiology and strategies of neutralization. Eur J Cell Biol90, 495-504.2. Scheller, J., Chalaris, A., Schmidt-Arras, D., and Rose-John, S. (2011) The pro- and antiinflammatoryproperties of the Cytokine Interleukin-6. Biochim Biophys Acta - Mol CellRes 1813, 878-888.3. Scheller, J., Chalaris, A., Garbers, C., and Rose-John, S. (2011) ADAM17: a molecularswitch controlling inflammatory and regenerative responses. Trends Immunol 32, 380-387.4. Nechemia-Arbely, Y., Shriki, A., Denz, U., Drucker, C., Scheller, J., Raub, J., Pappo, O.,Rose-John, S., Galun, E., and Axelrod, J. H. (2011) Early hepatocyte DNA syntheticresponse posthepatectomy is modulated by IL-6 trans-signaling and PI3K/AKT activation.J Hepatol 54, 922-929.5. Malchow, S., Thaiss, W., Jänner, N., Waetzig, G. H., Gewiese-Rabsch, J., Garbers, C.,Yamamoto, K., Rose-John, S., and Scheller, J. (2011) Essential role of neutrophilmobilization in concanavalin A-induced hepatitis is based on classic IL-6 signaling but noton IL-6 trans-signaling. Biochim Biophys Acta 1812, 290-301.6. Kruglov, A. A., Tumanov, A. V., Grivennikov, S. I., Shebzukhov, Y. V., Kuchmiy, A. A.,Efimov, G. A., Drutskaya, M. S., Scheller, J., Kuprash, D. V., and Nedospasov, S. A.(2011) Modalities of Experimental TNF Blockade In Vivo: Mouse Models. Adv Exp MedBiol 691, 421-431.7. Jones, S. A., Scheller, J., and Rose-John, S. (2011) Therapeutic strategies for the clinicalblockade of IL-6/gp130 signaling. J Clin Invest 121, 3375-3383.8. Garbers C, Thaiss W, Jones GW, Waetzig GH, Lorenzen I, Guilhot F, Lissilaa R, FerlinWG, Grötzinger J, Jones SA, Rose-John S, Scheller J. (2011) Inhibition of classicsignaling is a novel function of soluble GP130 which is controlled by the ratio ofinterleukin 6 and soluble interleukin 6 receptor.J Biol Chem. 286, 42959-42970.9. Garbers, C., Jänner, N., Chalaris, A., Moss, M. L., Floss, D. M., Meyer, D., Koch-Nolte,F., Rose-John, S., and Scheller, J. (2011) Species specificity of ADAM10 and ADAM7 inIL-6 transsignaling and novel role of ADAM10 in inducible IL-6R shedding. J Biol Chem286, 14804-14811.10. Chalaris, A., Garbers, C., Rabe, B., Rose-John, S., and Scheller, J. (2011) The solubleInterleukin 6 receptor: Generation and role in inflammation and cancer. Eur J Cell Biol 90,484-494.11. Barkhausen, T., Tschernig, T., Rosenstiel, P., van Griensven, M., Vonberg, R. P., Dorsch,M., Mueller-Heine, A., Chalaris, A., Scheller, J., Rose-John, S., Seegert, D., Krettek, C.,and Waetzig, G. H. (2011) Selective blockade of interleukin-6 trans-signaling improvessurvival in a murine polymicrobial sepsis model. Crit Care Med 39, 878-888.12. Conrad, U., Plagmann, I., Malchow, S., Sack, M., Floss, D. M., Kruglov, A. A.,Nedospasov, S. A., Rose-John, S., and Scheller, J. (2011) ELPylated anti-human TNFtherapeutic single-domain antibodies for prevention of lethal septic shock. PlantBiotechnol J 9, 22-31.7.88114.7764.73310.37ImpactFactor3.6304.7339.5339.3345.2111.37914.1525.5815.5813.6306.2544.88617

Total impact factors 2010/2011: 153.326Impact factors 2010: 79.422Impact factors 2011: 73.904EGrantsE.1 Jürgen Scheller and Stefan Rose-JohnDer lösliche Interleukin-6-Rezeptor: Entstehung und physiologische BedeutungenSonderforschungsbereich 415, Teilprojekt B5Fördersumme (2007 – 2010) 248.700 €E.2 Stefan Rose-John and Jürgen SchellerThe function of the gp130-signaling-family for sleep and plasticitySonderforschungsbereich 654, Teilprojekt C5Fördersumme (2009 – 2013) 387.200 €E.3 Jürgen Scheller and Werner SolbachInfluences of sleep, hormones and cytokines on circadian rhythm of T cell activitySonderforschungsbereich 654, Teilprojekt C8Fördersumme (2009 – 2013) 353.600 €E.4 Jürgen SchellerADAM-protease activation in the context of IL-6R biologySonderforschungsbereich 877, Teilprojekt A2Fördersumme (2010 – 2011) 473.600 €E.5 Jürgen Scheller (2011-2014)ADAM-protease activation in the context of IL-6R biologyDFG-Einzelantrag SCHE 907/2-1 Fördersumme (2011-2014) 348.800 €18

Research Report Biochemical Institute, Christian-Albrechts-University Kiel5. Research Group Dr. Athena ChalarisA Group Leader: Dr. Athena ChalarisBLab Members:Ph.D. Students:Gerina VollmersJeanette SchwarzMedical doctoral studentsStephanie BlumDiploma studentsStefanie SchmidtTechnicians:Melanie BossC Research ReportC.1 Analysis of the role of the shedding protease ADAM17 in vivoUp to 10% of all cell surface proteins are proteolytically cleaved and released into the extracellular spaceShedding of integral membrane proteins is mostly observed for type I and type II transmembrane or GPIanchoredproteins, and the cleavage site is generally located in close proximity to the outer surface of the cellmembrane. Ectodomain shedding of transmembrane proteins provides a mechanism for protein down-regulationon the cell surface. In addition, production of soluble, functional protein ectodomains, including cytokines andsoluble cytokine receptors, can serve to initiate or inhibit autocrine and paracrine signaling. Members of theADAM family have emerged as major ectodomain shedding proteases. ADAMs are type I transmembraneproteins that consist of multiple domains. Most cleavage events examined are mediated by ADAM17 and itsclosest relative, ADAM10. 76 factors have been identified as substrates for ADAM17, among them importantimmuno-regulatory cytokines such as TNFα, ErbB-ligands and their receptors, Interleukin-6 Receptor (IL-6R)and cell adhesion molecules such as L-selectin (CD62L) or ICAM-1.The goal of our group is to elucidate the physiological role of the protease ADAM17 in vivo and to analyze itsrelevance for inflammatory ans neoplastic disease states.ADAM17-deficient mice have defects in the mammary epithelium, vascular system, lung, eye, hair, heart andskin and as a result die between embryonic day 17.5 and the first few days after birth. To study ADAM17-deficiency in vivo we have used a novel approach to generate mice with minimal ADAM17 expression in alltissues. These ADAM17 ex/ex mice are viable, and reach a normal life span. We use this mouse model in ourlaboratory to study the function of ADAM17 in inflammatory disorders like Crohn's disease, Multiple Sclerosisas well as epithelial cancer development. Recently we could show that, although ADAM17 ex/ex mice showed noTNFα shedding, they were highly susceptible to DSS-induced experimental colitis. Therefore we hypothesizethat ADAM17 balances inflammatory pathways and regeneration in injured tissues. Inhibition of ADAM17during inflammatory processes might result in a severe blockade of regenerative processes, resulting in unwantedside effects.19

D Publications 2010/2011Publications 20101. Waetzig, G. H., Chalaris, A., Rosenstiel, P., Suthaus, J., Holland, C.,Karl, N., Uriarte, L. V., Till, A., Scheller, J., Grötzinger, J., Schreiber,S., Rose-John, S., and Seegert, D. (2010) N-linked glycosylation isessential for the stability, but not the function of the interleukin-6signal transducer gp130. J Biol Chem 285, 1781-17892. Chalaris, A., Gewiese, J., Paliga, K., Fleig, L., Schneede, A., Krieger,K., Rose-John, S., and Scheller, J. (2010) ADAM17-mediated sheddingof the IL6R induces cleavage of the membrane stub by gammasecretase.Biochim Biophys Acta 1803, 234-2453. Vahdat, A.M., Reiners, K.S., Simhadri, V.L., Eichenauer, D.A., Büll,B., Chalaris, A., Simhadri, V.R., Wiegmann, K., Krell, H.W., Rose-John, S., Engert, A., von Strandmann, E.P., Hansen, H.P. (2010) TNFalpha-convertingenzyme (TACE/ADAM17)-dependent loss of CD30induced by proteasome inhibition through reactive oxygen species.Leukemia 24, 51-574. Bender, M., Hofmann, S., Stegner, D., Chalaris, A., Bösl, M., Braun,A., Scheller, J., Rose-John, S., and Nieswandt, B. (2010) Differentiallyregulated GPVI ectodomain shedding by multiple platelet-expressedproteinases. Blood 116, 3347-33555. Chalaris, A., Adam, N., Sina, C., Rosenstiel, P., Lehmann, J.,Schirmacher, P., Hartmann, D., Cichy, J., Gavrilowa, O., Schreiber, S.,Jostock, T., Matthews, V., Häsler, R., Becker, C., Neurath, M. F., Reiß,K., Scheller, J., and Rose-John, S. (2010) Critical role of the disintegrinmetalloprotease ADAM17 for intestinal inflammation and regenerationin mice. J Exp Med 207, 1617-1624Publications 20111. Scheller, J., Chalaris, A., Schmidt-Arras, D., and Rose-John, S. (2011)The pro- and anti-inflammatory properties of the Cytokine Interleukin-6. Biochim Biophys Acta - Mol Cell Res 1813, 878-8882. Scheller, J., Chalaris, A., Garbers, C., and Rose-John, S. (2011)ADAM17: a molecular switch controlling inflammatory andregenerative responses. Trends Immunol 32, 380-3873. Garbers, C., Jänner, N., Chalaris, A., Moss, M. L., Floss, D. M., Meyer,D., Koch-Nolte, F., Rose-John, S., and Scheller, J. (2011) Speciesspecificity of ADAM10 and ADAM7 in IL-6 transsignaling and novelrole of ADAM10 in inducible IL-6R shedding. J Biol Chem 286,14804-148114. Chalaris, A., Garbers, C., Rabe, B., Rose-John, S., and Scheller, J.(2011) The soluble Interleukin 6 receptor: Generation and role ininflammation and cancer. Eur J Cell Biol 90, 484-4945. Barkhausen, T., Tschernig, T., Rosenstiel, P., van Griensven, M.,Vonberg, R. P., Dorsch, M., Mueller-Heine, A., Chalaris, A., Scheller,J., Rose-John, S., Seegert, D., Krettek, C., and Waetzig, G. H. (2011)Selective blockade of interleukin-6 trans-signaling improves survival ina murine polymicrobial sepsis model. Crit Care Med 39, 878-888Impact Factor5.5814.7338.96610.3714.776Impact Factor4.7339.5335.5813.6306.254Total impact factors 2010/2011: 74.258Impact factors 2010: 44.527Impact factors 2011: 29.73120

Research Report Biochemical Institute, Christian-Albrechts-University KielEGrantsE.1 Athena Chalaris and Stefan Rose-JohnAnalysis of the role of the shedding protease ADAM17 in vivoSonderforschungsbereich 877, Teilprojekt A1Fördersumme (2010 – 2013) 433.760 Euro21

Research Report Biochemical Institute, Christian-Albrechts-University Kiel6. Research Group Dr. Dirk Schmidt-ArrasA Group Leader: Dr. rer. nat. Dirk Schmidt-ArrasB Lab Members: PhD students:Antje SchüttMarija StevanovicMiryam MüllerBachelor student:Ruven WilkensTechnicians:Silke HornNico Schneider(technician trainee)CResearch Report:C.1 Spatio-temporal signalling of oncogenic cytokine receptorThe cytokine interleukin-6 (IL-6) plays important roles in inflammation and regeneration. In complex with theIL-6 receptor it binds to signalling subunit gp130 which is expressed in all cell types. Recently deletion mutantsof gp130 have been identified in inflammatory hepatocellular adenomas. The deletions occured predominantlywithin the IL-6 binding loop in the D2 domain of the gp130 extracellular domain. These mutants rendered gp130constitutively active in a ligand-independent manner. So far it is not understood how these mutations can lead togp130 activation.Based on the IL-6/IL-6R/gp130 hexamerix crystal structures we hypothesized that (I) destabilization of IL-6binding loop conformation leads to gp130 activation and (II) activation of gp130 involves destabilization of ahydrophobic core connecting D2 and D3 domain. We therefore generated different point mutants in thebackground of gp130 wildtype and gp130 DY186-Y190 (gp130 DYY). Interestingly mutation of Asp-218 andCys-172 which position the IL-6 binding loop lead to activation of gp130, confirming our hypothesis (see Fig.1).Substitution of Val-252 by the small residue glycine also lead to activation of gp130 pointing to a role of thehydrophobic core in gp130 activation. Furthermore when we deleted the N-terminal D1 domain or only the firstN-terminal five residues, gp130 DYY signalling was completely abrogated. Using two differently taggedversions of the gp130 DYY DD1 or gp130Furthermore when we deleted the N-terminal D1 domain or only the first N-terminal five residues, gp130 DYYsignalling was completely abrogated. Using two differently tagged versions of the gp130 DYY DD1 or gp130Cell biological analysis of gp130 DYY revealed that downstream signal activation is not solely dependend onJAK kinases, but also on Src kinases. Interestingly downstream signalling was blocked by Geldanamycin,indicating that gp130 DYY or downstream kinases are dependent on the chaperone HSP90.Furthermore we found that gp130 DYY displays impaired maturation and predominantly exist in a highmannoseform which resides in the endoplasmic reticulum (ER), which we could show by biochemical analysis andconfocal microscopy (see Fig.1). In addition to the ER, gp130 DYY is also found in Rab5+ early endosomes.23

Destabilization of IL-6 binding loop leads to gp130 activation. right panel: gp130 DYY displays delayed maturation asrevealed by 35S-cysteine/methionine metabolic labeling and localizes predominantly inside the endoplasmic reticulum.Using artificially ER-retained gp130 DYY and internalization deficient gp130 DYY mutants we're currentlyinvestigating the contribution of these two gp130 DYY pools to the oncogenic signalling. In order to find outthe physiological consequences of gp130 deletion mutants in vivo, we generated transgenic mice expressinggp130 DYY as EYFP fusion under the control of the hepatocyte-specific Albumin promotor. Initial analysis ofthese mice revealed increased acute-phase protein levels in the serum, as well as increased STAT3phosphorylation in the liver. Histological analysis revealed that in two lines an increased lipid deposition wasobserved. In all transgenic lines a mild inflammatory phenotype was observed. Further analysis are ongoing alsoin respect to Adenoma formation.C.2 The impact of IL-6 trans-signalling on liver regeneration and cancer formationAn artificially constitutive active L-gp130 construct has been published previously. Hereby the extracellulardomain of gp130 has been replaced by the Leucin-zipper of c-Jun and an additional disulfide bridge. Wegenerated transgenic mice expressing L-gp130 under the control of a bidirectional tetracycline-inducible TREpromotor allowing parallel expression of firefly luciferase. These mice were crossed to LAP-tTA (TET-offsystem), LAP-rtTA (TET-on) and CMV-rtTA(TET-on) mice. Doxycyclin was administered via food or drinkingwater. Although we could detect the rtTA transcript in the liver, as well as doxycyclin in the serum, there was noonset of luciferase activity or Lgp130 expression in the liver detectable. CMV-rtTA/Lgp130 mice displayedluciferase activity in other organs like heart and stomach indicating that the promotor might not be activatable inhepatocytes.C.3 The impact of ADAM proteases on liver regeneration and cancer formationThe family of ADAM (A disintegrin and metalloprotease) family has been implicated in a variety of signallingprocesses. ADAM10 has been identified as sheddase for the Notch receptor. In collaboration with the group ofPaul Saftig we're currently establishing an inducible hepatocyte-specific knock-out of ADAM10 in mice. Wetherefore generated triple-transgenic mice harboring two floxed ADAM10 alleles, Cre recombinase expressionunder the control of the Tet-repressor element (TRE), as well as the epxression of the Tet-repressor tTA underthe control of the liver-specific LAP-promotor. After initial characterization of the knock-out mice which iscurrently ongoing, we will perform partial hepatectomy on these mice in collaboration with Juri Bergmann andanalyse the regenerative potential. In a recent report it has been shown that metastasizing tumor cells stimulatemetastasis-associated macrophages via the secretion of extracellular matrix proteins. Furthermore tumor growthand metastasis was strongly dependent on TNFa. Given the fact that ADAM17 has been shown to be the majorsheddase of TNFa (tumor necrosis factor) we hypothesized that ADAM17 hypomorphic mice might be protectedfrom tumor metastasis. As expected, when we treated murine bone-marrow derived macrophages with theconditioned supernatant of murine tumor cell lines, TNFa secretion was diminished in ADAM17 hypomorphicmice as compared to its wildtype counterpart. We're currently establishing a tumor metastasis model using theC57BL/6 syngeneic tumor cell lines LLC (lung carcinoma), CMT93 (colon carcinoma) and Hep56.1D(hepatoma). We will inject these cell lines in C57BL/6 wt and ADAM17 hypomorphic mice in order to analysethe role of ADAM17 in the tumor and metastasis stroma. For the analysis of the role of ADAM17 within thetumor itself, we generated stable shRNA-mediated knock-down of ADAM17 in the above mentionned cell lines.24

Research Report Biochemical Institute, Christian-Albrechts-University KielWe could see that migration of ADAM17kd CMT93 cells was reduced in a scratch wound healing assay. We'recurrently analysing the underlying molecular mechanisms.C.4 Regulation of ADAM proteases by TetraspaninsAs the name indicates tetraspanins are a family of transmembrane proteins containing four transmembranehelices. Tetraspanins are involved in adhesion, motility, matrix modulation and cell fusion. Members of thetetraspanins have been shown to regulate the function of ADAM proteases. We could contribute to the analysisof the regulation of ADAM10 by Tetraspanin 15 which have been mainly performed by Johannes Prox in thegroup of Paul Saftig. Expression of Tetraspanin 8 (Tspan8) has been correlated with tumor growth andmetastasizing potential. Normally barely expressed in hepatocytes it is upregulated in hepatocellular carcinoma.Given the fact that Tspan8 is found in a complex with the cell adhesion molecule EpCAM, which has beenidentified as ADAM17 substrate, we hypothesize that Tspan8 regulates ADAM17 activity. In collaboration withMargot Zölller we are currently investigating the functional interplay of these three proteins in hepatocytes andcolon epithelial cells using FACS analysis and co-immunoprecipitation. Furthermore we generated a geneticallyencoded FRET-based activity sensor of ADAM17 which we are currently characterizing. This sensor will helpus to analyse the impact of Tetraspanins on ADAM17 activity of endogenously expressed ADAM17. In the nearfuture we will establish a HepaRG cell line with tetracycline-inducible expression of Tspan8 in order to analyseits effect on non-tumors hepatocytes. Moreover we're currently generating a tetracycline-induciblemicroRNA30-based expression construct to supress Tspan8 in human tumor cell lines. In order to findinteracting partners of Tspan8 and ADAM17 in hepatocellular carcinoma we're currently setting up a veryrecently published Luciferase complementation-based interaction assay in collaboration with Yves Jacob fromthe Institut Pasteur Paris.D. Publications 2010/2011:Publications 20101. Yau WL, Blisnick T, Taly JF, Helmer-Citterich M, Schiene-Fischer C,Leclercq O, Li J, Schmidt-Arras D, Morales MA, Notredame C, RomoD, Bastin P, Späth GF. (2010) Cyclosporin A treatment of Leishmaniadonovani reveals stage-specific functions of cyclophilins in parasiteproliferation and viability. PLoS Negl Trop Dis. 4(6):e729.Publications 20111. Schmidt-Arras D, Leclercq O, Gherardini PF, Helmer-Citterich M,Faigle W, Loew D, Späth GF. (2011) Adaptation of a 2D in-gel kinaseassay to trace phosphotransferase activities in the human pathogenLeishmania donovani. J Proteomics. 2011 74;1644-51.2. Scheller J, Chalaris A, Schmidt-Arras D, Rose-John S.: The pro- andanti-inflammatory properties of the cytokine interleukin-6. (2011)Biochim Biophys Acta. 1813, 878-88.Impact Factor4.69Impact Factor5.0804.733Total impact factors 2010/2011: 14.50Impact factors 2010: 4.69Impact factors 2011: 9.8125

Research Report Biochemical Institute, Christian-Albrechts-University Kiel7. Research Group Prof. Dr. Paul SaftigA Lab Leader: Prof. Dr. Paul SaftigB Lab Members: Group leaders:Michael SchwakeBernd SchröderJudith BlanzPostDoc:Andrea RittgerAlexander SchneedePh.D. Students:Silvio WeberJohannes ProxTelly SavallasJohann GrothChristina ZachosMichelle DanaharJanna SchneppenheimChristian RaabFrederike ZunkeSebastian WetzelSandra KissingMedical Students:Meike LüdemannMiriamWagnerJohannes KnabbeDiploma Students:Jörg BehmkeNur GüneliTechnician:Marlies RuschMaike Langer27

Inez GöttingSebastian HeldTobias LehmannTechnician traineeMerjem SenkaraRaphael KurzRouven BahnMarvin MurowskiSecretaryGundula HohnCResearch ReportC.1 Biology of lysosomes: Lysosomes, lysosomal membrane proteins, hydrolases andlysosomal storage disease:Lysosomes are ubiquitous organelles that constitute the primary degradative compartments of the cell. Theyreceive their substrates through endocytosis, phagocytosis or autophagy. The catabolic function of lysosomes iscomplemented by lysosome-related organelles (LROs), such as melanosomes, lytic granules, majorhistocompatibility complex (MHC) class II compartments and platelet dense granules. LROs share manyproperties with lysosomes, but they also contain celltype specific proteins and might require additional cellularmachinery for their biogenesis. Lysosomes and LROs are involved in various physiological processes, such ascholesterol homeostasis, plasma membrane repair, bone and tissue remodelling, pathogen defence, cell death andcell signalling. These complex functions make the lysosome a central and dynamic organelle and not simply thedead end of the endocytic pathway. Two classes of proteins are essential for the function of lysosomes: solublelysosomal hydrolases (also referred to as acid hydrolases) and integral lysosomal membrane proteins (LMPs).Each of the 50 known lysosomal hydrolases targets specific substrates for degradation, and their collective actionis responsible for the total catabolic capacity of the lysosome. In addition to bulk degradation and proproteinprocessing, lysosomal hydrolases are involved in antigen processing, degradation of the extracellular matrix andinitiation of apoptosis.Cartoon of a lysosome (from TIBS, 1086): The lysosome is a central, acidic organelle that is involved in the degradation ofmacromolecules through the activity of lysosomal hydrolases. Lysosomes are crucial for the maturation of phagosomes tophagolysosomes in phagocytosis, which is important for cellular pathogen defence. The macroautophagy pathway mediatesthe turnover of cytoplasmic components, such as organelles and large complexes, and is involved in cell death andproliferation. Macroautophagy depends on the fusion of lysosomes with autophagosomes to create autolysosomes, in whichdegradation occurs. Macroautophagy and chaperone-mediated autophagy, a direct lysosomal transport process for cytosolicproteins, are regulated by lysosome-associated membrane proteins (LAMPs). Lysosomal exocytosis and plasma membranerepair are Ca2+ and synaptotagmin 7 (SYT7)-dependent fusion events, which are possibly involved in pathogen entry,autoimmunity and neurite outgrowth. The lysosomal cell death pathway is triggered by a release of lysosomal cathepsinsthrough an unknown mechanism. Cellular cholesterol homeostasis is controlled by lysosomal cholesterol efflux throughNiemann–Pick C1 protein (NPC1). Major histocompatibility complex (MHC) class II-dependent antigen presentationrequires lysosomal proteases and membrane proteins. The release of exosomes is thought to be involved in adaptive immuneresponses. Lysosomal membrane proteins are also involved in the transport of newly synthesized hydrolases to the lysosome(for example, lysosomal integral membrane protein 2 (LIMP2)) and across the lysosomal membrane (for example, the V-typeH+-ATPase complex and chloride channel protein 7 (CLC7).28

Research Report Biochemical Institute, Christian-Albrechts-University KielThe mammalian lysosome contains ~25 LMPs, but additional LMPs are being revealed. LMPs reside mainly inthe lysosomal limiting membrane and have diverse functions, including acidification of the lysosomal lumen,protein import from the cytosol, membrane fusion and transport of degradation products to the cytoplasm. Themost abundant LMPs are the lysosome associated membrane protein (LAMP1), LAMP2, lysosome integralmembrane protein 2 (LIMP2; also known as SCARB2) and the tetraspanin CD63.Lysosome biogenesis requires integration of the endocytic and biosynthetic pathways of the cell. Lysosomaltargeting of newly synthesized lysosomal proteins can be direct, from the trans-Golgi network (TGN) to theendosomal system, or indirect, involving transport to the plasma membrane and subsequent endocytosis. Thebest understood direct pathway is the mannose 6 phosphate receptor (M6PR) mediated transport of lysosomalhydrolases. By contrast, remarkably little is known about the structural and molecular machinery for thetransport of LMPs to lysosomes.The significance of tightly regulated LMP trafficking is illustrated by recent findings that describe new andunexpected roles for LMPs in cellular physiology. It is becoming apparent that LMPs can impose specificfunctions onto the organelles through which they are transported or in which they reside, such as theendoplasmic reticulum (ER), lysosomes and the plasma membrane. Their importance is further highlighted bythe discovery of an increasing number of gene mutations that lead to lysosomal dysfunction and disease. Inaddition, various knockout mice and non mammalian model organisms have highlighted the role of LMPs in cellphysiology. We are continuing our efforts to characterize the molecular functions of these proteins for health anddisease.Towards a new therapy for the lysosomal storage disorder alpha-Mannosidosis:The European HUE-MAN network is coordinated through activities in our lab. Basic scientists and clinicians contribute tothe development of an enzyme replacement therapy fort he human alpha-Mannosidosis disease.The lysosomal storage disorder (LSD) alpha-Mannosidosis is a rare genetic disease affecting less than 500people worldwide and according to the EU regulations, designated as an "orphan" disease. Alpha-Mannosidosisis caused by an enyzme defect due to mutations in the gene for lysosomal acid alpha-Mannosidase (LAMAN)affecting the lysosomal and cellular glycoprotein catabolism with severe consequences for the organism. Inhumans, LAMAN deficiency results in progressive mental retardation, skeletal changes, hearing loss andrecurrent infections and many patients die during early childhood. Today, the most promising therapy forlysosomal storage disorders including alpha-Mannosidosis is Enzyme Replacement Therapy (ERT) where therespective enzyme lacking in the patient is produced by recombinant approaches and then introduced into theblood stream, from where it is internalized by the cells and reaches the lysosomes replacing the missingendogenous enzyme. ERT products are on the market today for a number of LSD including Gaucher, Fabry,Pompe disease and the Mucopolysaccharidoses MPSI, II and VI and clinical trials are underway for a number ofothers. To date, no real treatment for alpha-Mannosidosis is available. Since children are born healthy, an earlyinitiated therapy shortly after birth could dramatically improve their life expectancy and quality of life.Since pharmaceutical interest in this disease is low, two EU sponsored projects (EURAMAN and HUE-MAN)within the 5th and 6th framework program, respectively have worked towards developing the recombinanthuman enzyme (rhLAMAN) as a therapeutic agent for patients suffering from alpha-Mannosidosis and are nowthe basis for clinical trials in alpha-Mannosidosis. The enzyme has received Orphan Drug Designation in Europein January 2005 (EU/3/04/260).The main objectives of the ALPHA-MAN network are to transfer and expand the information and knowledgegained from the many years of work from the previous EURAMAN and HUE-MAN projects, to enable toperform “First in Man” clinical trials in alpha-Mannosidosis patients, using the medicinal enzyme productrhLAMAN as the therapeutic agent and furthermore to improve the knowledge regarding i) long term “chronicdosing” and ii) mechanism of lysosomal enzyme transfer across the Blood Brain Barrier, in a newly establishedimmune-tolerant mouse model. The final goal of ALPHA-MAN is to make a future treatment for ALL alpha-29

Mannosidosis patients available and thereby dramatically improve their life expectancy and quality of life. Inaddition, ALPHA-MAN will greatly increase the knowledge about the mechanism of how lysosomal enzymescan cross the blood brain barrier, which is also of great medical importance for the treatment of otherneurodegenerative disorders.Biology of the newly identified lysosomal sorting receptor LIMP-2:We (PI: Michael Schwake) have identified an unexpected role for the lysosomal membrane protein LIMP-2 as asorting receptor required for the delivery of the lysosomal enzyme beta-glucocerebrosidase to lysosomes. TheMannose-6-Phosphate-receptor pathway has been very well characterized as a major route for the sorting oflysosomal enzymes; however, the mechanism for the intracellular targeting ofbeta-glucocerebrosidasetolysosomes has been unclear until now.Our findings that beta-glucocerebrosidase associates with LIMP-2, thatthese proteins colocalize in intracellular vesicular compartments, and that the activity, levels, and localization ofbeta-glucocerebrosidase exhibit a dramatic correlation with the presenceor absence of LIMP-2 reveal that betaglucocerebrosidaseindependence of Mannose-6-Phosphate-based sorting mechanisms is almost certainly aconsequence of its routing via LIMP-2 through the lysosomalmembrane protein delivery pathway.The classical and the non-classicalLIMP-2 dependent lysosomal targeting pathwayAction myoclonus-renal failure syndrome (AMRF) is caused by mutations in LIMP-2. To date, six AMRFcausingmutations have been described, including splice site, missense and nonsense mutations. All mutationslead to a retention of LIMP-2 in the endoplasmic reticulum (ER) but affect the binding to b-GC differentially.The LIMP-2 segment 145–288, comprising the nonsense mutations, contains a highly conserved coiled-coildomain, which we suggest determines beta-glucocerebrosidase binding. Disruption of the helical arrangementand amphiphatic nature of the coiled-coil domain abolishes beta-glucocerebrosidase binding. In contrast to thereduced binding properties of the nonsense mutations, the only missense mutation (H363N) found in AMRFleads to increased binding of beta-glucocerebrosidase to LIMP-2, indicating that this highly conserved histidinemodifies the affinity of LIMP-2 to its ligand. We suggest that disruption of the coiled-coil structure or AMRFdisease-causing mutations abolish beta-glucocerebrosidase binding, indicating the importance of an intact coiledcoilstructure for the interaction of LIMP-2 and beta-glucocerebrosidase.The graduate research training school (GRK1459): Sorting and interaction betweenproteins of subcellular compartmentsThe DFG-Research Training Group 1459 is co-coordinated by our group and scientists from the UniversityMedical Center Hamburg-Eppendorf and the Bernhard-Nocht-Institute for Tropical Medicine in Hamburg. Theprogram is open to students with a diploma/master in natural sciences and medical students. The general topic ofthe Research Training Group is sorting and transport of selected proteins within the Golgi apparatus andendosomal compartments. In these organelles the decision is made whether a newly synthesized protein reachesits target via the secretory/biosynthetic pathway, or a recently internalized molecule (or bacterium) reaches itsintracellular destination via the endocytic/phagocytic pathway. Missorted proteins may lead to loss of function intheir target organelles, that may affect the well being of the cell and the organism as a whole.30

Research Report Biochemical Institute, Christian-Albrechts-University KielTherefore, the experimental approaches are related to diseases. By focussing on selected model proteins, basicmechanisms of the biogenesis of intracellular compartments as well as the balance of membrane transportbetween organelles and the interplay between cytosolic and membrane proteins will be investigated. Themajority of projects addresses sorting and transport processes under pathological conditions in cells derived frompatients or mouse models of human diseases, or cells infected by bacteria or in parasite cells. New insights intothe interactions between resident proteins of endosomes and the Golgi apparatus with components of thevesicular transport machinery and the actin cytoskeleton will be expected. A better understanding of cellularresponses to endogenous mutant proteins or exogenous pathogens will enable the development of noveltherapeutic strategies. Different experimental approaches such as ultrastructural analysis of cellularcompartments, genomics, biochemistry, time-resolved imaging, and structural biology will be applied andimprove our understanding of spatial and dynamic aspects of membrane transport or translocation. Students willgo through a three year curriculum of academic as well as non-academic courses in molecular and cellularbiology, biochemistry, infectiology, microbiology, and molecular biomedicine. The Research Training Groupoffers a continuous educational program with lectures, practical courses, seminars, regular report meetings andan international symposium every two years. The practical courses consist of several three-day hands trainingunits attended by up to 4 students. The company OLYMPUS is associated to the Research Training Group andoffers additional seminars on new developments in microscopy and practical courses. Seminars will be given byleading scientists and will foster a broad view on current topics of molecular life sciences. It is expected thateach student spends 1-3 months abroad in a laboratory cooperating in the research field. The program provides abroad education, not just on the specific topic of the thesis, but also in research topics of the other participatinggroups. The GRK has recently be reevaluated and is likely to be continued until 2017.Discovery and elucidation of the functions of new and unknown lysosomal membraneproteins:The identification of new lysosomal membrane proteins by subproteomic approaches (PI: Bernd Schröder), inwhich new members of the lysosomal membrane are being investigated is another focus in the lab. Thefunctional characterization of these new members of the lysosomal membrane will be done using biochemicaland mouse genetic approaches. Of special interest in these context are two proteins of hitherto unknownfunction, DIRC2 and TMEM192.Example of a new putative lysosomal transporter protein DIRC2. The putative topology and the lysosomaltargeting motifsare indicatedThe concept of regulated intramembrane proteolysis has emerged over the last decades as a novel concept ofcellular signalling. One group among the proteases being capable of cleaving substrates within the phospholipidbilayer are the signal-peptide-peptidase (SPP) and its homologues, the signal peptide-peptidase-like proteins(SPPL2a, -2b, -2c, -3). SPPL2a is present in membranes of lysosomes/late endosomes whereas SPPL2b was31

eported to reside at the plasma membrane as well as in endosomal compartments. To date, only TNFα, FasLigand (FasL) and Bri2(Itm2b) have been identified as substrates of SPPL2a/b. In the case of TNFα it wasdemonstrated that the proteolytic release of the TNFα intracellular domain influences gene expression andinduces the synthesis of the pro-infammatory cytokine IL12. Two of the known substrates suggest a regulatoryfunction of SPPL2a and SPPL2b in the context of the immune system. In order to analyse functions of SPPL2aand SPPL2b in a complex in vivo system, mouse lines deficient in either of the two proteases have beengenerated. Preliminary immunological phenotyping has revealed major abnormalities in SPPL2a -/- mice.C.2 Proteolysis in or at membranesProteolytic ectodomain release, a process known as “shedding”, has emerged as a key mechanism for regulatingthe function of a diversity of cell surface proteins. Shedding of integral membrane proteins is to our knowledgelimited to type I and type II transmembrane proteins or GPI-anchored molecules, in which the cleavage site isgenerally located close to the membrane surface. A Disintegrin And Metalloproteinases (ADAMs) have emergedas the major proteinase family that mediates ectodomain shedding. ADAM-mediated shedding is important in anumber of biological processes such as the interaction of sperm and egg, cell fate determination, cell migration,wound healing, neurite and axon guidance, heart development, immunity, cell proliferation and angiogenesis. Itis estimated that up to 4% of the proteins on the cell surface undergo ectodomain shedding affecting functionallydiverse proteins, such as cadherins, L-selectin, Fas ligand, TNF-α, EGFR ligands, ErbB2, ErbB4, AmyloidPrecursor Protein (APP), Notch receptor, Notch ligands and many others. ADAMs as proteins of about 750amino acid length, are characterized by a conserved domain structure, consisting of an N-terminal signalsequence followed by a prodomain, a metalloproteinase domain, a disintegrin domain with a cysteine-richregion, a transmembrane domain and a cytoplasmic tail. ADAM-mediated shedding is both constitutive andinducible, dependent on G-protein coupled receptors, protein kinase C, intracellular Ca 2+ levels, membrane lipidcomposition and other experimental and natural stimuli. Also modulation of ADAM activity by removal of theinhibitory prodomain, by changing their intracellular distribution and by interaction of proteins, and/orposttranslational modifications of their cytoplasmatic tails play a role in the regulation of ectodomain shedding.Dysregulation of ectodomain shedding is associated with autoimmune and cardiovascular diseases, infection,inflammation and cancer. Therefore, ADAMs are attractive targets for novel therapies. It becomes increasinglyclear that further research especially on the regulation and control of ADAM activity, ADAM redundancy insubstrate processing, ADAM structure, interaction of ADAM with regulatory proteins and the physiologicalelucidation of the relevance of ectodomain shedding is needed. Regarding the latter aspect, the use ofconditionally targeted mice in conjugation with disease models will be extremely helpful to elucidate the role ofthese proteins in selected tissues and developmental stages.ADAM10 conditional knockout in adult skin (see Weber et al. 2011; Development)Making use of our conventional knockout mice and cell lines derived from these embryos, we could identify anumber of ADAM10-specific substrates. We could show that ADAM10-mediated ectodomain sheddingmodulates the function of cell adhesion molecules and represents an essential process in the regulation ofparacrine, juxtacrine and autocrine signaling. We demonstrated that neuronal cadherin (N-cadherin) is cleavedspecifically by ADAM10 in its ectodomain. The ADAM10-induced N-cadherin cleavage resulted in changes inthe adhesive behaviour of cells and also in a dramatic redistribution of beta-catenin from the cell surface to thecytoplasmic pool, thereby influencing the expression of b-catenin target genes. Analysis of ADAM10-deficientfibroblasts, inhibitor studies, and RNA interference-mediated down-regulation of ADAM10 demonstrated thatADAM10 is also responsible for the constitutive and regulated shedding of E-cadherin in fibroblasts andkeratinocytes. ADAM10-mediated E-cadherin release is also regulated by proinflammatory cytokines therebymodulating keratinocyte cohesion in eczematous dermatitis. Interestingly, gamma-Protocadherins (Pcdh), whichare enriched at synapses and involved in synapse formation, specification, and maintenance, are also substratesfor ADAM10. Our results demonstrated that ADAM10-mediated Pcdh shedding represents the prerequisite forfurther processing through g-secretase activity leading to the accumulation of Pcdh fragments in the nucleus. Itwas also revealed by us that ADAM10 regulates the endothelial permeability and T-cell transmigration byshedding of vascular endothelial cadherin.32

Research Report Biochemical Institute, Christian-Albrechts-University KielWe were also able to show that ADAM10 and ADAM17 play an important functional role by regulating L1-dependent neuronal cell adhesion, cell migration, and neurite outgrowth. The emerging critical role of ADAM10for membrane proteolysis is underlined by the fact that we were able to identify in different collaborativeprojects a number of further important transmembrane proteins which are susceptible for ADAM10-mediatedectodomain shedding and cell signaling. The CXC-Chemokin-ligand 16, CX3CL1 (fractalkine) (24,25), CD44,betacellulin, Axl, desmoglein2, receptor tyrosine phosphatase, RAGE, Klotho, CD23 and ephrin are examplesfor the growing list of ADAM10 specific substrates. Interestingly, Bri2 (Itm2b), as a type II-orientedtransmembrane protein which is associated with familial British and Danish dementia, is first shed by ADAM10and subsequently processed by SPPL2A/SPPL2B. Also the apoptosis-inducing Fas ligand (FasL) is a type IItransmembrane protein that is involved in the downregulation of immune reactions by activation-induced celldeath (AICD) as well as in T cell-mediated cytotoxicity. Using pharmacological approaches in 293T cells, invitro cleavage assays as well as loss and gain of function studies in MEF cells, we could show that the ADAM10is critically involved in the shedding of FasL which is supposed to be a prerequisite to further intramembranecleavage by SPPL2 proteases. Interestingly, we could demonstrate that ADAM10 itself is also subject toregulated intramembrane proteolysis. ADAM9 and -15 were identified as the proteases responsible for releasingthe ADAM10 ectodomain, and presenilin (gamma-secretase) as the protease responsible for the release of theADAM10 intracellular domain (ICD). This domain then translocates to the nucleus and localizes to nuclearspeckles, thought to be involved in gene regulation. We concluded that ADAM10 performs a dual role in cells,as a metalloprotease when it is membrane-bound, and as a potential signaling protein once cleaved byADAM9/15 and the gamma-secretase. In collaborative studies with Carl Blobel, New York, we used ourADAM10/ADAM17 chimeric expression constructs to show that for PMA-stimulated TGF-alpha shedding, theintact ectodomain of ADAM17, but not its cytoplasmic and transmembrane domains are required. We could alsoshow that Ca ++ influx and stimulation of the P2X7R signaling pathway activate ADAM10 as sheddase of manyADAM17 substrates in Adam17 -/- fibroblasts and primary B cells.D Publications in 2010/2011Publications 20101. Ha, S.D., Ham, B., Mogridge, J., Saftig, P., Lin, S., Kim, S.O. (2010)Cathepsin B-mediated autophagy flux facilitates the anthrax toxin receptor 2-mediated delivery of anthrax lethal factor into the cytoplasm. J. Biol. Chem.,285, 2120-21292. Bai L, Beckers L, Wijnands E, Lutgens SP, Herías MV, Saftig P, Daemen MJ,Cleutjens K, Lutgens E, Biessen EA, Heeneman S. (2010) Cathepsin K genedisruption does not affect murine aneurysm formation. Atherosclerosis209:96-103.3. Arndt, V., Dick,N., Tawo,R., Dreiseidler,M., Wenzel,D.,Hesse,M.,Fürst,D.O., Saftig,P., Saint, R., Fleischmann,B.K., Hoch,M., Höhfeld, J.(2010) Chaperone-assisted selective autophagy is essential for musclemaintenance. Current Biology, 20, 143-1484. Le Gall, S.M., Maretzky, T., Issuree, P.D., Niu, X.,Reiss, K., Saftig, P.,Khokha, R., Lundell, D., Blobel, C.P. (2010) ADAM17 is regulated by arapid and reversible mechanism that controls access to its catalytic site. J. CellScience, 23, 3913-22.5. Mendelson, K., Swendeman, S., Saftig, P., Blobel, C.P. (2010) Stimulation ofthe PDGFRâ activates ADAM17 and promotes metalloproteinase-dependentcrosstalk between the PDGFRbeta and EGFR signaling pathways. J. Biol.Chem., 285, 25024-32.6. Blanz, J., Groth, J., Zachos, C., Wehling, C., Saftig, P., Schwake, M. (2010)Disease causing mutations within the lysosomal integral membrane proteintype 2 (LIMP-2) reveal the nature of binding to its ligand b-glucocerobrosidase. Hum. Mol. Genet., 19, 563-5727. Jorissen, E., Prox, J., Bernreuther, C., Weber, S., Schwanbeck, R., Serneels,L., Snellinx, A., Craessaerts, K., Ththiah, A., Tesseur, I., Bartsch, U.,Weskamp, G., Blobel, C.P., Glatzel, M., de Strooper, B., Saftig, P. (2010) Thedisintegrin/metalloproteinase ADAM10 is essential for the establishement ofthe brain cortex. J. Neurosci., 30:4833-4844.Impact Factor5.3284.08610.0266.2905.3288.0587.27133

8. Schröder, B., Wrocklage, C., Hasilik, A., Saftig, P. (2010) Molecularcharacterisation of „Transmembrane protein 192“ (TMEM192), a novel 3.603protein of the lysosomal membrane. Biol. Chem., 391:695-704.9. Shevtsova, Z., Garrido, M., Weishaupt, J., Saftig, P., Bähr, M., Lühder, F.,Kügler, S. (2010) CNS-expressed cathepsin D prevents lymphopenia in a 5.224murine model of congenital ceroid lipofuscinosis. Am. J. Pathol., 177:271-9.10. Schröder B, Saftig P. (2010) Molecular insights into mechanisms ofintramembrane proteolysis through signal peptide peptidase (SPP). Biochem 5.016J. 427:e1-3.11. Kim, J., Lilliehook, C., Dudak, A., Prox, J., Saftig, P., Federoff, H.J., Lim,S.T. (2010) Activity-dependent alpha-cleavage of nectin-1 is mediated by a5.328disintegrin and metalloproteinase 10 (ADAM10). J. Biol. Chem., 285:25024-32.12. Chalaris, A., Adam, N., Sina, C., Rosenstiel, P., Lehmann-Koch, J.,Schirmacher, P., Hartmann, D., Cichy, J., Gavrilova, O., Schreiber, S., Jostoc,T., Matthews, V., Häsler, R., Becker, C., Neurath, M.F., Reiß, K., Saftig,P.,14.776Scheller, J., Rose-John, S. (2010) Critical role of the disintegrinmetalloprotease ADAM17 for intestinal inflammation and regeneration inmice. J. Exp.Med. , 207, 1617-162413. Schröder, B., Wrocklage, C., Hasilik, A., Saftig, P. (2010) The proteome oflysosomes. Proteomics, 10, 4053- 76 4.81514. Saftig P, Schröder B, Blanz J. (2010) Lysosomal membrane proteins: lifebetween acid and neutral conditions. Biochem Soc Trans. 38(6):1420-3. 3.989Publications 20111. Saftig, P., Reiß, K. (2011) The "A Disintegrin And Metalloproteases"ADAM10 and ADAM17: Novel drug targets with therapeutic potential? Eur.J. Cell Biol., 90, 527-35.2. Weber, S., Niessen,M., Prox,J., Lüllmann-Rauch,R., Schmitz,A.,Schwanbeck,R., Blobel,C.P., Jorissen,E., de Strooper, B., Niessen, C., Saftig,P. (2011) The disintegrin/metalloproteinase ADAM10 is essential for theepidermal integrity and Notch mediated signaling. Development, 138, 495-5043. Tamboli, I.Y., Hampel, H., Tien, N.T., Tolksdorf, K., Breiden, B., Mathews,P.M., Saftig, P., Sandhoff, K., Walter, J. (2011) Sphingolipid storage impairsautophagic degradation of the amyloid precursor protein and promotes Abetageneration. J. Neurosci. 31,1837-18494. Carrasco-Marín, E., Fernández-Prieto, L., Rodriguez-Del Rio, E., Madrazo-Toca, F., Reinheckel, T., Saftig, P., Alvarez-Dominguez, C. (2011) LIMP-2links late phagosomal trafficking with the onset of the innate immuneresponse to Listeria monocytogenes: a role in macrophage activation. J BiolChem. 286:3332-3341.5. Huth, T., Rittger, A., Saftig, P., Alzheimer, C. (2011) beta-site APP-cleavingenzyme 1 (BACE1) cleaves cerebellar Na+ channel beta-subunit andpromotes Purkinje cell firing by slowing the decay of resurgent Na+ current.Pflug. Arch. Eur. J. Phy., 461, 355-3716. Damme, M., Stroobants, S., Walkley, S.U., Lüllmann-Rauch, R., D'Hooge,R., Fogh, J., Saftig, P., Lübke, T., Blanz, J. (2011) Cerebellar alterations andgait defects as therapeutic outcome measures for enzyme replacement therapyin α-mannosidosis. J Neuropathol Exp Neurol. 70, 83-94.7. Behnke, J., Eskelinen,E.L., Saftig, P., Schröder, B.A. (2011) Two dileucinemotifs mediate late endosomal/lysosomal targeting of “Transmembraneprotein 192” (TMEM192) and a C-terminal cysteine is responsible fordisulphide bond formation in TMEM192 homodimers. Biochem. J., 434, 219-31.8. Behnke J, Schneppenheim J, Koch-Nolte F, Haag F, Saftig P, Schröder B.2011 Signal-peptide-peptidase-like 2a (SPPL2a) is targeted to lysosomes/lateendosomes by a tyrosine motif in its C-terminal tail. FEBS Lett. 585:2951-7.Impact Factor3.6306.8987.2715.3283.6954.5645.0163.60134

Research Report Biochemical Institute, Christian-Albrechts-University Kiel9. Dauth, S., Sirbulescu, R.F., Jordans, S., Rehders, M., Avena, L., Oswald, J.,Lerchl, A., Saftig, P., Brix, K. (2011) Cathepsin K deficiency in mice inducesstructural and metabolic changes in the central nervous system that areassociated with learning and memory deficits. BMC Neuroscience, 12:74.10. van Niel, G., Charrin, S., Simoes, S., Romao, M., Rochin, L., Saftig, P.,Marks, M.S., Rubinstein, E., Raposo, G. (2011) The tetraspanin CD63regulates ESCRT-independent and -dependent endosomal sorting duringmelanogenesis. Dev Cell. 21:708-721.11. Desmond MJ, Lee D, Fraser SA, Katerelos M, Gleich K, Martinello P, Li YQ,Thomas MC, Michelucci R, Cole AJ, Saftig P, Schwake M, Stapleton D,Berkovic SF, Power DA (2011) Tubular proteinuria in mice and humanslacking the intrinsic lysosomal protein SCARB2/Limp-2. Am J Physiol RenalPhysiol. , i 300, F1437-47.12. Sulis, M.L., Saftig, P., Ferrando, A. (2011) Redundancy and specificity of themetalloproteinase system mediating oncogenic NOTCH1 activation in T-ALL. Leukemia, 25, 1564-9.13. Glomski,K., Monette, S., Manova, K., de Strooper, B., Saftig, P., Blobel, C.P.(2011) Deletion of Adam10 in endothelial cells leads to defects in organspecificvascular structures. Blood, 118(4):1163-74.14. Altmeppen, H., Prox, J., Puig, B., Kluth, M.A., Bernreuther, C., Thurm, D.,Jorissen, E., Petrowitz, B., Bartsch, U., de Strooper, B., Saftig, P., Glatzel, M.(2011) Lack of a-disintegrin-and metalloproteinase ADAM10 leads tointracellular accumulation and loss of shedding of the cellular prion protein invivo. Mol Neurodegen, 6, 36.15. Zhang, D., Leung, N., Saftig, P., Brömme, D. (2011) The effect of cathepsinK deficiency on airway development and TGF-ß1 degradation. RespiratoryResearch, 12,72.16. Savalas, L.R., Gasnier, B., Damme, M., Lübke, T., Wrocklage, C., Debacker,C., Jezegou, A., Reinheckel, T., Hasilik, A., Saftig, P., Schröder, B.A. (2011)"Disrupted in renal carcinoma 2" (DIRC2) - a novel transporter of thelysosomal membrane - is proteolytically processed by cathepsin L. Biochem.J. , 439, 113-28.17. Evans, S.F., Irmady, K., Ostrow, K., Kim, T., Nykjaer, A., Saftig, P., Blobel,C., Hempstead, B.L. (2011) Neuronal BDNF is synthesized in excess, withlevels regulated by sortilin mediated trafficking and lysosomal degradation. J.Biol. Chem., 286, 29556-67.18. Doyle, E.L., Ridger, V., Ferraro, F., Turmaine, M., Saftig, P., Cutler, D.F.(2011) CD63 is an essential co-factor to leukocyte recruitment by endothelialP-selectin. Blood, 118, 4265-73.19. Rabe, S., Reichwald, J., Ammaturo, D., de Strooper, B., Saftig, P., Neumann,U., Staufenbiel, M. (2011) The swedish APP mutation alters the effect ofgenetically reduced BACE1 expression on the APP processing. J.Neurochem., 119, 231-9.20. Cullen V, Sardi SP, Ng J, Xu YH, Sun Y, Tomlinson JJ, Kolodziej P, Kahn I,Saftig P, Woulfe J, Rochet JC, Glicksman MA, Cheng SH, Grabowski GA,Shihabuddin LS, Schlossmacher MG. (2011)Acid beta-glucosidase mutantslinked to Gaucher disease, Parkinson disease, and Lewy body dementia alter?-synuclein processing. Ann Neurol 69,940-53.21. Schneede A, Schmidt CK, Hölttä-Vuori M, Heeren J, Willenborg M, Blanz J,Domanskyy M, Breiden B, Brodesser S, Landgrebe J, Sandhoff K, Ikonen E,Saftig P, Eskelinen EL. (2011) Role for LAMP-2 in endosomal cholesteroltransport. J Cell Mol Med. 15, 280-95.3.0913.9463.5908.96610.5585.13.125.0165.32810.5584.33710.7464.608Impact factors 2010: 89. 138Impact factors 2011: 128. 966Total impact factors 2010/2011: 218. 10435

EGRANTSE.1 An integrated approach towards understanding the pathogenesis of CNS and PNS neurodegenerativedisordersIAP Network Project P6Fördersumme (2007 – 2011) 68.000,00 €E.2 Die in vivo Bedeutung lysosomaler Membranproteine bei der intrazellulären Verwertung von Parasitenund Bakterien nach InfektionDFG SA 683/6-3Fördersumme (2009 – 2010) 97.000,00 €E.3 Die Funktion von ADAM MetallproteasenSonderforschungsbereich 415, Teilprojekt B9Fördersumme (2007 – 2010) 377.400,00 €E.4 Analysis of the postnatal and tissue specific role of the protease ADAM10Sonderforschungsbereich 877, Teilprojekt A3Fördersumme (2010 – 2014) 569.000,00 €E.5 Characterization of the in vivo functions of signal-peptide-peptidase-like 2a/b in mouse modelsSonderforschungsbereich 877, Teilprojekt B7 (with Bernd Schröder)Fördersumme (2010 – 2014) 401.600,00 €E.6 Microscopy core facility: Analysis of protease structure, cell biology and functionSonderforschungsbereich 877, Teilprojekt Z3Fördersumme (2010 – 2014) 200.000,00 €E.7 Towards the development of an effective enzyme replacement therapy for human alpha-mannosidosisHUE-MAN LSHM-CT-2006-018692Fördersumme (2006 – 2009) 601.240,00 €E.8 Clinical Development of an enzyme replacement therapy in alpha-mannosidosis patients usingrecombinant human enzyme. – Coordinator-ALPHA-MANFördersumme (2010 – 2013) 650.000,00 €E.9 Excellenzcluster Inflammation at Interfaces TP3: Lysosomes and NLR Fördersumme (2009-2012);349.900 €; Miniproposal Fördersumme (2008) 50.000 €E.10 Sortierung und Wechselwirkung zwischen Proteinen subzellulärer Kompartimente; GraduiertenkollegGRK 1459/1 (DFG); Fördersumme (2008-2012) 374.855 €E.11 Design of zinc metalloenzyme targeted drugs using an integrated technology approach (DeZnit)Fördersumme (2007-2010) 150.000 €E.12 HBFG: Deutscher Forschungsgemeinschaft: Konfokales Laserscanmikroskop mit Live Cell ImagingDFG-INST-257/346-1 FUGG 200.000 € plus 200.000 € Landesmittel)36

Research Report Biochemical Institute, Christian-Albrechts-University KielFPRICEHans und Ilse Breuer Stiftung Alzheimer Award 2010 „100.000 €“The precise mechanism of development of Alzheimer's disease is still notfully understood. What is known is that small protein fragments thataccumulate in the brain, and which cluster together into so-calledplaques, play a devastating role. The surrounding nerves die off, andcognitive ability falls drastically. Paul Saftig from the University of Kielis interested in the ‘molecular scissors’ that produce these proteinfragments. Over the past twelve years, the biologist has focused hisattention on three genes in particular that are responsible for theformation of these Alzheimer's scissors. In a surprise revelation, thescissors, which cause so much damage in affected patients, also appear tohave a useful role. For this discovery, Saftig has been awarded withGermany’s highest endowed Alzheimer’s award.37

Research Report Biochemical Institute, Christian-Albrechts-University Kiel8. Research Group PD Dr. Michael SchwakeA Group Leader: PD Dr. Michael SchwakeBLab Members:PostdocDr. Meng Lin (2011)Ph.D. students:Christian RaabChristina ZachosFriederike ZunkeJohann GrothJudith PetersMichelle DanaherMD studentsJohannes KnabbeDiploma students:Sara HarkaiBachelor students:Gunnar ZochTechnicians:Maike LangerCResearch ReportEpilepsy is a common neurological disorder that is characterized by recurrent unprovoked seizures. There aremany different types of epilepsy that can be divided by seizure types and their presumptive cause. Progressivemyoclonus epilepsies (PMEs) are defined as progressive disorders, which present with action myoclonus, tonicclonicseizures and progressive neurological decline. They are predominantly autosomal recessive inherited andhave similar clinical manifestations but are genetically heterogeneous. For example, null-mutations in the geneencoding LIMP-2 were found to be responsible for Action Myoclonus-Renal Failure Syndrome (AMRF). AMRFis an autosomal-recessive human disorder with the combination of focal glomerulosclerosis and progressivemyoclonus epilepsy associated with storage material in the brain. The origin of the storage material is not knownso far. Recently, patients with myoclonic epilepsy, but no renal involvement have been described. To date, sixAMRF-causing mutations have been identified, including splice site, missense and nonsense mutations.We identified LIMP-2 as a lysosomal transport receptor for glucocerebrosidase (GC), the enzyme defective inGaucher disease (GD). It was known for a long time that in contrast to most lysosomal enzymes, GC is targeted39

to lysosomes independent of the mannose 6-phosphate receptors. We demonstrated that LIMP-2 is needed forlysosomal localization of GC. Apparently, the receptor-ligand complex is formed very early within the secretorypathway in the endoplasmic reticulum (ER). In contrast, all identified AMRF-causing mutations analyzed by usso far lead to retention of LIMP-2 in the endoplasmic reticulum (ER) and most likely to a mislocalization of GC.In another research area, we have recently identified a mutation in GOSR2 (p.G144W), which is a SNARE[soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] protein involved in ER to Golgitransport and functionally characterized the mutation. SNAREs are key elements responsible for membranefusion and can be classified as Q- and R-SNAREs. The determination the (neuro-)physiological role of GOSR2and the generation of a mouse model for this new form of progressive myoclonus epilepsy will be one of ourresearch focus in the near future.D Publications 2010/2011Publications 20101. Blanz J., Groth J., Wehling C., Saftig P. and Schwake M. (2010). Mutationswithin the lysosomal transport receptor lysosomal integral membrane proteintype 2 (LIMP-2) reveal the nature of binding to β-glucocerebrosidase. Hum.Mol. Gen. 19, 563–572.Publikationen 20111. Valkova C., Albrizio M., Röder I.V., Schwake M., Betto R., Rudolf R.,Kaether C. (2011). Sorting receptor Rer1 controls surface expression ofmuscle acetylcholine receptors by ER retention of unassembled a-subunits.Proc. Natl. Acad. Sci. U S A. 11, 621-5.2. Desmond M.J., Lee D., Fraser S.A., Katerelos M., Gleich K., Martinello P., LiY.Q., Thomas M.C., Michelucci R., Cole A.J., Saftig P., Schwake M.,Stapleton D., Berkovic S.F., Power D.A. (2011). Tubular proteinuria in miceand humans lacking the intrinsic lysosomal protein SCARB2/Limp-2. Am. J.Physiol. Renal. Physiol. 300, 1437-47.3. Corbett M.A.*, Schwake M.*, Bahlo M., Dibbens L.M., Lin M., GandolfoL.C., Vears D.F., O'Sullivan J.D., Robertson T., Bayly M.A., Gardner A.E.,Vlaar A.M., Korenke G.C., Bloem B.R., de Coo I.F., Verhagen J.M.,Lehesjoki A.E., Gecz J., Berkovic S.F. (2011). A mutation in the Golgi Qb-SNARE gene GOSR2 causes progressive myoclonus epilepsy with earlyataxia. Am. J. Hum. Genet. 88, 657-63. * equally contributed.Impact Factor8.1Impact Factor9.83.811.7Total impact factors 2010/2011: 33.4Impact factors 2010: 8.1Impact factors 2011: 25.3E40GrantsE.1 Michael Schwake and Paul SaftigLIMP-2: A fascinating lysosomal membrane protein with multiple functions. Analysis of its role in lateendocytotic compartments.Graduiertenkolleg 1459Fördersumme (2008 – 2012) 281.310 €E.2 Michael SchwakeAssemblierung und Transport von M-Strom vermittelnden Kv-Kanälen.Sachbeihilfe (SCHW866/4-1)Fördersumme (2008 – 2012) 126.000 €E.3 Michael SchwakeDeterminants of voltage - gated cation channels as substrates for regulated proteolysis.Sonderforschungsbereich 877, Teilprojekt B8Fördersumme (2010 – 2014) 405.760 €

Research Report Biochemical Institute, Christian-Albrechts-University Kiel9. Research Group Dr. Judith BlanzA Group Leader: Dr. Judith BlanzBLab Members:Post DocsDr. Alex SchneedePh.D. students:Michelle DanaherMirka AllerdingTechnicians:Inez GöttingMD studentsMaike LüdemannCResearch ReportLaboratory assistant (trainee)Meryem SenkaraC.1 Preclinical Enzyme Replacment Therapy studies in a mouse model for alpha-Mannosidosiswithin the EU sponsored project HUE-MAN and ALPHA-MAN.Lysosomal function depends on proper action of two classes of proteins, 1) lysosmal hydrolases that areresponsible for lysosomal degradation and lysosomal membrane proteins (LMP) that are involved inacidification of the lysosomal, transport processes across the lysosomal membrane and lysosomal maturation.Lysosomal Storage Disorders (LSD) are a group of rare human genetic diseases in which a defect in lysosomalhydrolysis of macromolecules such as lipids and glycoproteins leads to intralysosomal accumulation ofundegraded material with severe consequences for the organism and many patients suffering from LSD dieduring early childhood. In brain, the accumulation of storage material often leads to neurodegeneration andinflammation. Treatment of LSD is hardly working since most of the diseases including alpha-Mannosidosisshow severe neurological deficits.To date, Enzyme Replacement Therapy (ERT) is the most promising option for an efficient treatment of thesediseases even though recombinant enzymes are thought not to be able to cross the blood brain barrier andtherefore not to reach the central nervous system (CNS). In ERT the respective enzyme lacking in the patient isproduced by recombinant approaches and then introduced into the blood stream of the patient, from where it isinternalized by the cells replacing the missing endogenous enzyme.Lysosomal accumulation of mannosyl-linked oligosaccharides leads to the orphan and devastating LSD alpha-Mannosidosis that is caused by deficiency of the lysosomal hydrolase alpha-Mannosidase (LAMAN). To date,no real treatment for alpha-Mannosidosis is available. Since children are born healthy, an early initiated therapyshortly after birth could dramatically improve their life expectancy and quality of life.Since 2006, my group has worked together with European scientists and clinicians in two collaborative EUsponsored networks HUE-MAN (2006-2009) and ALPHA-MAN (since 2010) towards the development of41

ecombinant human enzyme (rhLAMAN) as a therapeutic agent for an effective treatment of patients sufferingfrom alpha-Mannosidosis.We have used alpha-Mannosidase KO mice – a valid mouse model for alpha-Mannosidosis - to establisheffective ERT in mice. Upon frequent intravenous injections of the human recombinant enzyme, alpha-Mannosidosis mice develop a severe humoral immune response associated with high mortality. Therefore, ERTstudies in this mouse model were limited to a maximum of four injections within two weeks. However, evenwith this short term treatment we could demonstrate a great reduction of stored sugars in a variety of tissuesincluding the CNS. The clearance of sugar storage was demonstrated by various methods such as morphologicalanalysis (dissapearance of storage vacuoles) and chromatographical methods (Thin Layer Chromatography andHPLC of sugar tissue extracts). Histological analysis of mouse brains before and after ERT and Thin LayerChromatography (TLC) of brain sugar extracts, respectively, is shown in Figure 1. Immunohistochemicalanalyses revealed lysosomal delivery of the injected enzyme, indicating correct targeting. Dose finding studiesrevealed clearance of storage in visceral tissues >70% after two injections of low doses ( 250U were needed. Four subsequent injections of 500U/kg led to a >50% clearance of sugarstorage in brain (Figure 1, right panel). Successful transfer across the blood-brain barrier was evident, since theinjected enzyme was found in hippocampal neurons, leading to a nearly complete disappearance of storagevacuoles within these neurons (Figure 1, left panel). However, the sugar reduction was not permanent since 12days after the last injection, sugars in brain start to accumulate again (Figure 1, right panel).Figure 1: Immunohistology (left panel) of brain sections and TLC analysis (right panel) of sugar extracts obtained from brains(CNS) and the G.trigemini (PNS) from KO mice before (KO*) and after treatment (KO_ERT). Mice have been treated 4x withhigh doses of rhLAMAN (500U/kg) and were analysed 4, 8 and 12 days (d) after the last injection.Our data suggest that rhLAMAN must be able to cross the Blood Brain Barrier but the mechanism howrhLAMAN enters the brain remains unclear and needs further investigations. The successful outcome of theHUE-MAN projects were the basis to apply for a subsequent EU network within framework 7 in which weplanned to realize clinical trials with alpha-Mannosidosis patients using rhLAMAN as a therapeutic drug. Thisnew EU consortium – ALPHA-MAN - has been successfully evaluated in March 2010 and is running sinceOctober 2010. First clinical trial data of alpha-Mannosidosis patients treated with rhLAMAN are very promisingsince patients show despite short term ERT (3 -6 months) significant improvements in motor and cognitiveparameters.C.2 Generation and Characterization of an immuntolerant alpha-Mannosidosis mouse model.As already mentioned, alpha-Mannosidosis mice develop a severe immune-reaction against the rhLAMAN afterfrequent injections precluding long-term ERT. Since chronic dosing studies will help us to find i) the mechanismby which the enzyme crosses the Blood Brain Barrier and ii) to see, to which extent, the observedneuropathology (see section D2) in alpha-Mannosidosis mice is reversible, we generated a transgenic mousemodel, that expresses an inactive form of the human enzyme in the alpha-Mannosidase KO background(Tg H72L /mLAMAN KO). The idea is, that a mouse model, that is deficient for the mouse LAMAN, but expressesan inactive form of the human enzyme, shows the “classical” KO phenotype but tolerates the injected humanenzyme, allowing chronic ERT studies. Indeed, our ERT experiments that were carried out with these42

Research Report Biochemical Institute, Christian-Albrechts-University KielTg H72L /mLAMAN KO mice showed that they are immunetolerant against the injected recombinant humanenzyme (unpublished data). Therefore we were able to perform first long term ERT studies that show promisingresults in terms of improving the observed neuropathology. Currently, using long term treated mice we areaddressing the question how the enzyme crosses the blood brain barrier which we hope to answer in the nearfuture.C.3 Neuropathology of the Lysosomal Storage Disorder alpha-MannosidosisTo better understand the efficiacy of ERT in terms of the underlying neuropathology we have analysed brains ofalpha-Mannosidosis mice in detail. Our biochemical and histological analyses revealed a specific cerebellarpathology of alpha-Mannosidosis mice. In addition to the well characterized sugar storage in brain of alpha-Mannosidosis mice, we found accumulation of free chlesterol and Gangliosides (GM1) specifically in themolecular layer of the cerebellum that is associated with macrophage infiltration, regional restriced astrogliosisand partial loss of Purkinje cells (Figure 2). The observation of the neuropathological abnormalities in alpha-Mannosidosis mice is of great importance since it can now be used as a “clinical” endpoint to study the efficacyof ERT in the CNS of alpha-Mannosidosis mice and will help us to understand the link between primary sugarstorage and the underlying behavioural deficits.Figure 2: (A-B, D) Vibratom sections of formalin fixed brains from 12 months old Wildtype (WT) and alpha-Mannosidosismice were analysed for activation of macrophages by either immunofluorescence (B, C) or the DAB technique (A) usingantibodies specific for macrophages like CD68 (A) and F4/80 (C). (B, D) Autofluorescence images were taken withexcitation wavelengths of 488nm and UV light. (A) In the KO brain, CD68 strongly labels activated microglial cells whichare found predominantly in the molecular layer (ml) of the cerebellum where abundant storage of autofluorescent materialoccurs (B, upper panel). The autofluorescence material that contains cholesterol as demonstrated by Filipin staining (B, lowerpanel in red) is taken up by macrophages labeled with F4/80 (C, green). ml=molecular layer; gl=granule cell layer;pcl=purkinje cell layer. Scale bars: (A) 400µm (upper panel), 50µm (lower panel); (B) 500µm (upper panel), 15µm (lowerpanel); (C) 15µm.C.4 Lysosomal Function of the lysosomal Membrane Protein LIMP-2.The lysosomal membrane is important for proper lysosomal function. One of the most abundant lysosomalmembrane proteins is the lysosome integral membrane protein 2 (LIMP-2) that was recently identified as adisease causing gene for the Action Myoclonus Renal Failure Syndrome (AMRF). AMRF is a rare kidneydisease associated with epilepsy and ataxia caused by mutations in LIMP-2 that are associated with lack of theLIMP-2 protein. Detailed morphological analyses of LIMP-2 deficient mice showed intracellular inclusions incerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes that were comparable to thepathological changes observed in AMRF patients. AMRF patients and LIMP-2 deficient mice suffer from ataxiawhich implies dysfunction of the cerebellum possibly as a consequence of the accumulated storage within thisbrain structure. Interstingly, expression profiling of LIMP-2 in brain revealed strong expression of LIMP-2 inPurkinje cells suggesting a specific function of LIMP-2 within these subtypes of CNS neurons. LIMP-2 deficientmice can serve as a mouse model for AMRF and can now be used to further investigate the AMRFneuropathology. These studies will be essential in clarifying the role of LIMP-2 in brain that is still elusive.In 2007, LIMP-2 was identified as a receptor for targeting the lysosomal hydrolase β-Glucocerebrosidase (βGC)to the lysosome. LIMP-2 is a type II transmembrane protein, spanning the membrane twice with a large luminaldomain. In vitro binding assays suggested that the binding of βGC to LIMP-2 occurs within the luminal domainof LIMP-2 and that a putative coiled coiled motif from amino acid 152-167 within this domain is important forthis interaction. To better understand the nature of the interaction between LIMP-2 to βGC we analyzed most ofthe known AMRF disease causing mutations of LIMP-2 and the resulting truncated proteins in more detail. Todate, six AMRF-causing mutations have been described, including splice site, missense and nonsense mutations.All mutations we have investigated lead to a retention of LIMP-2 in the endoplasmic reticulum (ER) but affectthe binding to βGC differentially. Using this approach we were able to narrow down the binding site to a region43

etween amino acid 145 and 288 within the luminal domain of LIMP-2. The LIMP-2 segment 145–288 containsa highly conserved coiled-coil domain, which we suggest determines βGC binding. In fact, disruption of thehelical arrangement and amphiphatic nature of the coiled-coil domain abolishes βGC binding, and a syntheticpeptide comprising the coiled-coil domain of LIMP-2 displays pH-selective multimerization properties. Withthis study we could demonstrate that disruption of the coiled-coil structure or AMRF disease-causing mutationsabolish βGC binding, indicating the importance of an intact coiled-coil structure for the interaction of LIMP-2and βGC. However, further structural analyses of LIMP-2 and βGC complexes are needed to understand theirmolecular interaction in more detail.D Publications 2010/2011Publications 20101. Saftig P, Schröder B, Blanz J., (2010) Lysosomal membrane proteins:life between acid and neutral conditions. Biochem Soc Trans. 38, 1420-3.2. Niemeyer MI, Cid LP, Sepúlveda FV, Blanz J, Auberson M, JentschTJ., (2010) No evidence for a role of CLCN2 variants in idiopathicgeneralized epilepsy. Nat Genet. 42, 3.3. Blanz J, Groth J, Zachos C, Wehling C, Saftig P, Schwake M., (2010)Disease-causing mutations within the lysosomal integral membraneprotein type 2 (LIMP-2) reveal the nature of binding to its ligand betaglucocerebrosidase.Hum Mol Genet19, 563-72..Publikationen 20111. Damme M, Stroobants S, Walkley SU, Lüllmann-Rauch R, D'Hooge R,Fogh J, Saftig P, Lübke T, Blanz J. (2011) Cerebellar alterations andgait defects as therapeutic outcome measures for enzyme replacementtherapy in α-mannosidosis. J Neuropathol Exp Neurol 70,83-94.2. Schneede A, Schmidt CK, Hölttä-Vuori M, Heeren J, Willenborg M,Blanz J, Domanskyy M, Breiden B, Brodesser S, Landgrebe J,Sandhoff K, Ikonen E, Saftig P, Eskelinen EL. (2011) Role for LAMP-2 in endosomal cholesterol transport. J Cell Mol Med. 15, 280-95.Impact Factor3.3836.388.058Impact Factor4.195.9Total impact factors 2010/2011: 57.92Impact factors 2010: 47.82Impact factors 2011: 10.1EGrantsE.1 ALPHA-MAN (EU #261331), 2010-2013, Fördersumme: 650 00044

Research Report Biochemical Institute, Christian-Albrechts-University Kiel10. Research Group Dr. Bernd SchröderA Group Leader: Dr. Bernd SchröderB Lab Members: Ph.D. Students:Janna SchneppenheimSusann HüttlTelly SavalasLaboratory Assistant:Sebastian HeldLaboratory Assistant in training:Rafael KurzCResearch ReportC.1. Physiological functions of the intramembrane proteases SPPL2a and SPPL2bThe concept of regulated intramembrane proteolysis (RIP) has emerged over the last decades as a novel conceptof cellular signalling. One group among the proteases being capable of cleaving substrates within thephospholipid bilayer are the signal-peptide-peptidase (SPP) and its homologues, the signal-peptide-peptidaselikeproteins (SPPL2a, -2b, -2c, -3). Whereas SPPL2a is present in membranes of lysosomes/late endosomes,SPPL2b was reported to reside at the plasma membrane as well as in endosomal compartments. To date, onlyTNFα, Fas Ligand (FasL) and Bri2 (Itm2b) have been identified as substrates of SPPL2a/b using in vitrooverexpression approaches. In agreement with the general concept of RIP it was demonstrated that the TNFαintracellular domain translocates into the nucleus after proteolytic release and influences gene expression therebyinducing the synthesis of the pro-infammatory cytokine IL12. Two of the known substrates suggest a regulatoryfunction of SPPL2a and SPPL2b in the context of the immune system. However, a relevance of these processesand proteolytic events in a complex in vivo system has not been analysed and established yet. It was alsosuggested that RIPping by SPPL2a and SPPL2b may have more generalized degradative functions beyondmediating reverse signalling by TNFα or other members of this superfamily - a hypothesis being supported bythe presence of SPPL orthologues in plants.45

In order to study functions of SPPL2a and SPPL2b in vivo, we have generated mouse lines deficient in either ofthe two proteases as well as mice being deficient in SPPLa and SPPL2b. Phenotypic analysis of these mice hasrevealed so far major immunological abnormalities associated with the deficiency of SPPL2a. These findingspoint to a fundamental role of SPPL2a mediated proteolysis in immunity that cannot be compensated for bySPPL2b. These in vivo approaches are combined with biochemical and cell biological experiments studying thedifferential processing of known and putative substrates in SPPL2a/b deficient cell lines. We are confident, thatchallenging these mice in infection and inflammation models will contribute to understanding the role ofSPPL2a/2b-mediated RIPping in pathophysiology and deepen our understanding of cellular and molecularprinciples of inflammation. The results of these studies will help us to decide whether SPPL2a and/or -2b mighteven be considered as potential drug targets and if specific inhibitors of these proteases might be capable ofmodulating the immune system in a therapeutic way. A central objective of the project is the unbiased search fornovel substrates cleaved by SPPL2a and/or –b and to ideally link the biochemical findings with the phenotypesobserved in the protease deficient mice.C.2 Functional characterisation of novel lysosomal membrane proteinsLysosomes play a crucial role in the degradation and turnover of different intra- and extracellularmacromolecules. Currently, extensive data are available about the proteins of the lysosomal matrix. On thecontrary only a minority of the lysosomal membrane proteins has been identified and substantially characterisedto date. This contrasts with the number of known and functionally described transport systems or enzymaticactivities that have been shown to be associated with this membrane.In a previous proteomic analysis of lysosomal membranes we have identified 16 novel enzyme and transporterproteins and 12 novel proteins of unknown functions not previously assigned to lysosomal membranes.Lysosomal localisation of several of these novel proteins could be confirmed by overexpression studies. Currentwork focusses on an in-depth biochemical characterisation of a selection of these proteins and will becomplemented by the generation of knock-out mice in order to unravel the functions of these novel lysosomalmembrane proteins.D Publications 2010/2011Publications 2010Impact Factor1. Schröder,B., Wrocklage,C., Hasilik,A., Saftig,P. (2010). Molecularcharacterisation of 'transmembrane protein 192' (TMEM192), a novel3.603protein of the lysosomal membrane. Biol Chem. 391, 695-7042. Schröder,B. Saftig,P. (2010). Molecular insights into mechanisms ofintramembrane proteolysis by signal peptide peptidase (SPP). Biochem. 5.016J. 427, 1-3.3. Schröder,B., Wrocklage,C., Hasilik,A., Saftig,P. (2010). The proteomeof lysosomes. Proteomics 10, 4053-4076. 4.81546

Research Report Biochemical Institute, Christian-Albrechts-University Kiel4. Saftig,P., Schröder,B., Blanz,J. (2010). Lysosomal membrane proteins:life between acid and neutral conditions. Biochem. Soc. Trans. 38,1420-1423.Publikationen 20111. Behnke, J., Eskelinen,E.L:, Saftig,P., Schröder,B. (2011). Twodileucine motifs mediate late endosomal/lysosomal targeting ofTransmembrane protein 192 (TMEM192) and a C-terminal cysteineresidue is responsible for disulfide bond formation in TMEM192homodimers. Biochem. J. 434, 219-231.2. Savalas,L.R.T., Gasnier,B., Damme,M., Lübke,T., Wrocklage,C.,Debacker,C., Jézégou,A., Reinheckel,T., Hasilik,A., Saftig,P.,Schröder,B. (2011). “Disrupted in renal carcinoma 2” (DIRC2) - anovel transporter of the lysosomal membrane - is proteolyticallyprocessed by cathepsin L. Biochem. J. 439, 113-128.3. Behnke,J., Schneppenheim,J., Koch-Nolte,F., Haag,F., Saftig,P.,Schröder,B. Signal-peptide-peptidase-like 2a (SPPL2a) is targeted tolysosomes/late endsosomes by a tyrosine motif in ist C-terminal tail.FEBS Lett. 585, 2951-2957.3.989Impact Factor5.0165.0163,601Total impact factors 2010/2011: 31.05Impact factors 2010: 17.42Impact factors 2011: 13.6347

Research Report Biochemical Institute, Christian-Albrechts-University Kiel11. Research Group Prof. Dr. Ursula JustA Group Leader: Prof. Dr. Ursula JustBCLab Members:Research ReportGroup leaders:Dr. Ralf SchwanbeckDr. Thomas Höfken (until March 2010)Postdocs:Dr. Meng Lin (until March 2010)Dr. Simone Martini (from July 2011)Ph.D. StudentsKristina BernothSimone Martini (until July 2011)Bachelor Students:Rebecca Grohmann (until August 2010)Technician:Silke Horn (until October 2010)Melanie Boss (until March 2011)Katarina Macha (from July 2011)Secretary:Iris PetrikatCarmen Janisch (until December 2010)C.1 Molecular basis of self-renewal and homing mutants of hematopoietic progenitorcells generated by insertional mutagenesis in vitroUsing retroviral insertional mutagenesis to generate hematopoietic stem cell lines, we have identified a numberof gene loci that are associated with increased self renewal of adult hematopoietic stem cells (Just et al., 2000and unpublished). Several integrations target the known oncogenes Evi1, Ccnd3 and Mll3 leading to aberrantexpression in about half of the hematopoietic stem cell lines. Some of these regions are also rearranged bychromosomal translocations in acute myeloid leukemias and myelodysplastic syndromes with concomitantaberrant expression (unpublished; collaboration with Prof. Dr. Reiner Siebert, Institut für Humangenetik, CAUKiel). Among the novel genes that we have identified as aberrantly expressed in most of the hematopoietic stemcell lines are H3Me3K4 Demethylases (cooperation with Prof. Dr. Ralf Huss and Dr. Burtscher, Roche,Penzberg, unpublished). Interestingly, in all cell lines analysed thus far, either a H3Me3K4 Demethyase is49

overexpressed or a H3Me3K4 Methylase is downregulated, suggesting that a reduction in specific H3Me3K4Methylation, a chromatin mark for accessible/active genes, is involved in maintaining the stem cell phenotypeand possibly longevity of the cells as suggested recently for worms and flies. Using functional approaches withsiRNA to downregulate expression of these genes in the stem cell lines and overexpression of these genes innormal stem cells as well as a detailed characterization of chromatin modifications, we are currently analysingthe role of enzymes mediating specific chromatin marks for self renewal of stem cells. Targeting these enzymesmay help to find ways for new therapeutic approaches.Brain metastasis, which occurs in 20-40% of all cancer patients is an important cause of neoplastic morbidityand mortality. Successful invasion into the brain by tumour cells must include attachment to microvesselendothelial cells, penetration through the blood-brain barrier (BBB), and a response to brain survival and growthfactors. Because it is surrounded by the BBB and lacks lymphatic drainage, the brain is a privileged site ofcancer metastasis. Under normal circumstances cells as well as drugs are prevented by the BBB to enter thebrain. In an earlier study, we have generated a mutant hematopoietic stem cell line that developed ahematological tumour in the brain that preferentially invades the mesencephalon and the medulla oblongata (Fig.1, unpublished).Fig. 1: Mutant FDCP-mix cells invade the brain after i.v. transplantationA-C: Five months old BDF1 mice 15 days after i.v. injection of 5 x 10 6 mutant FDCP-mix cells in 0.5 µl PBS were analysed.Shown are histological examples of three different mouse brains. Clinical signs of these diseased animals were a clear ataxiaand inducible, generalized seizures. (A) Mononuclear, hematopoietic cells including several cells with immature blastmorphology infiltrate the stratum moleculare of the CNS from the subarachnoid cavity and penetrating leptomenigeal vessels.(B) Infiltrating hematopoietic cells clearly differ from the neural stratum granulosum cells of the cerebellum by theireosinophilic cytoplasm and partially segmented, dark nuclei and seem to be connected to the perivascular space. LargePurkinje cells are also present separating the molecular from the granular layer of the cerebellar cortex. (C) Nodular, partiallyalready organized leukemic infiltrate of the leptomeninx and the subarachnoid cavity of a cistern at the base of the brain.Hematopoietic cells of several differentiation stages ranging from immature blast cells to mature myeloid cells are present.Staining: HE (Just, U., Löhler, J., Huss, R., unpublished)In a genome wide gene expression array comparing this mutant with the parental cells that are not able to crossthe blood-brain barrier, we detected increased expression of several signalling pathway molecules andtranscriptional regulators that may be involved in enhancing the production of ECM degrading enzymes such asmatrix metalloproteases and plasminogen activator that have been previously described to be upregulated inbrain metastatic tumour cells (cooperation with Prof. Dr. Ralf Huss and Dr. Burtscher, Roche, Penzberg,unpublished). Targeting these pathways may help to develop diagnostic assays for tumour stem cell metastasisinto the brain as well as novel therapeutic approaches. Additionally, knowledge of these mechanisms could leadto strategies specifically targeting cells or molecules into the brain.C.2 Role of the Notch pathway in development and diseaseIntracellular signalling through the Notch transmembrane receptors regulates proliferation and differentiation inmany developmental systems. Notch is activated by binding a member of the Delta and Serrate/Jagged family ofcell-surface proteins. Following activation, Notch is cleaved within the transmembrane domain, releasing theNotch intracellular domain (NIC) from the membrane. The NIC then translocates to the nucleus where it canmodulate gene expression via association with CSL proteins (RBP-J in mammals), and thereby affect cell fatechoice. Notch receptors and cognate ligands are expressed throughout development. To analyse the role of Notchsignaling in the development of mesoderm-derived cell lineages and in particular of hematopoietic cells, weexpressed a constitutive active form of murine Notch 1 in embryonic stem cells (ES) and in the multipotentialhematopoietic cell line FDCP-mix using a Tamoxifen-inducible expression system and looked at how activatedNotch1 affects lineage commitment, differentiation and proliferation of the cells. We found that Notch signalingplays a regulatory role in mesodermal development, cardiomyogenesis, hematopoietic development and thebalanced generation of blood vessel cell types (Schroeder and Just, EMBO J. 19, 2558-2568, 2000; Schroeder etal., PNAS 100, 4018-4023, 2003; Schroeder et al., Mech. Dev. 123, 570-579, 2006; Yurugi-Kobayashi et al.,Arterioscler. Thromb. Vasc. Biol. 26, 1977-1984, 2006; Henning et al., Cell Death Differ. 15, 398-407, 2008).To elucidate the molecular mechanisms by which Notch influences cell lineage decisions in a cell contextdependent manner, we have further identified cell-context dependent Notch target genes (Schwanbeck et al.,50

Research Report Biochemical Institute, Christian-Albrechts-University KielCTO 188, 91-102, 2008, Meier-Stiegen et al., 2010; see also research report of Dr. Ralf Schwanbeck).Interestingly, Notch directly upregulated transcription factors involved in cell lineage decisions strictlydependent on the cellular context, i.e. cell type and additional cell extrinsic signalling. Furthermore, Notchactivation correlated with the presence of an activating chromatin mark, H3 Lysine4 trimethylation (H3K4me3)or bivalent domains, which are characterized by an activating H3K4me3 chromatin mark and a repressingH3K27me3 mark, at the promotor regions of Notch target genes, suggesting that epigenetic marks maydetermine Notch signaling outcome (Schwanbeck et al. 2010). In collaboration with Prof. Dr. Urban Lendahl,Karolinska Institute, Sweden, and Prof. Dr. Jürgen Rohwedel, Universität Lübeck, we were analysing thefunction of Notch-mediated upregulation of specific transcription factors for cell lineage decisions that we hadidentified previously (Meier-Stiegen et al., 2010) during ectodermal and chondrocyte development. Using ansiRNA dependent strategy we could show that upregulation of Sox9 by activated Notch during early embryonicdevelopment along the neuro-ectodermal or chondrocyte lineage is critically involved in mediating neuroectodermallineage choice, neural stem cell self renewal, glial cell differentiation and early chondrocytedevelopment (Haller, R., Schwanbeck, R., Martini, S., Bernoth, K., Kramer, J., Just, U., Rohwedel, J. Notch1activation promotes induction of chondrogenic differentiation via direct activation of Sox9 expression. CellDeath Differ. Aug 26 [Epub ahead of print] 2011; Martini et al., in preparation; see also research report of Dr.Ralf Schwanbeck).In response to injury, the kidney reactivates embryonic developmental programs, among them the Notchsignaling pathway, which plays an essential role during early nephrogenesis, glomerulogenesis andtubulogenesis. In diabetic nephropathy, Notch signaling is activated in podocytes and leads to destruction of theglomerular barrier. Notch signaling is further activated after ischemia in acute tubular necrosis. In acute as wellas chronic renal failure, tissue hypoxia is an important pathomechanism. Notch and hypoxia interact at themolecular level by the induction of common target genes. Several of the Notch target genes we identified in oursystem are also activated under hypoxia or by combined hypoxia and Notch activation (collaboration with Prof.U. Lendahl, Karolinska Institute, Sweden). In collaboration with Prof. J. Rohwedel and Dr. J. Kramer,University of Lübeck, we were testing in a mouse model of ischemic kidney failure which Notch target genes areactivated, to get further insight into the pathogenesis of kidney failure. Our analyses thus far suggest that besidesHes1, Hey2 and HeyL, Id proteins, Hspb8 and EphrinA1 show an altered expression kinetics and may play a rolein mediating Notch effects in acute and chronic renal failure (Kramer, Rohwedel, Schwanbeck and Just,unpublished).C.3 Cell-context dependency of Notch1 target gene inductionSee Research Report of Dr. Ralf SchwanbeckC.4 Role of the Phosphatidylinositol-3-kinase subunit p85a in embryoid bodyformationSee Research Report of Dr. Ralf SchwanbeckD Publications in 2010/2011Publications 20101. Meier-Stiegen, F., Schwanbeck, R., Martini, S., Bernoth, K., Hieronymus, T., Ruau, D.,Zenke, M., Just, U. (2010) Activation of Notch target genes during embryonic celldifferentiation depend on the cellular context and include lineage determinants andinhibitors. PLoS One 5, e11481.2. Stocking C, Grez M, Fehse B, von Laer D, Itoh K, Prassolov V, Nowock J, Kühlcke K,Just U, Schröder T, Klump H, Schiedlmeier B, Grassman E, Meyer J, Li Z, Schambach A,Modlich U, Kustikova O, Galla M, Bode J, Zander A, Baum C. Cell and virus genetics atthe roots of gene therapy, retrovirology, and hematopoietic stem cell biology: WolframOstertag (1937-2010). Hum. Gene Ther. 21, 1501-1503, 2010Publikationen 20111. Schwanbeck, R., Martini, S., Bernoth, K., Just, U. (2011) The Notch signaling pathway:Molecular cular basis of cell context dependency. Eur. J. Cell Biol. 90, 572-581.2. Gurney, S., Forster, P., Just, U., Schwanbeck, R. (2011) Suppression of the PI3K subunitp85α delays embryoid body development and inhibits cell adhesion. J. Cell Biochem. 112,ImpactFactor4.4114.829ImpactFactor3.6303.12251

3573-3581, 2011.3. Schwanbeck, R., Just, U. (2011) The Notch signaling pathway in hematopoiesis andhematological malignancies. Haematologica 96, 1735-1737.6.532Total impact factors 2010/2011: 22.524Impact factors 2010: 9.240Impact factors 2011: 13.284EGRANTSE.1 SFB 415; Teilprojekt B8: 'Characterization of Notch/RBP-J signalling in dependence of thecellular context during mesodermal differentiation', Fördersumme (2007 – 2010) 241.200 €E.2 Hensel-Stiftung, Analyse der Rolle des Notch-Signaltransduktionswegs beim Nierenversagen,Fördersumme (2009-2011) 30.000 €FFUNCTIONSF.1 Member of the National Ethics Commitee for Stem Cell Research (ZES) Since July 2005F.2 Associate Editor for the Journal ‘Cells Tissues Organs’ Section: Stem Cells and TissueEngineering, Since 201152

Research Report Biochemical Institute, Christian-Albrechts-University Kiel12. Research Group Dr. Ralf SchwanbeckA Group Leader: Dr. Ralf SchwanbeckB Lab Members: Postdocs:Dr. Simone Martini(from July 2011)Ph.D. Students:Simone Martini (co-supervised with Prof.Dr. Ursula Just, until July 2011)Kristina Bernoth (co-supervised with Prof.Dr. Ursula Just)Technician:Silke Horn (until October 2010)Melanie Boss (until March 2011)Katarina Macha (from July 2011)CResearch ReportC.1 Cell-context dependency of Notch1 target gene inductionNotch1 is a pivotal cell-cell signaling pathway affecting a broad range of cellular processes like cell-lineagedecisions, differentiation, self-renewal, apoptosis, and proliferation. Therefore, this pathway is conservedthroughout all Metazoa. The general mechanism of Notch signal transduction seems to be rather simple at firstsight, since there are no second messenger or phosphorylation cascades involved. In the canonical pathway aneighboring cell present a Notch ligand (Delta or Serrate/Jagged family) so that it can dimerize with a Notch1receptor in the extracellular space, and subsequent proteolytic cleavages by the ADAM protease and γ-secretasetake place, releasing the intracellular domain of the Notch receptor (NICD). In skin and neuronal tissue ofembryos, ADAM 10 was shown to be responsible for the first cleavage. Due to nuclear localization signals theNICD moiety translocates into the nucleus, displaces a co-repressor protein from RBP-J/CSL complex renderingit to an activator. Then, Notch1 target genes like the Hes and Hey basic Helix-loop-Helix (bHLH) factors areinduced.Since the effects of Notch signaling on differentiation are rather complex and have sometimes even opposingresults depending on the respective cell-context, we were seeking for additional target genes of the pathway. In agenome-wide screen for Notch1 targets using microarray technology we recently described a series of noveltargets depending on the differentiation state of the cells used (Meier-Stiegen et al., 2010). The analysis revealedthat the target genes strongly varied at different stages of differentiation indicating that a much higher number ofregulated genes than initially thought are responsible for the strong cell-context dependency of Notch signaling.Among the new target genes we also found a number of transcription factors critically involved in earlydevelopment like Sox9, Pax6, Runx1, Id4 and Myf5. These were also regulated in a cell-context dependentmanner, e.g. Runx1 and Myf5 were only regulated in mesoderm, whereas Pax6 was induced in undifferentiatedand early neuroectodermal differentiation. Sox9 and Id4 were induced by activated Notch1 in all investigatedconditions, however, the strong Sox9 induction diminished at later stages of embryoid body differentiation,indicating the importance of the early stages of differentiation. One possible explanation for the cell-context53

dependency of Notch activation is the chromatin state of the target genes. Analysis of the novel characterizedtarget genes revealed that about 95% of the target genes carry a histone H3 Lysine 4 trimethylation (H3K4me3).Furthermore, the novel target genes were strongly enriched for the presence of bivalent domains (H3K4me3 andH3K27me3 at the same time) in their extended promoter region, indicating an involvement of the epigeneticstatus in Notch target gene regulation (Schwanbeck et al., 2010). The detailed characterization of the interplay ofchromosomal modifications and Notch activation is subject of further investigations.The function of the Notch1 target gene Sox9 for Notch mediated effects was characterized in more detail in thedifferentiation to chondrocytes. Interestingly, we found that a temporary activation of Notch1 during the earlystages of in vitro differentiation of embryonic stem cells into chondrocytes via embryoid body (EB) formationresulted in a strong increase in chondrogenic differentiation during later stages. This Notch1-induced increase incartilage development could be entirely reversed by the application of Sox9 siRNA, whereas the knownblockage of cardiac differentiation by Notch1 was unaffected by the reduced Sox9 levels (Haller et al., 2011). Inanother set of experiments we used the same Sox9 siRNA strategy to investigate the role of Sox9 in Notchmediatedcell lineage decisions during neuroectodermal development. Notch is known to play a role in thedecision between glial and neuronal cells as well as for the induction of neural crest differentiation, though themolecular basis of these effects is thus far not understood. Our experiments revealed that in neuroectodermaldifferentiation conditions Sox9 is also a direct target gene of Notch1. Notch1 induction led to a strong increasein glial cell formation while inhibiting the generation of neurons. Quenching of the Sox9 peak induced byNotch1 signaling using Sox9 siRNA led to a significant decrease of glial cells, demonstrating the pivotal role ofSox9 in mediating Notch1 signals also in this context.These experiments revealed that beside the well-known Notch target genes of the Hes/Hey family there are moredirectly regulated genes mediating the function of Notch signaling.C.2 HMGA proteins in stem cells and cancer gene regulationThe High Mobility Group (HMG) proteins are abundant non-histone proteins that are able to bind DNA andnucleosomes and induce structural changes in the chromatin fiber. The HMGA protein family contains three AThookmotifs, enabling the binding into the minor groove of AT-rich DNA. This binding, which is rather DNAstructure-specific than sequence-specific, introduce slight conformational changes in the DNA structure therebyfacilitating the subsequent interaction of specific transcription factors like NFkB and IRF3. Further recruitmentof p300/CBP and the general transcription factors enables then the assembly of a highly stereospecific complexas it is described in the case of the ‘Enhanceosome’ for the transcription of the IFN-b gene. Furthermore, HMGAproteins can interact also directly with transcriptions factors like NFkB, p53 or pRB and influence their activity.Further target genes of HMGA proteins are described including cyclins, interleukins and metalloproteases.HMGA proteins are abundant in undifferentiated und proliferating cells (like stem cells) whereas they are ratherundetectable in fully differentiated cells suggesting a role in self-renewal of stem cells. Along this line,misexpression of HMGA proteins in development or in adult cells was shown to be involved in neoplastictransformation and tumor formation, a fact that is most likely directly connected to the large number of targetgenes of HMGA proteins and interactions to transcriptions factors. Besides skin, mammary and lung carcinoma,misexpression of HMGA proteins was also shown in B-cell lymphomas and myeloid leukemias. There are twosplice variants called HMGA1a and HMGA1b with different biochemical properties and the expression patternsof these are mostly unknown in these tumors. Furthermore, the HMGA proteins are target of a variety ofposttranslational modifications like phosphorylation or acetylation. These modifications were previously shownto modulate the binding activity of the chromatin proteins, thereby facilitating a fine tuning of their function.Furthermore, HMGA proteins are important during early embryonic development. Using embryoid body culturesand siRNA-mediated knock-down we try to elucidate the role of HMGA proteins in mesodermal andneuroectodermal differentiation. Interestingly, HMGA2 was strongly induced during neuroectodermaldifferentiation around day 4 to 6, when Notch1 signaling was activated. Though HMGA2 seems not to be adirect Notch1 target gene, this may be one way of Notch signaling to exert its function, since HMGA2 isdescribed to be responsible for self-renewal in neural stem cells in the early development.C.3 The role of the Phosphatidylinositol-3-kinase subunit p85α in embryoid bodyformationEmbryoid bodies (EBs) are an accepted model of in vitro differentiation and embryogenesis. This process can beinfluenced by many internal and external factors. Since the phosphatidylinositol-3-kinases (PI3Ks) exert avariety of signaling functions in eukaryotes regulating cellular survival, proliferation, differentiation, migrationand trafficking, we tested this pathway regarding EB development. In eukaryotes there are three distinct classesof PI3Ks: class I (subdivided into IA and IB), class II and class III. The role of class IA PI3Ks, which we focuson, is to phosphorylate the second messenger lipid PIP2 to PIP3. Each of these complexes consists of onecatalytic subunit (p110α, p110β, or p110δ, encoded by three different genes) and one regulatory subunit (p50α,p55α, p85α, p85β, or p55γ). These α, β and γ regulatory subunits are also encoded by three different genes:Pik3r1, Pik3r2 and Pik3r3, respectively. We examined the role of the regulatory subunit p85α in undifferentiated54

Research Report Biochemical Institute, Christian-Albrechts-University KielES cells and in early embryoid body development. Therefore, we suppressed the PI3K regulatory subunit p85αusing a small interfering RNA (Pik3r1 siRNA) and examined the effects on EB development in hanging dropculture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negativecontrols. FACS assays showed that this increase in volume is not due to increased cellular proliferation. Instead,the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown byour observation that 40% of treated EBs form twin instead of single EBs, and that they have a significantlyreduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that theimpaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCRexpression analysis suggests that the adhesion molecule integrin-β1 (ITGB1) is transiently downregulated by thep85α suppression (Gurney et al., 2011). Up to now, the function of the regulatory subunit p85α was ratherpoorly understood. We provide first evidence that its gene Pik3r1 is involved in early embryoid development.We found that suppressing p85α leads to a delay in forming compact EBs, accompanied by a transient inabilityof the EBs to undergo normal cell-cell and cell-substrate adhesion.D Publications 2010/2011Publications 20101. Jorissen E., Prox J., Bernreuther C., Weber S., Schwanbeck R., Serneels L., Snellinx A.,Craessaerts K., Thathiah A., Tesseur I., Bartsch U., Weskamp G., Blobel C.P., Glatzel M.,De Strooper B., Saftig P. (2010) The disintegrin/metalloproteinase ADAM10 is essentialfor the establishment of the brain cortex. J. Neurosci. 30(14):4833-44.2. Meier-Stiegen F., Schwanbeck R. 1) , Bernoth K., Martini S., Hieronymus T., Ruau D.,Zenke M., Just U. 1) (2010) Activated Notch1 target genes during embryonic celldifferentiation depend on the cellular context and include lineage determinants andinhibitors. PLoS One. 5(7):e11481. ( 1) corresponding author)Publications 20111. Schwanbeck R., Bernoth K., Martini S., Just U. (2011) The Notch signaling pathway:Molecular basis of cell context dependency. Eur. J. Cell Biol. 90, 572-581.2. Weber S., Prox J., Niessen M., Lüllmann-Rauch R., Schmitz A., Schwanbeck R., BlobelC.P., Jorissen E., de Strooper B., Niessen C.M., Saftig P. (2011) The disintegrin/metalloproteinase ADAM10 is essential for the Notch-mediated development and integrityof the epidermis. Development 138(3):495-505.*) denotes equal contribution3. Gurney, S.M., Forster, P., Just, U., and Schwanbeck, R. (2011).Suppression of the PI3K subunit p85alpha delays embryoid body development and inhibitscell adhesion. J Cell Biochem. 112, 3573-35814. Schwanbeck, R., Just, U. (2011) The Notch signaling pathway inhematopoiesis and hematological malignancies. Haematologica, 96, 1735-1737.ImpactFactor7.2714.351ImpactFactor3.6306.8983.1226.532Total impact factors 2010/2011: 31.804Impact factors 2010: 11.622Impact factors 2011: 20.182EGrantsE.1 Analyse der architektonischen High-Mobility-Group A (HMGA) Chromatinproteine beihochmalignen lymphatischen Neoplasien der B-Zell-Reihe. Ralf Schwanbeck und LanaHarder; Hensel-Stiftung48500 €55

Research Report Biochemical Institute, Christian-Albrechts-University Kiel13. Research Group Prof. Dr. Roland SchauerAGroup Leader: Prof. (em.) Dr. med. Dipl.-Biochem. Roland SchauerCResearch ReportC.1 The significance of sialic acids and the study of their O-acetylationSialic acids are gaining much interest in cell biology, from fertilization to cell death, and they are undispensablefor maintaining the life of an eukaryotic cell of the deuterostome lineage. They were also found in some loweranimals and are expressed e.g. in Drosophila and cicada during a short period of development. Sialic acids alsooccur in many microorganisms, mainly in bacteria and protozoa where they represent strong virulence factors.Sialic acids mostly occupy the terminal positions of glycoproteins and glycolipids in monomeric or polymeric(polysialic acids) form on cell surfaces and thus are involved in the control of many cell functions, such ascellular interactions, transport and signalling events, learning, differentiation, innate and acquired immunity,proteolysis, phagocytosis, apoptosis, and control of the life-time of macromolecules and cells. They also appearto have antioxidative effects. Generally, sialic acids have a masking, antirecognition effect on receptors andantigens, thus hindering for example cell recognition, autoimmune reactions or the removal of macromoleculesand cells from the blood stream. On the other hand, they represent recognition sites, ligands, for sialic acidsbindingproteins (lectins). Prominent examples for the latter are the siglecs and selectins in mammalian cells,mainly involved in erythropoesis, leucocyte migration, and immunoregulation, and the hemagglutinins ofviruses. The binding of e.g. influenza viruses to sialic acids of erythrocytes and their release by virusneuraminidase led to the discovery of sialic acids and neuraminidase (sialidase) over 60 years ago. The bindingof influenza viruses to a mammalian cell depends on the type of sialic acid glycosidic linkage to the penultimatesugar of glycan chains on cell surfaces. This strongly influences the species specificity of virus infection.Presently, many other virus species, such as corona- and rotaviruses, are being recognized to adhere to sialicacids in the process of cell infection. These monosaccharides are also involved in other pathological processeslike cancer, bacterial and protozoal infections, e.g. malaria, sleeping sickness, Helicobacter pylori infection, andautoimmune diseases. Sialic acid derivatives were prepared which are potent inhibitors of virus propagation thuscuring, for example, influenza (flu). A rapidly expanding area concerns the involvement of sialic acid in tumors,inflammation and neurodegenerative diseases.There are more than 50 types of sialic acids known in nature with different N- and O-substituents (Fig. 1) as wellas dehydro, anhydro and lactone forms. All are formally derived from N-acetylneuraminic acid (Neu5Ac). Thebiosynthesis of sialic acids, their subcellular sites and some of the modification reactions are shown in Fig. 2.These chemical modifications influence the biology of cells, although only N-glycolylneuraminic acid (Neu5Gc)and O-acetylated derivatives of Neu5Ac like N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac 2 ) and N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac 2 ) have been studied in detail in this respect. They represent onco-fetalantigens and evoked much interest in tumor biology. O-Acetylated sialic acids are involved in growth anddifferentiation, for example of neuronal tissues, and inhibit apoptosis. Furthermore, Neu5Ac, Neu5Gc andNeu5,9Ac 2 were found to be specific receptors for many pathogenic microorganisms.57

Figure 1: The sialic acid familyWe focussed on the enzymology and molecular genetics of the O-acetylation of sialic acids. Due to the great(patho-)physiological significance of this sialic acid modification, knowledge of the regulation of the expressionof the corresponding AcCoA:sialate-O-acetyltransferase(s) and its distribution in human tissues and animalspecies is most important. Neither was a eukaryotic O-acetyltransferase isolated nor cloned or expressed so far,in spite of many attempts made in various laboratories, with the exception of a putative O-acetyltransferasegene/protein (Cas1 protein), which we detected in humans (S. Arming et al., 2011). With enzymatic studies ofthe O-acetylation of sialic acids in human serum and tonsillar lymphocytes as well as in lymphoblasts fromchildren suffering from acute lymphoblastic leukemia (ALL) (D. Wipfler et al., 2011 and C. Mandal et al., 2011)I concluded my experimental part of this area.C.2 Occurrence of N-glycolylneuraminic acidN-Acetyl hydroxylation yielding Neu5Gc is, like O-acetylation, a frequent sialic acid-modifying reaction, whichwe have studied for many years. Interest in this substance is increasing, since it was found that man does notexpress Neu5Gc, due to a gene (cmah) mutation which occurred in evolution during the human divergence fromgreat apes. This is of practical significance in microbial and non-microbial inflammation and one of the reasonswhy we are interested in the distribution of this monosaccharide in nature. The other reason are evolutionaryaspects. The loss of Neu5Gc expression also seems to be of nutritional significance, since Neu5Gc ingested fromfood, e.g. from red meat, is a xeno-antigen which is discussed to cause inflammation e.g. in intestinal mucosaleading to cancer. Correspondingly, the cooperation with Dr. Yann Guérardel (Lille) and Dr. Yasuro Shinohara(Sapporo) regarding the occurrence of Neu5Gc in animals and especially in sea food is continued58

Research Report Biochemical Institute, Christian-Albrechts-University KielFigure 2: Metabolism of sialic acidsC.3 Trypanosomal trans-sialidasesThese enzymes were found at the surface of pathogenic trypanosomes such as the African Trypanosoma bruceiand T. congolense, and the American T. cruzi, leading to sleeping sickness and Chagas Disease, respectively.Trans-Sialidases can act as sialidases or as sialyltransferases. In the case of the absence of suitable glycosylacceptors, trans-sialidases hydrolytically release sialic acids from their glycosidic bonds such as classicalsialidases (neuraminidases). They, however, prefer to transfer sialic acids from α2,3-glycosidic bonds ofglycans to terminal galactose residues of other glycoconjugate molecules yielding new α2,3-glycosidic linkages.In this way trypanosomes acquire sialic acids from the host (e.g. from erythrocytes, Fig. 3) and thus increasetheir virulence, especially by compromising the host's immune system.Therefore, inhibitors of trypanosomal trans-sialidases are expected to be suitable for therapy of these frequentand disastrous tropical diseases. We are testing, together with Dr. Silke Schrader (Köln), inhibitors of T. cruzitrans-sialidase synthesized by Prof. Joachim Thiem (Hamburg) and Prof. Teruo Yoshino (Tokyo).59

Figure 3: Reversible transfer of sialic acid by trypanosomal trans-sialidasesD Publications 2010/2011Publications 20101. Monti, E., Bonten, E., D'Azzo, A., Bresciani, R., Venerando, B.,Borsani, G., Schauer, R., and Tettamanti G. (2010) Sialidases invertebrates: a family of enzymes tailored for several cell functions.Adv. Carbohydr. Chem. Biochem. 64:403-479.Publications 20111. Arming, S., Wipfler, D., Mayr, Merling, A., Vilas, U., Schauer, R.Schwartz-Albiez, R. and Vlasak, R. (2010) The human Cas1 protein:A sialic-acid-specific O-acetyltransferase? Glycobiology 21: 553-564.2. Wipfler, D., Srinivasan, G.V., Sadick, H., Kniep, B., Arming, S.,Willhauck-Fleckenstein, M., Vlasak, R. Schauer, R. and Schwartz-Albiez, R. (2011) Differentially regulated expression of 9-O-acetylGD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiationand maturation of human T and B lymphocytes. Glycobiology21:1161-1172.3. Schauer, R., Srinivasan, G.V., Wipfler, D., Kniep, B. and Schwartz-Albiez, R. (2011) O-Acetylated sialic acids and their role in immunedefense. In: The Molecular Immunology of Complex Carbohydrates-3, A.M. Wu (ed.) Adv. Carbohydr. Chem. Biochem. 705:525-548.4. Schauer, R. and Kamerling, J.P. (2011) The chemistry and biology oftrypanosomal trans-sialidases: Virulence factors in Chagas Diseaseand Sleeping Sickness. ChemBioChem 12:2246-2264.Impact Factor2.67Impact Factor3.7913.7911.3793.945Impact factors 2010: 2.67Impact factors 2011: 12.906Total impact factors 2010/2011: 19.36760

Research Report Biochemical Institute, Christian-Albrechts-University KielAppendixSeminars 2010/201112. Januar 2010 Prof. Dr. Thomas Braun, MPI Bad NauheimMolecular circuits controlling cardiovascular remodelling26. Januar 2010 PD Dr. Hendrik Fuchs, BerlinRegulated shedding and intramembrane proteolysis of the humantransferrin receptor - signal transduction or degradation?02. Februar 2010 Prof. Dr. Eeva-Liisa Eskelinen, University HelsinkiAutophagosome biogenesis in three dimensions09. Februar 2010 Prof. Dr. Achim Gossler, HannoverCharacterization of Delta3, an atypical DSL protein11. Mai 2010 Prof. Dr. Henning Walczak, LondonA physiological role of linear ubiquitylation in TNF receptorsuperfamily signal transduction08. Juni 2010 Prof. Dr. Thomas Rudel, WürzburgADAM17-mediated shedding of tumor necrosis factor receptor Isupports Chlamydia trachomatis infection15. Juni 2010 Prof. Dr. B. Bechinger, StrassburgSolid-state NMR investigations of membrane-associated polypeptides:From antmicrobial peptides to apoptotic proteins29. Juni 2010 Prof. Dr. Christian Behl, MainzNeurons under pressure: How to deal with oxidative stress andaggregated proteins?13. Juli 2010 Prof. Dr. Hinrich Schulenburg, KielEvolutionary dynamics of C. elegans-pathogen interactions28. September 2010 Dr. Michael Willem, MünchenIs BACE1 a safe drug target for Alzheimer`s disease?02. November 2010 Prof. Dr. Eithan Galun, JerusalemTherapeutic targeting of platelet receptors: novel mechanisms25. Januar 2011 Prof. Dr. Markus Glatzel, UKE HamburgPrPC processing und exosome function22. Februar 2011 Prof. Dr. Bernhard Nieswandt, WürzburgTherapeutic targeting of plateletreceptors: novel mechanisms61

19. April 2011 Prof. Dr. Claus Pietrzik, Universität MainzThe role of the Lipoprotein Receptor LRP1 in Alzheimer's Disease17. Mai 2011 Prof. R. Michaela Kress, Universität Innsbruckgp130 cytokines and nociception: novel therapeutic strategies21. Juni 2011 PD Dr. Tobias Dechow, TU MünchenRole of gp130 and Notch in oncogenic signal transduction in lungcancer and multiple myeloma05. Juli 2011 Prof. Dr. Hana Algül, TU MünchenIL-6 as linking module between microenvironment and pancreaticoncogenesis13. Dezember 2011 Prof. Dr. Gerd Multhaupt, BerlinTowards the understanding of regulated intramembrane proteolysis inamyloid production62

Research Report Biochemical Institute, Christian-Albrechts-University KielPublications 2010/2011Original Papers and ReviewsAltmeppen, H., Prox, J., Puig, B., Kluth, M.A., Bernreuther, C., Thurm, D., Jorissen, E., Petrowitz, B., Bartsch,U., de Strooper, B., Saftig, P., Glatzel, M. (2011) Lack of a-disintegrin-and metalloproteinase ADAM10leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo. MolNeurodegen, 6, 36. 5.1Arming, S., Wipfler, D., Mayr, Merling, A., Vilas, U., Schauer, R. Schwartz-Albiez, R. and Vlasak, R. (2010)The human Cas1 protein: A sialic-acid-specific O-acetyltransferase? Glycobiology 21: 553-564. 3.791Arndt, V., Dick,N., Tawo,R., Dreiseidler,M., Wenzel,D.,Hesse, M.,Fürst,D.O., Saftig,P., Saint, R.,Fleischmann,B.K., Hoch,M., Höhfeld, J. (2010) Chaperone-assisted selective autophagy is essential formuscle maintenance. Current Biology, 20, 143-148 10.777Atreya R, Zimmer M, Bartsch B, Waldner MJ, Atreya I, H. Neumann H, Hildner K, Hoffman A, Kiesslich R,Rink AD, Rau T, Rose-John S, Kessler H, Schmidt J and Neurath MF (2011) Anti-TNF antibodies target T-cell apoptosis in inflammatory bowel diseases via TNFR2 and intestinal CD14+ macrophages. Gastroenterol,14, 2026-2038. 12.032Bai L, Beckers L, Wijnands E, Lutgens SP, Herías MV, Saftig P, Daemen MJ, Cleutjens K, Lutgens E, BiessenEA, Heeneman S. (2010) Cathepsin K gene disruption does not affect murine aneurysm formation.Atherosclerosis 209:96-103. 4.522Barkhausen, T., Tschernig, T., Rosenstiel, P., van Griensven, M., Vonberg, R. P., Dorsch, M., Mueller-Heine,A., Chalaris, A., Scheller, J., Rose-John, S., Seegert, D., Krettek, C., and Waetzig, G. H. (2011) Selectiveblockade of interleukin-6 trans-signaling improves survival in a murine polymicrobial sepsis model. CritCare Med 39, 878-888. 6.254Behnke J, Schneppenheim J, Koch-Nolte F, Haag F, Saftig P, Schröder B. 2011 Signal-peptide-peptidase-like 2a(SPPL2a) is targeted to lysosomes/late endosomes by a tyrosine motif in its C-terminal tail. FEBS Lett.585:2951-7. 3.39Behnke, J., Eskelinen,E.L., Saftig, P., Schröder, B.A. (2011) Two dileucine motifs mediate lateendosomal/lysosomal targeting of “Transmembrane protein 192” (TMEM192) and a C-terminal cysteine isresponsible for disulphide bond formation in TMEM192 homodimers. Biochem. J., 434, 219-31. 5.151Bender, M., Hofmann, S., Stegner, D., Chalaris, A., Bösl, M., Braun, A., Scheller, J., Rose-John, S., andNieswandt, B. (2010) Differentially regulated GPVI ectodomain shedding by multiple platelet-expressedproteinases. Blood 116, 3347-3355. 10.558Bens, S., Mohn, A., Yüksel, B., Kulle, A., Michalek, M., Chiarelli, F., Özbek, M.N., Leuschner, I., Grötzinger,J., Holterhus, P.M., Riepe, F.G. 2010. Congenital Lipoid Adrenal Hyperplasia: Functional characterization ofthree novel mutations in the STAT Gene. J Clin Endocrinol Metab. 95, 1301-1308. 6.495Blanz, J., Groth, J., Zachos, C., Wehling, C., Saftig, P., Schwake, M. (2010) Disease causing mutations withinthe lysosomal integral membrane protein type 2 (LIMP-2) reveal the nature of binding to its ligand b-glucocerobrosidase. Hum. Mol. Genet., 19, 563-572 7.249Bruhn, O., Cascorbi, I., Grötzinger, J., Jung, S. (2011) Antimicrobial peptides and proteins of the horse - insightsinto a well armed organism. Veterinary Research 42, 1-22. 3.765Carrasco-Marín, E., Fernández-Prieto, L., Rodriguez-Del Rio, E., Madrazo-Toca, F., Reinheckel, T., Saftig, P.,Alvarez-Dominguez, C. (2011) LIMP-2 links late phagosomal trafficking with the onset of the innateimmune response to Listeria monocytogenes: a role in macrophage activation. J Biol Chem. 286:3332-3341. 5.328Chalaris, A., Adam, N., Sina, C., Rosenstiel, P., Paliga, K., Lehmann, J., Schirmacher, P., Hartmann, D., Cichy,J., Gavrilowa, O., Schreiber, S., Jostock, T., Matthews, V., Häsler, R., Becker, C., Neurath, M. F., Reiß, K.,Scheller, J., and Rose-John, S. (2010) Critical role of the disintegrin metalloprotease ADAM17 for intestinalinflammation and regeneration in mice. J Exp Med 207, 1617-1624. 14.776Chalaris, A., Garbers, C., Rabe, B., Rose-John, S., and Scheller, J. (2011) The soluble Interleukin 6 receptor:Generation and role in inflammation and cancer. Eur J Cell Biol 90, 484-494. 3.630Chalaris, A., Gewiese, J., Paliga, K., Fleig, L., Schneede, A., Krieger, K., Rose-John, S., and Scheller, J. (2010)ADAM17-mediated shedding of the IL6R induces cleavage of the membrane stub by gamma-secretase.Biochim Biophys Acta 1803, 234-245. 4.733Chandan Mandal, Chhabinath Mandal, S. Chandra, R. Schauer and Chitra Mandal (2012) Regulation of O-acetylation of sialic acids by sialate-O-acetyltransferase and sialate-O-acetylesterase activities in childhoodacute lymphoblastic leukemia. Glycobiology 22:70-83. 3.791Conrad, U., Plagmann, I., Malchow, S., Sack, M., Floss, D. M., Kruglov, A. A., Nedospasov, S. A., Rose-John,S., and Scheller, J. (2011) ELPylated anti-human TNF therapeutic single-domain antibodies for prevention oflethal septic shock. Plant Biotechnol J 9, 22-31. 4.88663

Corbett M.A.*, Schwake M.*, Bahlo M., Dibbens L.M., Lin M., Gandolfo L.C., Vears D.F., O'Sullivan J.D.,Robertson T., Bayly M.A., Gardner A.E., Vlaar A.M., Korenke G.C., Bloem B.R., de Coo I.F., VerhagenJ.M., Lehesjoki A.E., Gecz J., Berkovic S.F. (2011). A mutation in the Golgi Qb-SNARE gene GOSR2causes progressive myoclonus epilepsy with early ataxia. Am. J. Hum. Genet. 88, 657-63. 11.7Cullen V, Sardi SP, Ng J, Xu YH, Sun Y, Tomlinson JJ, Kolodziej P, Kahn I, Saftig P, Woulfe J, Rochet JC,Glicksman MA, Cheng SH, Grabowski GA, Shihabuddin LS, Schlossmacher MG. (2011) Acid betaglucosidasemutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter ?-synucleinprocessing. Ann Neurol 69,940-53. 9.317Damme, M., Stroobants, S., Walkley, S.U., Lüllmann-Rauch, R., D'Hooge, R., Fogh, J., Saftig, P., Lübke, T.,Blanz, J. (2011) Cerebellar alterations and gait defects as therapeutic outcome measures for enzymereplacement therapy in α-mannosidosis. J Neuropathol Exp Neurol. 70, 83-94. 4.564Dauth, S., Sirbulescu, R.F., Jordans, S., Rehders, M., Avena, L., Oswald, J., Lerchl, A., Saftig, P., Brix, K.(2011) Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervoussystem that are associated with learning and memory deficits. BMC Neuroscience, 12:74. 3.09Desmond MJ, Lee D, Fraser SA, Katerelos M, Gleich K, Martinello P, Li YQ, Thomas MC, Michelucci R, ColeAJ, Saftig P, Schwake M, Stapleton D, Berkovic SF, Power DA (2011) Tubular proteinuria in mice andhumans lacking the intrinsic lysosomal protein SCARB2/Limp-2. Am J Physiol Renal Physiol. , i 300,F1437-47. 3.590Doyle, E.L., Ridger, V., Ferraro, F., Turmaine, M., Saftig, P., Cutler, D.F. (2011) CD63 is an essential co-factorto leukocyte recruitment by endothelial P-selectin. Blood, 118, 4265-73. 10.55Drucker, C., Gewiese, J., Malchow, S., Scheller, J., and Rose-John, S. (2010) Impact of interleukin-6 classicandtrans-signaling on liver damage and regeneration. J Autoimmun 34, 29-37. 7.881Evans, S.F., Irmady, K., Ostrow, K., Kim, T., Nykjaer, A., Saftig, P., Blobel, C., Hempstead, B.L. (2011)Neuronal BDNF is synthesized in excess, with levels regulated by sortilin mediated trafficking and lysosomaldegradation. J. Biol. Chem., 286, 29556-67. 5.3Febbraio MA, Rose-John S, Pedersen BK (2010) Is interleukin-6 receptor blockade the Holy Grail forinflammatory diseases? Clin Pharmacol Ther 87: 396-8. 6.378Fisher DT, Chen Q, Skitzki JJ, Muhitch JB, Zhou L, Appenheimer MM, Vardam TD, Unger E, Passanese J,Wang W-C, Dewhirst MW, Rose-John S, Repasky EA, Baumann H, Evans SS (2011) IL-6 trans-signalinglicenses murine and human tumor microvascular gateways for trafficking of cytotoxic T cells. J Clin Invest121: 2846-3859 14.152Floss DM, Schallau K, Rose-John S, Conrad U, Scheller J (2010) Elastin-like polypeptides revolutionizerecombinant protein expression and their medical. application. Trends Biotechnol 28: 37-45. 9.644Garbers C, Thaiss W, Jones GW, Waetzig GH, Lorenzen I, Guilhot F, Lissilaa R, Ferlin WG, Grötzinger J, JonesSA, Rose-John S, Scheller J. (2011) Inhibition of classic signaling is a novel function of soluble GP130which is controlled by the ratio of interleukin 6 and soluble interleukin 6 receptor. J Biol Chem. 286, 42959-42970. 5.328Garbers, C., Jänner, N., Chalaris, A., Moss, M. L., Floss, D. M., Meyer, D., Koch-Nolte, F., Rose-John, S., andScheller, J. (2011) Species specificity of ADAM10 and ADAM7 in IL-6 transsignaling and novel role ofADAM10 in inducible IL-6R shedding. J Biol Chem 286, 14804-14811. 5.328Gewiese-Rabsch J, Drucker C, Malchow S, Scheller J, Rose-John (2010) Role of IL-6 Transsignaling in CCl4induced liver damage, BBA-Mol Basis Disease 1802: 1054-61 5.211Giersberg, M., Floss, D., Kiprijanov, S., Conrad, U., and Scheller, J. (2010) Covalent dimerization between acamelidae anti-human TNF single domain antibody and the constant kappa light chain domain improvesneutralizing capacity. Biotechnol Bioeng 106, 161-166. 2.936Glomski,K., Monette, S., Manova, K., de Strooper, B., Saftig, P., Blobel, C.P. (2011) Deletion of Adam10 inendothelial cells leads to defects in organ-specific vascular structures. Blood, 118(4):1163-74. 10.55Greenhill CJ, Rose-John S, Lissilaa R, Ferlin W, Ernst M, Hertzog PJ, Mansell A, and Jenkins BJ (2011) IL-6trans-signaling modulates TLR4-dependent inflammatory responses via STAT3.J Immunol 186: 1199-1208 5.745Ha, S.D., Ham, B., Mogridge, J., Saftig, P., Lin, S., Kim, S.O. 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Research Report Biochemical Institute, Christian-Albrechts-University KielJones, S. A., Scheller, J., and Rose-John, S. (2011) Therapeutic strategies for the clinical blockade of IL-6/gp130signaling. J Clin Invest 121, 3375-3383. 14.152Jorissen, E., Prox, J., Bernreuther, C., Weber, S., Schwanbeck, R., Serneels, L., Snellinx, A., Craessaerts, K.,Ththiah, A., Tesseur, I., Bartsch, U., Weskamp, G., Blobel, C.P., Glatzel, M., de Strooper, B., Saftig, P.(2010) The disintegrin/metalloproteinase ADAM10 is essential for the establishement of the brain cortex. J.Neurosci., 30:4833-4844. 7.452Jung, S., Mysliwy, J., Spudy, B., Lorenzen, I., Gelhaus, C., Podschun, R., Leippe, M., Grötzinger, J. Human β-defensin2 and β-defensin3 chimeric peptides reveal the structural basis of the pathogen specificity of theirparent molecules. Antimicrob Agents CH 55, 954-960 (2011). 4.672Kim, J., Lilliehook, C., Dudak, A., Prox, J., Saftig, P., Federoff, H.J., Lim, S.T. 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Research Report Biochemical Institute, Christian-Albrechts-University KielAccumulated Impact Factors2002 = 220.152003 = 327.632004 = 320.062005 = 318.732006 = 380.8062007 = 258.4012008 = 292.8752009 = 311.3862010 = 303.3982011 = 386.49469