Contents · Contents Preface..... v Contributors ... Stefan R. Weisshaar, Gerrit J.K. Praefcke, and R. Jürgen Dohmen ... Christof Taxis and Michael Knop 44 PROTAC-Induced Proteolytic

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Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Three Decades of Studies to Understand the Functions of the Ubiquitin Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Alexander Varshavsky

Part I Enzymology and SubStratES of ubIquItIn famIly modIfIErS

2 Structure and Evolution of Ubiquitin and Ubiquitin-Related Domains . . . . . . . . . . 15A. Maxwell Burroughs, Lakshminarayan M. Iyer, and L. Aravind

3 Deciphering Tissue-Specific Ubiquitylation by Mass Spectrometry . . . . . . . . . . . . . 65Ugo Mayor and Junmin Peng

4 Analysis of Cellular SUMO and SUMO–Ubiquitin Hybrid Conjugates . . . . . . . . . . 81Marion Schnellhardt, Kristina Uzunova, Veronika N. Bade, Anke Krause, Stefan R. Weisshaar, Gerrit J.K. Praefcke, and R. Jürgen Dohmen

5 Recombinant Reconstitution of Sumoylation Reactions In Vitro . . . . . . . . . . . . . . . 93Annette Flotho, Andreas Werner, Tobias Winter, Andrea S. Frank, Heidi Ehret, and Frauke Melchior

6 Reconstitution of SUMO-Dependent Ubiquitylation In Vitro . . . . . . . . . . . . . . . . 111Kirstin Keusekotten and Gerrit J.K. Praefcke

7 Detection and Analysis of FAT10 Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125Annette Aichem and Marcus Groettrup

8 Isolation of NEDDylated Proteins in Human Cells . . . . . . . . . . . . . . . . . . . . . . . . . 133Orsolya Leidecker and Dimitris P. Xirodimas

9 The ISG15 Conjugation System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Larissa A. Durfee and Jon M. Huibregtse

10 Pupylation: Proteasomal Targeting by a Protein Modifier in Bacteria. . . . . . . . . . . . 151Kristin E. Burns and K. Heran Darwin

Part II rEcognItIon and chaIn formatIon of ubf modIfIErS

11 Role of UbL Family Modifiers and Their Binding Proteins in Cell Signaling . . . . . . 163Sjoerd J. L. van Wijk, Magda Bienko, and Ivan Dikic

12 Isolation of Ubiquitylated Proteins Using Tandem Ubiquitin-Binding Entities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173Fabienne Aillet, Fernando Lopitz-Otsoa, Roland Hjerpe, Mónica Torres-Ramos, Valérie Lang, and Manuel S. Rodríguez

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13 Using Linkage-Specific Monoclonal Antibodies to Analyze Cellular Ubiquitination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185Kim Newton, Marissa L. Matsumoto, Ronald E. Ferrando, Katherine E. Wickliffe, Michael Rape, Robert F. Kelley, and Vishva M. Dixit

14 Measuring Rates of Ubiquitin Chain Formation as a Functional Readout of Ligase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197Virginia P. Ronchi and Arthur L. Haas

15 Synthesis and Analysis of K11-Linked Ubiquitin Chains . . . . . . . . . . . . . . . . . . . . . 219Anja Bremm and David Komander

16 Synthesis and Analysis of Linear Ubiquitin Chains. . . . . . . . . . . . . . . . . . . . . . . . . . 229Kazuhiro Iwai

17 Detection and Quantitation of SUMO Chains by Mass Spectrometry . . . . . . . . . . . 239Ivan Matic and Ronald T. Hay

18 Bioinformatical Detection of Recognition Factors for Ubiquitin and SUMO. . . . . . 249Benjamin Vogt and Kay Hofmann

19 Surface Plasmon Resonance to Measure Interactions of UbFs with Their Binding Partners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263Julian Stingele, Uwe W. Roder, and Shahri Raasi

20 Identifying and Studying Ubiquitin Receptors by NMR . . . . . . . . . . . . . . . . . . . . . 279Xiang Chen and Kylie J. Walters

21 Exploring the Role of p97 and Its UBX-Domain Cofactors Through Identification of Their Interacting Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305Gabriela Alexandru

Part III ProtEaSomE bIogEnESIS, rEgulatIon and functIon

22 Assembly and Function of the Proteasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315Yasushi Saeki and Keiji Tanaka

23 Using Native Gel Electrophoresis and Phosphofluoroimaging to Analyze GFP-Tagged Proteasomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339Cordula Enenkel

24 Disulfide Engineering to Map Subunit Interactions in the Proteasome and Other Macromolecular Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349Mark Hochstrasser and Minoru Funakoshi

25 Using DNA Damage Sensitivity Phenotypes to Characterize Mutations Affecting Proteasome Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363Benoît Le Tallec and Anne Peyroche

26 Analysing Properties of Proteasome Inhibitors Using Kinetic and X-Ray Crystallographic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373Nerea Gallastegui and Michael Groll

27 Immunoproteasome-Specific Inhibitors and Their Application . . . . . . . . . . . . . . . . 391Michael Basler and Marcus Groettrup

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ixContents

28 Binding of Ubiquitin Conjugates to Proteasomes as Visualized with Native Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403Suzanne Elsasser, Yuan Shi, and Daniel Finley

29 Affinity Purification of Mammalian 26S Proteasomes Using an Ubiquitin-Like Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423Henrike C. Besche and Alfred L. Goldberg

30 Using siRNA Techniques to Dissect Proteasome Assembly Pathways in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433Takeumi Kaneko and Shigeo Murata

31 Reconstitution of PA700, the 19S Regulatory Particle, from Purified Precursor Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443George N. DeMartino

Part IV ProtEIn qualIty control

32 Cellular Responses to Misfolded Proteins and Protein Aggregates . . . . . . . . . . . . . . 455Scott A. Houck, Sangita Singh, and Douglas M. Cyr

33 Live-Cell Imaging of Ubiquitin–Proteasome System Function. . . . . . . . . . . . . . . . . 463Mark S. Hipp, Kirill Bersuker, and Ron R. Kopito

34 Analysis of Chaperone-Assisted Ubiquitylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473Michael Dreiseidler, Niko Dick, and Jörg Höhfeld

35 Use of CPY* and Its Derivatives to Study Protein Quality Control in Various Cell Compartments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489Alexandra Stolz and Dieter H. Wolf

36 Assays to Measure ER-Associated Degradation in Yeast . . . . . . . . . . . . . . . . . . . . . . 505Joseph R. Tran and Jeffrey L. Brodsky

37 SDS-PAGE Techniques to Study Ubiquitin-Like Conjugation Systems in Yeast Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519Hitoshi Nakatogawa and Yoshinori Ohsumi

38 Analysis of Ubiquitin-Dependent Proteolysis in Caenorhabditis elegans . . . . . . . . . . 531Alexandra Segref and Thorsten Hoppe

Part V cEllular and chEmIcal StratEgIES to Study and manIPulatE ubf rElatEd ProcESSES

39 Structural Insights into Functional Modes of Proteins Involved in Ubiquitin Family Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547Petra Hänzelmann, Antje Schäfer, Daniel Völler, and Hermann Schindelin

40 Identification and Application of NEDD8 E1 Inhibitors . . . . . . . . . . . . . . . . . . . . . 577Frank J. Bruzzese, Michael A. Milhollen, James M. Gavin, Helen R. Josephine, and James E. Brownell

41 Formation of Ubiquitin Dimers via Azide–Alkyne Click Reaction . . . . . . . . . . . . . . 589Silvia Eger, Martin Scheffner, Andreas Marx, and Marina Rubini

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42 Synthesis of Atypical Diubiquitin Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597Farid El Oualid, Dharjath S. Hameed, Dris El Atmioui, Henk Hilkmann, and Huib Ovaa

43 TIPI: TEV Protease-Mediated Induction of Protein Instability . . . . . . . . . . . . . . . . 611Christof Taxis and Michael Knop

44 PROTAC-Induced Proteolytic Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627Kimberly Cornish Carmony and Kyung-Bo Kim

45 Formation of Nondegradable Forked Ubiquitin Conjugates by Ring-Finger Ligases and Its Prevention by S5a . . . . . . . . . . . . . . . . . . . . . . . . . . 639Hyoung Tae Kim and Alfred L. Goldberg

46 S5a/Rpn10, a UIM-Protein, as a Universal Substrate for Ubiquitination . . . . . . . . 653Hyoung Tae Kim and Alfred L. Goldberg

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

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Contributors

annEttE aIchEm • Biotechnology Institute Thurgau, Kreuzlingen, Switzerland; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

fabIEnnE aIllEt • Proteomics Unit and Proteomics Platform, CIC bioGUNE CIBERehd, ProteoRed Technology Park of Bizkaia, Derio, Spain

gabrIEla alExandru • Protein Ubiquitylation Unit, The Scottish Institute for Cell Signalling (SCILLS), Dundee, Scotland, UK; University of Dundee, Dundee, Scotland, UK

l. araVInd • National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA

drISElatmIouI • Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

VEronIka badE • Center for Molecular Medicine Cologne (CMMC), Institute for Genetics, University of Cologne, Cologne, Germany

mIchaEl baSlEr • Department of Biology, Division of Immunology, University of Konstanz, Konstanz, GermanyM. Basler Biotechnology Institute Thurgau, Kreuzlingen, Switzerland

kIrIll bErSukEr • Department of Biology, Stanford University, Stanford, CA, USAhEnrIkE c. bESchE • Department of Cell Biology, Harvard Medical School,

Boston, MA, USAmagda bIEnko • Frankfurt Institute for Molecular Life Sciences and Institute

of Biochemistry II, Goethe University School of Medicine, Frankfurt (Main), Germany; Departments of Physics and of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

anja brEmm • Protein and Nucleic Acid Chemistry Division, MRC Laboratory of Molecular Biology, Cambridge, UK

jEffrEy l. brodSky • Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA

jamES E. brownEll • Discovery, Millennium Pharmaceuticals Inc., Cambridge, MA, USA

frank j. bruzzESE • Discovery, Millennium Pharmaceuticals Inc., Cambridge, MA, USA

krIStIn E. burnS • Department of Microbiology, New York University School of Medicine, New York, NY, USA

a. maxwEll burroughS • Omics Science Center (OSC), RIKEN Yokohama Institute, Kanagawa, Japan

kImbErly cornISh carmony • Department of Pharmaceutical Science, College of Pharmacy, University of Kentucky, Lexington, KY, USA

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xIang chEn • Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA

douglaS m. cyr • Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

k. hEran darwIn • Department of Microbiology, New York University School of Medicine, New York, NY, USA

gEorgE n. dEmartIno • Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

nIko dIck • Institut für Zellbiologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany

IVan dIkIc • Frankfurt Institute for Molecular Life Sciences and Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt (Main), Germany

VIShVa m. dIxIt • Physiological Chemistry Department, Genentech, Inc., South San Francisco, CA, USA

jürgEn dohmEn • Institute for Genetics, University of Cologne, Cologne, GermanymIchaEl drEISEIdlEr • Institut für Zellbiologie, Rheinische Friedrich-Wilhelms-

Universität Bonn, Bonn, GermanylarISSa a. durfEE • Section of Molecular Genetics and Molecular Biology,

Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA

SIlVIa EgEr • Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany

hEIdI EhrEt • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

SuzannE ElSaSSEr • Department of Cell Biology, Harvard Medical School, Boston, MA, USA

cordula EnEnkEl • Department of Biochemistry, University of Toronto, Toronto, ON, Canada

ronald E. fErrando • Pathology Department, Genentech, Inc., South San Francisco, CA, USA

danIEl fInlEy • Department of Cell Biology, Harvard Medical School, Boston, MA, USA

annEttE flotho • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

andrEa S. frank • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

mInoru funakoShI • Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA; Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan

nErEa gallaStEguI • Department Chemie, Technische Universität München, Garching, Germany

jamES m. gaVIn • Discovery, Millennium Pharmaceuticals Inc., Cambridge, MA, USAalfrEd l. goldbErg • Department of Cell Biology, Harvard Medical School,

Boston, MA, USA

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marcuS groEttruP • Department of Biology, Division of Immunology, University of Konstanz, Konstanz, Germany; Biotechnology Institute Thurgau, Kreuzlingen, Switzerland

mIchaEl groll • Department Chemie, Technische Universität München, Garching, Germany

arthur l. haaS • Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine and the Stanley S. Scott Cancer Center, New Orleans, LA, USA

dharjath S. hamEEd • Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

PEtra hänzElmann • Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany

ronald t. hay • Wellcome Trust Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, Scotland, UK

hEnk hIlkmann • Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

mark S. hIPP • Department of Biology, Stanford University, Stanford, CA, USA; Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany

roland hjErPE • Proteomics Unit and Proteomics Platform, CIC bioGUNE CIBERehd, ProteoRed, Technology Park of Bizkaia, Derio, Spain

mark hochStraSSEr • Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

kay hofmann • Bioinformatics Group, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany

jörg höhfEld • Institut für Zellbiologie, Rheinische Friedrich-Wilhelms- Universität Bonn, Bonn, Germany

thorStEn hoPPE • Institute for Genetics Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne,

Scott a. houck • Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

jon m. huIbrEgtSE • Section of Molecular Genetics and Molecular Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA

kazuhIro IwaI • Cell Biology and Metabolism Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan; Department of Biophysics and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Japan

lakShmInarayan m. IyEr • National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA

hElEn r. joSEPhInE • Discovery, Millennium Pharmaceuticals Inc., Cambridge, MA, USA

takEumI kanEko • Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

robErt f. kEllEy • Antibody Engineering Department, Genentech, Inc., South San Francisco, CA, USA

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kIrStIn kEuSEkottEn • Center for Molecular Medicine Cologne (CMMC), Institute for Genetics, University of Cologne, Cologne, Germany

kyung-bo kIm • Department of Pharmaceutical Science, College of Pharmacy, University of Kentucky, Lexington, KY, USA

mIchaEl knoP • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), ZMBH-DKFZ Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany

daVId komandEr • Protein and Nucleic Acid Chemistry Division, MRC Laboratory of Molecular Biology, Cambridge, UK

ron r. koPIto • Department of Biology, Stanford University, Stanford, CA, USAankE krauSE • Center for Molecular Medicine Cologne (CMMC),

Institute for Genetics, University of Cologne, Cologne, GermanyValérIE lang • Proteomics Unit and Proteomics Platform, CIC bioGUNE CIBERehd,

ProteoRed, Technology Park of Bizkaia, Derio, SpainorSolya lEIdEckEr • Wellcome Trust Centre for Gene Regulation and Expression,

College of Life Sciences, University of Dundee, Dundee, Scotland, UKfErnando loPItz-otSoa • Proteomics Unit and Proteomics Platform,

CIC bioGUNE CIBERehd, ProteoRed, Technology Park of Bizkaia, Derio, SpainandrEaS marx • Department of Chemistry, Konstanz Research School Chemical

Biology, University of Konstanz, Konstanz, GermanyIVan matIc • Wellcome Trust Centre for Gene Regulation and Expression,

Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, Scotland, UK

marISSa l. matSumoto • Antibody Engineering Department, Genentech, Inc., South San Francisco, CA, USA

ugo mayor • Proteomics Unit and Proteomics Platform, CIC bioGUNE CIBERehd, ProteoRed, Technology Park of Bizkaia, Derio, Spain

fraukE mElchIor • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

mIchaEl a. mIlhollEn • Discovery, Millennium Pharmaceuticals Inc., Cambridge, MA, USA

ShIgEo murata • Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

hItoShI nakatogawa • Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan

kIm nEwton • Physiological Chemistry Department, Genentech, Inc., South San Francisco, CA, USA

yoShInorI ohSumI • Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan

farIdEloualId • Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

huIb oVaa • Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

junmIn PEng • Department of Structural Biology, St. Jude Proteomics Facility, St. Jude Children’s Research Hospital, Memphis, TN, USA

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xvContributors

annE PEyrochE • CEA, iBiTecS, SBIGeM, Laboratoire du métabolisme de l’ADN et réponses aux génotoxiques, Gif-sur-Yvette, France

gErrIt j. k. PraEfckE • Center for Molecular Medicine Cologne (CMMC), Institute for Genetics, University of Cologne, Cologne, Germany

ShahrI raaSI • Laboratory of Cellular Biochemistry, Department of Biology, University of Konstanz, Konstanz, Germany

mIchaEl raPE • Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

uwE w. rodEr • GE Healthcare Europe GmbH, Freiburg, GermanymanuEl S. rodríguEz • Proteomics Unit and Proteomics Platform, CIC bioGUNE

CIBERehd, ProteoRed, Technology Park of Bizkaia, Derio, Spain; Biochemistry Department, University of the Basque Country, Leioa, Spain

VIrgInIa P. ronchI • Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine and the Stanley S. Scott Cancer Center, New Orleans, LA, USA

marIna rubInI • Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany

yaSuShI SaEkI • Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

antjE SchäfEr • Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany

martIn SchEffnEr • Department of Biology, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany

hErmann SchIndElIn • Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany

marIon SchnEllhardt • Institute for Genetics, University of Cologne, Cologne, Germany

alExandra SEgrEf • Institute for Genetics and Cologne Excellence Cluster on Cellu-lar Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

yuan ShI • Department of Cell Biology, Harvard Medical School, Boston, MA, USASangIta SIngh • Department of Cell and Developmental Biology, University of North

Carolina at Chapel Hill, Chapel Hill, NC, USAjulIan StIngElE • Department of Molecular Cell Biology, Max-Planck Institute

of Biochemistry, Martinsried, GermanyalExandra Stolz • Institute of Biochemistry, University Stuttgart,

Stuttgart, GermanybEnoît lE tallEc • CEA, iBiTecS, SBIGeM, Laboratoire du métabolisme

de l’ADN et réponses aux génotoxiques, Gif-sur-Yvette, France; Institut Curie, Centre de Recherche, Paris, France

kEIjI tanaka • Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

chrIStof taxIS • Philipps Universität Marburg, Fakultät Biologie – Genetik, Marburg, Germany

mónIca torrES-ramoS • Proteomics Unit and Proteomics Platform, CIC bioGUNE CIBERehd, ProteoRed, Technology Park of Bizkaia, Derio, Spain

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xvi Contributors

joSEPh r. tran • Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA; Graduate Program in Biochemistry and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

krIStIna uzunoVa • Institute for Genetics, University of Cologne, Cologne, Germany; IMP – Research Institute of Molecular Pathology, Vienna, Austria

alExandEr VarShaVSky • Division of Biology, California Institute of Technology, Pasadena, CA, USA

bEnjamIn Vogt • Institute for Genetics, University of Cologne, Cologne, Germany; Bioinformatics Group, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany

danIEl VöllEr • Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany

kylIE j. waltErS • Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA

StEfan r. wEISShaar • Center for Molecular Medicine Cologne (CMMC), Institute for Genetics, University of Cologne, Cologne, Germany

andrEaS wErnEr • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

kathErInE E. wIcklIffE • Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

SjoErd j. l. Van wIjk • Frankfurt Institute for Molecular Life Sciences and Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt (Main), Germany

tobIaS wIntEr • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

dIEtEr h. wolf • Institute of Biochemistry, University Stuttgart, Stuttgart, GermanydImItrIS P. xIrodImaS • Wellcome Trust Centre for Gene Regulation and Expression,

College of Life Sciences, University of Dundee, Dundee, Scotland, UK; Centre de Recherche de Biochimie Macromoléculaire – UMR 5237, CNRS, Montpellier, France

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R. Jürgen Dohmen and Martin Scheffner (eds.), Ubiquitin Family Modifiers and the Proteasome: Reviews and Protocols, Methods in Molecular Biology, vol. 832, DOI 10.1007/978-1-61779-474-2_15, © Springer Science+Business Media, LLC 2012

Chapter 15

Synthesis and Analysis of K11-Linked Ubiquitin Chains

Anja Bremm and David Komander

Abstract

Much has been learned about protein ubiquitination by studying the structural, biochemical, and bio-physical properties of ubiquitin chains in vitro. However, these analyses were limited to K48-, K63-linked, and linear ubiquitin chains. Only recently, enzymatic and chemical assembly systems for the remaining chain types have been developed. Here, we describe a method to synthesise K11-linked ubiquitin chains in vitro by exploiting the intrinsic K11-specificity of the E2 enzyme UBE2S.

Key words: Ubiquitin, K11 linkage, Atypical ubiquitin chains, UBE2S

Ubiquitin is attached to substrates via an isopeptide linkage between its C terminus and the e-amino group of a lysine residue. Additionally, ubiquitin can be ligated to one of the seven lysine residues or the N-terminal amino group of another ubiquitin molecule, thereby forming polymers of eight different linkage types. It has been demonstrated that all polyubiquitin chains are present in vivo (1). The best-characterised chain types are K48-linked and K63-linked ubiquitin polymers, which have degradative and nondegradative functions, respectively (2, 3). Enzymatic assembly systems have enabled access to large quantities of these polyubiquitin chains. The remaining “atypical” ubiquitin chains have not been studied in great detail and this is partly due to the lack of in vitro assembly systems.

Protein ubiquitination is catalysed by a three-step enzymatic cascade, including an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and E3 ligases (4). Whereas E3s determine substrate specificity, E2s are able to mediate the switch from ubiquitin chain initiation to elongation, regulate the processivity of chain

1. Introduction

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assembly and define the type of chain linkage (5). E2s specific for K48 or K63 linkages have been identified and are used for the generation of large amounts of these chain types in vitro.

K11-linked ubiquitin chains have been implicated in important biological processes, including cell cycle regulation (6), endoplasmic reticulum-associated degradation (ERAD) (1), and TNFa signalling (7). The RING-E3 anaphase-promoting complex (APC/C) uses the E2 enzymes UBE2C and UBE2S to promote the degradation of mitotic regulators by assembling K11-linked ubiquitin chains (6, 8, 9).

UBE2S also specifically generates free K11-linked diubiquitin in vitro in the absence of an E3 ligase (10). For structural and biophysical studies, the amount of unattached dimers obtained in a chain assembly reaction using wild-type (wt) UBE2S was not sufficient. Therefore, we engineered a UBE2S variant with increased efficiency. We found that wt UBE2S performs autoubiquitination in a cis reaction on its lysine rich C-terminal tail (10). Hence, we replaced the C terminus of UBE2S with a ubiquitin-binding domain (UBD), namely the zinc finger (ZnF) UBP domain of USP5/IsoT (11). This increases the local concentration of free ubiquitin near the active site and enhances formation of free K11-linked chains. The advantage of the ZnF UBP domain is that it binds ubiquitin with nanomolar affinity and that it interacts with the free C termi-nus of ubiquitin, leaving lysine 11, and the TEK box (6) accessible for chain elongation. This engineered UBE2S-UBD fusion protein generates free K11-linked ubiquitin dimers more efficiently than the wt enzyme and also allows the purification of trimers and tetramers in vitro. However, UBE2S-UBD is less specific and incor-porates also K63-linkages. Formation of contaminating K63-linkages can be counteracted by including the K63-specific deubiquitinase (DUB) AMSH in the assembly reactions. This method allows specific generation and purification of K11-linked ubiquitin chains. Taken together, in addition to UBE2R1 (Cdc34) and UBE2N/UBE2V1 (Ubc13/UEV-1), which specifically assemble K48-linked and K63-linked ubiquitin chains (5), respectively, an enzymatic system is now available for the generation of K11-linked ubiquitin chains in vitro.

Prepare all buffers using prechilled ultrapure water, for example Milli-Q water.

1. Plasmids: pGEX6P1-UBE2S to generate pGEX6P1-UBE2S-UBD (see Subheading 3.1), pGEX6P1-AMSH, and pRSETB-IsoT (12).

2. Materials

2.1. Affinity Purification of GST-Fusion Proteins

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2. Competent cells for plasmid DNA preparation (for example, OneShot® Mach1™-T1® cells, Invitrogen) and protein expression (Rosetta™ 2(DE3)pLacI cells, Novagen).

3. LB medium and selective plates containing 100 mg/ml ampicillin.

4. 1 M IPTG stock solution. Dissolve IPTG in H2O, sterile filter, and store aliquots at −20°C.

5. Lysis buffer: 270 mM sucrose, 50 mM Tris–HCl (pH 7.5), 50 mM NaF. Sterile filter and store at 4°C. Before usage add 0.1% v/v b-mercaptoethanol, protease inhibitor cocktail (for example, Complete, Mini, EDTA-free tablets, Roche), 1 mg/ml lysozyme and 0.1 mg/ml DNase I.

6. Glass chromatography columns (Bio-Rad). 7. Glutathione Sepharose™ 4B (GE Healthcare). Store at 4°C. 8. High-salt buffer: 500 mM NaCl, 25 mM Tris–HCl (pH 7.5),

5 mM DTT (see Note 1). 9. Low-salt buffer: 150 mM NaCl, 25 mM Tris–HCl (pH 7.5),

5 mM DTT. 10. PreScission™ protease (GE Healthcare). Store at −20°C (see

Note 2).

1. Ubiquitin storage buffer: 50 mM Tris–HCl (pH 7.5). 2. Ubiquitin from bovine red blood cells (Sigma-Aldrich). Dissolve

ubiquitin in ubiquitin storage buffer and store at −20°C. 3. Recombinant E1 enzyme can be purchased from Ubiquigent,

ENZO Life Sciences or Boston Biochem (see Note 3). 4. 0.1 M ATP stock solution: Dissolve ATP in H2O and adjust

pH to 7.0 with NaOH. Store at −20°C. 5. 10× ligation buffer: 400 mM Tris–HCl (pH 7.5), 100 mM

MgCl2, 6 mM DTT. Store at −20°C. 6. Recombinant UBE2S-UBD and AMSH (see Subheading 3.2). 7. 1 M DTT stock solution. Dissolve DTT in H2O and store

small aliquots at −20°C.

1. Fast protein liquid chromatography (FPLC) system: For exam-ple, the ÄKTApurifier™ system including a Mono S™ 5/50 cation exchange column and a 50-ml superloop (GE Healthcare).

2. Buffer A: 50 mM NH4Ac (pH 4.5). 3. Buffer B: 50 mM NH4Ac (pH 4.5), 1 M NaCl. Adjust pH of

buffer A and B with HCl.

2.2. Large-Scale K11-Linked Ubiquitin Chain Assembly Reaction

2.3. Fast Protein Liquid Chromatography

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Human UBE2S (NM_014501.2) was cloned into the BamHI/NotI restriction sites present in pGEX6P1. The UBE2S sequence has a naturally occurring unique NcoI restriction site just before the lysine-rich tail (residue 196).

1. Digest the pGEX6P1-UBE2S expression plasmid with NcoI and NotI (restriction enzymes for example from New England Biolabs Inc.). Incubate at 37°C for 2 h and purify cleaved plasmid DNA by gel extraction. This releases the lysine-rich C-terminal tail of UBE2S.

2. Amplify the USP5/IsoT ZnF UBP domain (residues 173–289) from cDNA or from pRSETB-IsoT by PCR. Use primers that generate a 5¢ NcoI site and a 3¢ NotI site in the PCR product so that it can be ligated into the NcoI/NotI restriction sites present in pGEX6P1-UBE2S (see Note 4). Purify the PCR product by gel extraction. Elute DNA in 30 ml H2O.

3. Digest the purified PCR product with NcoI and NotI. Incubate at 37°C for 2 h and purify the cleaved DNA by gel extraction.

4. Ligate the cleaved PCR product into linearized pGEX6P1-UBE2S by using for example a Rapid DNA Ligation kit (Roche).

5. Use the ligation reaction mixture for the transformation of competent Mach1 cells. Spread cells on selective ampicillin plates and grow overnight at 37°C.

6. Pick single bacterial colonies and grow cells in 2 ml LB/ampi-cillin medium.

7. Purify plasmid DNA (for example, QIAprep Spin Miniprep Kit, QIAGEN) and verify clones by sequencing.

See Note 5 for additional information.

1. Transform Rosetta cells (see Note 6) with pGEX6P1-UBE2S-UBD or pGEX6P1-AMSH.

2. For each construct, pick a single colony from a selective ampicillin plate to inoculate 50 ml LB/ampicillin medium. Grow cultures at 37°C, 220 rpm, and overnight.

3. Inoculate 1 l LB medium in a 2-l flask with 10 ml overnight culture and grow cultures at 37°C, 220 rpm until OD600 of 0.6 is reached. Take a 5 ml aliquot of the noninduced culture (sample NI) and grow separately.

4. Induce expression of recombinant proteins with 250 mM IPTG and grow cultures at 20°C, 220 rpm, overnight. Take a 5 ml aliquot (IND) and analyse it together with 5 ml of sample NI by SDS-PAGE and Coomassie staining to check induced expression of the recombinant protein (see Fig. 1a).

3. Methods

3.1. Cloning of UBE2S-UBD Fusion Construct

3.2. Expression and Affinity Purification of GST-Fusion Proteins

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5. Harvest cells by centrifugation, for example in a Beckman JLA-8.1000 fixed-angle rotor at 5,000 × g and 4°C for 15 min. Transfer the pellet into a 50-ml Falcon tube and flash-freeze it in liquid nitrogen.

6. Thaw bacterial pellet and lyse cells by resuspending the pellet in 30 ml lysis buffer per litre of culture. Incubate 10 min on ice.

7. Sonicate bacterial cells to support lysis. Keep samples on ice during sonication to avoid heating of the lysate.

8. Clear the cell lysate by centrifugation, for example in a Beckman JA-25.50 fixed-angle rotor at 25,000 × g and 4°C for 30 min. Take samples of the supernatant (SN) and the pellet (P) to check if the expressed protein is soluble and detectable in the supernatant.

9. Transfer 1 ml Glutathione Sepharose 4B (80% slurry in ethanol) per litre of culture into a class chromatography column. Remove the bottom cap and allow the column to drain. Wash the beads with two bed volumes of H2O to remove the 20% ethanol storage solution and equilibrate with two bed volumes of lysis buffer. Allow the column to drain and replace the bottom cap.

10. Filter the supernatant through a 0.45-mm syringe filter and transfer it into the column containing the equilibrated Glutathione Sepharose 4B. Close the top cap of the column and incubate with agitation at 4°C for 1 h to immobilize soluble GST-fusion proteins.

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11. Remove the top and bottom cap of the column and elute unbound proteins. Take a sample of the flow-through (SN/B).

12. Wash the Glutathione Sepharose 4B with 1 l high-salt buffer and subsequently with 500 ml low-salt buffer to remove non-specifically bound proteins. Allow the column to drain and replace the bottom cap of the column. Take a sample of the beads (B). Samples SN/B and B show if the GST-fusion protein is bound to the Glutathione Sepharose 4B (see Note 7).

13. To cleave the GST tag of the immobilized proteins add 30 mM PreScission protease in 5 ml low-salt buffer to the column, close the top cap, and incubate with agitation at 4°C overnight.

14. Elute cleaved proteins into a fresh tube. This fraction contains most of the cleaved protein (~70%). Wash the beads with 5 ml low-salt buffer to elute the remaining proteins. Repeat this step until all cleaved protein is eluted. This can be monitored by Bradford protein assay. Take an aliquot of each fraction (EL1, EL2, etc.) and analyse them together with samples SN, P, SN/B, and B by SDS-PAGE and Coomassie staining (see Fig. 1b).

15. Pool protein-containing fractions and concentrate them (see Note 8) using for example Vivaspin 15 centrifugal concentrators (Sartorius Stedim Biotech) with a molecular weight cut-off (MWCO) of 10,000 Da (for UBE2S-UBD) or 30,000 Da (for AMSH). Flash-freeze proteins in liquid nitrogen and store them at −80°C (see Note 9).

1. Thaw all enzymes and solutions on ice. 2. Set up a 1 ml reaction: Mix 250 nM E1 enzyme (see Note 3),

4.8 mM UBE2S-UBD, 2.94 mM ubiquitin, 400 nM AMSH, 10 mM ATP, and 10× ligation buffer (400 mM Tris–HCl (pH 7.5), 100 mM MgCl2, 6 mM DTT), and incubate at 37°C for 3 h (see Table 1).

3. After 1 h add another 400 nM AMSH to the reaction to coun-teract the formation of K63-linked ubiquitin chains. Repeat this step after 2 h.

4. Stop the reaction after 3 h by adding 50 mM DTT. Incubate for 10 min on ice. This releases any ubiquitin (monomers or polymers) covalently bound to the active site cysteine of UBE2S-UBD or the E1 enzyme.

5. Subsequently, dilute the reaction with 14 ml buffer A (50 mM NH4Ac [pH 4.5]) and incubate for at least 30 min on ice to precipitate the E1 enzyme and UBE2S-UBD (see Note 10).

6. Filter the diluted assembly reaction through a 0.2-mm syringe filter to remove precipitated enzymes.

3.3. Large-Scale Assembly Reaction

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1. Load the filtrate into a 50-ml superloop. 2. Purify K11-linked ubiquitin chains by cation exchange chroma-

tography to separate the chains by length. Equilibrate a Mono S™ column (GE Healthcare) with buffer A, load the diluted assembly reaction, and elute bound ubiquitin chains by running a gradient of buffer B (50 mM NH4Ac [pH 4.5], 1 M NaCl) from 0 to 50% over 96 column volumes. Collect 1 ml fractions and monitor protein elution by measuring the absorbance of the fractions at 280 nm (Fig. 2a, see Note 11).

3.4. Ubiquitin Chain Purification by FPLC

Table 1K11 chain assembly reaction

Final concentration Stock (e.g.) 1 ml reaction

E1 enzyme 250 nM 10 mM 25 ml

UBE2S-UBD 4.8 mM 480 mM 10 ml

Ubiquitin 2.94 mM 5.88 mM 500 ml

AMSH 400 nM 20 mM 20 ml

ATP 10 mM 0.1 M 100 ml

10× ligation buffer 1× 10× 100 ml

H2O 245 ml

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3. Analyse 5 ml of the peak fractions by SDS-PAGE and Coomassie staining (Fig. 2b, see Notes 12 and 13).

4. Pool fractions by chain length and concentrate them to >5 mg/ml if possible (see Note 11). Use for example a Vivaspin 2 (MWCO: 5,000 Da) centrifugal concentrator (Sartorius Stedim Biotech). Either dialyse purified ubiquitin chains against ubiquitin storage buffer (50 mM Tris–HCl [pH 7.5]) at 4°C overnight, or exchange buffer while concentrating the samples (i.e., repeatedly dilute the concentrated sample with ubiquitin storage buffer and concentrate again, see Notes 14 and 15). Flash-freeze the ubiquitin chains in liquid nitrogen and store them at −20°C.

1. Prepare high- and low-salt buffer freshly each time and add DTT to protect free SH-groups from oxidation during the iso-lation of proteins. The pH was adjusted by taking the theo-retical isoelectrical point of AMSH and UBE2S-UBD into account.

2. PreScission™ protease (GE Healthcare) is used for low-temperature, site-specific separation of the GST tag from proteins expressed using pGEX6P vectors. Since PreScission™ protease contains a noncleavable GST-tag, it is retained on the Glutathione Sepharose 4B resin and only the cleaved protein elutes from the column.

3. We purified recombinant human E1 enzyme using a baculovirus expression system (13). The concentration of E1 enzyme in the K11-chain assembly reaction can be reduced if commercial enzyme is being used.

4. Primer sequence for USP5/IsoT ZnF UBP domain amplification:

UBP-FW 5-CCAAGGTTCCATGGTACGGCAGGTGTCT AAGCATGCC-3;

UBP-RV 5-GCCTAGCGGCCGCTTATGTCTTCTGCAT CT-TCAGCATGTCGATG-3.

5. Different UBDs were fused to UBE2S, but neither the ubiq-uitin-associated (UBA) domain of Cezanne (an OTU-family deubiquitinase) nor the first ubiquitin-interacting motif (UIM) of the 26S proteasome regulatory subunit S5A improved free K11-linked ubiquitin chain assembly by UBE2S.

6. GST-fusion proteins were expressed in Rosetta 2 (DE3) pLacI cells from Novagen. These cells are BL21 derivates and carry a

4. Notes

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plasmid with tRNA genes that decode seven rare codons. The additional tRNAs can significantly improve the yield of recom-binant human proteins.

7. To instantly check if the GST-fusion protein bound to the Glutathione Sepharose 4B, a small aliquot (1 ml) of resin can be mixed with 1 ml Bradford protein assay reagent.

8. Concentration of GST-fusion proteins is important since the 5 mM DTT in the low-salt buffer can interfere with the ubiquitin chain assembly reaction. Purified UBE2S variants and AMSH are stable at high concentrations such as ~10 mg/ml, (295 mM UBE2S-UBD and 200 mM AMSH). From 1 l cultures roughly 5–15 mg protein can be purified.

9. Freshly purified UBE2S-UBD can be tested in small-scale autoubiquitination reactions using the set of ubiquitin K-only mutants (10). It is also advisable to test the large-scale assem-bly reaction at 20-fold smaller scale, which should result in visible bands of di- and triubiquitin in Coomassie stained SDS-PAGE gels.

10. Dilution of the reaction with buffer A drops the pH to 4.5 and causes precipitation of all proteins except mono- and polyubiquitin.

11. The absorbance of ubiquitin at 280 nm is very low since the molecule contains only one tyrosine residue and no tryptophan or cysteine residues. An extinction coefficient of 0.16 should be used to estimate ubiquitin concentration from OD280 measurements, e.g. using a Nanodrop spectrophotometer.

12. We experienced that the separation of K11-linked ubiquitin chains by cation-exchange chromatography was not always complete and that for example ubiquitin monomers and dim-ers eluted together from the Mono S™ column in the same fractions (see Fig. 2b). In this case, all fractions containing dimers were pooled, concentrated, diluted in buffer A to reduce salt concentration, and repurified by cation-exchange chromatography.

13. Analysing the eluted peak fractions by SDS-PAGE and Coomassie staining reveals the quality of the assembled K11-linked ubiquitin chains. Double bands for tri- and tetraubiq-uitin indicate contaminating K63 linkages as differently linked ubiquitin chains show distinct electrophoretic mobility (10).

14. Ubiquitin chains generated by the UBE2S-UBD fusion protein can be verified by tryptic digest and LC-MS/MS to verify the K11 linkage (10).

15. To obtain larger amounts of K11-linked tetramers, it is possible to use the purified dimers as input material in a second chain assembly reaction.

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16. K11-linked ubiquitin chains can be synthesised from mutant K63R ubiquitin. This avoids the generation of contaminating K63 linkages by UBE2S-UBD without including AMSH in the assembly reaction.

References

1. Xu P, et al (2009) Quantitative proteomics reveals the function of unconventional ubiq-uitin chains in proteasomal degradation. Cell 137:133–145

2. Chen ZJ, Sun LJ (2009) Nonproteolytic func-tions of ubiquitin in cell signaling. Mol Cell 33: 275–286

3. Hershko A, Ciechanover A (1998) The ubiq-uitin system. Annu Rev Biochem 67:425–479

4. Dye BT Schulman BA (2007) Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu Rev Biophys Biomol Struct 36: 131–150.

5. Ye Y, Rape M (2009) Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol 10:755–764

6. Jin L, et al (2008) Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133:653–665

7. Dynek JN, et al (2010) c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. EMBO J 29(24): 4198–209

8. Williamson A, et al (2009) Identification of a physiological E2 module for the human ana-phase-promoting complex. Proc Natl Acad Sci U S A 106:18213–18218

9. Garnett MJ, et al (2009) UBE2S elongates ubiq-uitin chains on APC/C substrates to promote mitotic exit. Nat Cell Biol 11: 1363–1369

10. Bremm A, Freund SM, Komander D (2010) Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydro-lyzed by the deubiquitinase Cezanne. Nat Struct Mol Biol 17:939–947

11. Reyes-Turcu FE, et al (2006) The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiq-uitin. Cell 124: 1197–1208

12. Reyes-Turcu FE, et al (2008) Recognition of polyubiquitin isoforms by the multiple ubiq-uitin binding modules of isopeptidase T. J Biol Chem 283:19581–19592

13. Komander D, et al (2008) The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Mol Cell 29:451–464

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Documents

Contents · Contents Preface..... v Contributors ... Stefan R. Weisshaar, Gerrit J.K. Praefcke, and R. Jürgen Dohmen ... Christof Taxis and Michael Knop 44 PROTAC-Induced Proteolytic