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Methods in Molecular Biology 2602 Manuel S. Rodriguez Rosa Barrio Editors The Ubiquitin Code M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. The Ubiquitin Code Edited by Manuel S. Rodriguez LCC-UPR 8241-CNRS Equipe M, Laboratoire de Chimie de Coordination, Toulouse, France Rosa Barrio CIC bioGUNE, Derio, Spain Editors ManuelS.Rodriguez RosaBarrio LCC-UPR8241-CNRS CICbioGUNE EquipeM Derio,Spain LaboratoiredeChimiedeCoordination Toulouse,France ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2858-4 ISBN978-1-0716-2859-1 (eBook) https://doi.org/10.1007/978-1-0716-2859-1 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. 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Preface Protein post-translational modification (PTM) by members of the ubiquitin family, also known as ubiquitin-like (UbL) proteins, efficiently contributes to regulate most biological processes.Defectsinthesemodificationsareattheoriginofdiversehumandiseasesinclud- ingcancer,neurodegeneration,inflammationor multipleinfections,amongmanyothers. Ubiquitylation is a multi-step process implicating multiple enzymes and cofactors that act on unmodified or partially modified substrates. The regulation of these modifications takes place first at the level of the ubiquitin activating (E1), conjugating (E2) or ligase (E3) enzymes. These enzymes mediate a thiol-ester cascade of reactions ending by cova- lently attaching a ubiquitin moiety on a target protein or on ubiquitin itself. Once the substrate is modified, the reverse reaction is mediated by de-conjugating enzymes that determinetheconnectionsthatwillbeestablishedwithproteinsthatrecognisethesechains throughreceptorregions.Inthisway,alargediversityofubiquitinchainscanconnectwith multiple effector functions. All these enzymes, cofactors, receptors and members of the ubiquitinfamilyareregulatedbydistinctPTMsthatintegratesignallingpathwaysdetermin- ingubiquitinchainarchitecture,localisationoractivityofthemodifiedtargets. Exploringabiologicallanguageisalwayschallengingsincemultiplelayersofcomplexity aresuperposedtoguarantyplasticityandabettercontrolofcrucialcellularevents.Unravel- lingthiscomplexityimposestheuseofmultipleapproachesincludingbiochemical,chemis- try, structural, biophysical, cellular and molecular techniques to uncover its meaning. To understandtheimpactofubiquitinconjugationtoproteintargetsanditsrelationshipwith otherPTMs,amajorbottleneckhasalwaysbeenthelackoftechnologythatwillhelpusto understand/decode these messages. In this book, we include some of the most novel and popular approaches to decipher the so-called Ubiquitin Code that was developed by members of many laboratories, including those integrating the UbiCODE network and colla- borators (http://ubicode.eu). Each of the protocols and model systems presented in this bookexploresfrontierquestionsthatshouldcontributetoobtaininsightsondistinctlayers ofcomplexityofthisintriguingandversatilecode. Our book starts with a couple of reviews that illustrate part of the ubiquitin chain complexityandthebiochemistrybehinditandalsorecapitulatesomeofthecurrentlyused methods used to explore these chains. Ferri-Blazquez recapitulates the ester-mediated cascade mediating the covalent conjugation of ubiquitin on target proteins (Chap. 1). While the acceptor residue is in most cases a lysine or the amino-terminal methionine in the client protein, ubiquitin can also be efficiently attached to cysteine, serine, and threo- nine. Conjugation on these less frequent residues was initially observed in a virus-encoded ubiquitinligase,whichtargetsacysteineresidueinahostproteintoinitiateitsdegradation. Theseubiquitylationeventsexpandthecomplexityanddiversityofubiquitinsignallingand broaden the capability of cellular messages in the Ubiquitin Code. Still, questions on the prevalence, relevance, and involvement in physiological and cellular functioning remain open. Anita Waltho et al (Chap. 2) revise the current methods to explore the 28 different possible ubiquitinbranches.Dedicatedmethodologiestoexploremorethantwoubiquitin- linkage types and highly complex chains composed by distinct branched ubiquitin chains generatingcomplexstructures,aretobeestablishedinthenear future. v vi Preface Some of the most original tools to explore the meaning of chain complexity are those generated by chemical synthesis. Non hydrolysable ubiquitin-UbL hybrid chains allow the possibilitytoidentifyreceptorproteinsandregulatorsofthosechains,amongotheraspects. Perez-Berrocaldescribesamethodtogeneratenon-hydrolysableubiquitin-NEDD8(Neu- ral Precursor Cell Expressed, Developmentally Down-Regulated 8)-based chains in all possible combinations of K48 hybrid chain dimers (Chap. 3). In addition, Huppelschoten et al (Chap. 4) describe the first fully synthetic method for the linear synthesis of fluores- centlylabelledrhodamineLC3AandLC3B.IncontrasttootherUbLproteins,LC3/GA- BARAP proteins are conjugated to lipids and are involved in autophagy-lysosome proteolysis. Theregulationofubiquitinconjugatingenzymesoftenrepresentsthefirstleveltosense thedistinctstimulireceivedbythecell.Therefore,measuringtheiractivityallowstoidentify thefirstcrucialsensorsofubiquitylation.Recasens-Zorzoetalreporttheuseofmicrobead- basedflowcytometryassaytoevaluatetheactivityofUbL-conjugatingenzymespurifiedor present in cell extracts (Chap. 5). The assay assesses the transfer of the UbL onto target substrates immobilised on colour-coded beads under physiological or pathological situa- tions. Monitoring E3 ligases is often the strategy chosen by many scientists since these enzymes greatly contribute to provide specificity to this system. Taibi et al develop a protocol to monitor HECT-mediated ubiquitylation in vitro (Chap. 6). Ubiquitylation is monitored in real time using the Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) system. This single step technique represents an excellent tool to study the enzymatic activity during chain elongation or to set up high throughput screenings for enzymaticactivityactivators/inhibitors,amongotherapplications. DUBs represent another important level of regulation that is frequently preferred to identify targets, monitor or control ubiquitylation levels. Elu et al present in this book an RNAi-basedscreeningtoidentifyDUBssubstratesbyspecifictargets’pull-downandimage analysis of the ubiquitylated fraction (Chap. 7). The possibilities to monitor ubiquitin and UbL hydrolases is diverse, and multiple methodologies have been implemented. Saad et al presenthereaprotocoltomonitorYuh1activity(Chap.8).Yuh1(AKAUCHL3)isahighly conserved ubiquitin C-terminal hydrolase regulating ubiquitin and NEDD8 (Rub1). The methodallowstoevaluateYuh1activityexpressedinbacteriaor yeastbytrimmingofUBB +1andRub1+1throughimmunoblotting,andfluorescencereadout. SinceubiquitylationandUbLconjugationregulatealmostallcellularprocesses,notall functions and response to distinct stimuli have been explored. We are still at the stage of identifyingtargetproteinsandregulatorsimplicatedinverydiversefunctionsandsituations. Six protocols describe distinct approaches based on unique tools associated to mass spectrometry identification. Gonzalez-Santamarta reports the use of ubiquitin K48 and K63 chain specific nanobodies to isolate endogenous chains from mammalian cells (Chap. 9). This protocol has been optimised to preserve ubiquitylated proteins until the mass spec- trometryanalysisstep. ThecomplexitytostudyproteinNEDDylationresidesnotonlyinthefactthatNEDD8 andubiquitinshareahighhomologybutalsointhedi-glycinesignaturethatbothmolecules leave in the modified targets. Oliveira et al report a protocol to map modification sites for NEDD8usingcelllinesthatstablyexpresstheNEDD8R74Kmutant(Chap.10).Digestion ofsampleswithLysylendopeptidasegeneratespeptideswithadi-glycineremnantonlyfrom proteins modified with NEDD8R74K but not with ubiquitin or ISG15. Cardoso Delgado etalusebiotintaggedversionsofNEDD8togeneratemammaliangeneticmodelstostudy in vivo liver diseases such as non-alcoholic fatty liver and fibrosis (Chap. 11). The biotin- Preface vii avidin interaction, which can undergo denaturing lysis conditions and stringent washes, ensures that only proteins conjugated to ubiquitin or NEDD8 are isolated. The NEDDy- lomeandubiquitomeisolatedunderthesameconditionscanthenbecompared.Despitethe accumulated evidence on the specific roles of NEDD8 we still need to better understand how this UbL molecule connects with effector molecules. This knowledge could be expanded by the identification of NEDD8 binding domains present in cofactors and key molecules regulating protein NEDDylation. Santonico reports here a protocol to identify bindingdomainsforubiquitinorNEDD8(Chap.12).Usually,bindingdomainsrecognise definedsurfacepatchesthroughtypicallyweakinteractions.Thereportedprotocolidentifies ubiquitin and NEDD8-binding domains by panning a human brain cDNA library whose products are displayed on the surface of lambda capsid. This approach proved to be very effectivefor thediscoveryofthepreviouslyunidentifiedubiquitinbindingdomains. A crucial step for our better understanding of ubiquitin-regulated functions is the way these chains and/or modified targets connect with the effector functions and how these processes are regulated. Barroso-Gomila et al developed a UbL-ID method to identify proximalinteractors(Chap.13).Theprotocolreportedherespecificallyfocussesonprotein SUMOylation but there is a huge potential of this protocol to be used to study proteins interacting with other members of the ubiquitin family. For SUMO, non-covalent interac- tions are mediated by SUMO Interacting Motifs (SIMs) found in some proteins. This technology merges promiscuous proximity-biotinylation by TurboID enzyme and protein-fragment complementation strategy to specifically biotinylate SUMO-dependent interactorsofparticularsubstrates. One last methodology to identify processes regulated by the UbL proteins LC3/GA- BARAP is reported by Quinet et al. The protocol is based on the use of LC3-Interacting Regions (LIR)-disposed in tandem to develop a capturing system to isolate endogenous ATG8 proteins and their interactors in order to facilitate the study of selective autophagy events (Chap. 14). This protocol was optimised to prepare affinity columns, reduce back- ground and improve the protein recovery to be analysed by immunodetection with anti- bodies recognising proteins of interest. The protocol can be easily adapted to identify cellular factorswhenassociatedtomassspectrometryanalysis. Theidentificationofcellularfactorsundermultiplephysiologicandpathologicsituation requires important computational data analysis to better integrate and identify crucial cellular events regulating vital processes. Matthiesen et al report a computational tool to facilitatemassspectrometryanalysisofubiquitin-enrichedsamplesincludingtheidentifica- tionofrelevantubiquitinbranches,sequencelogosandfunctionalenrichmentofubiquitin proteomesystemproteinsets(Chap.15). Oneofthemajor functionsregulatedbyubiquitylationisproteasome-mediateddegra- dation.Evenifthisfunctionwasreportedmorethan30yearsago,stilltherearemanyopen questions, as proteasome are highly dynamic complexes with distinct composition and distributed in most cellular compartments or associated to organelles. The function and regulation of some of these complexes are still in intensive investigation as they represent possibletargetsfortherapeuticintervention.Giventhecomplexityofthesemacromolecular entities, model organisms are often used. Panagiotidou et al developed a protocol to measure in plate and in gel proteasome activity in Caenorhabditis elegans (Chap. 16). The “in-plateactivity”providesaquantitativemeasurementofproteasomeactivitiesfromlysates bymeansofakineticreactionina96-wellplate.The“in-gelactivity”involvestheseparation lysatesinanativepolyacrylamidegelandtheassessment oftheactivityofeachproteasome viii Preface form.Downstreamimmunoblottingalsoallowsthesemi-quantitativeassessmentofprotea- someassembly. Analysing intracellular peptides generated by proteasomes is highly informative to understand the spatio-temporal regulation of protein homeostasis. Understanding the rules that govern proteolytic specificity and product diversity is of relevance not only to biochemistryandproteostasisbutalsotophysiologyandimmunology.Sahuetal(Chap.17) describedarapidmethodtoisolatepeptidesthatarecloselyassociatedwithproteasomesor trapped inside the core particle of proteasomes in eukaryotic cells. This approach, termed PTPs for proteasome-trapped peptides, requires a limited number of cells as starting materials compared to other publishedmethods,yet still providessufficient yields for mass spectrometry-basedproteomicanalysis. Altogether these protocols should contribute to expand our knowledge to better understand cellular plasticity and in particular the role of the ubiquitin family members in thecontrolofkeycellularevents.TheUbiquitinCodeasanewbiologicallanguagewilloffer newpossibilitiestobetterstratifyanddiagnosediseasesanddevelopadaptedtreatmentsfor diseasesthatwestillneedtounderstand. We acknowledge the COST Action CA20113 “ProteoCure” supported by COST (EuropeanCooperationinScienceandTechnology)andthefundingreceivedbythegrants 765445-EU(UbiCODEProgram)andSAF2017-90900-REDT(UBIRedProgram). Toulouse,France ManuelS.Rodriguez Derio,Spain RosaBarrio Contents Preface ..................................................................... v Contributors................................................................. xi PART I CHAIN DIVERSITY 1 ThioesterandOxyesterLinkagesintheUbiquitinSystem..... ....... ........ 3 AlbaFerri-Blazquez,ErnstJarosch,andThomasSommer 2 GettingtotheRootofBranchedUbiquitinChains:AReview ofCurrentMethodsandFunctions ......... ....... ........ ....... ........ 19 AnitaWalthoandThomasSommer PART II UB AND UBL CHEMICAL TOOLS 3 ChemicalSynthesisofNon-hydrolyzableUbiquitin (-Like)HybridChains...... ........ ....... ....... ........ ....... ........ 41 DavidA.Pe´rezBerrocal,GerbrandJ.vanderHedenvanNoort, andMoniqueP.C.Mulder 4 TotalLinearChemicalSynthesisofLC3AandLC3B....... .. ...... ......... 51 YaraHuppelschoten,JensBuchardt,ThomasEilandNielsen, andGerbrandJ.vanderHedenvanNoort PART III METHODS TO STUDY UBIQUITIN CONJUGATING ENZYMES (E3S) 5 AMicrobead-BasedFlowCytometryAssaytoAssesstheActivity ofUbiquitinandUbiquitin-LikeConjugatingEnzymes ...... ....... ........ 65 ClaraRecasens-Zorzo,PierreGaˆtel,Fre´de´riqueBrockly, andGuillaumeBossis 6 MonitoringHECTUbiquitinationActivityInVitro ......... ....... ........ 81 VincenzoTaibi,SimonaPolo,andElenaMaspero PART IV METHODS TO STUDY DUBS 7 RNAi-BasedScreeningfor theIdentificationofSpecific Substrate-DeubiquitinasePairs ...... ....... ....... ........ ....... ........ 95 NagoreElu,NataliaPresa,andUgoMayor 8 StrategiesforMonitoring“UbiquitinC-TerminalHydrolase1” (Yuh1)Activity ..... ....... ........ ....... ....... ........ ....... ........ 107 ShahafSaad,EdenBerda,YuvalKlein,SuhaIssa,andElahPick ix