Table Of ContentMethods in
Molecular Biology 2602
Manuel S. Rodriguez
Rosa Barrio Editors
The Ubiquitin
Code
M M B
ETHODS IN OLECULAR IO LO GY
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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
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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 mem-
bers 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 spec-
trometry 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
