TheUbiquitinProteasomeSystem
MethodsandProtocols
Editedby
ThibaultMayor
DepartmentofBiochemistryandMolecularBiology,MichaelSmithLaboratories, UniversityofBritishColumbia,Vancouver,BC,Canada
GaryKleiger
DepartmentofChemistryandBiochemistry,UniversityofNevadaLasVegas,LasVegas,NV,USA
Editors
ThibaultMayor DepartmentofBiochemistry andMolecularBiology
MichaelSmithLaboratories
UniversityofBritishColumbia Vancouver,BC,Canada
GaryKleiger DepartmentofChemistry andBiochemistry
UniversityofNevadaLasVegas LasVegas,NV,USA
ISSN1064-3745ISSN1940-6029(electronic)
MethodsinMolecularBiology
ISBN978-1-4939-8705-4ISBN978-1-4939-8706-1(eBook) https://doi.org/10.1007/978-1-4939-8706-1
LibraryofCongressControlNumber:2018952458
© SpringerScience+BusinessMedia,LLC,partofSpringerNature2018
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Preface
In1975,apaperwaspublishedinthe ProceedingsoftheNationalAcademyofSciences describinganewlydiscoveredpolypeptidecapableofinducingthedifferentiationof Tcells[1].Theauthorsnamedthisproteinubiquitousimmunopoieticpolypeptide (UBIP)“becauseitiswidespreadandperhapsevenuniversallyrepresentedinliving cells... .”Itsoonbecameclearthatthispolypeptide,laterrenamedubiquitin,isakey signalingmoleculeineukaryoticcellbiology.Indeed,approximately5%ofhumangenes arededicatedtowardstheexpressionofenzymesandfactorsthatcontroltheubiquitin proteasomesystem(UPS),amassivenetworkofintegratedpathwaysresponsibleforregulatingproteindegradation,proteinlocalization,andenzymeactivity.Thepioneering researchofAaronCiechanover,AvramHershko,IrwinRose,andAlexVarshavskyhasled toafieldthatcontinuestomakenewandimportantdiscoveriesregardinghowcells function.Greaterthan54,000articleshavebeenindexedintheUSNationalLibraryof MedicinePubMeddatabasethatcontaintheword“ubiquitin”ineitherthetitleorabstract (Fig. 1).TheUPShasgiftedbothbiotechandpharmaceuticalcompanieswithfresh,new targetsforthetreatmentofvarioushumandiseases.Somefivedrugshavebeenapprovedin theUnitedStatesbytheFederalDrugAdministrationthattargetenzymesintheUPS,with thepromiseofmanymoreinthenottoodistantfuture.Theubiquitinfieldcontinuesto growandchangeatadizzyingpacewithsignificantadvancesforbothnewermethodologies—suchasmassspectrometry-basedproteomicsandcryo-electronmicroscopy—andthe moretraditionalbiochemical,cellbiological,andstructuralanalysesandassays.Thisvolume isdesignedforbothveteransinthefieldwantingtoexpandtheirbagoftricksandfornovices justgettingstarted.
Themethodshavebeenorganizedinfiveparts.Inthefirstpart,fivemethodsare describedthatcanbeappliedtothedeterminationofthemechanismsofactionfor ubiquitin-conjugatingenzymes(E2s),ubiquitinligases(E3s),andde-ubiquitylating
(DUB)enzymes.Thereareapproximately35E2s,andperhapsasmanyas600E3sand 90DUBsinthehumanproteome,withthevastmajorityawaitingin-depthcharacterization. ThesechapterswillbehelpfulforanyresearcheraimingtoreconstituteinvitroubiquitylationasameansofuncoveringE2and/orE3function,ortoinfertheaffinityofaDUBforits substrate.
Inthesecondpartofthisvolume,sixmethodsaredescribedfornewtechnological advancesthatwillenabletheresearcherto(1)assembleandpurifypoly-ubiquitinchainsthat areuniformwithrespecttolinkagetypeinquantitiessufficientforbiochemicalandstructuralanalyses;(2)discoverthebindingpartnersofessentiallyanyproteinintheUPS; (3)screenforubiquitinvariantswithuniquepropertiesthatcanmodulateenzymesinthe UPSwithinthecontextoflivingcells;(4)discovertheuniqueaminoacidsequence signaturesthattargetproteinstoberecognizedanddegradedbytheUPS;(5)useflow cytometrytowardsidentifyingtheUPSenzymesresponsibleforproteinturnoverinahighthroughputmanner;and(6)identifyandcharacterizeubiquitylationpathwaysusingthe easytoworkwithmodelorganism E.coli that,lackingtheUPS,optimizestheresearcher’s chancesforsuccess.
Inthethirdpart,cutting-edgemethodsaredescribedforthreeareasinstructural biology:X-raycrystallography,small-angleX-rayscattering(SAXS),andcryo-electron microscopy.Methodstotrapdynamicmulti-subunitcomplexesforstructuralcharacterizationusingproteincross-linkingaredescribed(whilethismethodisspecifictotheubiquitinlikeproteinSUMO,thesimilaritiesbetweentheubiquitinandSUMOcascadesaresogreat thatthesemethodswillundoubtedlyproveusefulformembersoftheubiquitinfield).Itis oftendesirabletocharacterizethestructuresofUPSenzymesinsolution,andSAXSisa powerfulandrelativelysimplemethoddescribedheretoachievethisgoal.Whilecryoelectronmicroscopyhasenabledthedeterminationofthestructuresoflarge,multi-subunit complexestonearoratatomicresolution,onechallengehasbeenthepreparationofthe gridswheremacromoleculesareplacedpriortofreezing.Adetailedprotocolforthis procedurehasbeenincluded.
Inthefourthpart,sevenchaptersarededicatedtomethodologiestostudythe26S proteasome,themulti-subunitcomplexthatrecognizesanddegradesubiquitylatedproteins.Thesechapterscover(1)theexpressionandpurificationofthe19Sproteasome regulatorycomplexin E.coli,aswellastheabilitytointroducechemicalmodificationsto surfaceresiduesonanygivensubunit;(2)theuseofnativegelelectrophoresistoseparate andstudytheproteasomeduringvariousstagesofassembly;(3)theuseofisotopicpulsechasetodirectlymeasuretheratesofproteindegradationwithinlivingcells;(4)three uniqueandcomplementarymethodstopurifyproteasomesfromcellseithergrownin isolationorfromintacttissues;(5)severalquantitative invitro methodsforassayingthe sequentialactivitiesofthe26Sproteasome;(6)methodstoassessthefunctionalimpactof proteasomephosphorylation;and(7)novelproteasomesubstratesthatenablethemeasurementofproteindegradationinahigh-throughputmanner.
Thefifthpartisdedicatedtocutting-edgeproteomictechniquesthatarefirstsummarizedinareviewandthencoveredinthreechaptersdescribingmethodsto(1)identify ubiquitinationsitesonendogenousproteinsfromanyeukaryoticorganismortissue; (2)enrichforpoly-ubiquitinchainswithspecificlinkagetypesformassspectrometry analysis;and(3)employcross-linkingtodeterminedomaintopologywithinamulti-subunit complexsuchastheproteasome.
Weare,firstandforemost,mostgratefultoallofourcolleaguesthatcontributedtothis volume.Whileitisundoubtedlytruethatmanyresearchersconsidertheirresearchareasas
special,weareconstantlytakenabackbythegenerosityandgoodwilloftheinvestigators thatmaketheubiquitinfieldtheirhome.WearegratefultoSpringerfortheopportunityto featureourworkthroughthisbookseriesandtoJohnWalkerforhisdedicatedhelpin assemblingthisbook.Lastly,wewouldliketodedicatethisvolumetoourpostdoctoral advisor,RayDeshaies,aswellashislong-timeresearchassociate,RatiVerma.Thisvolume wouldnothavebeenpossiblewithouttheirexpertmentorshipandfriendship.
Vancouver,BC,CanadaThibaultMayor LasVegas,NV,USAGaryKleiger
Reference
1.GoldsteinG,ScheidM,HammerlingU,SchlesingerDH,NiallHD,BoyseEA(1975)Isolationofa polypeptidethathaslymphocyte-differentiatingpropertiesandisprobablyrepresenteduniversallyin livingcells.ProcNatlAcadSciUSA72(1):11–15
Preface.. ...................................................................v Contributors .................................................................ix
PART IMETHODSTO UNCOVERTHE MECHANISMSOF ACTION FOR ENZYMESTHAT ASSEMBLEOR DISASSEMBLE POLY-UBIQUITIN CHAINS
1CharacterizationofRING-Between-RINGE3Ubiquitin TransferMechanisms ....................................................3
KatherineH.ReiterandRachelE.Klevit
2Single-TurnoverRING/U-BoxE3-MediatedLysineDischargeAssays ........19 LoriBuetow,MadsGabrielsen,andDannyT.Huang
3MethodsforNAD-DependentUbiquitinationCatalyzed by Legionellapneumophila EffectorProteins.. ..............................33
JiazhangQiuandZhao-QingLuo
4UsingInVitroUbiquitylationAssaystoEstimatetheAffinities ofUbiquitin-ConjugatingEnzymesforTheirUbiquitinLigasePartners .......39 SpencerHill,ConnorHill,andGaryKleiger
5CompetitionAssayforMeasuringDeubiquitinatingEnzyme SubstrateAffinity... ....................................................59
MichaelT.MorganandCynthiaWolberger
PART IITOOLSTO STUDY UBIQUITYLATION
6EnzymaticAssemblyofUbiquitinChains ..................................73 MartinA.Michel,DavidKomander,andPaulR.Elliott
7Ubiquitin-ActivatedInteractionTraps(UBAITs):ToolsforCapturing Protein-ProteinInteractions. .............................................85 HazelF.O’Connor,CalebD.Swaim,LarissaA.Canadeo, andJonM.Huibregtse
8GeneratingIntracellularModulatorsofE3LigasesandDeubiquitinases fromPhage-DisplayedUbiquitinVariantLibraries... .......................101 WeiZhangandSachdevS.Sidhu
9IntegratedProteogenomicApproachforIdentifyingDegradationMotifs inEukaryoticCells.. ....................................................121 YifatGeffen,AlonAppleboim,RichardG.Gardner,andTommerRavid 10AMethodtoMonitorProteinTurnoverbyFlowCytometry andtoScreenforFactorsthatControlDegradation byFluorescence-ActivatedCellSorting ....................................137 SophieA.ComynandThibaultMayor ix
11
E.coli-BasedSelectionandExpressionSystemsforDiscovery, Characterization,andPurificationofUbiquitylatedProteins.. ...............155
OlgaLevin-Kravets,TalKeren-Kaplan,IlanAttali,ItaiSharon, NetaTanner,DarShapira,RituRathi,AvinashPersaud, NoaShohat,AnnaShusterman,andGaliPrag
PART IIISTRUCTURAL APPROACHESAS APPLIEDTO ENZYMES THAT PARTICIPATEINTHE UBIQUITIN PROTEASOME SYSTEM
12StrategiestoTrapEnzyme-SubstrateComplexesthatMimicMichaelis IntermediatesDuringE3-MediatedUbiquitin-LikeProteinLigation.. ........169 FrederickC.StreichJr.andChristopherD.Lima
13Small-AngleX-RayScatteringfortheStudyofProteins intheUbiquitinPathway... .............................................197
Jean-Franc¸oisTrempeandKalleGehring
14MethodsforPreparingCryo-EMGridsofLargeMacromolecular Complexes. ............................................................209
LeifuChangandDavidBarford
PART IVMETHODSTO STUDY 26SPROTEASOME FUNCTION
15RecombinantExpression,UnnaturalAminoAcidIncorporation, andSite-SpecificLabelingof26SProteasomalSubcomplexes. ...............219
JaredA.M.BardandAndreasMartin
16NativeGelApproachesinStudyingProteasomeAssembly andChaperones ........................................................237 JeroenRoelofs,AnjanaSuppahia,KenrickA.Waite,andSoyeonPark
17MeasuringtheOverallRateofProteinBreakdowninCells andtheContributionsoftheUbiquitin-Proteasome andAutophagy-LysosomalPathways. .....................................261 ZheSha,JinghuiZhao,andAlfredL.Goldberg
18MethodstoRapidlyPrepareMammalian26SProteasomes forBiochemicalAnalysis .................................................277 Chueh-LingKuo,GalenAndrewCollins,andAlfredL.Goldberg
19MeasurementoftheMultipleActivitiesof26SProteasomes... ...............289 HyoungTaeKim,GalenAndrewCollins,andAlfredL.Goldberg
20ExploringtheRegulationofProteasomeFunctionbySubunit Phosphorylation ........................................................309 JordanJ.S.VerPlankandAlfredL.Goldberg
21ScalableInVitroProteasomeActivityAssay.. ..............................321 AmitKumarSinghGautam,KirbyMartinez-Fonts, andAndreasMatouschek
PART VPROTEOMIC METHODSTO STUDYTHE UBIQUITIN PROTEASOME SYSTEM
22ExploringtheRampantExpansionofUbiquitinProteomics..
AmaliaRoseandThibaultMayor
23UbiquitindiGLYProteomicsasanApproachtoIdentify andQuantifytheUbiquitin-ModifiedProteome ............................363 AmitFulzeleandEricJ.Bennett
24InterpretingtheLanguageofPolyubiquitinwithLinkage-Specific AntibodiesandMassSpectrometry... .....................................385 MarissaL.Matsumoto,ErickR.Castellanos,YiJimmyZeng, andDonaldS.Kirkpatrick
25DissectingDynamicandHeterogeneousProteasomeComplexes UsingInVivoCross-Linking-AssistedAffinityPurification andMassSpectrometry ..................................................401 XiaorongWangandLanHuang
Contributors
ALON APPLEBOIM DepartmentofBiologicalChemistry,TheAlexanderSilbermanInstitute ofLifeSciences,TheHebrewUniversityofJerusalem,Jerusalem,Israel;SchoolofComputer ScienceandEngineering,TheHebrewUniversityofJerusalem,Jerusalem,Israel
ILAN ATTALI DepartmentofBiochemistryandMolecularBiology,InstituteofStructural Biology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel
JARED A.M.BARD DepartmentofMolecularandCellBiologyandCaliforniaInstitutefor QuantitativeBiosciences,UniversityofCaliforniaatBerkeley,Berkeley,CA,USA
DAVID BARFORD MRCLaboratoryofMolecularBiology,Cambridge,UK
ERIC J.BENNETT SectionofCellandDevelopmentalBiology,DivisionofBiologicalSciences, UniversityofCalifornia,SanDiego,LaJolla,CA,USA
LORI BUETOW CancerResearchUKBeatsonInstitute,Glasgow,UK;InstituteofCancer Sciences,UniversityofGlasgow,Glasgow,UK
LARISSA A.CANADEO DepartmentofMolecularBiosciences,UniversityofTexasatAustin, Austin,TX,USA
ERICK R.CASTELLANOS DepartmentofStructuralBiology,GenentechInc.,SouthSan Francisco,CA,USA
LEIFU CHANG MRCLaboratoryofMolecularBiology,Cambridge,UK
GALEN ANDREW COLLINS DepartmentofCellBiology,HarvardMedicalSchool,Boston, MA,USA
SOPHIE A.COMYN DepartmentofBiochemistryandMolecularBiology,MichaelSmith Laboratories,UniversityofBritishColumbia,Vancouver,BC,Canada
PAUL R.ELLIOTT DivisionofProteinandNucleicAcidChemistry,MRCLaboratoryof MolecularBiology,Cambridge,UK
AMIT FULZELE SectionofCellandDevelopmentalBiology,DivisionofBiologicalSciences, UniversityofCalifornia,SanDiego,LaJolla,CA,USA
MADS GABRIELSEN CancerResearchUKBeatsonInstitute,Glasgow,UK;Instituteof CancerSciences,UniversityofGlasgow,Glasgow,UK
RICHARD G.GARDNER DepartmentofPharmacology,UniversityofWashington,Seattle, WA,USA
YIFAT GEFFEN DepartmentofBiologicalChemistry,TheAlexanderSilbermanInstituteof LifeSciences,TheHebrewUniversityofJerusalem,Jerusalem,Israel;SchoolofComputer ScienceandEngineering,TheHebrewUniversityofJerusalem,Jerusalem,Israel
KALLE GEHRING DepartmentofBiochemistry,McGillUniversity,Montre´al,QC,Canada
ALFRED L.GOLDBERG DepartmentofCellBiology,HarvardMedicalSchool,Boston,MA, USA
CONNOR HILL DepartmentofChemistryandBiochemistry,UniversityofNevada,Las Vegas,NV,USA
SPENCER HILL DepartmentofChemistryandBiochemistry,UniversityofNevada,Las Vegas,NV,USA
DANNY T.HUANG CancerResearchUKBeatsonInstitute,Glasgow,UK;Instituteof CancerSciences,UniversityofGlasgow,Glasgow,UK
LAN HUANG DepartmentofPhysiologyandBiophysics,UniversityofCalifornia,Irvine, CA,USA
JON M.HUIBREGTSE DepartmentofMolecularBiosciences,UniversityofTexasatAustin, Austin,TX,USA
TAL KEREN-KAPLAN DepartmentofBiochemistryandMolecularBiology,Instituteof StructuralBiology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv, Israel
HYOUNG TAE KIM DepartmentofCellBiology,HarvardMedicalSchool,Boston,MA,USA
DONALD S.KIRKPATRICK DepartmentofMicrochemistry,ProteomicsandLipidomics, Genentech,Inc.,SouthSanFrancisco,CA,USA
GARY KLEIGER DepartmentofChemistryandBiochemistry,UniversityofNevada,Las Vegas,NV,USA
RACHEL E.KLEVIT DepartmentofBiochemistry,UniversityofWashington,Seattle,WA, USA
DAVID KOMANDER DivisionofProteinandNucleicAcidChemistry,MRCLaboratoryof MolecularBiology,Cambridge,UK
CHUEH-LING KUO DepartmentofCellBiology,HarvardMedicalSchool,Boston,MA,USA
OLGA LEVIN-KRAVETS DepartmentofBiochemistryandMolecularBiology,Instituteof StructuralBiology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv, Israel
CHRISTOPHER D.LIMA StructuralBiologyProgram,SloanKetteringInstitute,Memorial SloanKetteringCancerCenter,NewYork,NY,USA;HowardHughesMedicalInstitute, NewYork,NY,USA
ZHAO-QING LUO DepartmentofBiologicalSciences,PurdueInstituteforInflammation, ImmunologyandInfectiousDisease,PurdueUniversity,WestLafayette,IN,USA
ANDREAS MARTIN DepartmentofMolecularandCellBiologyandCaliforniaInstitutefor QuantitativeBiosciences,UniversityofCaliforniaatBerkeley,Berkeley,CA,USA;Howard HughesMedicalInstitute,UniversityofCaliforniaatBerkeley,Berkeley,CA,USA
KIRBY MARTINEZ-FONTS DepartmentofMolecularBiosciences,TheUniversityofTexasat Austin,Austin,TX,USA
ANDREAS MATOUSCHEK DepartmentofMolecularBiosciences,TheUniversityofTexasat Austin,Austin,TX,USA
MARISSA L.MATSUMOTO DepartmentofStructuralBiology,GenentechInc.,SouthSan Francisco,CA,USA
THIBAULT MAYOR DepartmentofBiochemistryandMolecularBiology,MichaelSmith Laboratories,UniversityofBritishColumbia,Vancouver,BC,Canada
MARTIN A.MICHEL DivisionofProteinandNucleicAcidChemistry,MRCLaboratoryof MolecularBiology,Cambridge,UK
MICHAEL T.MORGAN DepartmentofBiophysicsandBiophysicalChemistry,JohnsHopkins UniversitySchoolofMedicine,Baltimore,MD,USA
HAZEL F.O’CONNOR DepartmentofMolecularBiosciences,UniversityofTexasatAustin, Austin,TX,USA
SOYEON PARK DepartmentofMolecular,Cellular,andDevelopmentalBiology,Universityof ColoradoBoulder,Boulder,CO,USA
AVINASH PERSAUD DepartmentofBiochemistryandMolecularBiology,Instituteof StructuralBiology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv, Israel
GALI PRAG DepartmentofBiochemistryandMolecularBiology,InstituteofStructural Biology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel xivContributors
JIAZHANG QIU KeyLaboratoryofZoonosis,MinistryofEducation,CollegeofVeterinary Medicine,JilinUniversity,Changchun,China
RITU RATHI DepartmentofBiochemistryandMolecularBiology,InstituteofStructural Biology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel
TOMMER RAVID DepartmentofBiologicalChemistry,TheAlexanderSilbermanInstituteof LifeSciences,TheHebrewUniversityofJerusalem,Jerusalem,Israel
KATHERINE H.REITER DepartmentofBiochemistry,UniversityofWashington,Seattle,WA, USA
JEROEN ROELOFS DivisionofBiology,KansasStateUniversity,Manhattan,KS,USA
AMALIA ROSE DepartmentofBiochemistryandMolecularBiology,MichaelSmith Laboratories,UniversityofBritishColumbia,Vancouver,BC,Canada
ZHE SHA DepartmentofCellBiology,HarvardMedicalSchool,Boston,MA,USA
DAR SHAPIRA DepartmentofBiochemistryandMolecularBiology,InstituteofStructural Biology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel
ITAI SHARON DepartmentofBiochemistryandMolecularBiology,InstituteofStructural Biology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel
NOA SHOHAT DepartmentofBiochemistryandMolecularBiology,InstituteofStructural Biology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel
ANNA SHUSTERMAN DepartmentofBiochemistryandMolecularBiology,Instituteof StructuralBiology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv, Israel
SACHDEV S.SIDHU DonnellyCentreforCellularandBiomolecularResearch,Universityof Toronto,Toronto,ON,Canada;BantingandBestDepartmentofMedicalResearch, UniversityofToronto,Toronto,ON,Canada;DepartmentofMolecularGenetics, UniversityofToronto,Toronto,ON,Canada
AMIT KUMAR SINGH GAUTAM DepartmentofMolecularBiosciences,TheUniversityofTexas atAustin,Austin,TX,USA
FREDERICK C.STREICH JR StructuralBiologyProgram,SloanKetteringInstitute, MemorialSloanKetteringCancerCenter,NewYork,NY,USA
ANJANA SUPPAHIA DivisionofBiology,KansasStateUniversity,Manhattan,KS,USA
CALEB D.SWAIM DepartmentofMolecularBiosciences,UniversityofTexasatAustin, Austin,TX,USA
NETA TANNER DepartmentofBiochemistryandMolecularBiology,InstituteofStructural Biology,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,TelAviv,Israel
JEAN-FRANC¸OIS TREMPE DepartmentofPharmacologyandTherapeutics,McGillUniversity, Montre´al,QC,Canada
JORDAN J.S.VERPLANK HarvardMedicalSchool,Boston,MA,USA
KENRICK A.WAITE DivisionofBiology,KansasStateUniversity,Manhattan,KS,USA
XIAORONG WANG DepartmentofPhysiologyandBiophysics,UniversityofCalifornia, Irvine,CA,USA
CYNTHIA WOLBERGER DepartmentofBiophysicsandBiophysicalChemistry,JohnsHopkins UniversitySchoolofMedicine,Baltimore,MD,USA
YI JIMMY ZENG DepartmentofMicrochemistry,ProteomicsandLipidomics,Genentech,Inc., SouthSanFrancisco,CA,USA
WEI ZHANG DonnellyCentreforCellularandBiomolecularResearch,Universityof Toronto,Toronto,ON,Canada
JINGHUI ZHAO DepartmentofCellBiology,HarvardMedicalSchool,Boston,MA,USA; AbbVie,Cambridge,MA,USA
MethodstoUncovertheMechanismsofActionforEnzymes thatAssembleorDisassemblePoly-UbiquitinChains
CharacterizationofRING-Between-RINGE3Ubiquitin TransferMechanisms
KatherineH.ReiterandRachelE.Klevit
Abstract
Proteinubiquitinationisanessentialposttranslationalmodificationthatregulatesnearlyallcellularprocesses.E3ligasescatalyzethefinaltransferofubiquitin(Ub)ontosubstratesandthusareimportant temporalregulatorsofubiquitinmodificationsinthecell.E3sareclassifiedbytheirdistincttransfer mechanisms.RINGE3sactasscaffoldstofacilitatethetransferofUbfromE2-conjugatingenzymes directlyontosubstrates,whileHECTE3sformanE3~UbthioesterintermediatepriortoUbtransfer.A thirdclass,RING-Between-RING(RBR)E3s,areclassifiedasRING/HECThybridsbasedontheirability toengagetheE2~UbconjugateviaaRING1domainwhilesubsequentlyforminganobligateE3~Ub intermediatepriortosubstratemodification.RBRscomprisethesmallestclassofE3s,consistingofonly 14familymembersinhumans,yettheirdysfunctionhasbeenassociatedwithneurodegenerativediseases, susceptibilitytoinfection,inflammation,andcancer.Additionally,theiractivityissuppressedbyautoinhibitorydomainsthatblocktheircatalyticactivity,suggestingtheirregulationhasimportantcellular consequences.Here,weidentifytechnicalhurdlesfacedinstudyingRBRE3sandprovideprotocolsand guidelinestoovercomethesechallenges.
Keywords Ubiquitin,Transthiolation,UbiquitinE3ligase,RBR,HHARI,Parkin,HOIP,RINGBetween-RING
1Introduction
Ubiquitin(Ub)modificationsaregeneratedbythesequential transferofUbbetweenatrioofenzymes(E1-activating,E2-conjugating,andE3-ligase)ontosubstrates.Theresultingproducts havedramaticeffectsonproteinfunctionandhalf-life,andthereforetheirgenerationmustbetightlyregulated.E3ligasesprovide bothspatialandtemporalcontroloverthefinalubiquitintransfer stepbyactingasscaffoldsthatrecruitbothE2~Ub(~denotes thioester)andsubstrates.E3sareclassifiedintothreefamilies basedontheirdistincttransfermechanisms:RING(Really Interesting New Gene),HECT(homologousto E6AP carboxyl terminus),andRBR(RING-Between-RING).RINGE3susean eponymousRINGdomaintoengageE2~UbandpromoteUb
ThibaultMayorandGaryKleiger(eds.), TheUbiquitinProteasomeSystem:MethodsandProtocols,MethodsinMolecularBiology, vol.1844, https://doi.org/10.1007/978-1-4939-8706-1_1, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2018 3
Fig.1 DomainarchitectureofRBRenzymes.Domaincolorsrepresent:RING1-IBR-RING2module(blue),autoinhibitorydomains(purple),accessorydomains(grey)
transferdirectlyontosubstrateresidues,mostoftenalysine.HECT E3sformanobligateE3~UbthioesterintermediatepriortoUb transfer.RBRshavebeendubbedasRING/HECThybridsbased ontheiruseofaRING1domaintobindE2~Ubandanactive-site cysteinetomediatetransferofubiquitinthroughanE3~Ubintermediate[1–5].AllRBRsshareastructurallysimilarcatalyticmodule,composedofanE2-bindingdomain(RING1),anin-betweenRINGdomain(IBR)ofunknownfunction,andacatalyticdomain (RING2)(Fig. 1).Despitetheirsharedcatalyticcore,itisbecomingincreasinglyclearthatRBRE3spossessdistinctfeaturesand propertiesassociatedwiththeiruniquefunctionsthatdemand furtherinvestigation.
RBRE3ligasescompriseanimportantclassofenzymes involvedinneurodegeneration(Parkin),organogenesis (HHARI),andinflammation(HOIP),yetlittleisknownabout howtheyperformtheircellularfunctions[6–10].StudyofRBR E3shasbeengreatlyfacilitatedbythediscoverythattheseenzymes containanactive-sitecysteineresidue,allowingforthestraightforwardgenerationofstructurallyintactligase-deadformsofthe RBRs[1].Subsequentstructuralandbiochemicalstudiesofseveral RBRE3s(Parkin,HHARI,andHOIP)provideamechanistic foundationonwhichtofurtherunderstandRBRfunction,but muchremainstobelearnedfromthesewell-studiedcasesaswell asforothermembersofthefamily[11–19].Asoutlinedbelow, basicinformationislackingforvirtuallyeverymemberoftheRBR family.Inthisarticle,wedescribeavailableapproachesandtoolsto addresstheenzymaticE2:RBRpairsandtheiractivity.
Fig.2 FundamentalquestionssurroundingRBRubiquitintransfer.HHARIdomainstructureshownasan example.(1)WhatE2sworkwitheachRBR?(2)HowareRBRenzymesactivated?(3)Whatubiquitinproducts doeseachRBRform?(4)WhatsubstratesdoRBRstarget?
TofullyinvestigatethecellularfunctionofagivenRBRE3, answerstofourquestionsareneeded(Fig. 2):
WhichE2sworkwitheachRBR?
HowareRBRenzymesactivated? WhatubiquitinproductsdoesanRBRE3generate? WhatarethesubstratesforagivenRBRE3?
Eachquestionposesitsownsetofchallengesandtechnical hurdles,asdiscussedbelow.Furthermore,thereisachickenand eggsituationwhennoneoftheinformationisavailable:forexample,howcanonedeterminetheproductsofanRBR-catalyzed reactionifonedoesnotknowwithwhichofthe~36humanE2s itworks.Anadditionallayerofcomplexityandchallengearises fromthefactthatmostRBRE3sareauto-inhibited.Thus,one mustfirstdiscoverwaysinwhichauto-inhibitioncanbereleased beforeotherinvitroinvestigationscanbecarriedout.
1.1RBREnzymes
AreAuto-inhibited
UbtransferbyRBRE3sisgenerallysuppressedbyanautoinhibitorymechanism(forarecentreview, see [20]).Ofthe humanRBRE3swhoseauto-inhibitionhasbeenstudied,amajorityinvolvedomainsoutsidetheRING1-IBR-RING2catalytic modulethatphysicallyoccludetheRING2catalyticsite.These includeParkin,HHARI,TRIAD1,and,possiblyHOIP[2, 4, 5, 16, 21].Thus,invitrostudiesofRBRactivityrequirethereleaseof auto-inhibition.Insomecases,suchasHHARI(see below),inhibitioncanbereleasedbyremovaloftheinhibitorydomain [1, 16].SuchanapproachcangenerateconstructsofanRBRE3 thatcan(1)bindE2~Uband(2)generatetheE3~Ubintermediate,butitisnotcurrentlyknownwhetherremovalofanon-RBR
domainwillhaveanimpactoneitherthetypesofproductsgeneratedorontheabilityoftheRBRE3totransferUbtoasubstrate. Webrieflyreviewwhatiscurrentlyknownabouthowtogenerate activatedformsofRBRE3s,asthesemayserveasguidesforhowto proceedinsystemsforwhichtheinformationislacking.
HHARIauto-inhibitionismediatedbyitsC-terminalAriadne domain,whichblockstheRING2activesite(Fig. 1)[16].HHARI retainsitsabilitytobindanE2fromtheauto-inhibitedstate,but thatbindingdoesnotreleasetheinhibition[15, 22].Thisobservationisconsistentwithrecentstructuralstudiesinwhich UbcH7~Ubboundtoauto-inhibitedHHARIstillhas~50A ˚ separationbetweenE2andE3activesites,suggestingadditionalconformationalchangesarerequiredfortransthiolationtooccur [15–17].Onemechanismbywhichinhibitionisreleasedinvolves asecondE3ligase,acullin-RINGligase(CRL)[2, 22].Bindingof HHARItoaneddylatedformofeitherCUL-1orCUL-3activates HHARItoallowtransferofUbviaitsRING2activesite[2].This, inturn,promotesHHARI-mediatedmono-ubiquitinationofcullin substrates[22].Thismodeofactivationhasbeendemonstrated bothincellularandinvitrocontextsforknowncullinsubstrates, makingitaviable,iftechnicallydifficult,methodtogenerateactive HHARI[2, 7, 22].However,itisnotyetknownwhetherHHARI activatedinthismanneriscapableofmodifyingother(non-cullinspecific)substrates.ActivationofHHARIcanalsobeachievedby disruptingtheintramolecularinteractionbetweentheAriadne domainandRING2,eitherbydeletionoftheAriadnedomain,or bypointmutationsattheRING2:Ariadneinterface(Table 1) [16, 22].
Table1 ActiveRBRconstructsthatretainactivityintheabsenceofactivatingpartners
RING2 177-395Catalyticmodule.DeletionofautoinhibitoryAriadnedomain [1, 15, 37]
OpenAF430A,E431A, E503A PointmutationsdisrupttheRING2: Ariadnebindinginterface [16, 22]
OpenBR420A,N423A, E503A PointmutationsdisrupttheRING2: Ariadnebindinginterface [15, 16] Δariadne1-400Deletionofauto-inhibitoryAriadne domain [16, 17] ParkinRING1-IBR-REPRING2 217-465Catalyticmodule.DeletionofautoinhibitoryN-terminus [1, 13, 37]
HOIPRING1-IBRRING2-LDD 697-1072Catalyticmodule.DeletionofautoinhibitoryN-terminus [4, 18, 37] TRIAD1 Δariadne1-348Deletionofauto-inhibitoryAriadne domain [37]
AlthoughallRBRE3shaveaRING1-IBR-RING2modulein common,flexiblelinkersandadditionalidiosyncraticelementsgive risetouniqueinter-domainorientationsandrelationshipsand, therefore,distinctactivationrequirements.Someoftheseinterdomaininteractionsarethesourceofauto-inhibition:forexample, theParkinRING0domainoccludesitsRING2activesite,whilea smallrepressordomain(REP)thatislocatedbetweentheIBRand RING2blockstheE2bindingsiteinRING1(Fig. 1)[11, 14, 21].Parkinactivationiscoupledtophosphorylationeventsthat leadtotheonsetofmitophagy[23, 24].ThemitochondrialassociatedkinasePTEN-inducedkinase(PINK1)activatesParkin bytwodistinctmechanisms.Inresponsetomitochondrialdamage, PINK1phosphorylatestheParkinUBLdomain,releasingitfrom theRBRmoduleandincreasingParkinligaseactivity [25–28].Additionally,ubiquitinisphosphorylatedbyPINK1, andbindingofphospho-UbtoParkinallostericallyactivatesthe E3[29, 30].ThesephosphorylationeventsleadtothetranslocationofParkintodamagedmitochondria,whereitcantagthemfor removal.ThestudyofParkinisanactiveareaofinvestigation,given itsimmediatemedicalrelevance(Parkinson’sdisease),andthisis nowaidedbythecommercialavailabilityofrecombinantphosphoUbandPINK1.
RBRactivationbyE3:E3complexformationisnotlimitedto HHARI,asthelinearubiquitinchainassemblycomplex(LUBAC) formslinearchainsthroughtheactionoftwoRBR-containing subunits:HOIP,whichharborscatalyticactivity,andHOIL-1L. InteractionsbetweenHOIP’subiquitin-associateddomainandthe ubiquitin-likedomain(UBL)ofeitherHOIL-1Loranadditional subunit,Shank-associatedRHdomain-interactingprotein(SHARPIN),promotecomplexformationandactivation[31–34].Both HOIPandHOIL-1Lcontaincatalyticcysteineresidues;however, linearchainactivityresidesintheHOIPRING2domainanda C-terminallinearubiquitinchaindeterminingdomain(LDD). HOIPactivityisinhibitedbydomainsadjacenttotheHOIPRBR module.Whiletheexactmechanismofauto-inhibitionisunknown, HOIPcatalyticactivityisrestoredthroughbindingtotheHOIL-1 UBLdomainandisfurtherenhancedthroughinteractionswith full-lengthHOIL-1[5].Thisenhancedactivityisdependentonthe HOIL-1catalyticcysteine,eventhoughHOIL-1isnotintrinsically activeonitsown[5].Todate,invitrostudiesofHOIPactivityhave reliedon“active”constructscontainingtheRBRandC-terminal LDDdomain(see Table 1).Whileastructureoffull-lengthHOIP hasremainedelusive,severalcrystalstructureshavecapturedsnapshotsoftheHOIPcatalyticdomainpoisedforlinearchainassembly [18, 19].Thesestructureshelpedtodefinetheessentialroleofthe LDDdomaininlinearubiquitinchainformationbyorientingan acceptorubiquitinN-terminusproximaltoadonorubiquitinatthe HOIPactivesite.Additionally,astructureofHOIPRBR-LDD in
1.2RBRE3andTheir E2s
complexwithUbcH5~UbcapturedtheE2inanelongatedopen conformation[19].Notably,thereisanE2activesiteinclose proximitytoanE3activesiteinthiscrystal,suggestingamodel forE2-to-RBRubiquitintransfer[19].Inthefuture,revealing whetherthesemechanismsextendtoallRBRsandidentifying idiosyncraticdetailsofindividualRBRswillbeofgreatinterestto thefield.
LikeotherE3families,RBRswilllikelyinteractwithmultipleE2 enzymesinresponsetodistinctcellularsignalsandenzymeexpressionlevels.Todate,mostRBRsstudiedarefunctionalwithUbcH7 andUbcH5,yettheiraffinitiesfortheE2svarysubstantially,a factorwhichmayhaveimportantramificationsinthecell.Additionally,whileUbcH5isbothlysineandcysteinereactive,the reactivityprofileofUbcH7islimitedtoonlycysteines,makingit adedicatedE2forRBRandHECTligases.Todate,highthroughputE2screenshavebeenlimitedtoRINGandHECT ligases;thusthegeneralinteractionandreactivityofE2swith RBRsrequirefurtheranalysis.TherearereportsofotherE2s workinginconcertwithRBRE3s(e.g.,UBE2Kworkswith LUBAC[35]),butthecompletecadreofE2sforagivenRBRE3 hasyettobedefined.Additionally,thesequenceofeventsbywhich RBRsareactivatedandE2~Ubbindsisunknown,andtherefore theabilitytodetectE2interactionsmayrequireactiveRBRconstructs,asdescribedabove.UnderstandinghowRBRsinteractand functionwithaselectsetofE2sisafundamentalquestionthatmust beaddressedaswebegintoprobeRBRubiquitintransfer mechanisms.
Mechanistically,RING1domainsaredistinctfromtraditional RINGsintheirabilitytorestrictE2~Ubtransfertoacatalytic cysteine.CanonicalRINGdomainsgenerallyemploya“linchpin” residuethatrestrictstheUbofaboundE2~Ubconjugatetobeina “closedstate,”whichisintrinsicallymoreactivetowardlysines [36].RBRRING1s,however,lackalinchpinresidueandinstead promoteanopenconformationofE2~Ubtopreventaminolysis [15, 17, 19].InthecaseofHHARI,anextendedloopinRING1 servesasawedgethatdisfavorstheclosedE2~Ubconjugate, therebypromotinganopenstateandtransthiolationwiththeE3 activesite[15, 17].However,HOIPRING1doesnotcontainan extendedloop,yettheHOIP:UbcH5~Ubstructuredepictsan openconjugate,suggestingadditionalinteractionsmayrestrict closedE2~Ubconformations[19].Recentstudieshaveshown thatHHARI,Parkin,andHOIPRING2domainscontainaweak ubiquitinbindingsurfacethatisimportantfortransthiolation [37].AdonorubiquitinbindingsiteontheHOIPIBR-RING2 linkeridentifiedintheHOIP:UbcH5-Ubcrystalstructuresuggests thisinteractionmaybeacommonfeaturerequiredforUbtransfer totheRBRactivesite[19].
1.3RBRE2~Ub:E3 TransferMechanisms
1.4RBRProduct Formation
1.5RBRSubstrates
Itisnowagenerallyacceptedtenetthatthelastmoietyholdingan activatedUbpriortoitstransfertosubstratedeterminesthetypeof ubiquitinproductformedonthesubstrate.Inthecaseofcanonical RINGE3s,itistheE2thatdeterminesproduct,whiletheformationofanRBR~Ubintermediateshiftsthisimportantfunctionto theRBRE3.This“rule”predictsthatanE2’sactivitywithtraditionalRINGE3smaynotreflectthefinalproductformedwhen thatE2workswithRBRE3s.Sucharelationshipisillustratedby theUbe2K:LUBACinteraction.IntheabsenceofanyE3,orinthe presenceofRINGE3s,Ube2KbuildsK48-linkedpoly-Ubchains, butwhenpairedwithLUBAC,theRBRHOIPusestheUb providedbyUbe2KtoassemblelinearUbchainsdespitetheintrinsicUbe2Kpreference[35].Thetypeofubiquitinproductformedis notaconservedcharacteristicamongRBRs:HHARImodifies substrateswithmono-ubiquitin,HOIPformslinearubiquitin chains,andParkinformspoly-ubiquitinchains[15, 22, 38, 39].Inmanycases,ourunderstandingoftheRBRE3~Ubtransfer mechanismhasbeenlimitedtoauto-ubiquitinationoftheE3,but whetherproductpreferencesholdtrueforbonafidesubstrates remainstobedetermined.Producttypecanbedetermined invitroinseveralways.Ubiquitinvariants,suchaslysine-lessUb (K0)thatisunabletoformlysine-linkedchains,distinguishpolyUbchainsfrommultiple-mono-Ubevents,animportantdistinctionasbothproductsmayappearashigh-molecular-weightubiquitinsmearsonreducingSDS-PAGEgels.Additionally,chaintype maybeparsedoutusingUbmutantsthatcarryasinglelysine(with allothersmutated)todeterminelinkagespecificity.Asnotedabove, ubiquitininteractionswithRBRsplayanimportantroleinthe transfermechanism,soanyvariantofUbshouldbeusedwith caution,asmutationsthatdisrupttheseinteractionsmayconfound interpretationofresultsobtainedwiththem.Therefore,massspectrometricanalysisofproductsshouldbeperformedforarigorous identificationofproducttype.
InteractionsbetweenE3sandsubstratesareoftentransient.Consequently,theidentificationofE3:substratepairsbycommonbinding techniquessuchasyeasttwohybridsandco-immunoprecipitation canbechallengingandareoftennotfruitful.QuantitativeproteomicsstudieshaverevealednumerousbindingpartnersforHHARI andParkin,yetverificationofactualsubstrateshasbeenslow [22, 40].AkintostudyingE2~Ubtransthiolation,thestudyof RBRsubstrateinteractionsandmodificationsiscomplicatedby auto-inhibitionandalackofinformationregardingthesubstrate bindingdomainswithinRBRE3s.Forthemostcommonlystudied RBRs(Parkin,HHARI,HOIP),ithasbecomeroutinetousethe corecatalyticmodule(RING1-IBR-RING2)togenerateE3activity inassays;however,whetherthesedomainsaresufficientforspecific substratemodificationhasyettobedetermined.
1.6Future Approaches
AlthoughstructuralinformationrevealinganactivestateofafulllengthRBRhasbeenelusive,numerousstructuresofinhibited statesrevealthatinallcasessignificantconformationalchanges arenecessarytobridgethegapbetweenE2~UbandE3active sites.Thus,acleargoalistodeterminehowtheactivesitesare broughttogethertogeneratetheE3~Ubintermediatethatis requiredforRBRfunction.Inadditiontopurelystructural approaches,moresensitivetoolsarebeingdevelopedforthedetectionofE3~Ubconjugates.Electrophilicactivity-basedprobes, suchasUb-vinylmethylester(VME)havebeenwidelyusedin thefieldtotrapthioesters[41].However,theseprobesarestoichiometricsuicideinhibitorsandthereforelimittheabilitytoassess turnover.Recently,afluorescentUbprobe(UbFluor)wasusedto reportonthetransthiolationactivityofParkin[42].Termeda “bypasssystem”(ByS),giventhatE1,ATP,andE2arenotrequired fortheprobe’smodificationoftheRING2activesite,thisprobe showsgreatpromiseforusewithotherRBRstosimplifythestudy ofthecatalyticallyimportantintermediatestate.
Thebiochemicalassaysdescribedinthischapterareastarting pointtofurtherdefineRBRUbtransfersteps.Here,wedescribe methodstoscreenforRBR-reactiveE2saswellastocharacterize RBRproductformation,theoutputofwhichrequiresanactive RBRconstruct.Weprovideasummaryofactiveconstructs describedintheliterature(Table 1)asabasisfordesigningactive constructsforcloselyrelatedRBRs.Thesestudieswillnaturallybe iterativeatfirstandlimitedtotheuseofknownRBRE2s(UbcH5 andUbcH7).OnceanE2/RBRmolecularnetworkisestablished, onecanbegintouncoverthedetailsofhowtheseE3sorchestrate Ubtransfer.
2Materials
2.1Autoubiquitination Components
Prepareallsolutionsusingultrapurewater(resistivityof 18.2MΩ cmat25 C).Allstocksolutionsshouldbefiltered using0.22 μmfilterunits.
1.Reactionbuffer:0.02MHEPESpH7.5,0.1MNaCl, 0.5mMDTT.
2.RecombinanthumanHA-Ub(see Notes1 and 2).
3.RecombinanthumanE1(see Notes1 and 2).
4.RecombinanthumanUbcH5andUbcH7(see Notes1 and 2).
5.RecombinanthumanRBRenzymes(see Notes1 and 2).
6.ATPstocksolution:0.1Madenosinetriphosphate(ATP), 0.1MMgCl2.DissolveATPinH2OandadjustpHto7.0 withsodiumbicarbonate.Prepareimmediatelybeforeuse.
2.2Yeast Two-Hybrid(Y2H) Assays
7.1MDTTstocksolution.DissolveDTTinH2Oandstoresmall aliquotsat 20 C.
8.2 reducingSDSsamplebuffer.
9.PVDFfilterforimmunoblotting.
1.Y2Hvectors:Baitandpreyexpressionvectorsdesignedto expressfusionproteinsoftheGAL4DNA-bindingdomainor -activatingdomainwithyourbaitandpreyprotein,respectively.Vectorbackbonesareavailablecommercially,andE2 preycloneshavebeendepositedinAddgene[43].
2.Yeasthoststrain:AH109(Clonetech).
3.YPADmedium:Dissolve20gbactopeptone,10gbactoyeast extract,20gglucose,and40mgadeninesulfatedihydratein 1000mLH2O.Autoclaveandstoreatroomtemperatureor 4 C.
4.Syntheticdextroseminimalmedia(SD)-Leu/-Trpplates.SD minimalmediapacketsareavailablecommercially(see Note3). DissolvepacketsinH2Oandautoclave.Pourplatesandlet cool.Storeat4 C.
5.SelectivemediaSD-His/-Leu/-Trpplates.SDminimalmedia packetsareavailablecommercially(see Note3).PrepareasSD -Leu/-Trpplatesabove.Oncemediaiscooltothetouch, supplementwithfiltersterilized3-amino-1,2,4-triazole(3AT) to0–10mM(optimizedtoreducefalsepositives).Pourplates andletcool.Storeat4 C.
3Methods
3.1Designing anActiveRBR
Construct
ThestudyofRBRubiquitintransferrequiresactiveE3constructs, yetassessmentofactivityrequiresafunctionalE2:RBRpair.Two humanE2s,UbcH7andUbcH5,appeartobeactivewithevery RBRE3testedtodate,makingthemgoodchoicesforinitial studies.GiventhelimitedknowledgeofRBRactivation,active constructsaretypicallydesignedtolacktheauto-inhibitiondomain and,ataminimum,containtheRBRmodule.Duetointeractions betweentheRBRmoduleandauxiliarydomains,thepresenceof additionaldomainsmayberequiredtoachievesolubility.Table 1 includesalistofcommonlyusedactiveRBRconstructsthathave beensuccessfullypurifiedintheliterature.ForlessstudiedRBRs, activatedconstructswillneedtobedesigneddenovo.EachRBR seemstohaveitsownmechanismsforactivation;thus,inhibitory domainssurroundingeachRBRmodulewillneedtobeidentified bydomaindeletion.Additionally,theidentificationofpointmutationsaimedatdisruptinginhibitorydomaininteractionscan provideameansforworkingwithmorecompleteconstructs(see
3.2InVitroAutoubiquitinationAssay
Note4).RBRenzymesarehighlyactive;thusthetypesofproducts assembledduringauto-ubiquitinationassayswillneedtobefurther validated(see below).
ThefollowingmethodismodifiedfromourstudiesonHHARI auto-ubiquitination:
1.Setupa50 μLreactiononice:Mix1 μME1enzyme,5 μM UbcH7,20 μMHA-Ub,and2 μMactiveRBRenzymein 47.5 μL20mMHEPESpH7.5,100mMNaCl,and 0.5mMDTT(see Note5).Incubatereactionmixtureand 100mMATP/MgCl2 stockat37 Cfor1min.
2.Initiatereactionbyadding2.5 μLATPstockandincubate reactionat37 Cfor10min.
3.Take10 μLaliquotsat0,1,5,and10mintime-points,andadd 10 μLof2 reducingSDSsamplebuffertostopthereaction. Boilfor5min.
4.Separatethereactionmixtureby15%SDS-PAGE.
5.TransfertoaPVDFfilterandperformstandardimmunoblottinganalysiswithanti-HAantibody(see Note6).
3.3DefiningE2:RBR Pairs:Yeast Two-HybridScreening
OurunderstandingofRBRtransthiolationandsubstratemodificationhasbeenlimitedtothestudyofindividualE2:E3pairs.Togain insightintoRING1E2recruitmentandfunctionaltransthiolation reactions,high-throughputstudiesand/oruseofacompleteE2 panelcanidentifywhichE2sarecapableofpairingwithanRBRE3. ThesetypesofscreenshavebeenperformedforRINGandHECT E3sbuthaveyettobepursuedforRBRs.ManyE2:E3interactions areofmodestaffinityandarethereforedifficulttocapturebypulldownassays.Giventhereareonly14humanRBRs,yeast two-hybrid(Y2H)assaysprovideatractablewaytoexplorethe rangeofE2interactions,usingpreviouslycreatedE2Y2Hlibraries availableonAddgene[43].TargetedY2Hassayshavebeenusedto identifyE2:E3pairsbutmayrequiretheuseoflow-stringency conditionstoidentifypotentialpositivehits[43].Thesemustbe validatedbiochemically,asfalsepositivesarelikelytoappearunder suchliberalconditions.
GenerateY2Hclonesasdescribed[43].Briefly:
1.Designprimersforthefull-lengthandactivedeletioncoding sequencesforeachRBRofinterest(see Note7).Includethe endogenousstopcodonandrestrictionenzymesitesforligationintotheGal4-activationdomain(GAD)orGal4 DNA-bindingdomain(GBD)plasmids.
2.PCRamplifytheRBRcodingsequencefromhumancDNA(see Note8).
3.4Determining theTypesofRBRUb Products
3.DigestRBRamplificationandpGAD/pGBDplasmidwiththe appropriaterestrictionenzymes.
4.LigateRBRinsertintopGAD/pGBDplasmid.
5.Sequenceverifyfinalconstructs.
6.Co-transformE2baitclones(availablefromAddgene)and RBRpreyclonesintoyeaststrainAH109andgrowinYPAD mediafor12h.
7.SelectforpositivetransformantsonminimalSD-Leu/-Trp platesfor24h.
8.Inoculate100 μLofsterilewaterwithasinglecolonyandspot viaserialdilutionsontoselectivemedia(SD-His/-Leu/-Trp) supplementedwith0–10mM3-amino-1,2,4-triazole(3AT).
9.Incubateyeastat30 Candscoregrowthfor7days.
10.Verifypositiveinteractionsviaadditionalbindingstudies(e.g., co-immunoprecipitation,pulldowns,NMR)andassessactivity withinvitroauto-ubiquitinationassays.
EachRBRdictatesthetypesofUbproductsitformsonsubstrates. Thus,knowledgeoftheassociatedE2(s)isnotsufficienttopredict theRBRproductformation.Additionally,RBRsarehighlyactive, andasseenwithHHARI,canformmulti-mono-Ubproducts duringauto-ubiquitinationassays.Multiplemono-vs.poly-Ub canbedistinguishedusinglysine-lessubiquitinmutants(UbK0), whiledistinctchainlinkagescanbeassessedusinglysine-onlyubiquitinmutants(ex:UbK48),inwhichallotherlysineresiduesare mutatedtoarginine.Rigorouscharacterizationoflinkagetype shouldincludemassspectrometricanalysisofproducts.Additionally,whethersubstratesthemselvesplayaroleinRBRproduct formationisnotyetknownandwillneedtobeassessedona substratebasis.
1.Performauto-ubiquitinationassays,asdescribedabove,inthe presenceofoneofthefollowingubiquitinvariants:HA-UbK0, -UbK6,-UbK11,-Ub27,-Ub29,-Ub33,-Ub48,or-Ub63.
2.Multiplemono-ubiquitinationwillberevealedascollapsed chainformation(1–2 ubiquitinmodifications)inthepresenceofUbK0 (see Fig.4e[7]foranexampleUbpattern).
4Notes
1.RecombinantHA-Ub,humanandwheatE1enzymes, UbcH5,andUbcH7arecommerciallyavailable.Inourlab, wepurifytheseenzymesusingan E.Coli BL21(λDE3)expressionsystem[1, 15].
2.Auto-ubiquitinationassaysrequireactivationofRBRs. See Table 1 foralistofcommonlyusedactiveRBRconstructs. WhenmakingRBRsin-house,wesupplementourRBRexpressionmediawith0.2mMZnCl2.
3.SDminimaldropoutmediacanbemadein-houseifyourlab hasaccesstoindividualaminoacidstockreagents.
4.RBRstructuralstudieshaverevealedseveralinteractionsurfacesbetweentheRBRmoduleandauxiliarydomains,which likelyaffectthestabilityoftheprotein.Forexample,residues C-terminaltotheHOIPRBRmodulearerequiredforsolubility.Additionally,whenselectingactiveRBRconstructsfor substratemodificationassays,onemusttakeintoconsideration thatRBRsubstratebindingdomainsareunknown.
5.WeusehumanE1andUbcH7inourHHARIubiquitination assays;however,wheatE1andUbcH5canalsobeusedwith minimalimpacttoauto-ubiquitinationresults.
6.Whenpossible,auto-ubiquitinationassaysshouldincludea comparisontoauto-inhibitedandcatalyticallydeadE3s.Additionally,theuseofT7-taggedRBRconstructsallowsforthe directassessmentofmodifiedE3species.
7.Severalauto-inhibitedandactiveconstructsshouldbepursued foreachRBR,asthesequenceofE2bindingandRBRrelease fromauto-inhibitionhasyettobeparsedoutforallRBRs.
8.HumancDNAlibrariesarecommerciallyavailableorcanbe purifiedfromcelllines.Y2HkitswithpremadecDNAlibraries areavailablethroughcommercialvendors.
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But, with two-handed wrath, If baseness or pretension crossed his path, Struck once nor needed to strike more. 2.
His magic was not far to seek,— He was so human! Whether strong or weak, Far from his kind he neither sank nor soared, But sate an equal guest at every board: No beggar ever felt him condescend, No prince presume; for still himself he bare At manhood’s simple level, and where’er He met a stranger, there he left a friend. How large an aspect! nobly unsevere, With freshness round him of Olympian cheer, Like visits of those earthly gods he came; His look, wherever its good-fortune fell, Doubled the feast without a miracle, And on the hearthstone danced a happier flame; Philemon’s crabbed vintage grew benign; Amphitryon’s gold-juice humanized to wine.
III. 1.
The garrulous memories
Gather again from all their far-flown nooks, Singly at first, and then by twos and threes, Then in a throng innumerable, as the rooks Thicken their twilight files
Tow’rd Tintern’s gray repose of roofless aisles: Once more I see him at the table’s head When Saturday her monthly banquet spread To scholars, poets, wits, All choice, some famous, loving things, not names, And so without a twinge at others' fames; Such company as wisest moods befits, Yet with no pedant blindness to the worth Of undeliberate mirth, Natures benignly mixed of air and earth, Now with the stars and now with equal zest Tracing the eccentric orbit of a jest.
2.
I see in vision the warm-lighted hall, The living and the dead I see again, And but my chair is empty; ’mid them all ’Tis I that seem the dead: they all remain Immortal, changeless creatures of the brain: Well nigh I doubt which world is real most, Of sense or spirit, to the truly sane; In this abstraction it were light to deem Myself the figment of some stronger dream; They are the real things, and I the ghost That glide unhindered through the solid door, Vainly for recognition seek from chair to chair, And strive to speak and am but futile air, As truly most of us are little more.
Him most I see whom we most dearly miss, The latest parted thence, His features poised in genial armistice And armed neutrality of self-defence Beneath the forehead’s walled preëminence, While Tyro, plucking facts with careless reach, Settles off-hand our human how and whence; The long-trained veteran scarcely wincing hears The infallible strategy of volunteers Making through Nature’s walls its easy breach, And seems to learn where he alone could teach. Ample and ruddy, the board’s end he fills As he our fireside were, our light and heat, Centre where minds diverse and various skills Find their warm nook and stretch unhampered feet; I see the firm benignity of face, Wide-smiling champaign, without tameness sweet, The mass Teutonic toned to Gallic grace, The eyes whose sunshine runs before the lips While Holmes’s rockets curve their long ellipse, And burst in seeds of fire that burst again To drop in scintillating rain. 4.
There too the face half-rustic, half-divine, Self-poised, sagacious, freaked with humor fine, Of him who taught us not to mow and mope
About our fancied selves, but seek our scope
In Nature’s world and Man’s, nor fade to hollow trope, Content with our New World and timely bold To challenge the o’ermastery of the Old; Listening with eyes averse I see him sit Pricked with the cider of the Judge’s wit (Ripe-hearted homebrew, fresh and fresh again), While the wise nose’s firm-built aquiline Curves sharper to restrain The merriment whose most unruly moods Pass not the dumb laugh learned in listening woods Of silence-shedding pine:
Hard by is he whose art’s consoling spell Hath given both worlds a whiff of asphodel, His look still vernal ’mid the wintry ring Of petals that remember, not foretell, The paler primrose of a second spring.
5.
And more there are: but other forms arise And seen as clear, albeit with dimmer eyes: First he from sympathy still held apart By shrinking over-eagerness of heart, Cloud charged with searching fire, whose shadow’s sweep Heightened mean things with sense of brooding ill, And steeped in doom familiar field and hill,— New England’s poet, soul reserved and deep, November nature with a name of May, Whom high o’er Concord plains we laid to sleep, While the orchards mocked us in their white array And building robins wondered at our tears, S t h d i hi i th h t
Snatched in his prime, the shape august
That should have stood unbent ’neath fourscore years, The noble head, the eyes of furtive trust, All gone to speechless dust. And he our passing guest, Shy nature, too, and stung with life’s unrest, Whom we too briefly had but could not hold, Who brought ripe Oxford’s culture to our board, The Past’s incalculable hoard,
Mellowed by scutcheoned panes in cloisters old, Seclusions ivy-hushed, and pavements sweet With immemorial lisp of musing feet; Young head time-tonsured smoother than a friar’s, Boy face, but grave with answerless desires, Poet in all that poets have of best, But foiled with riddles dark and cloudy aims, Who now hath found sure rest, Not by still Isis or historic Thames, Nor by the Charles he tried to love with me, But, not misplaced, by Arno’s hallowed brim, Nor scorned by Santa Croce’s neighboring fames, Haply not mindless, wheresoe’er he be, Of violets that to-day I scattered over him; He, too, is there, After the good centurion fitly named, Whom learning dulled not, nor convention tamed, Shaking with burly mirth his hyacinthine hair, Our hearty Grecian of Homeric ways, Still found the surer friend where least he hoped the praise.
Yea truly, as the sallowing years Fall from us faster, like frost-loosened leaves Pushed by the misty touch of shortening days, And that unwakened winter nears, ’Tis the void chair our surest guest receives, ’Tis lips long cold that give the warmest kiss, ’Tis the lost voice comes oftenest to our ears; We count our rosary by the beads we miss: To me, at least, it seemeth so, An exile in the land once found divine, While my starved fire burns low, And homeless winds at the loose casement whine Shrill ditties of the snow-roofed Apennine.
1.
Now forth into the darkness all are gone, But memory, still unsated, follows on, Retracing step by step our homeward walk, With many a laugh among our serious talk, Across the bridge where, on the dimpling tide, The long red streamers from the windows glide, Or the dim western moon Rocks her skiff’s image on the broad lagoon, And Boston shows a soft Venetian side In that Arcadian light when roof and tree, Hard prose by daylight, dream in Italy; Or haply in the sky’s cold chambers wide Shivered the winter stars, while all below, As if an end were come of human ill, The world was wrapt in innocence of snow And the cast-iron bay was blind and still; These were our poetry; in him perhaps Science had barred the gate that lets in dream, And he would rather count the perch and bream Th ith th t’ idl f l
IV.
Than with the current’s idle fancy lapse; And yet he had the poet’s open eye That takes a frank delight in all it sees, Nor was earth voiceless, nor the mystic sky, To him the life-long friend of fields and trees: Then came the prose of the suburban street, Its silence deepened by our echoing feet, And converse such as rambling hazard finds; Then he who many cities knew and many minds, And men once world-noised, now mere Ossian forms Of misty memory, bade them live anew As when they shared earth’s manifold delight, In shape, in gait, in voice, in gesture true, And, with an accent heightening as he warms, Would stop forgetful of the shortening night, Drop my confining arm, and pour profuse Much worldly wisdom kept for others' use, Not for his own, for he was rash and free, His purse or knowledge all men’s, like the sea. Still can I hear his voice’s shrilling might (With pauses broken, while the fitful spark He blew more hotly rounded on the dark To hint his features with a Rembrandt light) Call Oken back, or Humboldt, or Lamarck, Or Cuvier’s taller shade, and many more Whom he had seen, or knew from others' sight, And make them men to me as ne’er before: Not seldom, as the undeadened fibre stirred Of noble friendships knit beyond the sea, German or French thrust by the lagging word, For a good leash of mother-tongues had he. At last, arrived at where our paths divide, “Good night!” and, ere the distance grew too wide, “Good night!” again; and now with cheated ear I half hear his who mine shall never hear.
Sometimes it seemed as if New England air For his large lungs too parsimonious were, As if those empty rooms of dogma drear Where the ghost shivers of a faith austere Counting the horns o’er of the Beast, Still scaring those whose faith in it is least, As if those snaps o' th' moral atmosphere That sharpen all the needles of the East, Had been to him like death, Accustomed to draw Europe’s freer breath In a more stable element; Nay, even our landscape, half the year morose, Our practical horizon grimly pent, Our air, sincere of ceremonious haze, Forcing hard outlines mercilessly close, Our social monotone of level days, Might make our best seem banishment; But it was nothing so; Haply his instinct might divine, Beneath our drift of puritanic snow, The marvel sensitive and fine Of sanguinaria over-rash to blow And trust its shyness to an air malign; Well might he prize truth’s warranty and pledge In the grim outcrop of our granite edge, Or Hebrew fervor flashing forth at need In the gaunt sons of Calvin’s iron breed, As prompt to give as skilled to win and keep; But, though such intuitions might not cheer, Yet life was good to him, and, there or here, With that sufficing joy, the day was never cheap; Thereto his mind was its own ample sphere, And, like those buildings great that through the year Carry one temperature, his nature large Made its own climate, nor could any marge
Traced by convention stay him from his bent: He had a habitude of mountain air; He brought wide outlook where he went, And could on sunny uplands dwell Of prospect sweeter than the pastures fair High-hung of viny Neufchâtel; Nor, surely, did he miss Some pale, imaginary bliss
Of earlier sights whose inner landscape still was Swiss. V. 1.
I cannot think he wished so soon to die With all his senses full of eager heat, And rosy years that stood expectant by To buckle the winged sandals on their feet, He that was friends with earth, and all her sweet Took with both hands unsparingly: Truly this life is precious to the root, And good the feel of grass beneath the foot; To lie in buttercups and clover-bloom, Tenants in common with the bees, And watch the white clouds drift through gulfs of trees, Is better than long waiting in the tomb; Only once more to feel the coming spring As the birds feel it when it bids them sing, Only once more to see the moon Through leaf-fringed abbey-arches of the elms Curve her mild sickle in the West Sweet with the breath of hay-cocks, were a boon Worth any promise of soothsayer realms Or casual hope of being elsewhere blest; To take December by the beard And crush the creaking snow with springy foot, While overhead the North’s dumb streamers shoot, Till Winter fawn upon the cheek endeared, Then the long evening-ends Lingered by cosy chimney-nooks, With high companionship of books Or slippered talk of friends And sweet habitual looks, Is better than to stop the ears with dust: Too soon the spectre comes to say, “Thou must!”
When toil-crooked hands are crost upon the breast, They comfort us with sense of rest; They must be glad to lie forever still; Their work is ended with their day; Another fills their room; ’tis the World’s ancient way, Whether for good or ill; But the deft spinners of the brain, Who love each added day and find it gain, Them overtakes the doom
To snap the half-grown flower upon the loom (Trophy that was to be of life-long pain), The thread no other skill can ever knit again. ’Twas so with him, for he was glad to live, ’Twas doubly so, for he left work begun; Could not this eagerness of Fate forgive Till all the allotted flax were spun? It matters not; for, go at night or noon, A friend, whene’er he dies, has died too soon, And, once we hear the hopeless He is dead, So far as flesh hath knowledge, all is said.
VI. 1.
I seem to see the black procession go: That crawling prose of death too well I know, The vulgar paraphrase of glorious woe; I see it wind through that unsightly grove, Once beautiful, but long defaced With granite permanence of cockney taste And all those grim disfigurements we love: There, then, we leave him: Him? such costly waste Nature rebels at: and it is not true
Of those most precious parts of him we knew: Could we be conscious but as dreamers be, ’Twere sweet to leave this shifting life of tents S k i th h l l f D it
Sunk in the changeless calm of Deity; Nay, to be mingled with the elements, The fellow-servant of creative powers, Partaker in the solemn year’s events, To share the work of busy-fingered hours, To be night’s silent almoner of dew, To rise again in plants and breathe and grow, To stream as tides the ocean caverns through, Or with the rapture of great winds to blow About earth’s shaken coignes, were not a fate To leave us all-disconsolate; Even endless slumber in the sweetening sod Of charitable earth
That takes out all our mortal stains, And makes us cleanlier neighbors of the clod, Methinks were better worth Than the poor fruit of most men’s wakeful pains, The heart’s insatiable ache: But such was not his faith, Nor mine: it may be he had trod Outside the plain old path of God thus spake, But God to him was very God, And not a visionary wraith Skulking in murky corners of the mind, And he was sure to be Somehow, somewhere, imperishable as He, Not with His essence mystically combined, As some high spirits long, but whole and free, A perfected and conscious Agassiz. And such I figure him: the wise of old Welcome and own him of their peaceful fold, Not truly with the guild enrolled Of him who seeking inward guessed Diviner riddles than the rest, And groping in the darks of thought Touched the Great Hand and knew it not; Rather he shares the daily light, From reason’s charier fountains won,
,
Of his great chief, the slow-paced Stagyrite, And Cuvier clasps once more his long-lost son.
2.
The shape erect is prone: forever stilled
The winning tongue; the forehead’s high-piled heap, A cairn which every science helped to build, Unvalued will its golden secrets keep: He knows at last if Life or Death be best: Wherever he be flown, whatever vest The being hath put on which lately here So many-friended was, so full of cheer
To make men feel the Seeker’s noble zest, We have not lost him all; he is not gone To the dumb herd of them that wholly die; The beauty of his better self lives on In minds he touched with fire, in many an eye He trained to Truth’s exact severity; He was a Teacher: why be grieved for him Whose living word still stimulates the air? In endless file shall loving scholars come The glow of his transmitted touch to share, And trace his features with an eye less dim Than ours whose sense familiar wont makes numb.
F , I , February, 1874.
TO HOLMES
ON HIS SEVENTY-FIFTH BIRTHDAY
D Wendell, why need count the years Since first your genius made me thrill, If what moved then to smiles or tears,
Or both contending, move me still?
What has the Calendar to do With poets? What Time’s fruitless tooth With gay immortals such as you Whose years but emphasize your youth?
One air gave both their lease of breath; The same paths lured our boyish feet; One earth will hold us safe in death, With dust of saints and scholars sweet.
Our legends from one source were drawn, I scarce distinguish yours from mine, And don’t we make the Gentiles yawn With “You remembers?” o’er our wine!
If I, with too senescent air, Invade your elder memory’s pale, You snub me with a pitying “Where Were you in the September Gale?”
Both stared entranced at Lafayette, Saw Jackson dubbed with LL. D. What Cambridge saw not strikes us yet As scarcely worth one’s while to see.
Ten years my senior, when my name In Harvard’s entrance-book was writ, Her halls still echoed with the fame Of you, her poet and her wit.
’Tis fifty years from then to now: But your Last Leaf renews its green, Though, for the laurels on your brow (So thick they crowd), ’tis hardly seen.
The oriole’s fledglings fifty times Have flown from our familiar elms;
Have flown from our familiar elms; As many poets with their rhymes Oblivion’s darkling dust o’erwhelms.
The birds are hushed, the poets gone Where no harsh critic’s lash can reach, And still your wingëd brood sing on To all who love our English speech.
Nay, let the foolish records be That make believe you’re seventy-five: You’re the old Wendell still to me,— And that’s the youngest man alive.
The gray-blue eyes, I see them still, The gallant front with brown o’erhung, The shape alert, the wit at will, The phrase that stuck, but never stung.
You keep your youth as yon Scotch firs, Whose gaunt line my horizon hems, Though twilight all the lowland blurs, Hold sunset in their ruddy stems.
You with the elders? Yes, ’tis true, But in no sadly literal sense, With elders and coevals too, Whose verb admits no preterite tense.
Master alike in speech and song Of fame’s great antiseptic—Style, You with the classic few belong Who tempered wisdom with a smile.
Outlive us all! Who else like you
Could sift the seedcorn from our chaff, And make us with the pen we knew Deathless at least in epitaph?
W , August 29, 1884.
IN A COPY OF OMAR KHAYYÁM.
T pearls of thought in Persian gulfs were bred, Each softly lucent as a rounded moon; The diver Omar plucked them from their bed, Fitzgerald strung them on an English thread.
Fit rosary for a queen, in shape and hue, When Contemplation tells her pensive beads Of mortal thoughts, forever old and new. Fit for a queen? Why, surely then for you!
The moral? Where Doubt’s eddies toss and twirl Faith’s slender shallop till her footing reel, Plunge: if you find not peace beneath the whirl, Groping, you may like Omar grasp a pearl.
ON RECEIVING A COPY OF MR. AUSTIN DOBSON’S “OLD WORLD IDYLLS.”
At length arrived, your book I take To read in for the author’s sake; Too gray for new sensations grown, Can charm to Art or Nature known This torpor from my senses shake?
Hush! my parched ears what runnels slake? Is a thrush gurgling from the brake? Has Spring, on all the breezes blown, At length arrived?
Long may you live such songs to make, And I to listen while you wake, With skill of late disused, each tone Of the Lesboum barbiton, At mastery, through long finger-ache, At length arrived. II.
As I read on, what changes steal O’er me and through, from head to heel? A rapier thrusts coat-skirt aside, My rough Tweeds bloom to silken pride,— Who was it laughed? Your hand, Dick Steele!
Down vistas long of clipt charmille Watteau as Pierrot leads the reel; Tabor and pipe the dancers guide As I read on.
While in and out the verses wheel The wind-caught robes trim feet reveal, Lithe ankles that to music glide, But chastely and by chance descried; Art? Nature? Which do I most feel
As I read on?
TO C. F. BRADFORD
ON THE GIFT OF A MEERSCHAUM PIPE.
T pipe came safe, and welcome too, As anything must be from you; A meerschaum pure, ’twould float as light As she the girls call Amphitrite. Mixture divine of foam and clay, From both it stole the best away: Its foam is such as crowns the glow Of beakers brimmed by Veuve Clicquot; Its clay is but congested lymph Jove chose to make some choicer nymph; And here combined,—why, this must be The birth of some enchanted sea, Shaped to immortal form, the type And very Venus of a pipe
And very Venus of a pipe.
When high I heap it with the weed From Lethe wharf, whose potent seed Nicotia, big from Bacchus, bore And cast upon Virginia’s shore, I’ll think,—So fill the fairer bowl And wise alembic of thy soul, With herbs far-sought that shall distil, Not fumes to slacken thought and will, But bracing essences that nerve To wait, to dare, to strive, to serve.
When curls the smoke in eddies soft, And hangs a shifting dream aloft, That gives and takes, though chance-designed, The impress of the dreamer’s mind, I’ll think,—So let the vapors bred By Passion, in the heart or head, Pass off and upward into space, Waving farewells of tenderest grace, Remembered in some happier time, To blend their beauty with my rhyme.
While slowly o’er its candid bowl
The color deepens (as the soul That burns in mortals leaves its trace Of bale or beauty on the face),
I’ll think,—So let the essence rare Of years consuming make me fair; So, ’gainst the ills of life profuse, Steep me in some narcotic juice; And if my soul must part with all That whiteness which we greenness call, Smooth back, O Fortune, half thy frown, And make me beautifully brown!
Dream-forger, I refill thy cup
With reverie’s wasteful pittance up
With reverie s wasteful pittance up, And while the fire burns slow away, Hiding itself in ashes gray, I’ll think,—As inward Youth retreats, Compelled to spare his wasting heats, When Life’s Ash-Wednesday comes about, And my head’s gray with fires burnt out, While stays one spark to light the eye, With the last flash of memory, ’Twill leap to welcome C. F. B., Who sent my favorite pipe to me.
BANKSIDE.
(HOME OF EDMUND QUINCY.)
D , M 21, 1877.
I.
I christened you in happier days, before These gray forebodings on my brow were seen; You are still lovely in your new-leaved green; The brimming river soothes his grassy shore; The bridge is there; the rock with lichens hoar; And the same shadows on the water lean, Outlasting us. How many graves between That day and this! How many shadows more Darken my heart, their substance from these eyes Hidden forever! So our world is made Of life and death commingled; and the sighs Outweigh the smiles, in equal balance laid: What compensation? None, save that the All-wise So schools us to love things that cannot fade.
