2012分子医学课件及复习题蛋白质修饰

ProteinModification

蛋白质修饰Posttranscriptionalmodificationsincluding:RemovalofN-MetDisulfidebondformationProteolyticprocessingChemicalmodifications:

Phosphorylation(磷酸化) Acetylation(乙酰化) Methylation(甲基化) Ubiquitination(泛素化) SUMOylation(smallubiquitin-relatedmodifier) Glycosylation(糖基化) Myristylation(十四烷基化)andFarnesylation(法尼基化) ……Formoreinformation,seeFormoreinformation,seeRegulationofactivity

turnactivityonturnactivityoffgenerateadifferentfunction

Protein-proteininteraction

modificationsitemaybeabindinginterfaceSubcellularlocalization

modificationsitemaybeatargetingsignalmodificationmaybeamembraneanchor

Degradationidentifytheproteinfordegradation……Whyareproteinsmodified?ProteinsneedtobeproperlymodifiedMethodstodetectproteinmodifications1Dor2DgelelectrophoresisWesternBlot):needspecificantibodyChIP:ChIP-on-chip;ChIP-SeqMS(massspectrometry)Bioinformatics……Phosphorylation(磷酸化)MostcommonposttranslationalmodificationtoproteinsineukaryotesEnzymesandregulatorsareturned‘on’and‘off’EnergyfromATPPhosphorylationandsignalingtransductionProteinkinaseProteinphosphatasePhosphorylationsitesinaproteinPhosphorylationchangesPIoftheprotein,whichcanbeeasilydetectedon2DgelsMSanalysisofC/EBPβafterinvitrophosphorylationbyMAPKand/orGSK3β

ProcNatlAcadSciUSA.2005,102(28):9766-71WesternblottingofErkMethodsusedtoanalyzeproteinphosphorylationAcetylation&DeacetylationDefinition:theadditionorremovalofanacetylgroup,eitherattheN-terminusoftheproteinoratlysineresiduesN-alpha-terminalAcetylation:80-90%humanproteins

enzyme:N-alpha-acetyltransferases(NATs) associatedwiththeribosome,wheretheyacetylate nascentpolypeptidesco-translationally

LysineAcetylationandDeacetylation:histoneandnon- histoneproteins enzyme:histoneacetyltransferase(HAT) histonedeacetylase(HDAC)Histonechiefproteincomponentsofchromatin

H1,H2A,H2B,H3,H4,andH5

posttranslationalmodifications:altertheirinteractionwithDNAandnuclearproteins.

H3&H4:longtails;canbemodifiedatseveralplaces,includingmethylation,acetylation,phosphorylation,ubiquitination,sumoylation,citrullinationandADP-ribosylation. ThecoreofthehistonesH2AandH3canalsobemodified.HistoneCode:hypothesizedtobeacodeconsistingofcovalenthistonetailmodifications

→epigeneticcode

PatternofHistoneModificationHistoneacetylation/deacetylationchangesitsinteractionwithDNAHistoneAcetylation/deacetylationAcetylationenhancestranscription

H3K9,H3K14DeacetylationrepressestranscriptionThebiologicalconsequenceofmodificationdepends,andisacoordinatedresultNon-histoneAcetylation/Deacetylationasignificantpost-translationalregulatorymechanism

analogoustophosphorylation

functions:modifyproteinactivity;crosstalkwithphosphorylation,methylation,ubiquitination,sumoylation,andothersfordynamiccontrolofcellularsignaling(seefurtherreading)ProteinAcetylation&SignalTransductionStatAcetylation--AKeyFacetofCytokineSignaling?

supportingdata(Science,2005,307(5707):269-73)

Suspect?(Science,2005,307(5707):217–8)

Figurelegend:(Bottom)crystalstructureofStat3Showingthelocationoflysine685(red)inthetyrosine-phosphorylatedStat3dimer.Thefourrecognizeddomainsarecoiled-coildomain(green);theDNAbindingdomain(red),thelinkerdomain(orange),andtheSH2domain(cyan).Adisorderedloop(blackdottedcurve)connectstheSH2domainonthelefttoTail1(magenta).TwostrandsofDNAareshowninwhiteandblack.(Top)Detailedviewofthedimerinterfaceofthetwotyrosine-phosphorylatedSH2domains(cyan)ofStat3.Twotailsegmentsareshowninmagentaandyellow,andthedottedcurveisadisorderedloop(residues689to701)connectingtheSH2domainonthelefttoitstailsegment.Lysine685isdepictedinredandtyrosine705isshowninmagenta.[Science,2005]ProteinAcetylation&SignalTransductionStatAcetylation--AKeyFacetofCytokineSignaling?

supportingdata(Science,2005,307(5707):269-73) Suspect?(Science,2005,307(5707):217–8)Acetylation-dependentsignaltransductionfortypeIinterferonreceptor

Cell,2007,131(1):93-105

Methylation(甲基化)

DNAmethylation:typicallyoccursatCpGsites

invertebrates catalyzedbytheenzymeDNAmethyltransferase.

Proteinmethylation:typicallyonarginineorlysine Argininecanbemethylatedonceortwiceby

Peptidylargininemethyltransferases(PRMTs)

Lysinecanbemethylatedonce,twiceorthreetimesbylysinemethyltransferases.

DNAMethylationAnalysis

BisulfiteGenomicSequencing

PyrosequencingTheHELPassay(CCGGHpaII-CCGGMspI)ChIP-on-chipassaysMethylatedDNAimmunoprecipitation(MeDIP)MolecularbreaklightassayforDNAadeninemethyltransferaseactivity……Formoreinformation,seeBisulfite-mediatedconversionofC

U

BisulfiteGenomicSequencingNucleotidesinblueareunmethylatedcytosinesconvertedtouracilsbybisulfite,whilerednucleotidesare5-methylcytosinesresistanttoconversion.

DNAmethylationanalysismethodsnotbasedonmethylation-specificPCR

Followingbisulfiteconversion,thegenomicDNAisamplifiedwithPCRthatdoesnotdiscriminatebetweenmethylatedandnon-methylatedsequences.Thenumerousmethodsavailablearethenusedtomakethediscriminationbasedonthechangeswithintheampliconasaresultofbisulfiteconversion.Formoreinformation,see

MethylatedDNAimmunoprecipitation(MeDIP)MethylatedAminoAcidArgLysHistoneMethylationcatalyzedbyhistonemethyltransferases(HMT)

servesinepigeneticgeneregulation:Methylationatdifferentsitesofhistonesmayhave

differentfunctionongeneexpression

monomethylation:mostlyactivation(Cell,2007,129:823–37)

dimethylation:H3K79me2,activation(Mol.Cell.Biol,2008,28:2825-39)

trimethylation:mostlyrepression(Cell,2007,129:823–37)

HistoneDemethylation

Jumonji-domain-containingproteinsLSD1isagenewhichcodesaflavin-dependentmonoamineoxidase,whichcandemethylatemono-anddimethylatedlysines,specificallyH3K4andH3K9.theJumonjidomain-containing(JmjC)histonedemethylasesareabletodemethylatemono-,di-,ortri-methylatedlysines.

HistoneMethylationCell.2007Oct5;131(1):93-105Acetylation-dependentsignaltransductionfortypeIIFNreceptorNature.2007,450(7167):309-13.ChIPprocedureJHDM1B/FBXL10isanucleolarproteinthatrepressestranscriptionofribosomalRNAgenesMono-ubiquitination-Regulation:

endocytosis,geneexpression,proteinsorting,subnucleartraffickingMulti-ubiquitination-ProteindegradationUbiquitination(泛素化)TheoncoproteinSS18-SSX1promotesp53ubiquitinationanddegradationbyenhancingHDM2stability.TransfectionExampleMolCancerRes.2008Jan;6(1):127-38.SUMOylationSUMO(smallubiquitin-relatedmodifier)proteinsaresmallproteintagsthatareconjugatedtoproteinstomodifytheirfunction.TheubiquitinsystemtagsproteinsfordegradationbytheproteosomebutSUMOconjugationhasarangeofotherfunctions,stabilizingsomeproteinsandalteringtheirsubcellularlocalization.Sumoylationmayalsoinfluenceubiquitinationandproteinstabilityindirectly.ThreedifferentSUMOproteinsareconjugatedtoproteins,SUMO-1,SUMO-2andSUMO-3.Glycosylation(糖基化)MajorformofproteinmodificationN-linkedglycosylation(Asn)

InitialglycosylationintheER,processingintheGolgiO-linkedglycosylation(Ser,Thr)happenedintheGolgiManydifferentformsandfunctionsN-linkedglycosylationAprecursoroligosaccharideisformedonadolichollipidThisistransferredtothegrowingproteinProcessofN-linkedglycosylationGlycosylationandproteinfoldingbyglucosidaseO-linkedglycosylationFunctionsofglycosylationProperfolding

正确折叠Stabiliseproteinsagainstproteolysis

防止降解FormECM(extracellularmatrix)胞外基质Modulationofimmuneresponse

免疫反应

Selectinsbindtooligosaccharides,cell-cellinteractionsAnchorproteinsonmembrane

锚定作用GPI(Glycosylphosphatidylinositol糖基磷脂酰肌醇)anchoredproteinsGPIanchoredproteinsTheT-cellmarkerThy-1,acetylcholinesterase,intestinalandplacentalalkalinephosphataseMyristylation(十四烷基化)andFarnesylation(法尼基化)AttachescytosolicproteinstotheplasmamembraneProteinusuallyinvolvedinsignaltransductionFirstidentifiedin1993Total14members(11inhuman),andcaspase-2,3,6,7,8,9,and10areknowntobeinvolvedinapoptosisAmilestoneinapoptosisstudyKnownastheexecutionmachinenaryofapoptosisCaspases(cysteine-asparticacidproteases)TwoApoptosisSignalingPathwaysBycelldeathReceptors(extrinsic)Bymitochondria(intrinsic)NCAsp-XAsp-X32-53kDaPredomain(3-24kDa)Largesubunit(17-21kDa)Smallsubunit(10-13kDa)CellDeath/ProgrammedCellDeath

necrosis(notprogrammed)

apoptosis(TypeIcelldeath)

autophagy(TypeIIcelldeath)

characterizedbytheformationoflargevacuoleswhich eatawayorganellesinaspecificsequencepriortothe nucleusbeingdestroyed.

caspase-independentprogrammedcell-death

NatureMedicine,2005,11:725–30.

necroptosis

Cell,2008,135:1311-23.

ApoptosisPhenomenonOverviewofsignaltransductionpathwaysinvolvedinapoptosisTheInterfacebetweenApoptosisandProgrammedNecrosisKey:activationoftheserine/threoninekinaseRIP1(receptor-interactingproteinkinase1),Cel