生物化学与分子生物学学习课件：蛋白质的生物合成 2

PARTI:ProteinSynthesisComponentsPARTII:ProteinSynthesisProcessPARTIII:ProteinSynthesisRegulationPARTIV:PosttranslationalProcessingandTargetingPARTV:ProteinSynthesisinMedicinePARTI:ProteinSynthesisComp1PARTIV:PosttranslationalProcessingandTargeting生物化学与分子生物学学习课件：蛋白质的生物合成22FromDNAtoproteinFromDNAtoprotein34FateofnascentpolypeptidesAssociationofnascentpolypeptidewithvariouschaperonesystemscommitsthemtofoldingpathwaysIfproteinsfailtofold(onethirdofthenascentpolypeptides),theyarerecognizedandtargetedfordegradation4FateofnascentpolypeptidesAThemacromoleculesassistingtheformationofproteinsecondarystructureincludeMolecularChaperonProteinDisulfideIsomerase(PDI)PeptideProlylCis-transIsomerase(PPI)4.1ProteinFoldingNewlysynthesizedpolypeptidesmustformhigherorderstructurebeforetheybecomefunctionalproteinsThemacromoleculesassistingt56ProteinsareassistedinfoldingbymolecularchaperonesHeatshockproteins,Hsp60,Hsp70andHsp90arethreemainclassesHsp70recognizesexposed,unfoldedregionsofnewproteinchains-especiallyhydrophobicregionsItbindstotheseregions,protectingthemuntilproductivefoldingreactionscanoccur4.2.1Chaperons6ProteinsareassistedinfoldMechanismhollowcylinderHsp60Mechanismhollowcylinder7GroELformstwostacked7-memberedringsof60kDsubunits;GroESisadomeonthetopTheGroES-GroELComplex(Hsp60)GroELformstwostacked7-memb89ModeloftheGroELcylinder(blue)inactionNascentproteinapparentlybindsreversiblymanytimestothewallsofthedonutstructure,eachtimedrivenbyATPhydrolysis,eventuallyadoptingitsfoldedstructure,thenbeingreleasedfromtheGroES-GroELcomplex9ModeloftheGroELcylinder(10EucaryoticHsp90ChaperonesHsp90saccountfor1-2%oftotalcytosolicproteinSignaltransductionmoleculessuchastyrosinekinasereceptors,steroidhormonereceptors,non-receptortyrosinekinasesareclientsforHsp9010EucaryoticHsp90ChaperonesH生物化学与分子生物学学习课件：蛋白质的生物合成2114.2.2ProteindisulfiteisomerasespatterndisulfidebondsCellularenvironmentisreducingCysteinesareoxidizedtoformS-SbondsIneukaryotes,proteindisulfideisomerase,PDI,usedforoxidationintheendoplasmicreticulum(ER)4.2.2Proteindisulfiteisomera12Thecorrectivedisulfideisveryimportantforthestructureofsecretedproteinsandmembraneproteins.Thecorrectivedisulfideisve134.2.3Peptidylprolylisomerization14IsomerizationofprolinesMostpeptidebondsaretrans(100xmorestablethancis).Whensecondresidueisproline,transformisonly4xmorestableInnativeproteins,cis-prolinepeptidesarestabilizedbytertiarystructurebutinunfoldedstatethereisanequilibriumbetweencisandtransisomersCis-transisomerizationofprolinepeptides,catalyzedbyPPI,istherate-limitingstepinfoldingforsomeproteins4.2.3PeptidylprolylisomerizaCyclosporineAandCyclophilinFK506andFKBP12CyclosporineAandCyclophilin154.3PosttranslationalProcessingandModificationsofNewlySynthesizedPolypeptidesN-andC-terminusprocessingand/ormodificationTrimmingofpeptidesthroughproteolyticcleavageCovalentmodificationofsomeaminoacids(phosphorylation,methylation,acetylation,glycosylation…)4.3PosttranslationalProces16N-andC-terminusprocessingand/ormodificationRemovingN-fMet,(inprokaryotes)orN-Met(ineukaryotes),orseveralaminoacidresiduesattheN-terminal(inbothprokaryotesandeukaryotes)AcetylationofN-terminalaminoacidresidueinmorethan50%eukaryoticproteinC-terminalprocessingmayalsooccursN-andC-terminusprocessinga17Methionineaminopeptidase2FumagillinAnti-angiogenesisacitivityMethionineaminopeptidase2Fum18POMC,apolypeptidehormoneprecursor,iscleavedintodifferentpeptidehormonesindifferenttissues.NCSignalpeptideKRKR103peptideACTH

-LT-MSH

-MSHEndophinPolypeptidesmayundergoproteolyticprocessingtoproduceseveralpeptidesPOMC,apolypeptidehormonepr19PosttranslationalModificationWhatisit?AdditionofgroupsordeletionofpartstomakeafinishedproteinWhatgroups?Howmuch?Where?-methyl-acetyl-glyco-phosphoAndmanymore…Whatpurpose?-targeting(eg.somelipoproteins)-stability(eg.secretedglycoproteins)-function(eg.surfaceglycoproteins)-controlofactivity(eg.clottingfactors,caspases)PosttranslationalModification20HistoneModificationHistoneModification21HistoneCodeHypothesisHistonescanbemodifiedbypost-translationalmodifications(PTMs),includingacetylation,methylation,phosphorylationandubiquitination(mainlyinN-terminal)Thehistonecodehypothesis:specificPTMsregulategeneexpressionbytwomechanisms:(1)changingthechromatinstructureintoactivatedorrepressedtranscriptionalstate(2)actingasadockingsitefortranscriptionalregulatorsH3K4meH3K36meH3K9meH3K27meEuchromatinHeterochromatinLowacetylationH3K9me,H3K27me,H4K20meHighacetylationH3K4me,H3K36me,H3K79meHistoneCodeHypothesisHistone22PhosphorylationP-SITE:S/T/YPhosphorylationP-SITE:23Amplificationorover-expressionofHER2in30%breastcancerTherapy:monoclonalantibodytrastuzumab(Herceptin)Pertuzumab,whichinhibitsdimerizationofHER2andHER3receptorstamoxifenAmplificationorover-expressi24ProteinGlycosylation

CommoninEukaryoticProteins25NitrogenAsparagineSerineandThreonineProteinGlycosylation

Commoni生物化学与分子生物学学习课件：蛋白质的生物合成226N-LinkedGlycansN-linkedglycansarecovalentlyattachedtoAsnresidueswithinaconsensussequence(Asn-Xaa-Ser/Thr),enablingpredictionofthemodificationsitesbyproteinsequenceanalysisAllN-linkedglycansshareacommonpentasaccharidecore(GlcNAc2Man3)recognizedbylectinsandN-glycanaseenzymes(PNGaseF)ThesereagentshavebeenusedtovisualizeproteinsbearingN-linkedglycansfromcellortissuelysatesandtoenrichthemformassspectrometryanalysisN-LinkedGlycansN-linkedglyca27O-LinkedGlycansComparabletoolsarelackingforthestudyofproteinsbearingO-linkedglycans.Mucin-type,themostprevalentO-linkedglycosylationischaracterizedbyanN-acetylgalactosamine(GalNAc)residue-linkedtothehydroxylgroupofSerorThr.GalNAcresidueisinstalledbyafamilyof24N-acetyl-galactosaminyltransferases,thenfurtherelaboratedbyaseriesofglycosyltransferasestogeneratehigher-orderO-linkedstructures.Becauseofthecomplexbiosyntheticorigin,O-linkedglycansarenotinstalledatadefinedconsensusmotifandtheirpresencecannotbeaccuratelypredictedbasedontheprotein'sprimarysequenceO-LinkedGlycansComparabletoo28Thecorrectlyfoldedproteinsneedtobetransportedtospecialcellularcompartmentstoexertdesiredbiologicalfunctions.AAssequenceontheN-terminusthatdirectsproteinstobetransportedtopropercellulartargetsitesiscalledsignalsequence.4.4ProteinTargetingThecorrectlyfoldedproteins29SignalsequencesAllthesecretoryproteinshavethesignalsequences.Consistof13-36AAinthreeregionsPositivelychargedAAatN-terminusHydrophobiccoreof10-15AAinthemedialregionSmallpolarAAatC-terminusSignalsequencesAllthesecret30SignalsequenceforERCleavagesiteSignalsequenceforERCleavage31a.SecretedproteinintoERSignalrecognitionparticle(SRP)a.SecretedproteinintoERSig32生物化学与分子生物学学习课件：蛋白质的生物合成233生物化学与分子生物学学习课件：蛋白质的生物合成234ERmembraneERmembrane35生物化学与分子生物学学习课件：蛋白质的生物合成236生物化学与分子生物学学习课件：蛋白质的生物合成237RibosomedissociatesRibosomedissociates38生物化学与分子生物学学习课件：蛋白质的生物合成239SecretoryproteinsaretransportedtothecellperipherySecretoryproteinsaretranspo40b.MitochondrialproteinMitochondrialproteinsincytosolarepresentinprecursorforms.Signalsequenceof20-25AAatN-terminusarerichinSer,Thr,andbasicAA.b.MitochondrialproteinMitoch41b.Mitochondrialproteinb.Mitochondrialprotein42c.NuclearproteinK-K/R-X-K/RNuclearLocalizationSequencec.NuclearproteinK-K/R-X-K/R43D.NuclearExportSignalLXXXLXXLXLD.NuclearExportSignalLXXXLX44PARTV:ProteinSynthesisinMedicinePARTV:451.MoleculardiseasesareassociatedwithdefectiveproteinsMutationsongenecodingregionsSilentmutations:codeforthesameaminoacid.Missensemutations:codeforadifferentaminoacid.Nonsensemutations:introduceastopcodonandcantruncatetheprotein.AbnormalproteinfoldingCysticfibrosiscausedbydeletionofPhe508inCFTR,resultinginimproperproteinfolding1.Moleculardiseasesareasso46OnlineMendelianInheritanceinMan(OMIM)isadatabasethatcataloguesalltheknowndiseaseswithageneticcomponentOnlineMendelianInheritancei47Uniprot

()Uniprot

(http://www.uniprot.or48RibosomopathiesAtleast50%ofpatientswithDiamond-Blackfananemia(先天性再生障碍性贫血)carrymutationsinribosomalproteinsTreacher-Collinssyndrome,NorthAmericanIndianchildhoodcirrhosis,chromosome5q-syndromeisolatedcongenitalaspleniaRibosomopathiesAtleast50%of49CongenitalAspleniaInisolatedcongenitalasplenia,themostrecentlydiscoveredribosomopathy,haploinsufficiencyoftheribosomal

proteinRPSApreventssplenicdevelopment.

Thesepatientsare

pronetoseverebacterialinfectionsbecausetheylackaspleen,

buttheyareotherwisehealthy

andhavenootherobservabledevelopmental

anomaliesCongenitalAspleniaInisolated50Diamond-Blackfananemia

(戴-布二氏贫血)InthecaseofDiamond-Blackfananemia,mutationsinanyof11differentribosomalproteinsleadtobonemarrowfailure.Diamond-Blackfananemia

(戴-布二氏贫51CirrhosisInNorthAmericanIndianchildhoodcirrhosis,amutationintheribosomebiogenesisfactorhUTP4/Cirhincausesbiliarycirrhosis,forwhichtheonlytreatmentislivertransplantationrequiredbyearlyadolescence.Littleisknownaboutthemolecularmecha-nismsthatleadtothisdisease.CirrhosisInNorthAmericanInd52Shwachman-Bodian-Diamondsyndrome(许氏症)Shwachman-Bodian-DiamondsyndromearisesfrommutationsintheSBDSprotein,whichisinvolvedinlargeribosomalsubunitmaturation.Patientswiththisdisordersuffernotonlyfromdysfunctionofthepancreas,butalsofrombonemarrowfailure,skeletalabnormalities,andanenlargedliver.Shwachman-Bodian-Diamondsyndr53Treacher-Collinssyndrome

(颌面部骨发育不全综合征)Treacher-CollinssyndromeoftenresultsfromhaploinsufficiencyinTCOF1,thegenethatencodesTreacle,anucleolarproteininvolvedinpre-rRNAsynthesis.Treacher-Collinssyndrome

(颌面部542.Manyvirusesusethehostcellproteinsynthesismachinery

(1)VirusmRNAismoreefficientlytranslatedthanhostcellmRNA.(2)VirusesmakeabundantmRNA.(3)SomevirusescaninhibithostcellmRNAbindingto40Ssubunit.2.Manyvirusesusethehostc553.ProteinSynthesiscanbeinhibitedbyantibioticsandtoxinsTheproteinsynthesisishighlyregulated.Thisprocesscanalsobetheprimarytargetformanytoxins,antibioticsandinterferons.TheinterferonsinteractspecificallywithproteinsandRNAstointerrupttheproteinsynthesis.3.ProteinSynthesiscanbein56AntibioticsAntibiotics57nametargetfunctionTetracycline四环素30SblocktheAsitetopreventbindingofAA-tRNAwith30SStreptomycin链霉素30SInhibitsinitiationChloromycetin氯霉素50Sblockthepeptidyltransferase,andinhibittheelongationCycloheximide放线菌酮60S(Eu)blockthepeptidyltransferase,andinhibittheelongationPuromycin嘌呤霉素50S(Pro)and60S(Eu)releasetheprematuredpeptide,andterminateselongationErythromycin红霉素50SInhibitthetranslocaseAntibioticsnametargetfunctionTetracycline58IthasasimilarstructuretoTyr-tRNA.Itworksforbothprokaryotesandeukaryotes.PuromycinIthasasimilarstructureto59Sometoxins,suchasplantproteinRicin(篦麻毒素),isamongthemosttoxicsubstanceknown,whichactson60ssubunits.ToxinsRicinusCommuniscastor-oilplantSometoxins,suchasplantpro60rRNAN-glycosylaseactivityThericintargetsA4324thatiscontainedinahighlyconservedsequenceof12nucleotidesuniversallyfoundineukaryoticribosomesof60srRNAN-glycosylaseactivity61DiphtheriatoxinDiphtheriatoxinisanexotoxinsecretedbyCorynebacteriumdiphtheriae,thepathogenbacteriumthatcausesdiphtheria.Unusually,thetoxingeneisencodedbyabacteriophage(avirusthatinfectsbacteria).Thetoxinc