蛋白質(zhì)領(lǐng)域新進(jìn)展與研究新技術(shù)

蛋白質(zhì)領(lǐng)域新進(jìn)展與研究新技術(shù)蛋白質(zhì)研究熱門領(lǐng)域蛋白質(zhì)結(jié)構(gòu)與（新）功能

Sirtuin

（SIRT1-7）：metabolism,aging,etc

TrendsEndocrinol

Metab.2014,25(3):138-45.蛋白質(zhì)（新）修飾及其功能組蛋白賴氨酸二羥基異丁?；捌鋵臃只恼{(diào)控NatChemBiol.2014,10(5):365-70.蛋白質(zhì)翻譯調(diào)控

Curr

Opin

StructBiol.2015,32:74-80.Science.2015,347(6228):1367-671.（一）蛋白質(zhì)修飾ProteinPosttranslationalModifications

(PTMs)PTMsinvolvingadditionoffunctionalgroupsPTMsinvolvingadditionofotherproteinsorpeptides

ISGylation;SUMOylation;ubiquitination;NeddylationPTMsinvolvingchangingthechemicalnatureofaminoacids

citrullination;deamidation;eliminylation;carbamylationPTMsinvolvingstructuralchangesdisulfidebridges;proteolyticcleavage;racemizationFormoreinformation,seehttp:///wiki/Posttranslational_modificationAdditionofFunctionalGroupstoProteinPTMsinvolvingadditionbyanenzymeadditionofhydrophobicgroupsformembranelocalizationadditionofcofactorsforenhancedenzymaticactivityuniquemodificationsoftranslationfactorsadditionofsmallerchemicalgroupsPTMsinvolvingnon-enzymaticadditions:

glycationProteinModificationsProtein:

Histoneornon-histoneModificationsites:

Phosphorylation:

serine,threonine,andtyrosine(O-linked),orhistidine(N-linked)Acetylation:lysineMethylation:lysineorarginineLysineNewModificationslysinepropionylation丙酰化（MolCellProteomics,2007）lysinebutyrylation丁?；∕olCellProteomics,2007）lysine2-hydroxyisobutyrylation賴氨酸二羥基異丁?；?/p>

（NatChem

Biol,2014）lysinesuccinylation琥珀酰化（NatChem

Biol,2011；

MolCellProteomics,2012；MolCell,2013）lysinecrotonylation巴豆?；–ell,2011）lysinemalonylation丙二酰化（MolCellProteomics,2011，

2012）lysineglutarylation戊二?；揎棧–ellMetab,2014）ProteinacylationisbalancedbyKDACsandKATsMolCellProteomics.2015Formoreinformation,see/wiki/Posttranslational_modificationRegulationofactivity

turnactivityonturnactivityoffgenerateadifferentfunction

Protein-proteininteraction

modificationsitemaybeabindinginterfaceSubcellularlocalization

modificationsitemaybeatargetingsignalmodificationmaybeamembraneanchor

Degradationidentifytheproteinfordegradation……Whyproteinsneedtobemodified?Methodstodetectproteinmodifications1Dor2DgelelectrophoresisWesternblot:needspecificantibodyEasternblot:usingprobestodetectlipids,carbohydrate,phosphorylationoranyotherproteinmodificationChIP:ChIP-on-chip;ChIP-SeqMS(massspectrometry)Bioinformatics……PhosphorylationchangesPIoftheprotein,whichcanbeeasilydetectedon2DgelsMSanalysisofC/EBPβafterinvitrophosphorylationbyMAPKand/orGSK3β

ProcNatlAcadSciUSA.2005,102(28):9766-71WesternblottingofErkDetectionMethods:examples1、組蛋白修飾Histonechiefproteincomponentsofchromatin

H1,H2A,H2B,H3,H4,andH5

posttranslationalmodifications:altertheirinteractionwithDNAandnuclearproteins.

H3&H4:longtails;canbemodifiedatseveralplaces,includingmethylation,acetylation,phosphorylation,ubiquitination,sumoylation,citrullinationandADP-ribosylationetc. ThecoreofthehistonesH2AandH2Bcanalsobemodified.HistoneCode:hypothesizedtobeacodeconsistingofcovalenthistonetailmodifications

→epigeneticcode

HistoneVariants組蛋白修飾一般研究流程usetrypsintodigestthehistoneproteins(trypticpeptides)high-performanceLC(HPLC)/MS/MSanalyzewiththePTMapsoftware,anonrestrictivesequencealignmentalgorithmthatenablesdetectionofmassshiftcausedbyaPTMidentifythemodifiedHpeptide(s)andmassshiftvalueaccordingtothismassshift,proposethereasonableelementalcompositionaccordingtotheformula,proposepossiblestructuresforthemodificationverificationfunctionstudyStructorPredicationBasedonMassShiftValueNatChemBiol,2014

如何研究組蛋白修飾的功能？NatRevMolCellBiol.2014,15(8):509-24.點(diǎn)突變TE：phosphorylationmimicKQ：acetylationmimicSemisyntheticmethods+

peptideligationtheuseofsyntheticbiologytospecificallyintroducemodifiedresidues賴氨酸二羥基異丁酰化修飾位點(diǎn)多。組蛋白中已鑒定63個(gè)位點(diǎn)，遠(yuǎn)高于已知的組蛋白乙酰化位點(diǎn)數(shù)目。在乙?；瘓?bào)道后的40年多年里(1964-2006)，一共才鑒定了大約100個(gè)組蛋白翻譯后修飾位點(diǎn)。截至目前為止，趙英明課題組已鑒定200多個(gè)組蛋白修飾位點(diǎn)。分布廣。大多數(shù)乙?；稽c(diǎn)主要存在于組蛋白的N-端，而二羥基異丁?；粌H存在于組蛋白的N-端，而且也廣泛存在于組蛋白的C-端。豐度高?；赟ILAC技術(shù)的定量分析表明，組蛋白賴氨酸二羥基異丁?；稽c(diǎn)的豐度比乙?；稽c(diǎn)高。動態(tài)變化。二羥基異丁?；诩?xì)胞有絲分裂的不同時(shí)期、精子細(xì)胞減數(shù)分裂的不同階段等生物學(xué)過程中處于動態(tài)變化狀態(tài)。lysine2-hydroxyisobutyrylationPatternofHistoneModificationPatternofHistoneModificationSitesofHistoneModifications/nsmb/journal/v14/n11/images/nsmb1337-F1.gifHistoneCodeCell,2013,155:391.1組蛋白乙?；疕istoneAcetylation/deacetylationAcetylationenhancestranscription

H3K9,H3K14DeacetylationrepressestranscriptionThebiologicalconsequenceofmodificationdepends,andisacoordinatedresultHistoneacetylation/deacetylationchangesitsinteractionwithDNA1.2組蛋白甲基化ProteinMethylation

typicallyonarginineorlysinearginine

canbemethylatedonceortwiceby

Peptidylarginine

methyltransferases(PRMTs)lysine

canbemethylatedonce,twiceorthreetimesbylysinemethyltransferases.

MethylatedAminoAcidArg(R)Lys(K)HistoneMethylationcatalyzedbyhistonemethyltransferases(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)

HistoneMethylation1.3組蛋白修飾的功能Coordinatedrecruitmentofhistone-modifyingactivitiesThefunctionsofHistoneModificationsNatRevMolCellBiol.2014,15(8):509-24.Tailmodifications:regulateallDNA-templatedprocesses,includingreplication,transcriptionandrepairserveasplatformsfortherecruitmentofspecificeffectorproteins,suchastranscriptionalregulatorsorchromatinremodellersCoremodifications:haveadirecteffectonnucleosomalarchitectureaffectinghistone–histoneandhistone–DNAinteractions,aswellasthebindingofhistonestochaperonesOverviewofhistonecore-modificationsNatRevMolCellBiol.2014,15(8):509-24.Basicprincipleofmodification-inducedalterationsofnucleosomearchitectureandstabilityNatRevMolCellBiol.2014,15(8):509-24.Potentialeffectsofhistonemodificationsonhistone–DNAandhistone–histone

interactionsNatRevMolCellBiol.2014,15(8):509-24.HistonemethylationaffectschaperonebindingNatRevMolCellBiol.2014,15(8):509-24.HistoneModificaitonsandCancerHistoneModificationsandHumanDiseasesCoffin-Lowrysyndromeisararegeneticdisordercharacterizedbymentalretardationandabnormalitiesoftheheadandfacialandotherareas.ItiscausedbymutationsintheRSK2gene(histonephosphorylation)andisinheritedasanX-linkeddominantgenetictrait.Malesareusuallymoreseverelyaffectedthanfemales.Rubinstein-Taybisyndromeischaracterizedbyshortstature,moderatetosevereintellectualdisability,distinctivefacialfeatures,andbroadthumbsandfirsttoes.ItiscausedbymutationsinCREB-bindingprotein(histoneacetylation)Non-histoneproteinmethylationaprevalentpost-translationalmodificationanimportantregulatorofcellularsignaltransductionmediatedbytheMAPK,WNT,BMP,HippoandJAK-STATsignallingpathwayscrosstalkbetweenmethylationandothertypesofPTMs,andbetweenhistoneandnon-histoneproteinmethylation.affectscellularfunctionssuchaschromatinremodelling,genetranscription,proteinsynthesis,signaltransductionandDNArepairNatRevMolCellBiol.2015,16(1):5-17.Mono-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.Homework了解細(xì)胞內(nèi)主要的蛋白質(zhì)修飾方式及其功能FurtherReadingHersheyMD,ZhaoY.Metabolicregulationbylysinemalonylation,

succinylationandglutarylation.MolCellProteomics.2015Feb25.[Epubaheadofprint]TessarzP,KouzaridesT.Histonecoremodificationsregulatingnucleosomestructureanddynamics.NatRevMolCellBiol.2014,15(11):703-8.3.KimuraH.Histonemodificationsforhumanepigenomeanalysis.JHumGenet.2013,58(7):439-45.4.CarlsonSM,GozaniO.Emergingtechnologiestomaptheprotein

methylome.JMolBiol.2014,426(20):3350-62.（二）蛋白質(zhì)翻譯調(diào)控Proteinsynthesis(Translation)Initiation:Smallsubunitandlargesubunitform80Sribosomeatstartcodon.Elongation:RibosomedecodesmRNA.Termination:Releaseofribosomeatthestopcodon.AAAAAAAAAAAAInitiation

Elongation

TerminationPolysome–Poly-ribosomeSeveralribosomescantranslateanmRNAatthesametime,formingwhatiscalledapolysome.ItcanproducemanypolypeptidessimultaneouslyfromasinglemRNA.Moreribosomes=EfficientThemoreribosomesonspecificmRNA,thehighertranslationefficiencyLiu,B.etal.Adv

Nutr.2011.However,wedon’tknowthepositionsofribosmesonmRNA!PolyribosomeprofilingRibosomeprofiling(Ribosomefootprinting)Ingolia,NT.etal.Science.2009.FreezeribosomebyCHXIngolia,NT.etal.NatRevGenet.2014.RibosomeprofilingapplicationsIngolia,NT.etal.NatRevGenet.2014.Evaluatetranslationalefficiency(ribosomedensity)1RibosomeprofilingapplicationsIngolia,NT.etal.NatRevGenet.2014.Monitorribosomestalling/pausingonmRNA2Whatistheeffectofproteotoxicstressonproteintranslation?LiuB.etal.MolCell.2013IntracellularproteotoxicstresstriggersearlyelongationpausingofribosomesIngolia,NT.etal.NatRevGenet.2014.3Detectco-translationalevents(selectiveribosomeprofiling)RibosomeprofilingapplicationsNatRevMolCellBiol.2010.11:113-27.4IdentifytranslationinitiationsitesInitiationisthelimitingstepoftranslationRibosomeprofilingapplicationsInitiationdoesnotnecessarilystartfromAUGconservedbasesaroundtheinitiationcodonScience.2012;336(6089):1719-23.Alternativeinitiationmaygenerateproductswithdifferentlocalization,functionet.alProperselectionofthetranslationinitiationsite(TIS)onmRNAsiscrucialfortheproductionofdesiredproteinproductsAlternativeinitiation:differentisoformsfromonetranscriptScience.2013;341(6144):399-402.Howtoidentifytranslationinitiationsite?Globaltranslationinitiationsequencing(GTI-seq)GlobalIdentificationofTISsbyGTI-seqAnnotatedTISMis-annotatedTISDownstreamTISUpstreamTISLeeS,etal.

ProcNatlAcadSciUSA.2012GlobalIdentificationofTISsbyGTI-seqModifiedGTI-seqforinvivoNatureMethods12,147–153(2015)SmallProteinsTranslatedfrom:shortORFsinsupposedlynon-codingRNAsuntranslatedregions(UTRs)oftranscriptsalternativeORFs(AltORFs)withincanonicalmRNAsFunctions:distinctroles:chemokines(8-10kD);humanin(24aa);…regulateotherproteinsandtheirinteractionsCurrentOpinioninStructuralBiology2015,32:74–80PossibleOriginsofSmallProteinsCurrentOpinioninStructuralBiology2015,32:74–80FunctionalrolesoftranscriptsandproteinsCurrentOpinioninStructuralBiology2015,32:74–80FurtherReadingLandryCR,ZhongX,Nielly-ThibaultL,RoucouX.Foundintranslation:functionsandevolutionofarecentlydiscoveredalternativeproteome.Curr

Opin

StructBiol.2015,32:74-80.GaoX,WanJ,LiuB,MaM,ShenB,QianSB.Quantitativeprofilingofinitiatingribosomesinvivo.NatMethods.2015,12(2):147-53.LiuB,QianSB.Translationalreprogrammingincellularstressresponse.WileyInterdiscipRevRNA.2014,5(3):301-15.（三）蛋白質(zhì)結(jié)構(gòu)研究技術(shù)蛋白質(zhì)結(jié)構(gòu)測定方法X射線晶體衍射分析X-rayCrystallography核磁共振技術(shù)Nuclearmagneticresonance質(zhì)譜技術(shù)電鏡三維重構(gòu)技術(shù)：

掃描電子顯微鏡

冷凍電子顯微鏡Cryo-electronmicroscopy(cryoEM)蛋白質(zhì)結(jié)構(gòu)數(shù)據(jù)庫（proteindatabank，PDB）Exp.Method

Proteins

NucleicAcids

Protein/NAComplexes

Other

Total

X-RAY8062115024194486321NMR90241072197710300ELECTRONMICROSCOPY508511700729HYBRID5732163other1554613178Total903652