中山大学蛋白质组学总结.docx
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中山大学蛋白质组学总结.docx
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中山大学蛋白质组学总结
I.Introduction
Structure
•Buildingblocks——aminoacids
•Peptidebondandpeptides
•Primarystructureofprotein
•Thethree-dimensionalstructureofproteins
•Gettingproteinstructure
•Integralmembraneproteins
•Relationshipbetweenstructureandfunctionofproteins
Function
•Principleingeneral
•Proteinfolding,misfoldinganddisease
Regulation
*Expression
*Transportation&Localization
*Modification(Phosphorylation,glycosylation,methylation,acetylation,andubiquitin)
*Interaction
*Degradation
Levelsofstructure
1°:
Aminoacidresiduesequence
2°:
Structuralelements
3°:
Specific3-dimensionalstructure
4°:
Arrangementofsubunitsinmulti-subunitprotein
Note
• 16%N
•Metformetaboliclabelling
•Trp,Tyr,andtoalesserextentPhe,absorbultravioletlight.Thisaccountsforthecharacteristicstrongabsorbanceof•lightbyproteinsatawavelengthof280nm.
•Counttheaminoacidnumbersinprotein. 138 128 110
•symbol
•Nonstandardaminoacids (hydroxyproline,hydroxylysine)
•Someadditionalaminoacids selenocysteine(硒半胱氨酸)andpyrrolysine(吡咯赖氨酸)
Chemicalsynthesisofpeptides
• Liquid(Solution)phasepeptidesynthesis
• Solidphasepeptidesynthesis(SPPS)
• Pre-treatmentofaminoacids
• Formationofpeptidebond
• Deprotect,hydrolyzeandpurification
Differencebetweenbiological andchemicalsynthesisofpeptides
∙Template
∙Orientation生物NC化学CN
∙Efficiency(Aproteinwith100aminoacids:
Chemicalsynthesis--about4days;Biologicalprocess--about5secondsinE.coli)
∙Condition
∙size
Sequence
∙Sanger1953胰岛素
∙反推DNA(简并性)
∙抗原抗体核糖体测mRNA
∙测一部分比对数据库
Motifs(units)
structuremotifs
1)Helix-loop-helix2αhelicesjoinedbybridge
2)βhairpin2adjacentantiparallelβstrandsconnectedbyaβ-turn.
3)β−α−βloopAnαhelixservesastheconnectionbetween2parallelβstrands.
functionmotifs Thr-Gly-Tyr,Thr-Pro-Tyrmotif
Domains
1)Allαdomainsrepeatofhelix-loop-helixmotif
2)Allβdomainsrepeatofβhairpingivesanti-parallelβstructures
3)α/βdomainsrepeatofβ−α−βmotiftogiveparallelβstructures
4)α+βdomainsTheα-helicesandβ-sheetsarerelativelyseparate.
介于2级与三级结构modular evolutionunionofnewfunction
分子识别 Molecularrecognition
1.不同组分识别(四级结构)
2.内吞等受体、配体标记
3.抗原抗体
4.酶与底物、酶抑制剂
5.疫苗
地中海贫血
胰蛋白酶Trypsin
II.Proteinfolding,misfoldinganddisease
能量最低的天然构象
Thirdstep
∙Folding
∙Modification
∙Assemblyandoligomerization
∙TransportationandLocalizaion
∙Thedeterminants:
secondarystructure
When:
Co-transationalPost-translational
Where:
Ribosome,Cytoplasm,Specificcompartments,MitochondrionorER
Molecularchaperines(分子伴侣)=Heatshockprotein(Hsp)
Function:
I.preventmisfolding
II.rescuemisfoldingandevenaggregatedproteins
ERhasacrucialquality-controlroleandhowtodescriminate
I.dependprimarilyonERchaperones
II.monitorstheirarchitecturaldesignthroughubiquitousfoldingsensors.
III.cell-specificfactorsandfacilitateexportofindividualproteinsorclassesofproteins
IV.aremarkableseriesofglycosylationanddeglycosylationreactions.
Relatedtodiseases
1.错误折叠影响细胞功能 囊性纤维化
2.aggregate沉淀 amyloid淀粉样蛋白
Alzheime'sandParkinson'sdiseases,thespongiformencephalopathiesandtypeIIdiabetes
Leadtorapiddegradation
I.Structuralalterations
II.Mutation
III.Errorsintranscriptionortranslation
Proteindegradation
1.ubiquitin–proteasomepathway
2.ER-associateddegradation
III.Regulationofproteinfunctions
Expression
Level TemporalSpatial
Localizationandtranslocation
Cellulardestinationofproteins
amino-terminalsignalsequence
nuclearlocalizationsequence,NLS
Modificationconsequence activationchange&signaltransduction
1.Conformationchange
2.Translocation
3.Proteininteraction
Interaction
Degradation
IV.ProteinPhosphorylation Regulation&Advantages
pk=proteinkinase
onethirdproteins Molecularswitch
Regulation:
1.
1.Increasingordecreasingthebiologicalactivityofanenzyme.byconformationchange
2.Helpingmoveproteinsbetweensubcellularcompartments.
3.Allowinginteractionsbetweenproteinstooccur.
4.Labelingproteinsfordegradation.
Target:
enzymes,structuralproteins,cellreceptors,ionchannelsandsignalingmolecules.
Advantages:
rapid,norequirenewprotein,easilyreversible
phosphorylationcascade:
Serine/threoninekinasesandTyrosinekinases
Classification:
aminoacidstobephosphorylated;substratespecificity; regulators
Serine/threonineproteinkinase:
PKA cAMP-dependent
PKB/Akt
PKC cyclicnucleotide-independent
PKD PKCmu growthfactor/stress-inducedsignaling
RegulationofReceptortyrosinekinases:
Dimerization Trans-autophosphorylation
Effectsofphosphorylationon adaptorproteinfunction:
1.
1.APthatundergoaconformationalchangeinresponsetoTP.
2.STPof APaltersitsconformationandsubsequentfunction.
3.STPof APrecruitsinabindingpartnerforit.
4.STPof APenableittobecomeabindingpartnerforAP.
5.APthatareSTPbutfunctionofphosphorylationremainstobedetermined.
V.Glycosylation
polysaccharides 原因、功能与意义 三种类型特点与修饰位点
第一个糖基是哪里什么糖,O-修饰:
单糖,位置
Thedonormoleculeisanactivatednucleotidesugar
Functions:
★
I.preventuncorrectfold(3.4th)
II.conferstability.(secretedglycoproteins)半衰期
III.cell-celladhesion粘附免疫系统细胞
IV.involvecellsignalling
Mechanismscommonfeatures
I.enzymaticprocess
II.sitespecific
III.donormolecule=activatednucleotidesugar
N-linkedglycosylation
consensussequenceAsn-Xaa-Ser/Thr
Modificationandmaturation
N-glycancommonpentasaccharidecore.GlcNAc
14-sugarprecursor(firstaddedtotheasparagine)=3glucose,9mannose,and2N-acetylglucosaminemolecules
Processofbiosynthesis
1.beginwithGlcNAc
2. coreissynthesizedfirstlinkedtoDol-Pandindependentoftheproteinthatwillbeglycosylatedinalaterstep.
3.precursormoleculestartsatthecytosolicsiteofRERwiththeattachmentofGlcNAcfromUDP-GlcNActoDol-P.
4.SubsequentlytherestofthecoresugarsbecomeaddedtothemoleculestepbysteptoyieldManp5-GlcNAcp2.
5.ThisstructurestaysthenattachedtoP-P-DolbutistransferredtothelumensiteoftheRERwhereadditionalprocessingoccursbeforetheactualglycosylationoftheproteintakesplacecotranslationally.
degreedependenton:
1.theavailableamountofcompletelyglucosylatedprecursors,
2.oligosaccharyltransferaseactivity
3.thenumberofavailableAsn-X-Thr/Sersitesintheproteinandtheirconformationalaccessibility.
O-linkedglycosylation
DifferenttypesofO-linkedoligosaccharidesarefoundinnature.
tissue-specific GalNAc-T
exposedSer/Thr
startingwithanGalNActothehydroxylsidechainofaSer/Thr
FunctionsofMucin-TypeO-LinkedGlycans:
1.inintramolecularinteractions,maintainingthe2ndand3rdstructureofglycoproteins.
2.inintermolecularinteractions,stabilizingthe4thstructureofglycoproteincomplexes.
3.inmediaterecognition.(ABObloodgroupantigens,recognitionofglycopeptidesbytheMHC-complexorbyantibodies)
4.inmodulationoftheactivityofenzymesandsignalingmolecules.
5.inproteinexpressionandprocessing.
O-GlcNAcMeetstheRequirementsforsignaltransductionmodifications
O-linkedGlcNAcglycan
∙monosaccharidemodification
∙onSer/Thr residues
∙nucleocytoplasmicproteins.
∙dynamic,inducible,Reversible
∙analternativetothephosphorylationofintracellularproteins
O-phosphorylation阴阳
VI.Ubiquitination&ProteinDegradation 降解途径、过程
Effect:
∙Tosupplyaminoacidsforfreshproteinsynthesis.
∙Toremoveexcessenzymes.
∙Toremovetranscriptionfactorsthatarenolongerneeded.
2majorintercellulardevices
Lysosomes dealprimarilywith
extracellularproteins, e.g.,plasmaproteins,takenintothecellbyendocytosis.
cell-surfacemembraneprotein, inreceptor-mediatedendocytosis
proteasomes dealprimarilywithendogenousproteins(selective)
∙Transcriptionfactors.
∙Cyclins(whichmustbedestroyedtoprepareforthenextstepinthecellcycle).
∙Proteinsencodedbyvirusesandotherintracellularparasites.
∙Proteinsthatarefoldedincorrectly.
hollowcylinder(空心圆筒):
regulatoryparticle+core(20S)+regulatoryparticle(19S)
activitiesareassociatedwiththreeoftheβsubunits
(chymotrypsin-like,trypsin-like,post-glutamylactivity)
ubiqutinasmallprotein:
76aa
Conservedthroughoutallthekingdomsoflife;
Totargetproteinfordestructionorotherfatestarget
Synthesizedaspolyprotein(cleavageofprecursors)
C-terminusformisopeptidebondwithaminogroupoflysinesidechain inatargetprotein.
Activationandconjugationofubiquitin
E1E2E3
∙Proteasomesdegradeproteinsthatarerecognizedbytheirubiquitintags.
∙Proteinsaretaggedfordegradationbyspecificubiquitinconjugatingsystems.
∙Regulationofubiquitinconjugationcanregulatetheamountsofspecificproteins,allowingthesetochangerapidly.
∙Proteinubiquitylationclearlyplaysafundamentalroleincellularfunction.
∙WhileLys48-linkedpolyubiquitinchainsarerequiredforcontrolledproteindegradation,otherubiquitinmodificationshavediversefunctionsinnumerouscellularprocesses.
∙Infact,itisnowbecomingevidentthatubiquitylationmaybeaspervasive(普遍的)asphosphorylationasamechanismforcontrollingproteinfunction.
彭宣宪
第一章.导论
I.keyconcept
理论基础体系+研究方法=学科
传统的蛋白质研究vs蛋白质组学
个体 整体
分析静态性质 比较动态变化
孤立个体 相互作用
结构 功能
小规模、非连续 高通量、自动化
II.development
后基因组时代标志主要任务
1860发现FriedrichMiescher
1967测序EDmanandBegg
1970双向电泳=分子聚焦+凝胶电泳 KenrickandMargolis
1972 X-raycrystallogr
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