生化-郑利民课件信号转导2－生化

1,Signal-TransductionPathways信号转导通路(2)郑利民2008珠海zhenglm,2,Biosignaling:Howaretheyregulated?,3,凋亡细胞,细胞凋亡：细胞死亡的主要方式；受机体严密调控。凋亡细胞的清除：主要被巨噬细胞（Mf）吞噬；摄取普通的凋亡细胞能主动抑制Mf活化安全清除凋亡细胞，维持组织自身稳态的机制,巨噬细胞摄取凋亡细胞后，通过分泌多种细胞因子（TGFb，IL10，PGE2等）并减少TNFa等，抑制免疫应答,细胞凋亡和免疫应答,4,Apoptoticcell,已知：粒细胞是清除入侵病原的主力军；多种病原均可引起粒细胞凋亡。Q：这类诱导凋亡是病原的致病策略?消灭这些关键免疫细胞，利用它们作为“特洛依木马”来感染并灭活M,5,UV照射或细菌感染诱导人粒细胞凋亡,UV,S.aureus,E.coli,6,7,大肠杆菌或金葡菌诱导的凋亡粒细胞刺激巨噬细胞产生TNF和上调CD64表达,8,采用多种不同方式诱导粒细胞凋亡，发现：照射或老化所致的凋亡细胞能抑制巨噬细胞活化而病原感染所致的凋亡细胞能显著激活巨噬细胞,抗原递呈细胞（APC）,UV或老化,凋亡,?,凋亡粒细胞,感染,趋炎症反应抗原递呈,凋亡,J.Immunol.2002,168:6358-65；2004,173:6319-26,9,这两类凋亡粒细胞对M的活化具截然相反的作用（细胞固有一死，但死的意义有不同）说明：粒细胞凋亡是调控免疫和炎症反应的新型防御机制早期，吞噬受感染的凋亡细胞可激活M，帮助控制病原；后期，过剩的粒细胞发生凋亡并抑制M活性，炎症消退。,Question：M如何识别这两类凋亡细胞并作出相反应答？,10,抗原递呈细胞（APC）,UV或老化,凋亡,凋亡,HSP,凋亡粒细胞,感染或热敏,趋炎症反应抗原递呈,1.凋亡细胞中的危险信号,如HSP，而不是这些细胞所相伴的细菌，决定了APC的免疫应答类型。2.它们可作为天然佐剂，来研制新型抗肿瘤和感染疫苗,J.Immunol.2004,173:6319-6326,11,病原菌经常是通过干扰细胞的信号通路而致病致病性沙门氏菌能侵入巨噬细胞并诱导其凋亡；非致病性沙门氏菌经调理后也能进入巨噬细胞，但不能诱导其凋亡,12,SopE,PKB/Akt,Rac,CDC42,PTK,PI-3K,R,调理后吞噬,病原侵袭,死亡信号,生存信号,细胞凋亡和粘膜屏障损坏,细胞存活,细胞凋亡是由：细胞内“死亡/生存”信号之间的精密平衡来决定干扰该平衡就可改变病原对细胞凋亡的最终影响,病原侵袭和吞噬对巨噬细胞凋亡的影响及其机制,13,细胞存活,细胞凋亡,Thebalancebetweenpro-andanti-apoptoticgenes/signalsdeterminethecellfate,细胞接受到“死亡信号”，不一定就会死亡若同时也接受到“生存信号”，就可继续存活,14,PART1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPART1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways,15,RegulatorymechanismsofBio-signals,3.1Phosphorylationasaregulatorymechanism3.2Regulationoftranscriptionbysteroidhormones3.3Regulationofthecellcyclebyproteinkinases,16,RegulationofSignalingPathways,Externalsignals,Secondmessengers,Modulatorproteins,Function,Targetproteins,HormonescAMPOdorantscGMPDrugsCa2+LightDAGIP3MitogenichormonesTyrosineGrowthkinasesfactors,proteinkinaseAndphosphatase,structuralproteinsAndenzymes,metabolicorphysiologicalresponses,Plasmamembrane,17,ProteinKinases1:GPCRs(5%),2:Kinases(2.8%genome),25%ofthemammalianintracellularproteinsundergoesreversiblephosphorylation,18,Proteinkinases(534humankinases),Ser/thrKinasescomprise80%ofallproteinkinases,AGC,19,Phosphorylation,32,Manyproteinsactastargetingoranchoringmediatorse.g.AKAPassociatestightlywiththeregulatorysubunitsofPKACellscontainmanydifferentisoformsofPKC,whicharelocalizedbyanchoringproteins,e.g.RACKsorreceptorsforactivatedPKCPhosphatasesarealsooftenlocalizedcloselytoensurethatintegratedcontrolispossible.,33,Mechanismsusedbyhormones,retinoids,andVit.Dtoregulategeneexpression,Hormone(H),carriedtothetargettissueonserumbindingprot,diffusesacrossthePMandbindstoitsspecificreceptorprotein(Rec)inthenucleus.,HbindingchangestheconformationofRec;formshomoorheterodimersandbindstospecificRregionscalledhormoneresponseelements(HREs)intheDNAadjacenttospecificgenes,Bindingregulatestranscriptionoftheadjacentgene(s),increasingordecreasingtherateofmRNAformation.,Alteredlevelsofthehormoneregulatedgeneproductproducethecellularresponsetothehormone,34,3.3.1Thecellcycle,Whatiscellcycle?Aprocessofcellularreproduction,35,36,Fourphases:G1,S,G2,MGap(G1andG2)phases:Respondtoproliferativeandanti-proliferativesignals,synthesizeRNAandproteinSynthetic(S)phase:ReplicationofthechromosomesMitotic(M)phase:segregationofchromosomesandothercellconstituentsintotwodaughtercells,37,Cellcycletransitsorderlyandirreversibly,38,Cellcycletransitsorderlyandirreversibly,Thecompletionofonephaseisrequiredforthebeginningofthenext.Periodicactivationofspecificcyclin-CdkcomplexesAcycleofproteindestructioneliminatesproteinsusedintheprecedingphaseaswellasproteinsthatwouldinhibitprogressionintothenextphase.,39,Cyclin-dependentkinases(Cdks),Cdksdrivecellcycleprogressionbyphosphorylatingagroupofsubstrates,-CdkspresentauniquepatternofactivityineachphaseofthecellcycleCdk1(Cdc2)Cdk2Cdk3Cdk4Cdk5Cdk6Cdk7,G1,G2,M,LateG1,S,G1,S,G2,M,40,RegulationofCdksactivityCyclin:Activatingsubunit.,Cyclinswerenamedbecauseoftheircyclicexpressionduringthecellcycle,41,FourmechanismsregulateCDKactivity:1phosphorylationordephosphorylation,2controlleddegradationofcyclinsubunit,3periodicsynthesisofCDKsandcyclins,4actionofspecificCDKinhibitingproteins.,42,RegulationofCdksactivityPhosphorylationanddephospho.inCdk1,Activatingphosphorylationsite:Thr161Inhibitoryphosphorylationsite:Thr14,Thr15,P,P,43,Growthcontrollingsignalsareconveyedbystimulatoryorinhibitorypathways,44,sometargetproteinsofCDKs:Laminin-BreakdownofnuclearenvelopebeforesegregationofsisterchromatidsinmitosisispartlyduetoPi-lamininbyCDKActinmyosincontractilemachinery-InvolvedinpinchingadividingcellintotwoequalpartsduringcytokinesisRetinoblastomaprotein,pRb-participatesinamechanismthatarrestscelldivisioninG1,CDKsRegulateCellDivisionbyPhosphorylatingCriticalProteins,45,Summary,FourbasiccharactersofbiosignalsFourschemesofintercellularsignalingFourgeneraltypesofsignaltransducersgatedionchannels;receptorenzymes;G-proteincoupledreceptors；SteroidReceptorsGproteins:HeterotrimericActivationanddeactivationmechanisms,46,Majorsecondmessengers:cAMP,IP3,DAG,Ca2+,cGMP.Usinganexampletoexplainthecascadeofevents:asinglemoleculeofhormoneactivatescatalystthatinturnactivatesanothercatalyst,andsoon,resultsinsignalamplificationandcellresponseThecellcycleanditsregulation,47,PART1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPART1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways,信号转导异常的原因和机制,一、信号转导异常1.生物学因素：干扰细胞内信号转导通路:如霍乱毒素2.理化因素：电离辐射或机械刺影响信号转导成分而致癌3.遗传因素：信号蛋白数量/功能改变（突变）4.免疫学因素：刺激型和阻断型抗受体抗体5.内环境因素,49,二、信号转导异常的发生环节,Gs,ADH,H2O,H2O,配体、受体或受体后信号通路的任何一个环节出现障碍都会影响到最终效应，使细胞增殖、分化、凋亡、代谢或功能失常，并导致疾病。以尿崩症为例,50,ADH作用的三个环节异常均可导致尿崩症：ADH分泌减少中枢性尿崩症ADH-V2受体变异肾小管上皮细胞水通道蛋白（AQP2）异常,集合管上皮细胞对ADH的反应性降低,家族性尿崩症,不同受体介导的信号转导通路存在cross-talk并非所有的信号转导蛋白异常都能导致疾病,51,细胞信号转导异常与疾病,受体、信号转导障碍与疾病受体数量减少受体亲和力降低受体阻断型抗体的作用协同因子或辅助因子缺陷受体功能缺陷受体后信号转导蛋白缺陷,特定信号转导过程减弱或中断,激素抵抗征,52,雄激素受体缺陷与雄激素抵抗征,原因和机制：AR减少或失活突变,男性假两性畸形特发性无精症症延髓脊髓性肌萎缩,53,胰岛素受体与胰岛素抵抗性糖尿病,1.遗传性胰岛素受体异常，包括受体的合成减少与配体的亲和力降低，如Arg735突变为SerTPK活性降低，如Gly1008突变等2.自身免疫性胰岛素受体异常血液中存在抗胰岛素受体的抗体,54,二、受体、信号转导过度激活与疾病,某些信号转导蛋白过度表达某些信号转导蛋白组成型激活突变刺激型抗受体抗体,生长激素（GH）分泌过多的垂体腺瘤中，30%是由于编码Gs的基因突变所致，从而抑制了GTP酶活性，使Gs处于持续激活状态，cAMP含量增多，垂体细胞生长和分泌功能活跃。,通路过度激活,如肢端肥大症和巨人症,55,霍乱毒素选择性的催化Gs亚基上的Arg201核糖化，使GTP酶活性丧失，不能将GTP水解成GDP，从而使Gs处于不可逆激活状态,不断刺激AC生成cAMP,胞浆中的cAMP含量可增加至正常的100倍以上，导致小肠上皮细胞膜蛋白构型改变，大量Cl-和水分子持续转运入肠腔，引起严重腹泻和脱水。,56,霍乱(Cholera),CT：CholeraToxin,57,三、多环节信号转导异常与疾病,肿瘤1.促细胞增殖的信号转导过强生长因子产生增多受体的改变生长因子受体表达异常增多突变使受体组成型激活细胞内信号转导蛋白的改变如Ras突变,58,2.抑制细胞增殖的信号转导过弱,生长抑制因子受体减少、丧失受体后信号转导通路异常,细胞的生长负调控机制减弱或丧失,59,Oncogene-encodeddefectiveEGFreceptor,60,GEF：GuaninenucleotideExchangeFactorGAP：GTPaseActivatingProtein,ActivationofRasprotein,61,GEF：GuaninenucleotideExchangeFactorGAP：GTPaseActivatingProtein,62,肿瘤形成必须是在单个细胞中发生多重遗传突变单个癌基因激活和抑癌基因失活不足以引起肿瘤细胞的显著扩增，因为细胞对凋亡的易感性也同时增加；凋亡抑制是肿瘤细胞能够生长到足以威胁宿主所必须的；例如：Rb的失活仅会促进p53依赖的凋亡，但不会引起肿瘤，除非细胞的凋亡过程被抑制；,Apoptosisandoncogenesis,63,3.3.3Oncogenes,TumorSuppressorGenes(TSGs),andProgram