生化鄭利民信號(hào)轉(zhuǎn)導(dǎo)2－生化課08第1頁(yè)/共70頁(yè)2Signal-TransductionPathways

信號(hào)轉(zhuǎn)導(dǎo)通路(2)

第2頁(yè)/共70頁(yè)3Biosignaling:Howaretheyregulated?第3頁(yè)/共70頁(yè)4凋亡細(xì)胞細(xì)胞凋亡：細(xì)胞死亡的主要方式；受機(jī)體嚴(yán)密調(diào)控。凋亡細(xì)胞的清除：主要被巨噬細(xì)胞（Mf）吞噬； 攝取普通的凋亡細(xì)胞能主動(dòng)抑制Mf活化安全清除凋亡細(xì)胞，維持組織自身穩(wěn)態(tài)的機(jī)制巨噬細(xì)胞攝取凋亡細(xì)胞后，通過(guò)分泌多種細(xì)胞因子（TGFb，IL10，PGE2等）并減少TNFa等，抑制免疫應(yīng)答細(xì)胞凋亡和免疫應(yīng)答第4頁(yè)/共70頁(yè)5Apoptoticcell已知：粒細(xì)胞是清除入侵病原的主力軍； 多種病原均可引起粒細(xì)胞凋亡。Q：這類誘導(dǎo)凋亡是病原的致病策略???

消滅這些關(guān)鍵免疫細(xì)胞，利用它們作為“特洛依木馬”來(lái)感染并滅活MΦ第5頁(yè)/共70頁(yè)6UV照射或細(xì)菌感染誘導(dǎo)人粒細(xì)胞凋亡UVS.aureusE.coli第6頁(yè)/共70頁(yè)7第7頁(yè)/共70頁(yè)8大腸桿菌或金葡菌誘導(dǎo)的凋亡粒細(xì)胞刺激巨噬細(xì)胞產(chǎn)生TNF和上調(diào)CD64表達(dá)第8頁(yè)/共70頁(yè)9采用多種不同方式誘導(dǎo)粒細(xì)胞凋亡，發(fā)現(xiàn)：照射或老化所致的凋亡細(xì)胞能抑制巨噬細(xì)胞活化而病原感染所致的凋亡細(xì)胞能顯著激活巨噬細(xì)胞抗原遞呈細(xì)胞（APC）UV或老化凋亡???凋亡粒細(xì)胞感染趨炎癥反應(yīng)抗原遞呈凋亡J.Immunol.2002,168:6358-65；2004,173:6319-26第9頁(yè)/共70頁(yè)10這兩類凋亡粒細(xì)胞對(duì)MΦ的活化具截然相反的作用（細(xì)胞固有一死，但死的意義有不同）說(shuō)明：粒細(xì)胞凋亡是調(diào)控免疫和炎癥反應(yīng)的新型防御機(jī)制早期，吞噬受感染的凋亡細(xì)胞可激活MΦ，幫助控制病原；后期，過(guò)剩的粒細(xì)胞發(fā)生凋亡并抑制MΦ活性，炎癥消退。Question：MΦ如何識(shí)別這兩類凋亡細(xì)胞并作出相反應(yīng)答？第10頁(yè)/共70頁(yè)11抗原遞呈細(xì)胞（APC）UV或老化凋亡凋亡HSP凋亡粒細(xì)胞感染或熱敏趨炎癥反應(yīng)抗原遞呈1.凋亡細(xì)胞中的危險(xiǎn)信號(hào),如HSP，而不是這些細(xì)胞所相伴的細(xì)菌，決定了APC的免疫應(yīng)答類型。2.它們可作為天然佐劑，來(lái)研制新型抗腫瘤和感染疫苗J.Immunol.2004,173:6319-6326第11頁(yè)/共70頁(yè)12病原菌經(jīng)常是通過(guò)干擾細(xì)胞的信號(hào)通路而致病致病性沙門(mén)氏菌能侵入巨噬細(xì)胞并誘導(dǎo)其凋亡；非致病性沙門(mén)氏菌經(jīng)調(diào)理后也能進(jìn)入巨噬細(xì)胞，但

不能誘導(dǎo)其凋亡第12頁(yè)/共70頁(yè)13SopEPKB/AktRac,CDC42PTKPI-3K

R調(diào)理后吞噬病原侵襲死亡信號(hào)生存信號(hào)細(xì)胞凋亡和粘膜屏障損壞細(xì)胞存活細(xì)胞凋亡是由：細(xì)胞內(nèi)“死亡/生存”信號(hào)之間的精密平衡來(lái)決定干擾該平衡就可改變病原對(duì)細(xì)胞凋亡的最終影響病原侵襲和吞噬對(duì)巨噬細(xì)胞凋亡的影響及其機(jī)制第13頁(yè)/共70頁(yè)14細(xì)胞存活細(xì)胞凋亡Thebalancebetweenpro-andanti-apoptoticgenes/signalsdeterminethecellfate細(xì)胞接受到“死亡信號(hào)”，不一定就會(huì)死亡若同時(shí)也接受到“生存信號(hào)”，就可繼續(xù)存活第14頁(yè)/共70頁(yè)15PARTⅠ1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPARTⅡ1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways第15頁(yè)/共70頁(yè)16RegulatorymechanismsofBio-signals3.1Phosphorylationasaregulatorymechanism3.2Regulationoftranscriptionbysteroidhormones3.3Regulationofthecellcyclebyproteinkinases第16頁(yè)/共70頁(yè)17RegulationofSignalingPathwaysExternalsignalsSecondmessengersModulatorproteinsFunctionTargetproteinsHormonescAMPOdorants

cGMPDrugsCa2+LightDAG IP3Mitogenichormones TyrosineGrowth kinasesfactorsproteinkinaseAndphosphatasestructuralproteinsAndenzymesmetabolicorphysiologicalresponsesPlasmamembrane第17頁(yè)/共70頁(yè)18ProteinKinases1:

GPCRs

(5%),

2:

Kinases

(2.8%genome)

~25%

ofthemammalianintracellularproteinsundergoesreversiblephosphorylation第18頁(yè)/共70頁(yè)19Proteinkinases(534humankinases)Ser/thrKinasescomprise80%ofallproteinkinasesAGC第19頁(yè)/共70頁(yè)20Phosphorylation&de-phosphorylationarethemostcommonregulatorymechanisms,mediatedbyproteinkinaseandphosphatase,respectively

蛋白激酶

nNTPnNDP蛋白質(zhì)蛋白質(zhì)-nPi

nPi 蛋白磷酸酶

H2O第20頁(yè)/共70頁(yè)21NobelPrizeinPhysiologyandMedicine1992EldwinG.KrebsEdmondH.Fischer

USA

“Reversibleproteinphosphorylationasabiologicalregulatorymechanism"J.Biol.Chem.216:121-132,1955第21頁(yè)/共70頁(yè)22cAMPPKAPhosporylatingcellularproteinsResponseActivationofGProtein–CoupledReceptors第22頁(yè)/共70頁(yè)23GEF：GuaninenucleotideExchangeFactorGAP：GTPaseActivatingProteinActivationofRasprotein第23頁(yè)/共70頁(yè)24SHCGrb2GEFRas-GTPFRas-GDPFPiGAPSHCGrb2GEFRas-GTPFRas-GDPFPiGAPRas蛋白的上游和下游信號(hào)通路第24頁(yè)/共70頁(yè)25PI3K的活化機(jī)制及PIP3磷酸酶(PI3KisadownstreamtargetofRas)第25頁(yè)/共70頁(yè)26PI3K－AKT途徑對(duì)細(xì)胞生存的調(diào)控機(jī)制第26頁(yè)/共70頁(yè)27Two-componentsystemorsignaling第27頁(yè)/共70頁(yè)28鈍化蛋白活化蛋白

激素受體PIP2

磷脂酶C

PKCDAG

G－蛋白

IP3

細(xì)胞反應(yīng)

Ca2+IP3敏感通道結(jié)合態(tài)IP3

內(nèi)質(zhì)網(wǎng)或液泡 細(xì)胞反應(yīng)

Ca2+細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)的雙信使系統(tǒng)

第28頁(yè)/共70頁(yè)29Mostofthesecomplicatedsignalingpathwaysaretransducedbythephosphorylationanddephosphoralationofsignalingprotein,regulatedbytheproteinkinasesandphosphatses第29頁(yè)/共70頁(yè)30蛋白激酶（proteinkinase，PK）植物和酵母中～2%的基因編碼蛋白激酶；而在哺乳動(dòng)物中，高達(dá)5－8％。根據(jù)磷酸化靶蛋白的氨基酸殘基的種類不同，蛋白激酶有絲氨酸/蘇氨酸激酶、酪氨酸激酶和組氨酸激酶等三類。部分蛋白激酶具有雙重底物特異性，既可使絲氨酸或蘇氨酸殘基磷酸化，又可使酪氨酸殘基磷酸化第30頁(yè)/共70頁(yè)31蛋白磷酸酶proteinphosphatase，PP蛋白磷酸酶的分類與蛋白激酶相對(duì)應(yīng)，分為絲氨酸/蘇氨酸型蛋白磷酸酶和酪氨酸型蛋白磷酸酶。有些酶具有雙重底物特異性對(duì)蛋白磷酸酶的研究還不如蛋白激酶那樣深入。但兩者的協(xié)同作用在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中的作用是不言而喻的。

第31頁(yè)/共70頁(yè)32PhosphorylationasaRegulatoryMechanismSignaltransductionAlteredkinaseactivitycGMPcAMPInsulinCa2+kinase

Thereare120kinasesinyeast–i.e. 2%oftheirentiregenomeSomekinasesarelocalizedtodiscretesubcellularregionsthatareincloseproximitytothespecifictargetprotein;第32頁(yè)/共70頁(yè)33

Manyproteinsactastargetingoranchoringmediatorse.g.AKAPassociatestightlywiththeregulatorysubunitsofPKACellscontainmanydifferentisoformsofPKC,whicharelocalizedbyanchoringproteins,e.g.RACKsorreceptorsforactivatedPKCPhosphatasesarealsooftenlocalizedcloselytoensurethatintegratedcontrolispossible.第33頁(yè)/共70頁(yè)34Mechanismsusedbyhormones,retinoids,andVit.DtoregulategeneexpressionHormone(H),carriedtothetargettissueonserumbindingprot,diffusesacrossthePMandbindstoitsspecificreceptorprotein(Rec)inthenucleus.HbindingchangestheconformationofRec;formshomoorheterodimersandbindstospecificRregionscalledhormoneresponseelements(HREs)intheDNAadjacenttospecificgenesBindingregulatestranscriptionoftheadjacentgene(s),increasingordecreasingtherateofmRNAformation.Alteredlevelsofthehormoneregulatedgeneproductproducethecellularresponsetothehormone第34頁(yè)/共70頁(yè)353.3.1ThecellcycleWhatiscellcycle?

Aprocessofcellularreproduction

第35頁(yè)/共70頁(yè)36第36頁(yè)/共70頁(yè)37Fourphases:G1,S,G2,MGap(G1andG2)phases:Respondto

proliferativeandanti-proliferative

signals,synthesizeRNAandproteinSynthetic(S)phase:Replicationofthe

chromosomesMitotic(M)phase:segregationof

chromosomesandothercellconstituentsintotwodaughtercells第37頁(yè)/共70頁(yè)38Cellcycletransitsorderlyandirreversibly第38頁(yè)/共70頁(yè)39CellcycletransitsorderlyandirreversiblyThecompletionofonephaseisrequiredforthebeginningofthenext.Periodicactivationofspecificcyclin-CdkcomplexesAcycleofproteindestruction

eliminatesproteinsusedintheprecedingphaseaswellasproteinsthatwouldinhibitprogressionintothenextphase.第39頁(yè)/共70頁(yè)40Cyclin-dependentkinases(Cdks)Cdksdrivecellcycleprogression

byphosphorylatinga

groupofsubstrates

-Cdkspresentauniquepatternofactivityineach

phaseofthecellcycle Cdk1(Cdc2) Cdk2 Cdk3 Cdk4Cdk5 Cdk6 Cdk7G1G2,MLateG1,SG1,S,G2,M第40頁(yè)/共70頁(yè)41RegulationofCdksactivity

Cyclin:Activatingsubunit.

CyclinswerenamedbecauseoftheircyclicexpressionduringthecellcycleMitogenStimulation第41頁(yè)/共70頁(yè)42FourmechanismsregulateCDKactivity:1phosphorylationordephosphorylation,2controlleddegradationofcyclinsubunit,3periodicsynthesisofCDKsandcyclins,4actionofspecificCDKinhibitingproteins.第42頁(yè)/共70頁(yè)43RegulationofCdksactivity

Phosphorylationanddephospho.inCdk1

Activatingphosphorylationsite:Thr161Inhibitoryphosphorylationsite:Thr14,Thr15PP第43頁(yè)/共70頁(yè)44Growthcontrollingsignalsareconveyedbystimulatoryorinhibitorypathways第44頁(yè)/共70頁(yè)45sometargetproteinsofCDKs:Laminin-BreakdownofnuclearenvelopebeforesegregationofsisterchromatidsinmitosisispartlyduetoPi-lamininbyCDKActinmyosincontractilemachinery----InvolvedinpinchingadividingcellintotwoequalpartsduringcytokinesisRetinoblastomaprotein,pRb--participatesinamechanismthatarrestscelldivisioninG1CDKsRegulateCellDivisionbyPhosphorylatingCriticalProteins第45頁(yè)/共70頁(yè)46Summary

FourbasiccharactersofbiosignalsFourschemesofintercellularsignalingFourgeneraltypesofsignaltransducers

gatedionchannels;receptorenzymes;G-proteincoupledreceptors；SteroidReceptorsGproteins:Heterotrimeric&monomeric; Activationanddeactivationmechanisms第46頁(yè)/共70頁(yè)47Majorsecondmessengers:cAMP,IP3,DAG,Ca2+,cGMP.Usinganexampletoexplainthecascadeofevents:asinglemoleculeofhormoneactivatescatalystthatinturnactivatesanothercatalyst,andsoon,resultsinsignalamplificationandcellresponseThecellcycleanditsregulation第47頁(yè)/共70頁(yè)48PARTⅠ1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPARTⅡ1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways第48頁(yè)/共70頁(yè)信號(hào)轉(zhuǎn)導(dǎo)異常的原因和機(jī)制一、信號(hào)轉(zhuǎn)導(dǎo)異常1.生物學(xué)因素：干擾細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)通路:如霍亂毒素2.理化因素：電離輻射或機(jī)械刺影響信號(hào)轉(zhuǎn)導(dǎo)成分而致癌3.遺傳因素：信號(hào)蛋白數(shù)量/功能改變（突變）4.免疫學(xué)因素：刺激型和阻斷型抗受體抗體5.內(nèi)環(huán)境因素第49頁(yè)/共70頁(yè)50二、信號(hào)轉(zhuǎn)導(dǎo)異常的發(fā)生環(huán)節(jié)V2RGsACcAMPATPPKAADHH2OH2O配體、受體或受體后信號(hào)通路的任何一個(gè)環(huán)節(jié)出現(xiàn)障礙都會(huì)影響到最終效應(yīng)，使細(xì)胞增殖、分化、凋亡、代謝或功能失常，并導(dǎo)致疾病。以尿崩癥為例第50頁(yè)/共70頁(yè)51ADH作用的三個(gè)環(huán)節(jié)異常均可導(dǎo)致尿崩癥：

ADH分泌減少中樞性尿崩癥

ADH-V2受體變異腎小管上皮細(xì)胞水通道蛋白（AQP2）異常集合管上皮細(xì)胞對(duì)ADH的反應(yīng)性降低家族性尿崩癥不同受體介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路存在cross-talk

并非所有的信號(hào)轉(zhuǎn)導(dǎo)蛋白異常都能導(dǎo)致疾病第51頁(yè)/共70頁(yè)52細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)異常與疾病受體、信號(hào)轉(zhuǎn)導(dǎo)障礙與疾病受體數(shù)量減少受體親和力降低受體阻斷型抗體的作用協(xié)同因子或輔助因子缺陷受體功能缺陷受體后信號(hào)轉(zhuǎn)導(dǎo)蛋白缺陷特定信號(hào)轉(zhuǎn)導(dǎo)過(guò)程減弱或中斷激素抵抗征第52頁(yè)/共70頁(yè)53雄激素受體缺陷與雄激素抵抗征原因和機(jī)制：AR減少或失活突變男性假兩性畸形特發(fā)性無(wú)精癥癥延髓脊髓性肌萎縮第53頁(yè)/共70頁(yè)54胰島素受體與胰島素抵抗性糖尿病1.遺傳性胰島素受體異常，包括受體的 合成減少 與配體的親和力降低，如Arg735突變?yōu)镾er TPK活性降低，如Gly1008

突變等2.自身免疫性胰島素受體異常 血液中存在抗胰島素受體的抗體第54頁(yè)/共70頁(yè)55二、受體、信號(hào)轉(zhuǎn)導(dǎo)過(guò)度激活與疾病某些信號(hào)轉(zhuǎn)導(dǎo)蛋白過(guò)度表達(dá)某些信號(hào)轉(zhuǎn)導(dǎo)蛋白組成型激活突變刺激型抗受體抗體生長(zhǎng)激素（GH）分泌過(guò)多的垂體腺瘤中，～30%是由于編碼Gsα的基因突變所致，從而抑制了GTP酶活性，使Gsα處于持續(xù)激活狀態(tài)，cAMP含量增多，垂體細(xì)胞生長(zhǎng)和分泌功能活躍。通路過(guò)度激活如肢端肥大癥和巨人癥第55頁(yè)/共70頁(yè)56霍亂毒素選擇性的催化Gsα亞基上的Arg201核糖化，使GTP酶活性喪失，不能將GTP水解成GDP，從而使Gsα處于不可逆激活狀態(tài),不斷刺激AC生成cAMP,胞漿中的cAMP含量可增加至正常的100倍以上，導(dǎo)致小腸上皮細(xì)胞膜蛋白構(gòu)型改變，大量Cl-和水分子持續(xù)轉(zhuǎn)運(yùn)入腸腔，引起嚴(yán)重腹瀉和脫水。第56頁(yè)/共70頁(yè)57腸腔GsCT霍亂(Cholera)ACcAMP↑↑↑Cl-H2ONa+CT：CholeraToxin第57頁(yè)/共70頁(yè)58三、多環(huán)節(jié)信號(hào)轉(zhuǎn)導(dǎo)異常與疾病㈠腫瘤1.促細(xì)胞增殖的信號(hào)轉(zhuǎn)導(dǎo)過(guò)強(qiáng) ⑴生長(zhǎng)因子產(chǎn)生增多 ⑵受體的改變 生長(zhǎng)因子受體表達(dá)異常增多 突變使受體組成型激活⑶細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)蛋白的改變 如Ras突變第58頁(yè)/共70頁(yè)592.抑制細(xì)胞增殖的信號(hào)轉(zhuǎn)導(dǎo)過(guò)弱生長(zhǎng)抑制因子受體減少、喪失受體后信號(hào)轉(zhuǎn)導(dǎo)通路異常細(xì)胞的生長(zhǎng)負(fù)調(diào)控機(jī)制減弱或喪失第59頁(yè)/共70頁(yè)60Oncogene-encodeddefectiveEGFreceptor第60頁(yè)/共70頁(yè)61GEF：GuaninenucleotideExchangeFactorGAP：GTPaseActivatingProteinActivationofRasprotein第61頁(yè)/共70頁(yè)62GEF：GuaninenucleotideExchangeFactorGAP：GTPaseActivatingProteinMutationofRasconstitutiveactivation第62頁(yè)/共70頁(yè)63腫瘤形成必須是在單個(gè)細(xì)胞中發(fā)生多重遺傳突變單個(gè)癌基因激活和抑癌基因失活不足以引起腫瘤細(xì)胞的顯著擴(kuò)增，因?yàn)榧?xì)胞對(duì)凋亡的易感性也同時(shí)增加；凋亡抑制是腫瘤細(xì)胞能夠生長(zhǎng)到足以威脅宿主所必須的；例如：Rb的失活僅會(huì)促進(jìn)p53依賴的凋亡，但不會(huì)引起腫瘤，除非細(xì)胞的凋亡過(guò)程被抑制；Apoptosisandoncogenesis第63頁(yè)/共70頁(yè)643.3.3Oncogenes,TumorSuppressorGenes(TSGs),andProgrammed