生物化學(xué)：信號(hào)轉(zhuǎn)導(dǎo)通路

1Signal-TransductionPathways

信號(hào)轉(zhuǎn)導(dǎo)通路

Howcouldtheinsideofthecellknow whatwashappeningontheoutside?腦：神經(jīng)信號(hào)警告身體各部位，釋放激素激活腎上腺眼：瞳孔放大，視野變窄心臟：心率加速肺：氣管擴(kuò)張，呼吸頻率加快肌肉：血量增加，肌肉收縮肝臟：糖原分解，糖被釋放到血液脂肪細(xì)胞：脂肪酸被釋放到血液腸胃：流入消化系統(tǒng)的血量減少Timetoflee!髓質(zhì)皮質(zhì)醇腎上腺素去甲腎上腺素皮質(zhì)3干細(xì)胞分化：根據(jù)組織局部微環(huán)境的差異而分化成相應(yīng)的細(xì)胞2011年，Science評(píng)出

本世紀(jì)前十年十大科學(xué)成就：5.細(xì)胞重編程：將充分發(fā)育的細(xì)胞進(jìn)行“重編程”，使其成為多能干細(xì)胞-具有成為其身體中任何類(lèi)型細(xì)胞的能力。Howcouldtheinsideofthecellknowwhatwashappeningontheoutside?signaltransduction!RobertJ.LefkowitzBrianK.Kobilka“forstudiesofG-protein-coupledreceptors”表彰他們對(duì)G蛋白耦聯(lián)受體的研究人高度緊張時(shí)“腎上腺素開(kāi)始大量分泌”2012年，諾貝爾化學(xué)獎(jiǎng)Forthediscoverythatmaturecellscanbereprogrammedtobecomepluripotent“發(fā)現(xiàn)成熟細(xì)胞可以重新編程而獲得多能性”ShinyaYamanakaJohnB.Gurdon2012年，諾貝爾生理/醫(yī)學(xué)獎(jiǎng)7信號(hào)

受體

反應(yīng):手觸摸就是刺激(信號(hào))，小葉合攏就是反應(yīng)。偶聯(lián)刺激到反應(yīng)之間的生化和分子途徑,就是這個(gè)反應(yīng)的信號(hào)轉(zhuǎn)導(dǎo)通路

觸摸含羞草后,小葉合攏8SignalTransductionPathway:Complicated9細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)網(wǎng)絡(luò)的簡(jiǎn)單模式（信號(hào)輸入）（信號(hào)輸出）10ImportantrolesofbiosignalingFunctionalintegrationofdistantorgans,tissuesandcellsrequirescommunication;Signalingisperhapsaprimalrequirementtorespondtoourenvironment;Thefoundationofanycomplexresponsepathwaylieswithcellularbiochemicals.BiosignalingIntercellular（細(xì)胞間）&Intracellular（細(xì)胞內(nèi)）11常見(jiàn)四種類(lèi)型：Endocrine(內(nèi)分泌）Paracrine(旁分泌）Autocrine(自分泌）MembraneattachedproteinIntercellularsignaling

（細(xì)胞間信號(hào)）12Fourschemesofintercellularsignaling(1)13Fourschemesofintercellularsignaling(2)14Intracellularsignaling

（細(xì)胞內(nèi)信號(hào)）15Electron-micrographofmacrophage(pink)attackingEscherichiacoli(green)16M

吞噬處理入侵細(xì)菌及提呈抗原的機(jī)制17FcR

CR3Ca++srcPI3kPKCMAPKRhoGTPasegelsolinArp2/3PLCPLDActinrearrangementPhagocytosis;OxidativeactivationSignalsforphagocytosisSignalReceptorAmplificationTransductionResponsessecondmessengers信號(hào)轉(zhuǎn)導(dǎo)要素：信號(hào)或配體,受體,信號(hào)放大(產(chǎn)生第二信使),應(yīng)答和反饋調(diào)節(jié)18PARTⅠ1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPARTⅡ1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways191Fourbasiccharacteristics:1.1Specificity1.2Amplification1.3Desensitization/Adaptation1.4Integration20SpecificitySignalmoleculefitsbindingsiteonitscomplementaryreceptor;othersignalsdonotfit.thrombin凝血酶21oftenshort-lived&lowconcentration22Desensitization/Adaptation

ReceptoractivationtriggersafeedbackcircuitthatshutsoffthereceptororremovesitfromthecellsurfaceProducearapidandmajorcellularresponsetoatransientsignal23IntegrationWhentwosignalshaveoppositeeffectsonametaboliccharacteristicsuchasconcentrationofasecondmessengerX,orthemembranepotentialVm,theregulatoryoutcomeresultsfromtheintegratedinputfrombothreceptors242.Fourtypesofsignaltransducers2.1GatedIonChannels

2.1.1Ligand-gatedionchannels 2.1.2Voltage-gatedionchannels2.2ReceptorEnzymes2.3GProtein–CoupledReceptors andSecondMessengers2.4SteroidReceptors25Fourgeneraltypesofsignaltransducers26WhyIonChannels?Restingpotential:Asymmetricion-distributionActingpotentialGatedIonChannels Ligand-gatedionchannels Voltage-gatedionchannels27Restingpotential28IonConc.inMammalianCellsandSerum(mM）IonCytoplasmBloodSerumK+1404Na+12145Cl-4116proteincharges1389Mg+20.81.5Ca+2<0.00021.8WhyIonChannels:asymmetricion-distribution29asymmetricion-distributionRestingpotential30Actingpotential31ActingpotentialVoltage-gatedNa+channels&K+channels32pumpandionleakchannelsCl

-leakchannel332.1.1Ligand-gatedionchannel:Bindingofsomesmallmoleculeforcesanallosterictransitioninprotein,open/closechannel.

acetylcholine(乙酰膽堿)receptorionchannel2.1.2Voltage-gatedionchannelAchargedproteindomainmovesrelativetothemembraneinresponsetoachangeintransmembraneelectricalpotential.

(voltage-gatedNa+，

Ca2+，K+channels)34乙酰膽堿受體離子通道1AchACh35BindingofAChtoRcauseconformationalchange.AsM2helicestwistslightly,theLeuresidues(yellow)rotateawayfromthechannelandarereplacedbysmallerpolarresidues(blue).Thisgatingmechanismopenschannel,allowingpassageofCa,Na,orK乙酰膽堿受體離子通道2Closed Open36Voltage-gatedNa+channels137Voltage-gatedNa+channels2382.Fourtypesofsignaltransducers2.1GatedIonChannels

2.1.1Ligand-gatedionchannels 2.1.2Voltage-gatedionchannels2.2ReceptorEnzymes2.3GProtein–CoupledReceptors andSecondMessengers2.4SteroidReceptors39

2.2ReceptorenzymesAligand-bindingdomain(胞外)andanenzymeactivesiteoncytosolicside,connectedbyasingletransmembranesegment.CommonlyakinasethatphosphorylatesTyrresiduesinspecifictargetproteins（insulinreceptor）Other:synthesizethei.c.secondmessengercGMPinresponsetoex.c.signals

(thereceptorforatrialnatriureticfactor)40Activationofreceptortyrosinekinases41InsulinreceptortyrosinekinaseInsulinstructure42Insulinreceptorbindsinsulinandundergoesautophosphorylationonitscarboxyl-terminalTyrresidues.InsulinreceptorphosphorylatesIRS-1onitsTyrresidues.SH2domainofGrb2bindstoP–TyrofIRS-1.SosbindstoGrb2,thentoRas,causingGDPreleaseandGTPbindingtoRas.ActivatedRasbindsandactivatesRaf-1.Raf-1phosphorylatesMEKontwoSerresidues,activatingit.MEKphosphorylatesERKonaThr&aTyrresidue,activatingit.ERKmovesintothenucleusandphosphorylatesNucleartranscriptionfactorssuchasElk1,activatingthem.PhosphorylatedElk1joinsSRFtostimulatethetranscriptionandtranslationofasetofgenesneededforcelldivision.43Activationofglycogensynthasebyinsulin44Regulationofbloodglucoselevel45Oncogene-encodeddefectiveEGFreceptor462.Fourtypesofsignaltransducers2.1GatedIonChannels

2.1.1Ligand-gatedionchannels 2.1.2Voltage-gatedionchannels2.2ReceptorEnzymes2.3GProtein–CoupledReceptors andSecondMessengers2.4SteroidReceptors472.3GPCRandSecondmessengersThreeessentialcomponents:1.aplasmamembranereceptorwithseventransmembranehelicalsegments2.anenzymeintheplasmamembranethatgeneratesanintracellular2ndmessenger3.aguanosinenucleotide–bindingprotein(Gprotein)GPCR：感受物化刺激，包括多種神經(jīng)遞質(zhì)、肽類(lèi)激素和趨化因子受體；超過(guò)半數(shù)的現(xiàn)代藥物靶向GPCR48NobelPrizeinPhysiologyandMedicine1994"fortheirdiscoveryofG-proteinsandtheroleoftheseproteinsinsignaltransductionincells"AlfredG.Gilman

1941-MartinRodbell

1925-199849ThreeessentialcomponentsofGProtein–CoupledReceptors5051AproteinbindsGuaninenucleotides(GDP,GTP);activatedinGTP-form,inactivatedinGDP-formIntegralmembraneprotein,heterotrimers(

);Havesimilar&subunits,butdifferin-subunitWhenG-proteinisactivated,thesubunitdissociatestointeractwithanenzymesthatgeneratesecondmessengers(e.g.cAMP)Others:smallG-proteins(~20-25kDa),e.g.Ras,Rho,Rac,etc,arenotmembranebound.Gprotein(GTP-bindingprotein)52Gprotein(discovery)M.Rodbell:atransducerprovidedthelinkbetweenreceptorandamplifier.A.G.Gilman:identify&purifytheGprotein.System:MutatedlymphomacellscontaininganormalreceptorandcAMP-generatingenzyme,wasyetunabletorespond(producecAMP),sinceitlackedthetransducermutatedcellnormalcell53“ON-OFF”switchisregulatedbyGTPorGDPboundform.AllG-proteinshasintrinsicGTPaseactivity,releasePiandinactivated.Activation:releaseofGDPandreplacedbyGTP54TheassociationofactiveGs

withadenylylcyclasestimulatesthecyclasetocatalyzecAMPsynthesisAdenosine3’,5’-cyclicmonophosphate(cAMP)55Taste-sensitive

areasoftongueFiveprimarytastes:BitterSweetSaltSourUmami

(Glutamate,delicious)TasteBack56Salty&sour:directlyviamembraneionchannels

Salt:detectedbypassagethoughsodiumchannelsSour:resultsfromtheeffectsofH+onvariouschannels.Umami:byamodifiedformofonebrainreceptorthatrespondstoglutamateasa

neurotransmitterDiversePathwaysDetectTastes(II)57Transductionforsweettastants58Twomajorsystems:2.3.1THEPKASYSTEM (cAMPasthesecondmessenger)

The-AdrenergicReceptorSystem2.3.2THEPKCSYSTEM

(DAG,IP3andCa2+asthesecondmessengers)RobertJ.LefkowitzBrianK.Kobilka“forstudiesofG-protein-coupledreceptors”表彰他們對(duì)G蛋白耦聯(lián)受體的研究人高度緊張時(shí)“腎上腺素開(kāi)始大量分泌”2012年，諾貝爾化學(xué)獎(jiǎng)6061G蛋白耦聯(lián)受體：感受物化刺激，包括多種神經(jīng)遞質(zhì)、肽類(lèi)激素和趨化因子受體超過(guò)半數(shù)的現(xiàn)代藥物靶向62synthesizedinadrenalmedulla;belongstocatecholamines(兒茶酚胺）;targetcellsincludeliver,skeletalmuscle,heartmuscleandadipose;releasedinresponsetoacutestressEpinephrine腎上腺素signal63Epinephrine腎上腺素signalingpathwaycAMP64Epinephrine腎上腺素signalingpathway(2)65ActivationofcAMP-dependentproteinkinase(PKA)InactivePKA:Regulatory(R)subunits:auto-inhibitorydomainsburiedcatalytic(C)subunits:substrate-bindingsitesblockedbyRsubunitsRsubunits:autoinhibitorydomainsburiedActivePKACsubunitsopensubstratebindingsites66Epinephrinetriggersaseriesofreactionsinhepatocytesinwhichcatalystsactivatecatalysts,resultingingreat“amplification”ofthesignalx分子10,000x分子67PKAregulatesanumberofenzymesTheproteinsphosohorylatedbyPKAsharearegionofsequencesimilarityaroundtheSerorThrresiduethatundergoesphosphorylation,asequencethatmarksthemforregulationbyPKA.ThecatalyticsiteofPKAinteractswithseveralresiduesneartheThrorSerresidueinthetargetprotein,whichdefinethesubstratespecificity.6869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。70腸腔GsCT霍亂(Cholera)ACcAMP↑↑↑Cl-H2ONa+CT：CholeraToxin71DesensitizationofthePKAsystem1desensitizingβ-AdrenergicReceptor2degradingthesecondmessenger72GsbgrecruitsbARKtothemembrane,whereitphospho-

SerattheC-terminusofRecpt.barrbindstothepi-C-terminusofRecpt.Receptor-arrestincomplexentersthecellbyendocytosis.73β-ArrestinuncouplesreceptorfromitsGproteinandbringstogether3enzymesofMAPKcascade.(Onestimulustriggerstwodistinctpathways:thepathactivatedbyGproteinandMAPKcascade)74degradingthesecondmessenger75RegulationoftranscriptionbysteroidhormonesSteroidreceptor76PARTⅠ1Basiccharacteristicsofsignaltransduction2FourgeneraltypesofsignaltransducersPARTⅡ1Regulatorymechanisms2Somediseasescausedbydefectsinthebiosignalingpathways77Biosignaling:Howaretheyregulated?78IntegrationWhentwosignalshaveoppositeeffectsonametaboliccharacteristicsuchasconcentrationofasecondmessengerX,orthemembranepotentialVm,theregulatoryoutcomeresultsfromtheintegratedinputfrombothreceptors79SopEPKB/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80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81RegulatorymechanismsofBio-signals3.1Phosphorylationasaregulatorymechanism3.2Regulationoftranscriptionbysteroidhormones3.3Regulationofthecellcyclebyproteinkinases82RegulationofSignalingPathwaysExternalsignalsSecondmessengersModulatorproteinsFunctionTargetproteinsHormonescAMPOdorants

cGMPDrugsCa2+LightDAG IP3Mitogenichormones TyrosineGrowth kinasesfactorsproteinkinaseAndphosphatasestructuralproteinsAndenzymesmetabolicorphysiologicalresponsesPlasmamembrane83NobelPrizeinPhysiologyandMedicine1992EldwinG.KrebsEdmondH.Fischer

USA

“Reversibleproteinphosphorylationasabiologicalregulatorymechanism"J.Biol.Chem.216:121-132,1955Page84ProteinPhosphorylationNobelPrize1992NobelPrize2000EdmondH.FischerandEdwinG.Krebs

J.Biol.Chem.216:121-132,1955ArvidCarlsson,PaulGreengardandEricKandel85ProteinKinases1:

GPCRs

(5%),

2:

Kinases

(2.8%genome)

~25%

ofthemammalianintracellularproteinsundergoesreversiblephosphorylation86Proteinkinases(534humankinases)Ser/thrKinasescomprise80%ofallproteinkinasesAGC87Phosphorylation&de-phosphorylationarethemostcommonregulatorymechanisms,mediatedbyproteinkinaseandphosphatase,respectively

蛋白激酶

nNTPnNDP蛋白質(zhì)蛋白質(zhì)-nPi

nPi 蛋白磷酸酶

H2O88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(lèi)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(lèi)。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89CalciumIsaSecondMessengerinManySignalTransductionsNormally,[Ca2]iis~100nM(而細(xì)胞外：>1mM)Hormonal,neural,orotherstimulicauseeitheraninfluxofCa2+intothecellthroughspecificCa2+channelsintheplasmamembraneorthereleaseofCa2+fromERormitochondria,Changesin[Ca2]iaredetectedbyCa2+-bindingproteinsthatregulateavarietyofCa2-dependentenzymes-Calmodulin(CaM)90SHCGrb2GEFRas-GTPFRas-GDPFPiGAPSHCGrb2GEFRas-GTPFRas-GDPFPiGAPRas蛋白的上游和下游信號(hào)通路91PI3K－AKT途徑對(duì)細(xì)胞生存的調(diào)控機(jī)制92Mostofthesecomplicatedsignalingpathwaysaretransducedbythephosphorylationanddephosphoralationofsignalingprotein,regulatedbytheproteinkinasesandphosphatses93蛋白磷酸酶proteinphosphatase，PP蛋白磷酸酶的分類(lèi)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。

94Mechanismsusedbyhormones,retinoids,andVit.DtoregulategeneexpressionHormone(H),carriedtothetargettissueonserumbindingprot,diffusesacrossthePMandbindstoitsspecificreceptorprotein(Rec)inthenucleus.HbindingchangestheconformationofRec;formshomoorheterodimersandbindstospecificRregionscalledhormoneresponseelements(HREs)intheDNAadjacenttospecificgenesBindingregulatestranscriptionoftheadjacentgene(s),increasingordecreasingtherateofmRNAformation.Alteredlevelsofthehormoneregulatedgeneproductproducethecellularresponsetothehormone95Summary

FourbasiccharactersofbiosignalsFourschemesofintercellularsignalingFourgeneraltypesofsignaltransducers

gatedionchannels;receptorenzymes;G-proteincoupledreceptors；SteroidReceptorsGproteins:Heterotrimeric&monomeric; Activationanddeactivationmechanisms96Majorsecondmessengers:cAMP,IP3,DAG,Ca2+,cGMP.Usinganexampletoexplainthecascadeofevents:asinglemoleculeofhormoneactivatescatalystthatinturnactivatesanothercatalyst,andsoon,resultsinsignalamplificationandcellresponse97

細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)調(diào)控與疾病防治Regulationofcellularsignalsinprevention&treatmentofdiseases以信號(hào)轉(zhuǎn)導(dǎo)蛋白為靶分子對(duì)疾病進(jìn)行防治STI571asaparadigm(范例）

forcancertherapyIn1960,describedthepresenceofaconsistentchromosomalabnormalityinCML（一種白血病)，so-calledPhiladelphiac