《生物技術(shù)藥物》之促紅細(xì)胞生成素課件

各論促紅細(xì)胞生成素Erytropoietin，EPO腎臟，間質(zhì)細(xì)胞（組織缺氧誘導(dǎo)產(chǎn)生EPO）刺激和調(diào)節(jié)紅細(xì)胞的生成：促進(jìn)紅系細(xì)胞的生長(zhǎng)，分化和增殖，促進(jìn)紅系池的擴(kuò)大；促進(jìn)紅細(xì)胞成熟；抗氧化作用臨床應(yīng)用：腎功能衰竭引起的貧血癌性貧血結(jié)締組織病貧血骨髓增生異常綜合癥貧血不良反應(yīng)：腎性/非腎性貧血EpoRisaproteinthatinhumansisencodedbytheEPORgene.EpoRisa59kDapeptideandisamemberofthecytokinereceptorfamily.EpoRpre-existsasdimerswhichuponbindingofa34kDaliganderythropoietin(Epo),changesitshomodimerizedstate.TheseconformationalchangesresultintheautophosphorylationofJak2kinasesthatarepre-associatedwiththereceptor(i.e.,EpoRdoesnotpossessintrinsickinaseactivityanddependsonJak2activity).TheJAK-STATsystemconsistsofthreemaincomponents:(1)areceptor(2)Januskinase(JAK)and(3)SignalTransducerandActivatorofTranscription(STAT).ManyJAK-STATpathwaysareexpressedinwhitebloodcells,andarethereforeinvolvedinregulationoftheimmunesystem.JAKs,whichhavetyrosinekinaseactivity,bindtosomecellsurfacecytokinereceptors.ThebindingoftheligandtothereceptortriggersactivationofJAKs.Withincreasedkinaseactivity,theyphosphorylatetyrosineresiduesonthereceptorandcreatesitesforinteractionwithproteinsthatcontainphosphotyrosine-bindingSH2domains.STATspossessingSH2domainscapableofbindingthesephosphotyrosineresiduesarerecruitedtothereceptors,andarethemselvestyrosine-phosphorylatedbyJAKs.ThesephosphotyrosinesthenactasbindingsitesforSH2domainsofotherSTATs,mediatingtheirdimerization.DifferentSTATsformhetero-orhomodimers.ActivatedSTATdimersaccumulateinthecellnucleusandactivatetranscriptionoftheirtargetgenes.STATsmayalsobetyrosine-phosphorylateddirectlybyreceptortyrosinekinases,suchastheepidermalgrowthfactorreceptor,aswellasbynon-receptortyrosinekinasessuchasc-src.TypeIcytokinereceptors,whosemembershavecertainconservedmotifsintheirextracellularamino-aciddomain.TheIL-2receptorbelongstothischain,whoseγ-chain(commontoseveralothercytokines)deficiencyisdirectlyresponsibleforthex-linkedformofSevereCombinedImmunodeficiency(X-SCID).TypeIIcytokinereceptors,whosemembersarereceptorsmainlyforinterferons.Immunoglobulin(Ig)superfamily,whichareubiquitouslypresentthroughoutseveralcellsandtissuesofthevertebratebodyTumornecrosisfactorreceptorfamily,whosemembersshareacysteine-richcommonextracellularbindingdomain,andincludesseveralothernon-cytokineligandslikereceptors,CD40,CD27andCD30,besidestheligandsonwhichthefamilyisnamed(TNF).Chemokinereceptors,twoofwhichactingasbindingproteinsforHIV(CXCR4andCCR5).TheyareGproteincoupledreceptors.TGFbetareceptorsAclassificationofcytokinereceptorsbasedontheirthree-dimensionalstructurehasbeenattempted.(Suchaclassification,thoughseeminglycumbersome,providesseveraluniqueperspectivesforattractivepharmacotherapeutictargets.)生長(zhǎng)因子類藥物胰島素樣生長(zhǎng)因子（IGF，IGF-I，IGF-II）肝臟（進(jìn)入血液）IGF-I：70個(gè)氨基酸，3個(gè)二硫鍵；IGF-II：67個(gè)氨基酸，3個(gè)二硫鍵。A+C+B構(gòu)成。生物學(xué)特性：胚胎發(fā)育生長(zhǎng)啟動(dòng)效應(yīng)提高腎功能影響生殖作用臨床應(yīng)用：治療胰島素受體紊亂(機(jī)理不甚明確)促生長(zhǎng)作用治療骨質(zhì)疏松硬化癥IGF-I型受體結(jié)構(gòu)與胰島素受體(Insulinreceptor,Ir)相似，由α和β兩個(gè)亞基構(gòu)成α2β2四聚體的糖蛋白，α亞基是配體結(jié)合部位，β亞單位具有內(nèi)在的酪氨酸激酶活性。表皮生長(zhǎng)因子Humanepidermalgrowthfactor，hEGF是一種多功能的生長(zhǎng)因子，在體內(nèi)體外都對(duì)多種組織細(xì)胞有強(qiáng)烈的促分裂作用來(lái)源：?jiǎn)魏思?xì)胞，外胚層，腎臟等結(jié)構(gòu)：由53個(gè)氨基酸組成的小分子多肽,分子內(nèi)有三對(duì)二硫鍵,不含糖基，對(duì)酸、堿、熱等理化因素均較穩(wěn)定。生物學(xué)特性：刺激皮膚和中胚層細(xì)胞生長(zhǎng)。對(duì)上皮細(xì)胞(內(nèi)皮)，有促生長(zhǎng)和增殖活性。能極強(qiáng)地促進(jìn)各種表皮組織生長(zhǎng)。加快K+和葡萄糖等小分子進(jìn)入細(xì)胞。作用原理：增加細(xì)胞移動(dòng)，分裂生長(zhǎng)，細(xì)胞間質(zhì)蛋白合成，促進(jìn)新血管形成，促進(jìn)核酸蛋白質(zhì)合成。臨床：治療潰瘍，燒傷，其他潰瘍是皮膚或黏膜表面組織的限局性缺損、潰爛，其表面常覆蓋有膿液、壞死組織或痂皮，愈后遺有瘢痕，可由感染、外傷、結(jié)節(jié)或腫瘤的破潰等所致，其大小、形態(tài)、深淺、發(fā)展過(guò)程等也不一致。常合并慢性感染，可能經(jīng)久不愈。hEGF與特異的跨細(xì)胞膜表面的EGFR結(jié)合后,刺激了EGFR復(fù)合物中的酪氨酸激酶的活性,通過(guò)EGFR復(fù)合物的自磷酸化作用,在細(xì)胞內(nèi)形成快速的信息傳遞網(wǎng)絡(luò),激活蛋白酶和磷酸酯酶等的一系列生化反應(yīng),促進(jìn)體內(nèi)Ca2+、K+和糖等低分子物大量進(jìn)入細(xì)胞內(nèi)(主動(dòng)運(yùn)輸),糖酵解量增大,RNA與蛋白質(zhì)合成增多,作用一段時(shí)間后,EGFR復(fù)合物開(kāi)始促進(jìn)DNA合成,并由此趨向刺激內(nèi)皮細(xì)胞、單核細(xì)胞等多種細(xì)胞分裂、增殖和分化,使之向創(chuàng)傷部位遷移,加速啟動(dòng)創(chuàng)傷組織再生、修復(fù)和胞外間質(zhì)形成。另一方面,hEGF能增加其他內(nèi)源性生長(zhǎng)因子,促進(jìn)羥脯氨酸合成,調(diào)節(jié)膠原酶和膠原的合成、分泌和沉淀,調(diào)節(jié)膠原降解,使膠原纖維以線性方式排列,增強(qiáng)創(chuàng)面抗張程度,減少疤痕形成,提高愈合質(zhì)量。Receptor-linkedtyrosinekinasessuchastheepidermalgrowthfactorreceptor(EGFR)areactivatedbyextracellularligands.Bindingofepidermalgrowthfactor(EGF)totheEGFRactivatesthetyrosinekinaseactivityofthecytoplasmicdomainofthereceptor.TheEGFRbecomesphosphorylatedontyrosineresidues.DockingproteinssuchasGRB2containsanSH2domainthatbindstothephosphotyrosineresiduesoftheactivatedreceptor.GRB2bindstotheguaninenucleotideexchangefactorSOSbywayofthetwoSH3domainsofGRB2.WhentheGRB2-SOScomplexdockstophosphorylatedEGFR,SOSbecomesactivated.ActivatedSOSthenpromotestheremovalofGDPfromamemberoftheRassubfamily(mostnotablyH-RasorK-Ras).RascanthenbindGTPandbecomeactive.ApartfromEGFR,othercellsurfacereceptorsthatcanactivatethispathwayviaGRB2includeTrkA/B,Fibroblastgrowthfactorreceptor(FGFR)andPDGFR.MAPK/ERKpathwayKinasecascadeActivatedRasactivatestheproteinkinaseactivityofRAFkinase.[4]RAFkinasephosphorylatesandactivatesMEK(MEK1andMEK2).MEKphosphorylatesandactivatesamitogen-activatedproteinkinase(MAPK).RAF,andMAPKarebothserine/threonine-selectiveproteinkinases.MEK(alsoknownasMAPKK)isatyrosine/threoninekinase.Inthetechnicalsense,RAF,MEK,andMAPKareallmitogen-activatedkinases,asisMNK(seebelow).MAPKwasoriginallycalled"extracellularsignal-regulatedkinases"(ERKs)and"microtubule-associatedproteinkinase"(MAPK).OneofthefirstproteinsknowntobephosphorylatedbyERKwasamicrotubule-associatedprotein(MAP).Asdiscussedbelow,manyadditionaltargetsforphosphorylationbyMAPKwerelaterfound,andtheproteinwasrenamed"mitogen-activatedproteinkinase"(MAPK).TheseriesofkinasesfromRAFtoMEKtoMAPKisanexampleofaproteinkinasecascade.Suchseriesofkinasesprovideopportunitiesforfeedbackregulationandsignalamplification.EGFRdimerizationstimulatesitsintrinsicintracellularprotein-tyrosinekinaseactivity.Asaresult,autophosphorylationofseveraltyrosine(Y)residuesintheC-terminaldomainofEGFRoccurs.Thisautophosphorylationelicitsdownstreamactivationandsignalingbyseveralotherproteinsthatassociatewiththephosphorylatedtyrosinesthroughtheirownphosphotyrosine-bindingSH2domains.Thesedownstreamsignalingproteinsinitiateseveralsignaltransductioncascades,principallytheMAPK,AktandJNKpathways,leadingtoDNAsynthesisandcellproliferation.柱狀上皮細(xì)胞：主要分布于鼻腔、鼻咽、器官、肺、胃、腸、子宮頸、子宮內(nèi)膜及輸卵管等部位。鱗狀上皮細(xì)胞：被覆于全身皮膚、口腔、喉部、鼻咽的一部分、食道、陰道的全部以及子宮頸。鱗狀上皮細(xì)胞分為基底層細(xì)胞、中層細(xì)胞和表層細(xì)胞。黏膜是指口腔、鼻腔、腸管、陰道等與外界相通體腔的濕潤(rùn)上皮細(xì)胞。AKTpathwayAktisinvolvedinthePI3K/AKT/mTORpathwayandothersignalingpathways.Bindingphospholipids：AktpossessesaproteindomainknownasaPHdomain,orPleckstrinHomologydomain,namedafterPleckstrin,theproteininwhichitwasfirstdiscovered.Thisdomainbindstophosphoinositideswithhighaffinity.InthecaseofthePHdomainofAkt,itbindseitherPIP3orPIP2Thisisusefulforcontrolofcellularsignalingbecausethedi-phosphorylatedphosphoinositidePIP2isonlyphosphorylatedbythefamilyofenzymes,PI3-kinases(phosphoinositide3-kinaseorPI3-K),andonlyuponreceiptofchemicalmessengerswhichtellthecelltobeginthegrowthprocess.Forexample,PI3-kinasesmaybeactivatedbyaGproteincoupledreceptororreceptortyrosinekinasesuchastheinsulinreceptor.Onceactivated,PI3-kinasephosphorylatesPIP2toformPIP3.Phosphorylation：OncecorrectlypositionedatthemembraneviabindingofPIP3,Aktcanthenbephosphorylatedbyitsactivatingkinases,phosphoinositidedependentkinase1(PDPK1atthreonine308)andthemammaliantargetofrapamycincomplex2(mTORC2atserine473),firstbymTORC2.mTORC2thereforefunctionallyactsasthelong-soughtPDK2molecule,althoughothermolecules,includingintegrin-linkedkinase(ILK)andmitogen-activatedproteinkinase-activatedproteinkinase-2(MAPKAPK2)canalsoserveasPDK2.PhosphorylationbymTORC2stimulatesthesubsequentphosphorylationofAktbyPDPK1.ActivatedAktcanthengoontoactivateordeactivateitsmyriadsubstrates(e.g.mTOR)viaitskinaseactivity.BesidesbeingadownstreameffectorofPI3-kinases,AktcanalsobeactivatedinaPI3-kinase-independentmanner.ACK1orTNK2,anon-receptortyrosinekinase,phosphorylatesAktatitstyrosine176residue,leadingtoitsactivationinPI3-kinase-independentmanner.StudieshavesuggestedthatcAMP-elevatingagentscouldalsoactivateAktthroughproteinkinaseA(PKA)inthepresenceofinsulin.c-JunN-terminalkinases(JNKs)Thec-JunN-terminalkinasesconsistoftenisoformsderivedfromthreegenes:JNK1(fourisoforms),JNK2(fourisoforms)andJNK3(twoisoforms)JNK,byphosphorylation,modifiestheactivityofnumerousproteinsthatresideatthemitochondriaoractinthenucleus.DownstreammoleculesthatareactivatedbyJNKincludec-Jun,ATF2,ELK1,SMAD4,p53andHSF1.ThedownstreammoleculesthatareinhibitedbyJNKactivationincludeNFAT4,NFATC1andSTAT3.Byactivatingandinhibitingothersmallmoleculesinthisway,JNKactivityregulatesseveralimportantcellularfunctionsincludingcellgrowth,differentiation,survivalandapoptosis.血小板衍生生長(zhǎng)因子Plateletd-erivedgrowthfactor，PDGF從血小板中發(fā)現(xiàn)，在損傷早期從血小板α顆粒釋放出來(lái)，啟動(dòng)并加速組織創(chuàng)傷修復(fù)，

主要由血小板合成，在組織受到損傷時(shí)巨噬細(xì)胞、血管平滑肌細(xì)胞、成纖維細(xì)胞、內(nèi)皮細(xì)胞、胚胎干細(xì)胞等也可以合成并釋放PDGF。結(jié)構(gòu)：是靠二硫鍵相連的A、B兩條多肽鏈組合成的二聚體。A鏈125/110個(gè)氨基酸，3個(gè)二硫鍵；B鏈109個(gè)氨基酸，3個(gè)二硫鍵生物學(xué)特性：刺激血管平滑肌細(xì)胞、成纖維細(xì)胞、膠質(zhì)細(xì)胞的分裂增生；誘導(dǎo)巨噬細(xì)胞與成纖維細(xì)胞的游走，對(duì)中性粒細(xì)胞、平滑肌細(xì)胞、成纖維細(xì)胞有趨化性；具有縮血管活性；誘導(dǎo)基因表達(dá)。臨床：傷口愈合MAPKpathwayTheadaptorproteinGrb2formsacomplexwithSosbytheGrb2SH3domain.Grb2(or)theGrb2/SoscomplexisrecruitedtothemembranebytheGrb2SH2domainbindingtoactivatedPDGFR-boundSHP2(AlsoknownasPTPN11,acytosolicPTP),therebyallowinginteractionwithRasandtheexchangeofGDPforGTPonRas.WhereastheinteractionbetweenGrb2andPDGFRoccursthroughinteractionwiththeSHP2protein,Grb2bindstoactivatedEGFRthroughShc,anotheradaptorproteinthatformsacomplexwithmanyreceptorsviaitsPTBdomain.Onceactivated,Rasinteractswithseveralproteins,namelyRaf.ActivatedRafstimulatesMAPK-kinase(MAPKKorMEK)byphosphorylatingaSerresidueinitsactivationloop.MAPKKthenphosphorylatesMAPK(ERK1/2)onTandYresiduesattheactivation-loopleadingtoitsactivation.ActivatedMAPKphosphorylatesavarietyofcytoplasmicsubstrates,aswellastranscriptionfactors,whentranslocatedintothenucleus.MAPKfamilymembershavebeenfoundtoregulatevariousbiologicalfunctionsbyphosphorylationofparticulartargetmolecules(suchastranscriptionfactors,otherkinasesetc.)locatedincellmembrane,cytoplasmandnucleus,andthuscontributetotheregulationofdifferentcellularprocessessuchascellproliferation,differentiation,apoptosisandimmunoresponses.PI3Kpathway

TheclassIAphospholipidkinase,PI-3kinase,isactivatedbythemajorityofRTKs.SimilarlytootherSH2domain-containingproteins,PI-3kinaseformsacomplexwithPYsitesonactivatedreceptors.ThemainfunctionofPI3KactivationisthegenerationofPIP3,whichfunctionsasasecondmessengertoactivatedownstreamtyrosinekinasesBtkandItk,theSer/ThrkinasesPDK1andAkt(PKB).ThemajorbiologicalfunctionsofAktactivationcanbeclassifiedintothreecategories–survival,proliferationandcellgrowth.Aktisalsoknowntobeimplicatedinseveralcancers,particularlybreast.PLCγisimmediatelyrecruitedbyanactivatedRTKthroughthebindingofitsSH2domainstophosphotyrosinesitesofthereceptor.Afteractivation,PLCγhydrolysesitssubstratePtdIns(4,5)P2andformstwosecondmessengers,diacylglycerolandIns(1,4,5)P3.Ins(1,4,5)P3stimulatesthereleaseofCa2+fromintracellularsupplies.Ca2+thenbindstocalmodulin,whichsubsequentlyactivatesafamilyofcalmodulindependentproteinkinases(CamKs).Inaddition,bothdiacylglycerolandCa2+activatemembersofthePKCfamily.ThesecondmessengersgeneratedbyPtdIns(4,5)P2hydrolysisstimulateavarietyofintracellularprocessessuchasproliferation,angiogenesis,cellmotility.轉(zhuǎn)化生長(zhǎng)因子Transforminggrowthfactor，TGF兩類多肽類生長(zhǎng)因子,轉(zhuǎn)化生長(zhǎng)因子-α和轉(zhuǎn)化生長(zhǎng)因子-β。TGFαisupregulatedinsomehumancancers.Itisproducedinmacrophages,braincells,andkeratinocytes,andinducesepithelialdevelopment.TGFβexistsinthreeknownsubtypesinhumans,TGFβ1,TGFβ2,andTGFβ3.TheseareupregulatedinMarfan'ssyndromeandsomehumancancers,andplaycrucialrolesintissueregeneration,celldifferentiation,embryonicdevelopment,andregulationoftheimmunesystem.轉(zhuǎn)化生長(zhǎng)因子-αItisproducedinmacrophages,braincells,andkeratinocyt