清道夫受体介导的脂质蓄积的分子机制讲座

烦恼有何惧怕，既然躲不掉，就调好心态与它共存。心向阳光，何惧风霜。

茫茫人海你我相遇就是缘分，欢迎下载！2021/8/121IntracellularAccumulationofLipidsviaScavengerReceptors2021/8/122FormationoffoamcellAthersclerosisisachronicinflammatoryreactioncausedbymacrophagesandlymphocytesthatuptakethepathogenicitylipidsinvadingthevessel.2021/8/123SR-AandCD36aretheprincipalreceptorsresponsibleforfoam-cellformationMacrophagecelltakesuplipidbySR-A2021/8/124StructureofSR-A

SixdomainsThreetypesTrimericmolecule2021/8/125

FoamcellformationMφadhesionHostdefenseandinnateimmunityAdaptiveresponsesDisordersofthenervoussystemPhagocytosisofapoptoticcellsFunctionsofSR-A

2021/8/126Thecytoplasmicdomainisthekeypartofthereceptor

2021/8/127

mediatetheinternalizationofligandssuchasox-LDL

participatetheregulationofcelladhesionandmigrationaccommodatetheexpressionandtransportofreceptorlink-coupledwithdownstreamofsignalingpathwayFunctionsofSR-Acytoplasmicdomain:2021/8/128

Cytoplasmicdomain：

Murine:MTKEMTENQRLCPHEREDADCSSESVKFDARSMTASLPHSTKNGPSVQEKLKSFK

(55)Rabbit:MAQWDSFTDQQEDTDSCSESVKFDARSNTALLPPNPKGPPLQEKLKSFK

(50)Bovine:MAQWDDFPDQQEDTDSCTESVKFDARSVTALLPPHPKNGPTLQERMKSYK(50)Human:MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFK

(50)

2021/8/129MSR-AAcLDLHSP90,HSP70,andGAPDHDirectlyInteractwiththeCytoplasmicDomainofMSRNakamuraT.etal.BBRC,290,858-864(2002)FunctionalandpossiblephysicalassociationofscavengerreceptorwithcytoplasmictyrosinekinaseLyninmonocyticTHP-l-derivedMacrophagesMikiS.etal.FEBSLetters,399,241-244(1996)HSP90HSP70GAPDHLyn2021/8/1210LigandBindingtoMacrophageScavengerReceptor-AInducesUrokinase-typePlasminogenActivatorExpressionbyaProteinKinase-dependentSignalingPathwayHsien-YehHsuetal.JBiolChem,273,1240-1246(1998)MSR-AAcLDLFucoidanPTKPLC-γPKCuPA2021/8/12119、人的价值，在招收诱惑的一瞬间被决定。2023/2/32023/2/3Friday,February3,202310、低头要有勇气，抬头要有低气。2023/2/32023/2/32023/2/32/3/20234:46:39PM11、人总是珍惜为得到。2023/2/32023/2/32023/2/3Feb-2303-Feb-2312、人乱于心，不宽余请。2023/2/32023/2/32023/2/3Friday,February3,202313、生气是拿别人做错的事来惩罚自己。2023/2/32023/2/32023/2/32023/2/32/3/202314、抱最大的希望，作最大的努力。03二月20232023/2/32023/2/32023/2/315、一个人炫耀什么，说明他内心缺少什么。。二月232023/2/32023/2/32023/2/32/3/202316、业余生活要有意义，不要越轨。2023/2/32023/2/303February202317、一个人即使已登上顶峰，也仍要自强不息。2023/2/32023/2/32023/2/32023/2/32021/8/1212MSR-AFucoidanHsp90,pp60SrcRac1PAKJNKp38PI3KproIL-1IL-1LigandsofMacrophageScavengerReceptorInduceCytokineExpressionviaDifferentialModulationofProteinKinaseSignalingPathwaysHsuHY.etal.JBiolChem,276,28719-28730(2001)2021/8/1213MSR-AAcLDLinternalization,adhesionGi/oRegulationofacetylatedlowdensitylipoproteinuptakeinmacrophagesbypertussistoxin-sensitiveGproteinsStewartCetal.JLipidRes,41,807-813(2000)ClassAscavengerreceptorsmediatecelladhesionviaactivationofGi/oandformationoffocaladhesioncomplexes

PostSR.etal.JLipidRes,43,1829-1836(2002)2021/8/1214phosphorylationofcytoplasmicdomain

signalsequenceincytoplasmicdomaininteractorswithcytoplasmicdomainFocusoncytoplasmicdomain：2021/8/1215BovineModelHumanModelMurineModelRabbitModel2021/8/1216Ser21（rat）increasesexpressionofreceptorpromotesmetabolismoflipidFongLGetal.JBiolChem,274,36088-36816(1999)phosphorylationofcytoplasmicdomain2021/8/1217phosphorylationofcytoplasmicdomainSer49（rat）reducesuptakeoflipidFongLGetal.JBiolChem,274,36088-36816(1999)2021/8/1218FDARS

internalizationofreceptor

FongLGetal.JBiolChem,274,36088-36816(1999)signalsequenceofcytoplasmicdomain2021/8/1219KLKSFKcelladhesionKosswigNetal.JBiolChem,278,34219-34225(2003)signalsequenceofcytoplasmicdomain2021/8/1220VXFD(rabbit)internalizationofreceptor,transferencetocellsurfaceMorimotoKetal.BiolPharmBull,22,1022-1026(1999)signalsequenceofcytoplasmicdomain2021/8/1221Ser48(human）expressionandinternalization

ofreceptor,cellmigration

HeiderHetal.FEBSLetters,505,185-190(2001)

phosphorylationofcytoplasmicdomain2021/8/1222Di-leucinemotif?signalsequenceofcytoplasmicdomain2021/8/1223

1.TheroleofDi-leucinemotifinthecytoplasmicdomainofSRA2021/8/1224(1)SR-Amutants/EGFPconstructs:WildtypeN3132LMEGFPSRASRA2021/8/1225(2)ExpressionpatternsofSRAmutant:*P<0.05,n=3EGFPSR-AN3132LMEGFPSR-AN3132LM*02004006008001000controlEGFPSR-AN3132LM2021/8/1226(3)TheinternalizationofDiI-AcLDLviaSRAmutant:DiI-acLDLSR-Aexpression**DiI-acLDL*P<0.05,n=3**P<0.05,n=3010203040EGFPWildtypeN3132LM*EGFPWildtypeN3132LM0.020.040.060.080.100.122021/8/1227(4)CelladhesionviaSRAmutant:00.10.20.30.40.50.60.70.80.9EGFPSR-AN3132LMratio%**P<0.05,n=3*2021/8/1228EGFPSR-AN3132LMImmunofluorescence:TheCHOcellsweretransfectedwithSRA/EGFPorSRAMutant/EGFP,(5)Di-leucinemotifandClathrin-coatedpit:GFPAnti-clathrin

overlay

2021/8/1229180KD97KDabca:EGFP-transfectedcellb:SR-A-transfectedcellc:N3132LMmutant-transfectedcellClathrinIP:2021/8/1230NΔ1-27NΔ1-27LM***P<0.05,n=3SR-ANΔ1-27NΔ1-27LM97KDABC(6)ExpressingoftheSRAmutantsinCHOcellEGFPSR-A4006008001000NΔ1-27NΔ1-27LM2002021/8/1231(7)TheinternalizationofDiI-AcLDLviaSRAmutant:*P<0.05,n=3*P<0.05,n=3****DiI-acLDLDiI-acLDLSR-Aexpression10203040EGFPSR-ANΔ1-27NΔ1-27LM0.120.100.080.060.040.020.000EGFPSR-ANΔ1-27NΔ1-27LM2021/8/1232*Di-leucinesignalmotifinthecytoplasmicdomainisrequiredforMSR-A-mediatedinternalization.

*Di-leucinesignalmotif-relatedendocytosisofAc-LDLmaydependontheclathrin-coatpit.*TheremightbemultiplesignalmotivesincytoplasmicdomainofMSR,whichadaptsthemultiplefunctionsofMSR.Di-LeucinemotifmaybeandistinctmotiffromtheVXFDinSR-Amediatedinternalizationandadhesion.2021/8/12332.IdentificationofpeptideligandsforthecytoplasmicdomainofSRA2021/8/1234(1)GST-cSRAfusionproteinexpressionandpurification:GSTfusionproteinwereseparatedona12%SDS.1,purifiedGSTfusionprotein;2,E.colisupernatantcontainingGSTfusionprotein;3and6,E.colisupernatant;4,molecularweightstandards;5,E.colisupernatantcontainingGST.123456Western-blotofthefusionproteinwithanti-GSTantibody.1,GST-cytoplasmicdomainofSRAfusionprotein;2,GST;3,E.colisupernatant;4,molecularweightstandards.12342021/8/1235(2)Screeningthephagepeptidelibrarywiththefusionprotein:Biopanningrounds

Inputphages（pfu*）

Outputphages（pfu）Relativeoutputratio**

14.4×1011

5.9×103

1.34×10-824.25×1011

3.45×1048.11×10-632.75×1011

6.4×1052.33×10-642.5×1011

3.25×1061.3×10-5Thepoolofphagebecomesenrichedinfavourofsequencesthatbindtothetargetafterfourroundsofbio-panning*phageformunit**outputphages/inputphages2021/8/1236BindingspecificityofthepositivephageclonesanalysedbyELISAusinganti-M13antibody.Theblack,bindingwiththefusionprotein(P);Thewhite,bindingwithGST(N).Thepositiveclones:P/N>3.(3)IsolationofbindingphageclonesbyPhageELISA:II4II5II6II7II9II10II11II12M1300.10.20.30.40.50.60.70.8二4二5二6二7二9二10二11二12M132021/8/1237(4)Sequencingtheselectedpositiveclones:*H11:TDRLFMNSIWPG(4,6,7,10,11,12)*H5:TFWDDGWLLLPR(5)*H9:YGDRWFMPVMRS(9)2021/8/1238(5)SearchingthePIR-PSDdatabase:H5:CaseinkinaseIIphosphorylationsite(TFWD)H11:ProteinkinaseCphosphorylationsite(TDR)2021/8/1239(6)ConstructionofexpressionplasmidsPEGFP-N1-H5PEGFP-N1-H9PEGFP-N1-H112021/8/1240EGFPPeptide/EGFP(7)Subcellularlocalizationofthepeptide/EGFPinCHOcellsexpressingSRA2021/8/1241(8)DiI-AcLDLuptakeassayEGFP-H11inhibitsSRA-media