mixed lineage kinase domain like蛋白向细胞质膜的迁移定位导致细胞坏死

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1、AbstractAbstractMixedlineagekinasedomain一1ikeprotein(Mlkl)wasidentifiedasdownstreamofReceptorinteractionprotein3(Rip3)inTumorNecrosisFactoralpha(TNFa)inducednecrosis．However，theexactfunctionofMlklinnecrosisislargelyunknown．ByreconstitutingexogenousMlklinMlkl—K0cell，

2、wefoundtheintegrityofN-terminusdomainofMlklwasrequiredforitSnormalfunction．01igomerizationofmlklwasessentialfornecrosis，asartificiallyforcedMlkloligomersbyfusinghormone—bindingdomain(HBD*)triggerednecrosis．Notably，artificiallyforcedoligomersofN—Terminaldomains(ND-ml

3、kl)butnotC—terminalkinase1ikedomaincausednecrosis．Furtherdeletionmutationsshowedthatthefour—G—helixbundleofMlkl(1—130aminoacids)iSsufficientfornecrosiS．BothTNFQinducedendogenousMlkloligomersinwildtypecellorHBD水mediatedND-mlkloligomersweretetramers．Bothofthehomo—olig

4、omerswereobservedtotranslocateto1ipidraftsoncellularplasmamembraneviaimmunofluorescenceconfocalmicroscopy．ThemembranetranslocatedsignalsequencewasininthejunctionofthefirstandsecondQ-helices．PlasmamembranetranslocationofMlklorND—mlklleadstoincreasedcellularplasmasodi

5、umconcentration，indicatedbyCoroNaGreensignalstrengthlargelyenhanceddetectedviaimmunofluorescenceconfocalmicroscopy．DepletionofsodiumincellculturemediuminhibitednecrosiS．A11oftheabovephenomenawerenotobservedinapoptosis．Thus，wecametoconclusionthat，Mlkloligomerizationl

6、eadstotranslocationto1ipidraftsoncellularplasmamembrane，afterthat，Mlklcomplexactseitherbyitselforviaotherproteinstoincreasesodiuminflux，whichincreasedcellularosmoticpressureandkeptcellintakingmorewater，eventuallyleadedtoplasmarupture．Keywords：Mlkl：necrosis：incresedp

7、lasmicsodiumconcentration：Rip3：translocationtoplasmamembrane．Ⅱ万方数据目录目录摘要．．．IAbst伯ct．．．．．．1I目录IIl第一章前言．．．．．．．．．．．．．．．．．．．11．1细胞坏死一．．11．2MlkI蛋白．31．2．1MIkI蛋白结构31．2．2tllkI蛋白在坏死细胞信号通路上的作用41．3细胞膜通透性和细胞死亡之间的关系．5第二章材料与方法．92．1药品。试剂92．2DNA相关实验及其操作方法102．2．1真核细胞表达质粒的构建。102．2．2大

8、肠杆菌感受态细胞的转化。132．2．3中等规模质粒DNA的提取(SDS-硐t变性法)。132．2．4质粒测序鉴定。132．3细胞株的构建、培养及相关实验操作方法．．142．3．1细胞培养。142．3．2基因敲除细胞株的构建．．142．3．3慢病毒包装。162．3．4细胞的慢病