目的观察创伤后应激障碍(PTSD)大鼠前额皮质(mPFC)神经元未折叠蛋白反应(unfolded protein response,UPR)标志物葡萄糖调节蛋白78(GRP78)和内质网调节蛋白57(ERP57)的表达变化,探讨内质网分子伴侣在PTSD发病机制中的作用。方法采用国际...目的观察创伤后应激障碍(PTSD)大鼠前额皮质(mPFC)神经元未折叠蛋白反应(unfolded protein response,UPR)标志物葡萄糖调节蛋白78(GRP78)和内质网调节蛋白57(ERP57)的表达变化,探讨内质网分子伴侣在PTSD发病机制中的作用。方法采用国际认定的PTSD动物模型-SPS大鼠模型,取80只成年雄性健康Wistar大鼠,随机分为正常组和SPS模型组(SPS1d,SPS4d,SPS7d),采用逆转录-聚合酶链式反应、免疫组织化学法和免疫印记检测PTSD-SPS大鼠m PFC神经元中GRP78和ERP57的表达变化。结果 SPS刺激后大鼠m PFC神经元细胞内GRP78和ERP57蛋白表达于1d开始逐渐升高,7d时表达最多;GRP78和ERP57的m RNA水平的变化与其蛋白表达变化相一致。结论 GRP78和ERP57在PTSD大鼠mPFC过表达可能参与SPS刺激诱导的UPR反应。展开更多
The protein disulfide isomerases（PDIs）family has a central function in the folding of proteins synthetized through the secretory pathway.ERp57,also known as Grp58 or PDIA3,is one of the main studied members of this ...The protein disulfide isomerases（PDIs）family has a central function in the folding of proteins synthetized through the secretory pathway.ERp57,also known as Grp58 or PDIA3,is one of the main studied members of this family.展开更多
Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induc...Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induce apoptosis as well as generate reactive oxygen species(ROS).Mitoxantrone(MXT)was used as a positive control for apoptosis.The SHSH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide(HyPer).The ERp57,also called GRP58(Glucose-regulated protein 58),is a stress protein induced in conditions like glucose starvation and viral infection.Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells.ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death.ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy.These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.展开更多
OBJECTIVE To identify the specific targets of a novel derivative of Danshensu ADTM as the protein disulfide isomerase(PDI)family proteins including ERp57.To further investigate the underlying mechanism of ADTM to modu...OBJECTIVE To identify the specific targets of a novel derivative of Danshensu ADTM as the protein disulfide isomerase(PDI)family proteins including ERp57.To further investigate the underlying mechanism of ADTM to modulate ERp57 to regulate platelet function with direct interaction withαⅡbβ3 integrin.METHODS To isolate the protein targets that bound to ADTM,a biotin-conjugated ADTM analogue(BAA)was designed and synthesized.BAA(300μmol·L-1)was incubated with rat blood platelet lysates and the BAA-protein complexes were pulled down with NeutrAvidin-agarose followed by protein profiling using LC-MS/MS.To determine platelet aggregation in vitro,rabbit platelets were incubated with the indicated concentrations of compounds and aggregation was induced by ADP(10μmol·L-1)or AA(200μmol·L-1)and measured using a platelet aggregometer.To determine platelet aggregation-induced by ADP in rat in vivo,ADTM(5-20mg·kg-1)in comparison with DSS(10mg·kg-1)and clopidogrel(18mg·kg-1)were administered daily by i.v.injection for 5d,respectively.To determine the action of ADTM on the ERp57/αⅡbβ3 interaction,it was examined by immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting.RESULTS BAA could bind to various proteins involved in platelet function.In particular,platelet aggregation-associated proteins were identified with>95% protein identification probability including ERp72,ERp57ERp5 and PDI,which are members of the protein disulfide isomerase(PDI)family related to platelet function and redox homeostasis.ADTM exhibited potent inhibition on the redox activity of ERp57 in a concentration-dependent manner(IC50=100 300μmol·L-1).In in vitro studies,ADTM exhibited concentration-dependent inhibition on ADP-induced and AA-induced platelet aggregation with comparable effects to aspirin and clopidogrel.In vivo study showed that ADP-induced platelet aggregation was significantly compromised(>40%reduction)in rats treated with ADTM(20mg·kg-1).Similarly,ADTM also exh展开更多
基金FONDECYT 1161284(SM),Millennium Institute No.P09-015-F,Fondo de Financiamiento de Centros de Investigación enáreas Prioritarias(FONDAP)15150012(CHSM)+3 种基金the Frick Foundation No.20014-15,ALS Therapy Alliance 2014-F-059,Muscular Dystrophy Association 382453,Comisión Nacional de Investigación Científica y Tecnológica(CONICYT)CONICYTUSA2013-0003the Michael J.Fox Foundation for Parkinson’s Research No.9277,Fundación Copec–Universidad Católica No.2013.R.40,Ecos-Conicyt C13S02,FONDECYT 1140549,Office of Naval Research–Global(ONR-G)N62909-16-1-2003Amyotrophic Lateral Sclerosis Research Program Therapeutic Idea Award AL150111(C.H.)LB is supported by a CONICYT fellowship
文摘The protein disulfide isomerases（PDIs）family has a central function in the folding of proteins synthetized through the secretory pathway.ERp57,also known as Grp58 or PDIA3,is one of the main studied members of this family.
文摘Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induce apoptosis as well as generate reactive oxygen species(ROS).Mitoxantrone(MXT)was used as a positive control for apoptosis.The SHSH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide(HyPer).The ERp57,also called GRP58(Glucose-regulated protein 58),is a stress protein induced in conditions like glucose starvation and viral infection.Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells.ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death.ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy.These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.
基金The project supported by National Natural Science Foundation of China(81403139)Research Committee of the University of Macao(MYRG2015-00161-ICMS-QRCM)
文摘OBJECTIVE To identify the specific targets of a novel derivative of Danshensu ADTM as the protein disulfide isomerase(PDI)family proteins including ERp57.To further investigate the underlying mechanism of ADTM to modulate ERp57 to regulate platelet function with direct interaction withαⅡbβ3 integrin.METHODS To isolate the protein targets that bound to ADTM,a biotin-conjugated ADTM analogue(BAA)was designed and synthesized.BAA(300μmol·L-1)was incubated with rat blood platelet lysates and the BAA-protein complexes were pulled down with NeutrAvidin-agarose followed by protein profiling using LC-MS/MS.To determine platelet aggregation in vitro,rabbit platelets were incubated with the indicated concentrations of compounds and aggregation was induced by ADP(10μmol·L-1)or AA(200μmol·L-1)and measured using a platelet aggregometer.To determine platelet aggregation-induced by ADP in rat in vivo,ADTM(5-20mg·kg-1)in comparison with DSS(10mg·kg-1)and clopidogrel(18mg·kg-1)were administered daily by i.v.injection for 5d,respectively.To determine the action of ADTM on the ERp57/αⅡbβ3 interaction,it was examined by immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting.RESULTS BAA could bind to various proteins involved in platelet function.In particular,platelet aggregation-associated proteins were identified with>95% protein identification probability including ERp72,ERp57ERp5 and PDI,which are members of the protein disulfide isomerase(PDI)family related to platelet function and redox homeostasis.ADTM exhibited potent inhibition on the redox activity of ERp57 in a concentration-dependent manner(IC50=100 300μmol·L-1).In in vitro studies,ADTM exhibited concentration-dependent inhibition on ADP-induced and AA-induced platelet aggregation with comparable effects to aspirin and clopidogrel.In vivo study showed that ADP-induced platelet aggregation was significantly compromised(>40%reduction)in rats treated with ADTM(20mg·kg-1).Similarly,ADTM also exh
