在口腔、食管和结直肠癌病灶中,TATA序列结合蛋白相关因子1(TATA-box binding protein associated factor 1 like,TAF1L)、stomatin家族蛋白1(stomatin-like protein 1,STOML1)和stomatin家族蛋白2(stomatin-like protein 2,STOML2)存...在口腔、食管和结直肠癌病灶中,TATA序列结合蛋白相关因子1(TATA-box binding protein associated factor 1 like,TAF1L)、stomatin家族蛋白1(stomatin-like protein 1,STOML1)和stomatin家族蛋白2(stomatin-like protein 2,STOML2)存在异常表达.为探究它们在消化系统癌变过程中的作用机理,利用6种消化道肿瘤(食道、胃、结肠、直肠、肝脏和胰腺)及癌旁组织的微陈列芯片,通过多重免疫组织化学染色,比对分析TAF1L、STOML1和STOML2这3种蛋白质在癌与癌旁组织的差异表达及与癌变的关联.结果发现,STOML2在食道、胃、结肠、直肠、肝脏和胰腺这6种消化系统癌组织中均呈显著性高表达(P<0.001),而同一家族成员STOML1则仅在食管癌和肝癌组织中表达明显增高(P<0.005).另外,除胰腺癌灶外,TAF1L在其余5种消化系统癌组织中也呈现高表达(P<0.005).同时,TAF1L、STOML1和STOML2这3种蛋白质均与上皮间充质转化的关键蛋白质MMP9有共定位现象.由此表明,3种蛋白质TAF1L、STOML1和STOML2在多种癌组织中可能具有广泛的促癌作用.由于3种蛋白质在多种癌组织的表达水平不同,说明它们各自存在着组织特异性,且具有不同的功能使命,需进一步研究TAF1L、STOML1和STOML2这3种蛋白质在消化系统恶性肿瘤发生发展过程中各自的分子病理学潜能,如能将它们打造成用于精准诊疗和预后判断的新型分子标志物,此研究结果将有重要的临床意义.展开更多
目的:初步研究78 k D的葡萄糖调节蛋白(the 78 k D glucose-regulated protein,GRP78)与乙型肝炎病毒(hepatitis B virus,HBV)的前S1蛋白(Pre S1)的相互作用位点。方法:利用PCR技术扩增GRP78的基因,将扩增的目的基因克隆至p W28载体质粒...目的:初步研究78 k D的葡萄糖调节蛋白(the 78 k D glucose-regulated protein,GRP78)与乙型肝炎病毒(hepatitis B virus,HBV)的前S1蛋白(Pre S1)的相互作用位点。方法:利用PCR技术扩增GRP78的基因,将扩增的目的基因克隆至p W28载体质粒,在大肠杆菌(Escherichia coli,E.coli)B834中表达,经过镍离子亲和层析柱纯化GRP78蛋白;将Pre S1 3个截短片段的重组质粒(p GST-Pre S1-X1/X2/X3)在B834中表达后,经过GST亲和层析柱纯化相应蛋白;利用蛋白质体外结合实验(pull down)、微量热泳动(microscale thermophoresis,MST)检测GRP78与Pre S1 3个截短片段的相互作用。结果:成功构建重组质粒p W28-GRP78;获得GRP78蛋白及Pre S1 3个截短片段的融合蛋白;pull down及MST实验验证了GRP78可以与Pre S1的3个片段结合,且GRP78与GST-Pre S1-X1结合效果最好。结论:利用分子克隆技术及蛋白质表达纯化技术,获得GRP78蛋白及Pre S1截短片段的融合蛋白,并初步筛选了Pre S1与GRP78的相互作用位点,为后续研究打基础。展开更多
Objective:PCBP1 is a family member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that belong to RNA-binding proteins and bear three KH domains. The protein plays a pivotal role in post-transcriptional regulatio...Objective:PCBP1 is a family member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that belong to RNA-binding proteins and bear three KH domains. The protein plays a pivotal role in post-transcriptional regulation for RNA metabolism and RNA function in gene expression. We hypothesized and were going to identify that the regulatory function of PCBP1 is performed through different complexes of proteins that include PCBP1. Methods:To test our hypothesis,approaches of protein wal-king with a yeast two-hybrid system (Y2H),pulling down in yeasts,co-immunoprecipitation and immunofluorescent microscopy assay were employed in this study. The PCBP1 was used as the initial "walker" to search for its interaction partner(s). Results:Candidate proteins including MYL6,PECAM1,CSH1,RAB7,p57KIP2,ACTG1,RBMS1 and PSG4-like were identified with selection mediums and preceding methods. Conclusion:With these candidate protein molecules,some protein complexes associating with PCBP1 are proposed,which may help in a better understanding of physiological functions of PCBP1 and proved evidence that PCBP1 is involved in variant biological pathways.展开更多
文摘泛素连接酶E4B通过U-box基序可将经泛素活化酶(Ubiquitin-activating enzyme, E1)和泛素结合酶(Ubiquitin-conjugating enzyme, E2)传递的泛素(Ubiquitin, UB)标记至底物蛋白质。探究3个蛋白质:金属蛋白酶M20家族蛋白质2(Peptidase M20 domain-containing protein 2,PM20D2)、多腺苷酸结合蛋白质1(Polyadenylate-binding protein 1,PABPC1)、细胞色素C氧化酶Ⅰ翻译激活剂(Translational activator of cytochrome C oxidase Ⅰ,TACO1)是否可被E4B介导泛素化。从HEK293细胞中提取总RNA,反转录为cDNA,以其为模板调取底物基因并构建重组质粒,利用大肠杆菌表达系统表达并纯化泛素化过程所需的各个相关蛋白质,通过蛋白质体外泛素化实验,对3个蛋白质进行泛素化验证。结果表明3个蛋白质均可以在蛋白质水平被E4B泛素化,证明3个蛋白质都是E4B的底物,为进一步在细胞中研究其泛素化的机理奠定基础。
文摘在口腔、食管和结直肠癌病灶中,TATA序列结合蛋白相关因子1(TATA-box binding protein associated factor 1 like,TAF1L)、stomatin家族蛋白1(stomatin-like protein 1,STOML1)和stomatin家族蛋白2(stomatin-like protein 2,STOML2)存在异常表达.为探究它们在消化系统癌变过程中的作用机理,利用6种消化道肿瘤(食道、胃、结肠、直肠、肝脏和胰腺)及癌旁组织的微陈列芯片,通过多重免疫组织化学染色,比对分析TAF1L、STOML1和STOML2这3种蛋白质在癌与癌旁组织的差异表达及与癌变的关联.结果发现,STOML2在食道、胃、结肠、直肠、肝脏和胰腺这6种消化系统癌组织中均呈显著性高表达(P<0.001),而同一家族成员STOML1则仅在食管癌和肝癌组织中表达明显增高(P<0.005).另外,除胰腺癌灶外,TAF1L在其余5种消化系统癌组织中也呈现高表达(P<0.005).同时,TAF1L、STOML1和STOML2这3种蛋白质均与上皮间充质转化的关键蛋白质MMP9有共定位现象.由此表明,3种蛋白质TAF1L、STOML1和STOML2在多种癌组织中可能具有广泛的促癌作用.由于3种蛋白质在多种癌组织的表达水平不同,说明它们各自存在着组织特异性,且具有不同的功能使命,需进一步研究TAF1L、STOML1和STOML2这3种蛋白质在消化系统恶性肿瘤发生发展过程中各自的分子病理学潜能,如能将它们打造成用于精准诊疗和预后判断的新型分子标志物,此研究结果将有重要的临床意义.
基金Supported in part by the “985” program(985-2-035-39) of the Chinese Ministry of Education“973” project(2007CB511902)of the Chinese Ministry of Sciences and Technology+1 种基金National Nature Science Foundation(30671157)the NewYork State Office of Mental Retardation and Developmental Disabilities(NYSOMRDD)~~
文摘Objective:PCBP1 is a family member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that belong to RNA-binding proteins and bear three KH domains. The protein plays a pivotal role in post-transcriptional regulation for RNA metabolism and RNA function in gene expression. We hypothesized and were going to identify that the regulatory function of PCBP1 is performed through different complexes of proteins that include PCBP1. Methods:To test our hypothesis,approaches of protein wal-king with a yeast two-hybrid system (Y2H),pulling down in yeasts,co-immunoprecipitation and immunofluorescent microscopy assay were employed in this study. The PCBP1 was used as the initial "walker" to search for its interaction partner(s). Results:Candidate proteins including MYL6,PECAM1,CSH1,RAB7,p57KIP2,ACTG1,RBMS1 and PSG4-like were identified with selection mediums and preceding methods. Conclusion:With these candidate protein molecules,some protein complexes associating with PCBP1 are proposed,which may help in a better understanding of physiological functions of PCBP1 and proved evidence that PCBP1 is involved in variant biological pathways.
