论文标题:SGT及其相关蛋白功能的研究
Studies of the Functions of SGT and Its Interaction Proteins
论文作者 申海莲
论文导师 顾建新,论文学位 博士,论文专业 生物化学与分子生物学
论文单位 复旦大学,点击次数 171,论文页数 99页File Size9207k
2004-05-07论文网 http://www.lw23.com/lunwen_1341727/ SGT蛋白;P53;Caspase;酵母双杂交系统;PIASI蛋白;β1,4-半乳糖基转移酶1/Ets/Ets1/E1AF
SGT ; P53 ; Caspase ; yeast two hybrid system ; PIASl ; GaT1 I ; Ets;Ets1;E1A
SGT(small glutamine-rich tetratricopeptiderepeat TPR)克隆于1998年,由313个氨基酸组成,其C末端有一段富含谷氨酰氨的序列,中间含有三个串联重复的TPR结构域。近年来的研究表明,SGT能够与热休克蛋白家族的Hsp70和Hsc70相互作用并调节其分子伴侣活性,而Hsp70和Hsc70的分子伴侣活性可以拮抗热休克、血清饥饿、化疗药物等引起的细胞凋亡,不仅如此,CHIP、TPR2、Strap这些含有TPR结构域的蛋白均以不同的方式参与了细胞凋亡的过程。为了弄清SGT是否也参与了细胞凋亡的过程,我们首先建立了SGT超表达的SMMC-7721稳转细胞株,用放线菌酮(CHX)处理稳转空载体的SMMC-7721和稳转SGT的SMMC-7721细胞后,分别用PI染色,AnnexinV和PI双染,DNA片段化降解检测,凋亡小体分析,Hoechest染色观察均证实SGT可以促进CHX诱导的7721细胞凋亡,进一步的研究表明这种促凋亡作用是Caspase依赖的,SGT可以促进Caspase家族蛋白的活化并将其底物PARP裂解,进一步的免疫荧光试验表明超表达SGT可使部分P53定位到线粒体。为了进一步探讨SGT的功能我们应用酵母双杂交的方法以SGT为诱饵筛选人胎肝cDNA文库鉴定出PIAS为它的一个新的结合蛋白,接下来用GST-pull down、免疫共沉淀,以及激光共聚焦的方法证实了二者在体内和体外均可相互作用。 β 1,4半乳糖基转移酶(GaIT Ⅰ)是一个最早克隆的糖基转移酶,长期以来被认为是一个管家基因,本实验室以前的工作表明GalT Ⅰ是SGT的一个相互作用蛋白,Ets转录因子家族的成员EIAF可调控其基因的转录。本文研究了Ets家族的另一成员Ets-1可否调控其转录。我们采用荧光素酶报告基因的方法证实Ets-1可激活GalT Ⅰ报告基因的表达,并具有量效关系。我们又采用突变分析法确定GaT Ⅰ启动子-215至-139核苷酸区域存在一个Ets-1的结合位点,进一步采用染色质免疫沉淀试验以及电泳凝胶迁移率改变试验证实Ets-1能够与此区域DNA直接结合,从而确定Ets-1是调控β 1,4半乳糖基转移酶基因的一个转录因子。
SGT (Small Glutamine-rich Teratricopeptiderepeat TPR) was cloned in 1998. It consists of 313 amino acids with a glutamine-rich sequence in its carboxyl terminal and three tandom TPR repeats in the center of the polypeptide chain. It"s reported that SGT interacts with Hsp70 and Hsc70 and regulates their chaperon activities, which antagonize against heat shock, serum depletion and the apoptosis induced by drugs used in chemical therapies. Further more, many TPR domain-containig proteins such as CHIP, TPR2 and Strap play important roles in cell apoptosis with different mechanisms. To clarify whether SGT participates in the progress of cell apoptosis, we first constructed the SMMC-7721 cell lines stably expressing SGT. SMMC-7721 cells and the stable transfected cells were treated with CHX, followed by PI staining, Annexin V and PI co-staining, DNA fragmentation test, and Hoechest 33258 staining, apoptosis bodies assay. It was found in all the experiments that SGT promoted the apoptosis of 7721 cells induced by CHX, and its role in the regulation of apoptosis was caspase-dependent. SGT potentiated the activity of caspases and facilitated the cleavage of PARP, a substrate of caspase3. Overexpression of SGT led to the localization of more p53 on mitochondria than control group. To further investigate the function of SGT protein, we screened the cDNA library of human fetal liver using SGT as the bait in yeast two-hybrid system. PIAS1 was identified as a new interacting partner of SGT, and their interactions in vitro and in vivo were confirmed by GST-pull down, co-immunoprecipitation and confocal assays.β-1,4-galactosyltransferase 1 (GalT I) is the first cloned glycosyltransferase. It had been regarded as a house-keeping gene for a long time. In our previous investigation, it was found that GaT I was an intraction protein of SGT, and EIAF, one of the Ets family members could regulat its expression. In this paper, we investigated whether Ets-1, another member of Ets family could regulate its expression. Cotransfection of GalT I promoter /luciferase reporter and Ets-1 expression plasmid increased the luciferase reporter activity in a dose dependent manner. By deletion analysis, we identified a region between nucleotide -215—139 in GalT I promoter which was critical for responsiveness to Ets-1. Site specific mutagenesis revealed this region contained an Ets binding site (-205—200) which was necessary for GalT I activation. We confirmed that Ets-1 coud bind to and activate GalT I promoter by EMSA and chromatin-immunoprecipitation. These data suggest that Ets-1 is a transcriptional factor of GalT I.
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