【作者】 刘栋；

【导师】 陈勇；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 硕士

【摘要】 内质网跨膜蛋白需肌醇酶1(IRE1)是内质网应激(ER stress)的核心感受器,可感受内质网管腔内未折叠蛋白的聚集并将这种刺激传递至细胞其他区域。近年研究发现内质网应激与多种肝脏疾病有关,如病毒感染(乙肝、丙肝),糖尿病-肥胖症相关的肝脏疾病,酒精性肝病,α1-抗胰蛋白酶缺乏症及缺血再灌注损伤等。内质网应激引起的未折叠蛋白反应(Unfolded Protein Response,UPR)可通过诱导分子伴侣表达上调,关闭蛋白翻译等降低内质网蛋白负荷,但持续过强的内质网应激反应则导致细胞凋亡。为阐明内质网应激与肝细胞凋亡的关系,本研究通过酵母双杂交系统筛选与IRE1相互作用的蛋白质,为进一步探索内质网应激在肝脏疾病中的作用奠定基础。1.酵母双杂交系统筛选IRE1相互作用蛋白质:采用酵母双杂交的方法,分别以hIRE1α(人IRE1α)N端段1～465位氨基酸及C端段468～977位氨基酸为诱饵,与人睾丸cDNA文库质粒顺序转化AH109酵母菌株,经三重/四重营养缺陷培养基及X-α-GAL半乳糖苷酶试验筛选,将PCR鉴定为阳性的酵母质粒转入大肠杆菌,提取质粒并测序,进行生物信息学分析。最后通过酵母回转杂交实验对获得的相互作用加以验证。结果:成功构建诱饵质粒pGBKT7-IRE1N及pGBKT7-IRE1C,并在酵母中正确表达融合蛋白,自激活检测及毒性检测均为阴性。分别筛选出8个及15个与IRE1相互作用的蛋白质;通过回转实验验证,确定了能与IRE1 C端段相互作用的蛋白质RACK1。RACK1是由SCOTIN基因编码的支架蛋白,可细胞内多种激酶相互作用,介导激酶与底物的结合。2.验证IRE1与RACK1在哺乳动物细胞及体外的相互作用:(1)构建真核表达载体3XFLAG-IRE1C, GFPC3-RACK1,双酶切及测序鉴定正确,以GFPC3空载体作为对照,共同转染293T细胞,利用免疫共沉淀技术验证IRE1与RACK1在哺乳动物细胞中的相互作用。( 2 )构建原核表达载体pGEX-6p2-RACK1,pGEX-6p1-IRE1C,分别转化大肠杆菌BL21,IPTG诱导融合蛋白表达并纯化,其中以precision酶切GST-RACK1融合蛋白,获得不带GST TAG的RACK1蛋白,与空GST蛋白为对照,分别与GST-IRE1C孵育,体外Pull-down实验验证IRE1与RACK1的相互作用。(3)构建真核表达质粒GFPC3-IRE1WT,转染人肝癌细胞系HepG2,同时以RACK1单抗及抗鼠-Rodamine为二抗进行免疫荧光实验,检测IRE1及RACK1在肝细胞中的定位。结果:通过体内及体外实验分别验证IRE1与RACK1相互作用的存在,且证明IRE1 C端为RACK1结合部位,免疫荧光证实IRE1与RACK1在肝细胞内有共定位,为其相互作用提供可靠依据。3.RNA干扰实验探讨RACK1对IRE1功能的影响:(1)转染RACK1 SiRNA入HepG2细胞,48小时后收取细胞裂解液,进行Western blot验证基因敲除效果。给予敲除RACK1的细胞以内质网应激诱导药物衣霉素(Thapsigargin,TG)处理,通过western blot检测IRE1的磷酸化及其下游内质网分子伴侣GRP78的表达,同时应用RT-PCR检测XBP-1 mRNA的表达及其剪切,确定RACK1敲除对IRE1功能的影响。(2)相同方法敲除HepG2细胞中RACK1的表达,给予不同剂量的TG处理,TUNEL法检测RACK1敲除对内质网应激时肝细胞凋亡的影响。结果:成功敲除HepG2细胞中RACK1的表达,Western blot及RT-PCR证实RACK1敲除可抑制内质网应激时IRE1的功能活化,同时抑制内质网应激对肝细胞凋亡的诱导。结论:本研究通过酵母双杂交系统首次发现IRE1与RACK1的相互作用,并通过体内、体外多种方法证实其相互作用的存在,最后通过RNA干扰及功能试验证实IRE1对ER stress的感受及活化,对分子伴侣的诱导及XBP-1的剪切,以及细胞凋亡的诱导,均依赖于RACK1的存在,提示RACK1是内质网应激反应信号通路中不可或缺的一个环节,与ER stress下肝细胞的凋亡密切相关,进一步的研究对内质网应激相关的肝脏疾病有重要意义。更多还原

【Abstract】 The Endoplasmic Reticulum(ER) resident protein Inosital Requiring Enzyme 1(IRE1) is the core sensor of ER stress, which senses the accumulation of unfolded protein in the ER lumen and transduces the signal to the rest part of the cell. Recent reports show that ER stress is associated with a lot of hepatic diseases, such as virus infection(HBV,HCV), diabetes-obese-related liver disease, alcoholic liver diseases,α1-antitrypsin deficiency and ischemia/reperfusion injury. The Unfolded Protein Response(UPR) induced by ER stress can reduce ER protein load through the induction of molecular chaperones and shut down of translation. However, persistent or intense ER stress result in apoptotic cell death. In order to elucidate the mechanism of ER stress induced apoptosis in hepatocytes, we screened the interacting proteins of IRE1 in a yeast-two-hybride system so as to give light to the investigation of ER stress in liver diseases.1. Screening of the interacting proteins of IRE1 in a yeast-two-hybrid system. Yeast-two-hybrid were employed to screen the interacting proteins for the N-termius(1-465aa) and C-terminus(468-977) of hIRE1αin a human testis cDNA library. The bait plasmids and the library cDNA were sequentially transfected into Saccharomyces cerevisiae AH109. Screening was carried out on a series of nutrition-selective plates, X-α-GAL assays and PCR assays. Yeast two-hybrid retransformation experiment was applied to verify the interactions. Results: Bait plasmids pGBKT7-IRE1N(1-465)and pGBKT7-IRE1C(468-977) were successfully constructed and the expression of fusion-proteins were correct. Tests for autonomous activation and toxicity were negative. The numbers of confirmed interactions were 8 for hIRE1α-N and 15 for hIRE1α-C. Retransformation experiment verified RACK1, transcript of the gene GNB2L1, a cytoplasmic located scaffold protein as a binding partner of the C terminus of IRE1.2. Verification of the interaction of IRE1 and RACK1 in mammalian cells and in vitro. (1) Construction of eukaryotic expression plasmids: 3XFLAG-IRE1C, GFPC3-RACK1. The construction was confirmed by restriction enzyme catalyzation and DNA sequencing. The plasmids were co-transfected into 293T cells and co-immunoprecipitation was applied to test the interaction of IRE1 and RACK1 in mammalian cells. (2) Prokaryote expression plasimds pGEX-6p2-RACK1,pGEX-6p1-IRE1C were constructed and transformed into E.coli BL21, the GST-fusion protein were purified by standard protocol. Fusion protein GST-RACK1 were catalyzed by PreCisionTM enzyme to produce the purified RACK1 protein. GST-Pull down assay were carried out to verify the in vitro binding of RACK1 to IRE1. (3) Eukaryotic expression plasmid GFPC3-IRE1WT were constructed for transfection HepG2 cells. The transfected HepG2 cells expressing fusion protein GFP-IRE1 WT were then used to do immunofluorescence experiment with anti-RACK1 mAb and Rodamine-conjugated anti-mouse secondary antibody. Results: In vivo and in vitro tests showed that RACK1 can bind to the C-terminus of IRE1. Immunofluorescence confirmed the co-locolization of IRE1 and RACK1, which give solid evidence for the interaction of the two proteins.3. RNAi tests for the function of the interaction between IRE1 and RACK1.(1)Transfect RACK1 SiRNA into HepG2 cells. Collect the cell lysates after 48h to test the interfering efficiency by western blot. The Si RNA treated and untreated cells were administered with thapsigargin to induce ER stress. Western blot were apllied to test the expression level of GRP78 and phosphorylated IRE1 to assess the activation of IRE1. RT-PCR assays were applied to test the expression and splice of XBP-1 mRNA. (2) TUNEL assays were applied to observe the effect on ER stress-mediated apoptosis by RACK1 SiRNA treatment. Results: The inhibition of RACK1 expression in HepG2 cells by SiRNA administration was successful. Our results confirmed that RACK1 depletion could suppress the activation of IRE1 under ER stress conditions, which inhibited ER stress mediated apoptosis of hepatocytes at the same time.Conclusion: Screening of interacting proteins of IRE1 by yeast-two-hybrid methods identified RACK1 as a novel binding partner. In vivo and in vitro assays confirmed the interaction. Immunofluorescence provided the direct evidence. Finally, RNAi experiments confirmed the function of RACK1’s binding to IRE1 in that it could mediate the activation of IRE1 and downstream pathways such as the upregulation of GRP78 and the catalyzation of XBP-1 as well as induction of apoptosis under ER stress conditions, indicating RACK1 as a component of IRE1 signalling network and closely related to ER stress mediated apoptosis of hepatocytes. Further studies are needed for the role of ER stress in liver diseases.更多还原