【作者】 潘传英；

【导师】 陈宏； 曹诚；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 人泛素类似蛋白hPLIC-2是酵母Dsk2和鼠PLIC(sproteins link IAP and cytoskeleton, PLICs)的同源类似物。在结构上具有两个重要的结构域,即N端的泛素类似结构域(UBL)和C端的泛素结合结构域(UBA)。hPLIC-2过表达时可特异性干扰依赖泛素的蛋白的降解,还可与蛋白酶体和E3泛素连接酶相互作用,在泛素化酶系和蛋白酶体间建立功能联系,但其在泛素蛋白酶体途径的具体功能未知。酵母双杂交实验发现:hPLIC-2可与c-Abl(cellular-Abelson gene),即Abelson鼠白血病病毒(murine leukemia virus) v-abl原癌基因在细胞内的同源基因,直接相互作用,而c-Abl可抑制蛋白酶体的降解活性。为此,本实验旨在研究hPLIC-2在泛素蛋白酶体途径中的调控及与c-Abl的相互作用。本研究采用RNA干扰技术,利用逆转录病毒载体和MCF-7细胞,结合细胞培养与转染、克隆筛选与免疫印迹实验,建立了稳定抑制内源性的hPLIC-2表达的细胞系;构建了pGEX-2T-hPLIC-2原核表达载体,制备了GST-hPLIC-2融合蛋白,融合蛋白与佛氏完全佐剂共同免疫新西兰大白兔,所得抗血清经纯化后制成了高滴度特异性的抗hPLIC-2多克隆抗体。以上实验为进一步研究hPLIC-2的功能打下基础。进一步的免疫印迹、免疫沉淀实验证明,hPLIC-2可促进细胞内总蛋白的泛素化;当hPLIC-2表达被抑制时(转染siRNA(hPLIC-2)质粒的DKO细胞和抑制细胞系中),细胞内总蛋白的泛素化水平明显降低;而hPLIC-2和c-Abl共同转染DKO细胞,细胞内总蛋白的泛素化水平又比单独转染hPLIC-2得到了更大程度的提高,提示hPLIC-2和c-Abl可能协同作用提高细胞总蛋白泛素化水平。采用同位素示踪技术检测hPLIC-2和c-Abl对细胞内总蛋白降解的调控时发现,hPLIC-2可抑制细胞内总蛋白的降解;而当hPLIC-2表达被抑制(转染siRNA(hPLIC-2)质粒的DKO细胞和抑制细胞系中)时,细胞内总蛋白的降解量显著升高;hPLIC-2和c-Abl共同转染DKO细胞时,细胞内总蛋白的降解比单独转染hPLIC-2时受到了更大程度的抑制;提示hPLIC-2和c-Abl协同作用抑制总蛋白质的降解。与此同时,为了确保结果的可靠性,我们转染了表达绿色荧光蛋白的pEGPF-C1质更多还原

【Abstract】 Human PLIC-2(hPLIC-2), the homologues of yeast Dsk2 and mouse PLICs (proteins link IAP and cytoskeleton, PLICs), contains an amino-terminal ubiquitin-like (ubl) domain and a carboxyl-terminal ubiquitin-associated (uba) domain. The ubiquitin-like hPLIC-2 protein can associate with proteasomes, and hPLIC-2 overexpression can specifically interfere with ubiquitin-mediated proteolysis. Because hPLIC-2 can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. However, the concrete function of hPLIC-2 in ubiquitin- proteasome pathway is unclear. It was shown by the yeast two-hybrid experiment using full length c-Abl (cellular-Abelson gene)as bait protein that hPLIC-2 may be one of the potential c-Abl. c-Abl, the ubiquitously expressed non-receptor tyrosine kinase, can inhibit the activity of proteasome. In this experiment, the function of hPLIC-2 in the ubiquitin- proteasome pathway and the interaction between hPLIC-2 and c-Abl were studied.Molecular biologic technology, such as RNA interference, cell transfection,protein purification, immunoblot, immunoprecipitation, were applied to this study. Firstly, a cell line in which endogenous hPLIC-2 was stably inhibited have been established. Anti-hPLIC-2 polyclonal antibody was prepared after expressing GST-hPLIC-2 fusion protein. Further study by Immunoblot and Immunoprecipitation authenticated that hPLIC-2 overexpression can advance total protein ubiquitination in vivo. While, when hPLIC-2 was inhibited, the degree of ubiquitination in vivo decreased clearly. The amount of hPLIC-2 binding ubiquitinated proteins was increased in the presence of c-Abl. The result suggested that hPLIC-2 and c-Abl act in concert to advance total protein ubiquitination in vivo. Isotopic更多还原