【作者】 王瑞瑛；

【导师】 罗贵民； 孙志贤；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 Survivin 是1997 年Ambrosini 等应用EPR-1 基因cDNA 作为探针去筛选人基因组文库而得到的,是IAP 家族的一个成员,以其独特的分子结构以及表达特点,在其被发现后的几年中得到广泛的研究与关注。本论文中,我们用蛋白合成抑制剂放线菌酮CHX处理宫颈癌细胞HeLaS3,并用Western-blot 检测,发现60min 后Survivin 蛋白的表达量明显降低。而用PKA 的抑制剂H89 预先处理细胞两个小时后,再用CHX 处理细胞,则Survivin蛋白量变化不明显。我们推断,Survivin 的稳定性可能与其潜在的PKA 磷酸化位点的磷酸化状态有关。进而通过序列比对,我们选择出三条针对Survivin 基因的siRNA 片段,并构建了相应的RNAi 载体,转染入HeLaS3 细胞后,瞬时转染的结果表明,Survivin 表达量在RNA 和蛋白水平上都得以下调,尤以S3 片段效果最为明显,并且用流式细胞术检测干涉载体转染对细胞产生的生物学效应,发现转染干涉载体pTet-U6-S3 后,细胞凋亡率得到很大的提高。另外,我们构建了Survivin 上潜在的pKA 磷酸化位点Ser81 的突变体S81D、S81A,用来检测该位点磷酸化不同状态对Survivin 稳定性的影响。但是把干涉载体转染至细胞,并把其培养成稳定株后,HeLaS3 细胞内源Survivin 量并没有被有效地抑制,这是很出乎预料的。更多还原

【Abstract】 There are two classes of important regulation proteins in apoptosis. One is the well known Bcl-2s protein family, the other is evolutionarily conserved IAPs(inhibitor of apoptosis protein) family. IAPs can not only directly inhibit the activation of procaspases, but inhibit activated caspases. To date, IAP proteins cloned from human cells have been identified as: XIAP、NAIP、c-IAP1、c-IAP2、ML-IAP/Livin、Survivin and apollon. The characteristic of IAP family proteins was a BIR domain contained about 70 amino acids rich in cysteine and histidine. Some of them also contained a RING zinc finger. An IAP protein—Survivin has caught the interest of cell biologists for its special architecture and expression manner. This gene was identified by Ambrosini et al. In 1997 in an accasional experiment. They have cloned Survivin by hybridization screening of a human P1 genomic library with the cDNA of effector cell protease receptor-1(EPR-1). The coding strand of Survivin gene is entirely complementary(antisense) to EPR-1. The human Survivin gene spans 14.7kb on telomeric position of chromosome 17 and is localized to band q25. It comprises three introns and four exons. The mature mRNA of Survivin was 1629 nucleotides in length. It encoded a protein of 142 amino acids, with an estimated molecular weight of 16.5kD. The organization of Survivin protein was unique as compare with that of other IAPs. Survivin contains only one partially conserved BIR domain and no RING zinc finger. But the more interesting thing was its expression specificity. Present during fetal development, Survivin is undetectable in terminally differentiated adult tissues. However, Survivin becomes prominently expressed in transformed cell lines and in all the most common human cancers. But it is expressed in almost all the tumors and about 50% highly differentiated non-Hodgkins lymphocyte tumor. And its high expression in cancer correlated with bad prognosis. Its expression is highly cell cycle-depended, and is the first molecular that is related with cell apoptosis and cycle regulation. Researches have demonstrated that the RING construct in the C terminated end has the function of protein-protein interaction and has ubiquitin ligase (E3) activity to ubiquitinate other proteins as well as itself for degradation. Intriguingly, while Survivin has no known E3 activity, Survivin can be ubiquitinated and degraded by the proteasome, and processed with the cell cycle-depended manner. Clearly, it will be valuable to identify the mechanism and the ubiquitination enzymes responsible for Survivin degradation. In the cells, besides the regulation of E3 activity, the ubiquitination modification of the substrate protein is also affected by the post-translation modification, and the most important one is phosphorilation modification. So we study that if the phosphorylation modification of Survivin is related to its degradation in ubiquitin-proteasome pathway. Through sequence analysis of Survivin protein, there are three potential PKC phosphorylation sites: Thr21, Ser88 and Thr127, two phosphorylation sites of Tyrosine kinase Ⅱ: Thr48 and Thr97, also a PKA phosphorylation site: Ser81.更多还原