【作者】 王茜；

【导师】 余龙；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 RLIM已知的功能是作为转录辅抑制蛋白或作为E3泛素连接酶,抑制某些LIM同源蛋白的转录,参与发育的调控。肿瘤抑制蛋白p53翻译后修饰的一种重要形式是乙酰化,由组氨酸乙酰转移酶介导。p53去乙酰化是与乙酰化相反的过程,由组氨酸去乙酰化酶介导。MDM2能够抑制组氨酸乙酰转移酶p300/CBP及PCAF介导的p53乙酰化,也能够促进组氨酸去乙酰化酶HDAC1介导的p53去乙酰化。p53是一个转录因子,具有转录激活活性,乙酰化可以提高p53的转录激活活性。我们发现,RLIM可以与组氨酸乙酰转移酶p300互作,促进p300介导的p53乙酰化,是一个新的能调节p53乙酰化的蛋白质。RLIM对p53乙酰化的调节是依赖于MDM2的,MDM2对p300介导的p53的乙酰化有抑制作用,RLIM可以消除这种抑制作用,从而促进p300介导的p53乙酰化。同时,MDM2可以协同HDAC1对p53去乙酰化,RLIM可以抑制MDM2对p53去乙酰化,从而促进p53乙酰化。RLIM促进p53乙酰化依赖于MDM2这一事实让我们联系到被人乳头瘤病毒(human papillomavirus,HPV)感染的细胞,在HPV阳性的宫颈癌细胞系中病毒蛋白E6会与人蛋白E6-AP形成E6/E6-AP复合物,介导p53泛素化及降解。MDM2在正常细胞内介导p53泛素化及降解,但在HPV阳性细胞中p53的泛素化及降解完全由E6/E6-AP复合物介导。RLIM依赖于MDM2促进p53乙酰化来提高p53转录激活活性对HPV阳性细胞的意义大于对正常细胞的意义,因为正常细胞里p53转录激活活性的提高还可以依赖于MDM2介导的p53泛素化减少来实现。我们发现RLIM能够促进HPV-18阳性宫颈癌细胞系HeLa细胞内p53乙酰化,这一过程依赖于MDM2。95%宫颈癌都感染了高危险性HPV,但是HPV的存在不足以使HPV造成的病灶恶化成为肿瘤,提示从HPV感染到宫颈癌的发生之间还存在未知的机制。p53在50%的肿瘤里突变,但是在宫颈癌中几乎没有突变。在HPV阳性的宫颈癌细胞系中p53蛋白量很低,因为E6/E6-AP复合物介导了p53泛素化及降解,尽管蛋白量低,但p53是野生型的并可以被诱导发生转录激活,导致细胞发生细胞周期抑制及凋亡。因此,p53蛋白量水平低下虽然可以从一定程度解释宫颈癌的发生,但是p53在转录激活活性上受到抑制可能也是导致宫颈癌发生的原因。我们发现RLIM提高了HeLa细胞中p53转录激活活性,并诱导HeLa细胞凋亡。我们还发现RLIM在我们检测的7例宫颈癌组织中的6例里下调。这些结果揭示出RLIM下调可能导致HPV感染细胞中p53乙酰化受到抑制,p53转录激活活性下降,细胞不能凋亡,走向癌化。我们的工作为从HPV感染到宫颈癌发生这一过程中的未知机制提供了有益的启示。更多还原

【Abstract】 RLIM (RING finger LIM domain-binding protein) is identified as a transcriptional co-repressor as well as an E3 ubiquitin ligase to inhibit the transcriptional activities of some LIM-hd (LIM homeodomain) proteins involved in development.Acetylation of tumor suppressor p53 is one of the posttranslational modifications of p53, which is mediated by histone acetyltransferases (HATs). Deacetylation of p53 is the contrary process of acetylation of p53, which is mediated by histone deacetylase (HDACs). MDM2 can inhibit HATs p300/CBP and PCAF mediated p53 acetylation. MDM2 also synergizes HDAC1 to mediate the deacetylation of p53. p53 is a transcription factor with transcriptional activity. Acetylation of p53 is beneficial to p53 transcriptional activation in response to stress.We have found that RLIM can interact with HAT p300 and promotes p300 mediated p53 acetylation. RLIM is a novel protein which can regulate p53 acetylation. The promotion of p53 acetylation by RLIM is dependent on MDM2. RLIM promotes p53 acetylation both through abrogating the inhibitory effect of MDM2 on p300-mediated acetylation of p53 and through inhibiting MDM2-mediated p53 deacetylation.We connet human papillomavirus (HPV) infected cells with the fact that RLIM targets MDM2 to promote p53 acetylation. In HPV positive cervical cancer cell lines the protein level of p53 is low, because viral E6 protein forms complex with human E6-AP protein to mediate ubiquitination and degradation of p53. p53 ubiquitination and degradation is mediated by MDM2 in normal cells. However, in HPV positive cells the ubiquitination and degradation of p53 is mediated by E6/E6-AP complex completely. Since MDM2 is replaced by E6/E6-AP on p53 ubiquitination and degradation in HPV positive cells, the fact that RLIM targets MDM2 to promote p53 acetylation is more important for p53 activation in HPV positive cells than in HPV negative cells. We have found RLIM promotes p53 acetylation in HPV-18 positive cervical cancer derived cell line HeLa cells. And the promotion is dependent on MDM2.95% of cervical cancer is related with HPV infection. However, the presence of HPV is not sufficient for malignant progression of HPV-containing lesions, suggesting that there are still unknown mechanisms existing in the progression from HPV infection to cervical cancer. p53 is mutated in 50% of human tumors while rarely mutated in cervical cancers. In HPV positive cervical cancer cell lines the protein level of p53 is low, because E6/E6-AP complex mediates ubiquitination and degradation of p53. However, p53 is wild type and can be induced to transactivation, leading to cell cycle arrest and apoptosis in these cells. The above suggests that both low protein level of p53 and inhibition of transcriptional activity of p53 may contribute to cervical carcinogenesis.We have found RLIM enhances the transcriptional activity of p53 in HeLa cells and induces apoptosis of the cells. RLIM is downregulated in six out of seven cervical carcinoma tissues we examined. These results indicate that downregulation of RLIM may impair the acetylation of p53 in HPV positive cells, resulting in decreasing of transcriptional activity of p53, leading to cells resistent to apoptosis and to be cancerous. Our work sheds some light in the progression from HPV infection to cervical cancer.更多还原