In vivo function of Mdm2 E3 ubiquitin ligase

描述(申请人提供)：转录因子P53通过诱导细胞周期停滞或凋亡来响应各种细胞应激源，在肿瘤抑制中发挥关键作用。破坏p53功能的p53基因突变出现在50%的人类癌症中，其余大多数癌症中两种p53抑制物MDM2和MDM4水平升高。目前的学说认为，MDM2主要通过其环指E3泛素连接酶调节P53的稳定性，而Mdm4主要通过隐藏P53转录激活结构域来控制P53的转录活性。体外研究表明，MDM2‘S环E3也参与了MDM4的降解和自身的降解，并提出了应激后P53由MDM2降解向自身降解转换的模型。许多p53诱导剂，包括抑癌基因p14ARF和核糖体蛋白L11，通过抑制mdm2‘S E3功能来稳定和激活p53。因此，从理论上讲，针对MDM2 E3功能的抗癌策略可以与针对MDM2-P53相互作用的策略合作，以激活每年数百万被诊断为P53阳性癌症患者的P53。然而，重要的是，为了实现这一目标，需要详细了解MDM2 E3调控的分子机制。我们最近产生了在MDM2环指区域带有单一残基替换的小鼠，取消了它的E3功能，而不影响P53结合。然而，出人意料的是，与目前的概念相反，我们的数据表明：1)在缺乏MDM2介导的P53泛素化的情况下，MDM2-P53的相互作用不能控制P53的活性；2)MDM2自身泛素不是体内MDM2降解的主要机制。我们的分析揭示了体外转基因研究产生的假说与小鼠模型之间的另一种脱节。在此小鼠模型的基础上，我们将检验三个假设：1)mdm2-mdm4相互作用增强或需要mdm2的S E3连接酶功能；2)mdm2的结合抑制P53‘S的凋亡但不抑制细胞周期停滞功能；3)体内存在一种未知的新的泛素连接酶来降解mdm2。目的：1.研究MDM2环E3在MDM4调控中的作用；2.研究MDM2和MDM4在P53调控中的非冗余作用。目的3.探讨MDM2在调控P53诱导的细胞周期停滞和细胞凋亡中的作用。与公共卫生相关：肿瘤抑制基因p53基因突变会损害p53功能，在50%的人类癌症中会发生突变，而在其余的大多数癌症中，两种p53抑制因子MDM2和MDM4的水平会升高。本项目计划利用新的小鼠模型来研究MDM2-MDM4-P53调控环的功能和机制。详细了解MDM2 E3泛素连接酶的分子机制和调控对于设计未来基于P53的抗癌策略至关重要。

DESCRIPTION (provided by applicant): The transcription factor p53 responds to variety of cellular stressors by inducing cell cycle arrest or apoptosis, playing a critical role in tumor suppression. Mutations in the p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of two p53 inhibitors Mdm2 and Mdm4 occur in most of the rest. Current dogma holds that Mdm2 mainly regulates p53 stability via its RING finger E3 ubiquitin ligase and Mdm4 mainly controls p53 transcriptional activity through concealing the p53 transcriptional activation domain. In vitro data have shown that Mdm2's RING E3 is also responsible for degradation of Mdm4 and itself, and a model is proposed that switch from Mdm2 degradation of p53 to self-degradation is responsible for p53 accumulation and activation after stress. Many p53 inducers including tumor suppressor p14ARF and ribosomal protein L11 stabilize and activate p53 through inhibition of Mdm2's E3 function. Thus, theoretically anticancer strategies targeting Mdm2 E3 function could cooperate with strategies targeting the Mdm2-p53 interaction to activate p53 in the millions of patients diagnosed with p53-positive cancers each year. Importantly however, detailed knowledge of the molecular mechanisms of Mdm2 E3 regulation will be required to achieve this goal. We have recently generated mice bearing a single-residue substitution in the Mdm2 RING finger domain abolishing its E3 function without affecting p53 binding. Unexpectedly however, in contrast to current notion our data have shown that 1) the Mdm2-p53 interaction, in the absence of Mdm2-mediated p53 ubiquitination, cannot control p53 activity, and 2) Mdm2 auto-ubiqutination is not the principle mechanism for Mdm2 degradation in vivo. Our analysis reveals yet another disconnect between hypotheses generated by in vitro transfection studies and mouse models. Based on this mouse model we will test three hypotheses: 1) Mdm2-Mdm4 interaction augments or necessitates Mdm2's E3 ligase function, 2) the binding of Mdm2 suppresses p53's apoptotic but not cell cycle arrest function, and 3) there is an unknown novel E3 ubiquitin ligase for Mdm2 degradation in vivo. Our specific aims are: Aim 1. To investigate the role of Mdm2 RING E3 in regulation of Mdm4 Aim 2. To investigate the non-redundant roles of Mdm2 and Mdm4 in regulation of p53. Aim 3. To investigate the role of Mdm2 in regulation of p53-induced cell cycle arrest and apoptosis. PUBLIC HEALTH RELEVANCE: Mutations in the tumor suppressor p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of two p53 inhibitors Mdm2 and Mdm4 occur in most of the rest. This project proposes to investigate the function and mechanism of the Mdm2- Mdm4-p53 regulatory loop using novel mouse models. Detailed knowledge of the molecular mechanisms and regulation of the Mdm2 E3 ubiqutin ligase is critically important for the design of future p53-based anticancer strategies.