In vivo function of Mdm2 E3 ubiquitin ligase

描述（申请人提供）：转录因子p53通过诱导细胞周期阻滞或凋亡来响应多种细胞应激源，在肿瘤抑制中起关键作用。在50%的人类癌症中，p53基因的突变损害了p53的功能，而在其余大多数癌症中，两种p53抑制剂Mdm2和Mdm4的水平升高。目前的观点认为，Mdm2主要通过其环指E3泛素连接酶调节p53的稳定性，Mdm4主要通过隐藏p53的转录激活域控制p53的转录活性。体外数据表明Mdm2的RING E3也负责Mdm4和自身的降解，并提出了一个模型，从Mdm2降解p53到自我降解的转变是应激后p53积累和激活的原因。许多p53诱导剂包括肿瘤抑制因子p14ARF和核糖体蛋白L11通过抑制Mdm2的E3功能来稳定和激活p53。因此，从理论上讲，靶向Mdm2 E3功能的抗癌策略可以与靶向Mdm2-p53相互作用的策略合作，在每年数百万被诊断为p53阳性癌症的患者中激活p53。然而，重要的是，要实现这一目标，需要详细了解Mdm2 E3调控的分子机制。我们最近产生了Mdm2环指结构域单残基替换的小鼠，在不影响p53结合的情况下取消了其E3功能。然而，出乎意料的是，与目前的观点相反，我们的数据表明：1)Mdm2-p53相互作用，在缺乏Mdm2介导的p53泛素化的情况下，不能控制p53的活性；2)Mdm2的自泛素化不是Mdm2在体内降解的主要机制。我们的分析揭示了体外转染研究和小鼠模型产生的假设之间的另一个脱节。基于该小鼠模型，我们将验证三个假设：1)Mdm2- mdm4相互作用增强或必须Mdm2的E3连接酶功能，2)Mdm2的结合抑制p53的凋亡但不抑制细胞周期功能，以及3)存在未知的新型E3泛素连接酶用于Mdm2的体内降解。我们的具体目标是：目标1。探讨Mdm2 RING E3在Mdm2 Aim 2调控中的作用。探讨Mdm2和Mdm4在p53调控中的非冗余作用。目标3。探讨Mdm2在p53诱导的细胞周期阻滞和细胞凋亡中的调控作用。公共卫生相关性：50%的人类癌症中发生肿瘤抑制因子p53基因突变，损害p53功能，其余大多数癌症中发生两种p53抑制剂Mdm2和Mdm4水平升高。本项目拟利用新型小鼠模型研究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.