In vivo function of Mdm2 E3 ubiquitin ligase

描述（由申请人提供）：转录因子p53通过诱导细胞周期停滞或细胞凋亡对各种细胞应激源作出反应，在肿瘤抑制中起关键作用。p53基因突变损害p53功能发生在50%的人类癌症中，其余大多数癌症中两种p53抑制剂Mdm 2和Mdm 4水平升高。目前的理论认为，Mdm 2主要通过其RING指E3泛素连接酶调节p53的稳定性，而Mdm 4主要通过隐藏p53转录激活结构域来控制p53的转录活性。体外数据表明，Mdm 2的RING E3也负责Mdm 4和自身的降解，并提出了一个模型，即从Mdm 2降解p53到自降解的转换负责应激后p53的积累和激活。许多p53诱导剂包括肿瘤抑制因子p14 ARF和核糖体蛋白L11通过抑制Mdm 2的E3功能来稳定和激活p53。因此，理论上，靶向Mdm 2 E3功能的抗癌策略可以与靶向Mdm 2-p53相互作用的策略合作，以激活每年数百万诊断为p53阳性癌症的患者中的p53。然而，重要的是，需要详细了解Mdm 2 E3调控的分子机制，以实现这一目标。我们最近产生的小鼠轴承单残基取代Mdm 2环指结构域废除其E3功能，而不影响p53结合。然而，出乎意料的是，与目前的观点相反，我们的数据表明：1）在不存在Mdm 2介导的p53泛素化的情况下，Mdm 2-p53相互作用不能控制p53活性，以及2）Mdm 2自身泛素化不是Mdm 2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，我们将测试三个假设：1）Mdm 2-Mdm 4相互作用增强或需要Mdm 2的E3连接酶功能，2）Mdm 2的结合抑制p53的凋亡但不抑制细胞周期阻滞功能，以及3）存在未知的新型E3泛素连接酶用于体内Mdm 2降解。我们的具体目标是：目标1。研究Mdm 2 RING E3在调节Mdm 4 Aim 2中的作用。研究Mdm 2和Mdm 4在p53调控中的非冗余作用。目标3。探讨Mdm 2在p53诱导的细胞周期阻滞和凋亡中的调控作用。公共卫生相关性：在50%的人类癌症中，肿瘤抑制基因p53的突变会损害p53的功能，而在其余的大多数癌症中，两种p53抑制剂Mdm 2和Mdm 4的水平会升高。本项目拟利用新型小鼠模型研究Mdm 2-Mdm 4-p53调控环的功能和机制。详细了解Mdm 2 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.