In vivo function of Mdm2 E3 ubiquitin ligase

Mdm2 E3 泛素连接酶的体内功能

• 批准号: 7667721
• 负责人: YANPING ZHANG
• 金额: $ 30.57万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-05 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7667721
• 关键词: Affect; Affinity; Apoptosis; Apoptotic; Binding; Biochemical; CDKN2A gene; Cell Cycle Arrest; Cells; Data; Diagnosis; Embryo; Fingers; Future; Goals; Human; In Vitro; Knock-in Mouse; Knowledge; Ligase; Malignant Neoplasms; Mediating; Modeling; Molecular; Mus; Mutation; Patients; Play; Regulation; Rest; Role; Stress; TP53 gene; Testing; Transcriptional Activation Domain; Transfection; Tumor Suppression; Tumor Suppressor Proteins; Ubiquitination; base; design; in vivo; inhibitor/antagonist; mouse model; novel; public health relevance; ribosomal protein L11; stressor; transcription factor; ubiquitin-protein ligase

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.

描述（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来响应多种细胞应激源，在肿瘤抑制中发挥关键作用。 50% 的人类癌症中出现了损害 p53 功能的 p53 基因突变，而其余大多数癌症中则出现了两种 p53 抑制剂 Mdm2 和 Mdm4 水平升高的情况。目前的理论认为，Mdm2 主要通过其环指 E3 泛素连接酶调节 p53 稳定性，而 Mdm4 主要通过隐藏 p53 转录激活结构域来控制 p53 转录活性。体外数据表明，Mdm2的RING E3也负责Mdm4及其自身的降解，并提出了一个模型，即p53从Mdm2降解转变为自降解负责应激后p53的积累和激活。许多 p53 诱导剂，包括肿瘤抑制因子 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，理论上，针对 Mdm2 E3 功能的抗癌策略可以与针对 Mdm2-p53 相互作用的策略配合，在每年诊断为 p53 阳性癌症的数百万患者中激活 p53。然而重要的是，要实现这一目标，需要详细了解 Mdm2 E3 调节的分子机制。我们最近生成了在 Mdm2 RING Finger 结构域中带有单残基取代的小鼠，废除了其 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）在没有 Mdm2 介导的 p53 泛素化的情况下，Mdm2-p53 相互作用无法控制 p53 活性，2）Mdm2 自动泛素化不是 Mdm2 的主要机制体内降解。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) Mdm2-Mdm4 相互作用增强或需要 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停滞功能，3) 存在一种未知的新型 E3用于 Mdm2 体内降解的泛素连接酶。我们的具体目标是： 目标 1. 研究 Mdm2 RING E3 在 Mdm4 调节中的作用 目标 2. 研究 Mdm2 和 Mdm4 在 p53 调节中的非冗余作用。目的 3. 研究 Mdm2 在调节 p53 诱导的细胞周期停滞和细胞凋亡中的作用。公共健康相关性：50% 的人类癌症中会出现损害 p53 功能的肿瘤抑制 p53 基因突变，而其余大多数癌症中都会出现两种 p53 抑制剂 Mdm2 和 Mdm4 水平升高的情况。该项目拟使用新型小鼠模型研究 Mdm2-Mdm4-p53 调节环路的功能和机制。详细了解 Mdm2 E3 泛素连接酶的分子机制和调控对于未来基于 p53 的抗癌策略的设计至关重要。