In vivo function and mechanism of the r-protein-Mdm2-p53 pathway

说明书(申请人提供)：r-蛋白-MDM2-P53通路的体内功能和机制MDM2与大量蛋白质相互作用，在调节细胞生长、增殖和凋亡方面发挥关键作用。在过去的几年中，mdm2研究中最重要的突破之一是mdm2‘S与核糖体蛋白(r-蛋白)的相互作用。我们发现，L5、L11和L23这三个r蛋白结合MDM2并阻断MDM2介导的P53降解，导致P53依赖的生长抑制。我们证明了抑制核糖体生物发生的事件会引起核仁压力并释放核仁r蛋白，核仁r蛋白与细胞核中的MDM2相互作用并触发p53的激活。然而，r-蛋白-MDM2相互作用的生理功能在体内还没有得到解决，这种相互作用的生物学作用也没有在小鼠模型中建立。许多MDM2抑制分子，包括三个r-蛋白和肿瘤抑制因子ARF，已知在中央酸性区域与MDM2结合。针对MDM2(中心酸性结构域)的突变已在人类癌症中被报道。我们最近发现，MDM2中央酸性结构域的突变破坏了r-蛋白质的相互作用，并减弱了P53‘S对核仁应激的反应。然而，尽管r-蛋白-MDM2-P53通路在人类癌症中的作用尚未在体内得到证实，小鼠模型对于进一步研究这些蛋白质是必不可少的。我们最近建立了一个表达r蛋白结合缺陷的突变MDM2的敲门小鼠模型，并开始剖析突变小鼠的生物学功能。在发现新的MDM2结合伙伴的过程中，我们确定线粒体Hep27是一个潜在的MDM2结合蛋白。Hep27与其他mdm2抑制剂结合在mdm2的S中心酸性区域，从机制上讲是mdm2功能的抑制剂。基于大量的生化、生物学和遗传学信息，我们提出了两个假设：1)r-蛋白-MDM2相互作用构成了对核糖体生物发生的监控，对P53的激活和肿瘤的抑制至关重要。2)Hep27-MDM2的相互作用代表了一种新的线粒体逆行信号通路，它通过MDM2转导线粒体应激，从而放大P53功能。我们的具体目标是：1.确定MDM2 C4锌指的生物学功能和生化机制。2.研究r-蛋白-MDM2-P53通路与其他P53信号通路的相互作用。3.确定线粒体应激激活的、由Hep27介导的P53通路。与公共卫生相关：肿瘤抑制因子p53基因的突变会损害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，p53抑制因子MDM2的水平会升高。该项目建议使用新的小鼠模型来研究MDM2-P53调控环的功能和机制。详细了解MDM2和P53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): In vivo function and mechanism of the r-protein-Mdm2-p53 pathway Mdm2 interacts with a large number of proteins and plays a critical role in regulating cell growth, proliferation and apoptosis. One of the most significant breakthroughs in the study of Mdm2 in the past several years is the demonstration of Mdm2's interaction with ribosomal proteins (r-proteins). We discovered that three r-proteins, L5, L11 and L23, bind Mdm2 and block Mdm2-mediated p53 degradation, leading to p53-dependent growth inhibition. We demonstrated that events inhibiting ribosomal biogenesis cause nucleolar stress and release nucleolar r-proteins, which interact with Mdm2 in the nucleus and trigger activation of p53. However, the physiological function of the r-protein- Mdm2 interaction has not been addressed in vivo, and the biological role of the interaction has not been established in a mouse model. A number of Mdm2 inhibiting molecules, including the three r-proteins and the tumor suppressor ARF, are known to bind Mdm2 in the central acidic domain. Mutations targeting Mdm2 the central acidic domain have been reported in human cancers. We recently found that mutations in the Mdm2' central acidic domain disrupt r-protein interaction and attenuate p53's response to nucleolar stress. However, despite implications in human cancer the role of the r-protein-Mdm2-p53 pathway in tumor suppression has not been proven in vivo, and mouse models are essential for the further study of these proteins. We have recently generated a knockin mouse model expressing a mutant Mdm2 deficient in r-protein binding and begun to dissect the biological functions of the mutant mice. During a discovery screen for novel Mdm2 binding partners we identified the mitochondrial Hep27 as a potential Mdm2 binding protein. Hep27 binds to Mdm2's central acidic region-in concert with the other Mdm2 inhibitors, and mechanistically is an inhibitor of Mdm2 function. Building upon a large amount of biochemical, biological and genetic information, we have formulated two hypotheses: 1) the r-protein-Mdm2 interaction constitutes surveillance for ribosomal biogenesis and is critical for the activation of p53 and suppression of tumorigenesis. 2) The Hep27- Mdm2 interaction represents a novel mitochondrial retrograde signaling pathway that transduces mitochondrial stress through Mdm2 to amplify p53 function. Our Specific Aims are: 1. To determine the biological function and biochemical mechanism of Mdm2 C4 zinc finger. 2. To investigate how the r-protein-Mdm2-p53 pathway interacts with other p53 signaling pathways. 3. To define a mitochondrial stress-activated, Hep27-mediated p53 pathway. PUBLIC HEALTH RELEVANCE: Mutations in the tumor suppressor p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of a p53 inhibitor Mdm2 occur in most of the rest. This project proposes to investigate the function and mechanism of the Mdm2-p53 regulatory loop using novel mouse models. Detailed knowledge of the molecular mechanisms and regulation of the Mdm2 and p53 is critically important for the design of future p53-based anticancer strategies.