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

描述（由申请人提供）：r-蛋白-Mdm 2-p53途径的体内功能和机制Mdm 2与大量蛋白相互作用，在调节细胞生长、增殖和凋亡中起关键作用。Mdm 2与核糖体蛋白（r-proteins）相互作用的研究是近年来Mdm 2研究的重大突破之一。我们发现三种r蛋白L5、L11和L23结合Mdm 2并阻断Mdm 2介导的p53降解，导致p53依赖性生长抑制。我们证明了抑制核糖体生物合成的事件会引起核仁应激并释放核仁r-蛋白，其与细胞核中的Mdm 2相互作用并触发p53的激活。然而，尚未在体内解决r-蛋白-Mdm 2相互作用的生理功能，并且尚未在小鼠模型中建立相互作用的生物学作用。许多Mdm 2抑制分子，包括三种r-蛋白和肿瘤抑制因子ARF，已知在中心酸性结构域结合Mdm 2。已经报道了在人类癌症中靶向Mdm 2中心酸性结构域的突变。我们最近发现Mdm 2 '中心酸性结构域的突变破坏了r-蛋白的相互作用并减弱了p53对核仁应激的反应。然而，尽管在人类癌症中的影响，r-蛋白-Mdm 2-p53通路在肿瘤抑制中的作用尚未在体内得到证实，小鼠模型对于这些蛋白质的进一步研究是必不可少的。我们最近产生了一个敲入小鼠模型表达的突变Mdm 2缺陷的r-蛋白结合，并开始解剖突变小鼠的生物学功能。在新的Mdm 2结合伴侣的发现筛选过程中，我们将线粒体Hep 27鉴定为潜在的Mdm 2结合蛋白。Hep 27与Mdm 2的中心酸性区域结合-与其他Mdm 2抑制剂一起，并且在机制上是Mdm 2功能的抑制剂。基于大量的生物化学、生物学和遗传学信息，我们提出了两个假设：1）r-蛋白-Mdm 2相互作用构成了对核糖体生物发生的监视，并且对于p53的激活和肿瘤发生的抑制至关重要。2)Hep 27-Mdm 2相互作用代表了一种新的线粒体逆行信号通路，其通过Mdm 2转导线粒体应激以放大p53功能。我们的具体目标是：1。目的探讨Mdm 2 C4锌指蛋白的生物学功能及作用机制。2.研究r蛋白-Mdm 2-p53信号通路与其他p53信号通路的相互作用。3.确定线粒体应激激活的Hep 27介导的p53通路。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，p53抑制剂Mdm 2的水平会升高。本项目拟利用新型小鼠模型研究Mdm 2-p53调控环的功能和机制。详细了解Mdm 2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.