The role of ribosomal proteins in the p53-MDM2 pathway

描述(申请人提供)：我们研究的长期目标是了解控制细胞增殖、分化和凋亡的化学、分子和细胞机制，通常发生在癌变的早期事件，涉及MDM2-P53反馈回路。由我之前资助的R01和许多其他小组支持的研究现在已经确定，核糖体蛋白(RP)，如RPL11、RPL5和RPL23，通过直接与MDM2结合并抑制其E3泛素连接酶对P53的泛素连接酶活性，在P53对各种细胞和环境致癌物或化学物质(包括可能导致核糖体应激的内源和外源化合物)的反应中发挥作用。此外，更多与MDM2结合的RPs包括RPL26、RPS7、RPS3和RPS14已被发现在这一途径中发挥作用。核糖体应激-P53途径在细胞生长、细胞凋亡和肿瘤发生中起重要作用，也与癌症和易患癌症的遗传性疾病，如5q综合征和钻石-布莱克凡贫血(DBA)密切相关。尽管在理解该通路在p53反应和肿瘤发生中的生物学重要性方面已经取得了巨大的进展，但其他的、非常重要的和具有挑战性的问题仍然有待解决。例如，仍然令人费解的是，这些RP如何机械地起作用来使MDM2失活。此外，特别耐人寻味的是，为什么需要如此多的RP来抑制MDM2并激活p53，以应对核糖体应激导致的化学物质、致癌物和细胞内异常。最后，作为5q综合征或DBA的发病机制，这些与MDM2结合的核糖体蛋白是否在P53激活中发挥作用？因此，我们将在这次更新应用中继续深入探讨这一信号通路。鉴于已有的信息，包括我们的初步研究，我认为上述MDM2结合的RPS可能作为一个动态的亚核糖体复合体发挥作用，而不是单独作用，以一种精心编排的方式抑制这种E3泛素连接酶的中心酸性和锌指域的MDM2活性，以响应由各种原因引起的RS或导致代谢紊乱或遗传病的遗传改变，导致P53激活。计划有两个特定的目标来验证这一假说：1)阐明RPS抑制MDM2的结构和功能；2)确定RPS是否作为核糖体应激诱导的动态亚核糖体复合体抑制细胞和动物中的MDM2活性。实现这些短期目标将使我们能够更好地了解这些核糖体蛋白如何在细胞或动物中作为动态复合体发挥作用，以抑制MDM2介导的p53泛素化，并在暴露于导致核糖体应激的内源和外源化合物时随后失活。拟议的实验结果不仅将为癌症或疾病相关表型提供可能的机制解释，还将为将MDM2的锌指结构域用作未来抗癌药物开发的潜在靶点提供一个框架。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the chemical, molecular and cellular mechanisms that govern cell proliferation, differentiation, and apoptosis and often occur as the early events of carcinogenesis, involving the MDM2-p53 feedback loop. Research supported by my previously funded R01 and by many other groups has now firmly established the role of ribosomal proteins (RP), such as RPL11, RPL5, and RPL23, in p53 response to various cellular and environmental carcinogens or chemicals, including endogenous and exogenous compounds, which may cause ribosomal stress, by directly binding to MDM2 and inhibiting its E3 ubiquitin ligase activity toward p53. Also more MDM2-binding RPs including RPL26, RPS7, RPS3 and RPS14 have been identified to play a role in this pathway. It is now acknowledged that this ribosomal stress-p53 pathway plays a vital role in cell growth, apoptosis, and tumorigenesis, and is also highly pertinent to cancers and cancer-prone genetic diseases, such as 5q-syndrome and Diamond-Blackfan anemia (DBA). Although a tremendous progress has been made in understanding the biological importance of this pathway in p53 response and tumorigenesis, additional, remarkably important and challenging questions still remain to be addressed. For example, it still remains puzzling how mechanistically these RPs function to inactivate MDM2. Also it is particularly intriguing why so many RPs are needed to suppress MDM2 and to activate p53 in response to ribosomal stress-causing chemical, carcinogens and intracellular abnormalities. Finally, would these MDM2-binding ribosomal proteins play a role in p53 activation as pathogenesis of 5q-syndrome or DBA? Thus, we will continue our in-depth interrogation into this signaling pathway in this renewal application. In light of available information including our preliminary studies, I propose that the aforementioned MDM2- binding RPs may work as a dynamic sub-ribosomal complex, instead of individually, to suppress MDM2 activity in a well choreographic way assembled at the central acidic and Zinc Finger domains of this E3 ubiquitin ligase in response to RS caused by various agents or genetic alterations including metabolic disorder or genetic diseases, leading to p53 activation. Two specific aims are planned to test this hypothesis: 1) To illustrate structural and functional insight into the mechanisms governing the inhibition of MDM2 by RPs; 2) To determine if RPs act as a ribosomal stress-induced dynamic sub-ribosomal complex to suppress MDM2 activity in cells and animals. Achieving these short term goals would allow us to better understanding how these ribosomal proteins function as a dynamic complex in cells or animals to suppress MDM2- mediated p53 ubiquitination and consequent inactivation upon exposure to endogenous and exogenous compounds that cause ribosomal stress. The outcomes from the proposed experiments would not only provide a possible mechanistic explanation for cancer- or disease-related phenotype, but also offer a framework for using the Zinc Finger domain of MDM2 as a potential target for the development of anti-cancer drugs in the future.