Biochemical Analysis of a p53 Isoform that Accelerates Mammalian Aging

描述（由申请人提供）：p53肿瘤抑制因子在癌症预防和衰老中起着关键作用。p53反应的激活对其肿瘤抑制功能很重要;相反，关闭活性p53对防止过早衰老至关重要。p53在癌症和衰老中的这种双重作用可能最好地通过称为Np 53（a.k.a. 40页53）。当Np 53在小鼠中过度表达时（与野生型p53一起），Np 53会导致p53靶基因的过度激活;尽管这会导致增强的肿瘤抑制，但小鼠也会迅速衰老并过早死亡。由Np 53引起的过度活跃的p53反应与转录激活有关。由于p53结合DNA并作为四聚体激活转录，因此p53/Np 53异源四聚体的形成被认为导致基因表达增强和加速衰老。然而，这种情况发生的分子机制在很大程度上尚未探索。在这个建议中，我们概述了一系列的实验，将开始，以确定的分子机制，其中Np 53的功能范围内的p53四聚体。这项工作的核心是一个详细的生物化学分析的混合p53/Np 53四聚体的背景下，一个天然的p53靶基因使用重建的人类转录系统。此外，我们将通过对28个已知的p53靶基因进行mRNA分析，确定哪些p53靶基因在人类细胞中受到p53/Np 53的差异影响。我们将特别关注与衰老相关的p53靶点，以确定哪些可能被Np 53亚型改变得最显著。总之，这些生物化学和细胞为基础的研究将开始，以确定分子机制，使Np 53亚型，以加速哺乳动物衰老。 公共卫生相关性：我们的研究分析了基因表达的基本机制--是什么在细胞中打开或关闭基因。了解这个过程是至关重要的，因为基因表达的适当调节几乎对每一个主要的生理过程都是必不可少的;此外，这种调节的破坏是人类疾病的标志，最明显的是癌症。在这个建议中，我们将研究一种被称为p53的不适当激活的蛋白质的功能。了解p53如何发挥作用，特别是在其异常的“过度活跃”状态下，是癌症和衰老问题的核心，因为我们正在研究的p53形式显示出预防癌症的能力增强，但同时缩短了寿命。发生这种情况的确切机制根本不清楚，将在本研究中进行探索。我们预计，通过我们的努力积累的信息将确定新的策略来控制p53的转录活性。

DESCRIPTION (provided by applicant): The p53 tumor suppressor plays critical roles in both cancer-prevention and aging. Activation of p53 response is important for its tumor suppressor function; conversely, shutting off active p53 is critical to prevent premature aging. This dual role of p53 in cancer and aging is perhaps best demonstrated by the activity of a naturally-occurring isoform of p53 called Np53 (a.k.a. 40p53). When over-expressed in mice (together with wild-type p53), Np53 causes hyper-activation of p53 target genes; although this results in enhanced tumor suppression, the mice also age rapidly and die prematurely. The hyper-active p53 response caused by Np53 has been linked to transcription activation. Because p53 binds DNA and activates transcription as a tetramer, the formation of p53/ Np53 hetero-tetramers is believed to cause the enhanced gene expression and accelerated aging. However, the molecular mechanism by which this occurs is largely unexplored. In this proposal, we outline a series of experiments that will begin to define the molecular mechanisms by which Np53 functions within the context of the p53 tetramer. Central to this work is a detailed biochemical analysis of mixed p53/ Np53 tetramers in the context of a native p53 target gene using a reconstituted human transcription system. In addition, we will identify which p53 target genes are differentially impacted by p53/ Np53 in human cells by performing mRNA analysis of 28 known p53 target genes. We will focus specifically on aging-related p53 targets in this screen to determine which might be altered most significantly by the Np53 isoform. Taken together, these biochemical and cell-based studies will begin to define the molecular mechanisms that enable the Np53 isoform to accelerate mammalian aging. PUBLIC HEALTH RELEVANCE: Our research analyzes the basic mechanisms of gene expression-what turns a gene "on" or "off" in a cell. Understanding this process is vital because proper regulation of gene expression is essential for virtually every major physiological process; furthermore, breakdown in this regulation is a hallmark of human disease, most notably cancer. In this proposal we will examine the function of an improperly activated protein called p53. Understanding how p53 functions, particularly in its abnormal, "hyper-active" state, lies at the heart of the cancer and aging problem because the form of p53 we are studying shows enhanced ability to prevent cancer yet concomitantly shortens lifespan. The precise mechanisms by which this occurs are not at all understood and will be explored in this study. We anticipate that the information accumulated by our efforts will identify new strategies for controlling the transcriptional activity of p53.