Biochemical Analysis of a p53 Isoform that Accelerates Mammalian Aging

描述(申请人提供)：P53肿瘤抑制因子在癌症预防和衰老方面都发挥着关键作用。P53反应的激活对于其肿瘤抑制功能是重要的；相反，关闭活跃的P53对于防止过早衰老至关重要。P53在癌症和衰老中的双重作用可能最好地体现在一种自然产生的P53亚型NP53(又名NP53)的活性上。40P53)。当Np53在小鼠体内过度表达时(与野生型P53一起)，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和过早死亡。Np53引起的高活性P53反应与转录激活有关。由于P53作为四聚体结合DNA并激活转录，P53/NP53异四聚体的形成被认为是导致基因表达增强和加速衰老的原因。然而，这种情况发生的分子机制在很大程度上还没有被探索。在这个提案中，我们概述了一系列实验，这些实验将开始定义Np53在P53四聚体的背景下发挥作用的分子机制。这项工作的中心是使用重组的人类转录系统在天然p53靶基因的背景下对混合的P53/NP3四聚体进行详细的生化分析。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/NP53的差异影响。在这个屏幕中，我们将特别关注与衰老相关的P53靶点，以确定哪些可能被NP53亚型改变得最明显。综上所述，这些生化和基于细胞的研究将开始定义使Np53异构体能够加速哺乳动物衰老的分子机制。 与公共健康相关：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了基因。了解这一过程是至关重要的，因为对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，这种调节的崩溃是人类疾病的一个标志，最明显的是癌症。在这项提案中，我们将研究一种名为P53的不正确激活的蛋白质的功能。了解p53是如何发挥作用的，特别是在其异常的、“极度活跃”的状态下，是癌症和衰老问题的核心，因为我们正在研究的p53的形式显示出增强的预防癌症的能力，但同时也缩短了寿命。发生这种情况的确切机制根本不被理解，将在这项研究中进行探索。我们预计，通过我们的努力积累的信息将确定控制p53转录活性的新策略。

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.