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中起着至关重要的作用。p53反应的激活对其抑瘤功能至关重要；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通过一种称为Np53（又名40p53）的自然存在的同种异构体的活性来证明。当Np53在小鼠中过度表达时（与野生型p53一起），会导致p53靶基因的过度激活；虽然这导致肿瘤抑制增强，但小鼠也会迅速衰老并过早死亡。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，因此p53/ Np53异四聚体的形成被认为是导致基因表达增强和衰老加速的原因。然而，发生这种情况的分子机制在很大程度上尚未被探索。在本提案中，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。这项工作的核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p53/ Np53四聚体进行详细的生化分析。此外，我们将通过对28个已知p53靶基因的mRNA分析，确定人类细胞中哪些p53靶基因受到p53/ Np53的差异影响。我们将在这个筛选中特别关注与衰老相关的p53靶点，以确定哪些靶点可能被Np53亚型改变得最显著。综上所述，这些生物化学和基于细胞的研究将开始定义使Np53亚型加速哺乳动物衰老的分子机制。

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.