The Sir2-p53-IGF link in mammalian life-span control

我们已经产生了一只转基因小鼠，在其中过表达了肿瘤抑制因子的短亚型 P53(P44)会导致健康和生命的过早丧失。我们打算通过以下方式来描述分子机制 在这种动物模型中，p44表达的增加加速了正常衰老的过程。具体而言 目的1，我们验证了p44/p53比例的改变可以调节哺乳动物寿命的假设。 为此，我们研究了p44在小鼠和人类正常衰老过程中的表达。 (实验1.1)，并使用RNAi确定其在全长p53的背景下与衰老速度的相关性 在细胞中(实验1.2)和动物中p44缺失的p53等位基因(实验1.3)中操纵p44 级别。在特定的AIM 2中，我们验证了P53通过偶联方式调节小鼠寿命的假设 Sir2和IGF-1通路。为此，我们提出了一系列的生化实验来阐明 P44干扰P53和Sir2相互作用的分子机制包括 改变P53与其已知结合伙伴的相互作用(实验2.1)；改变P53后 翻译修饰，包括磷酸化和乙酰化/去乙酰化(实验2.2)； 改变P53的亚细胞定位(实验2.3)；以及干扰P53的组装或功能- 依赖转录复合体(实验2.4)。如果P53是Sir2a和IGF信号之间的纽带 鼠标，然后p44分离链接，而这个链接如何正常工作的线索在于为什么它不是 当P44过多时，功能正常。最后，在特定的AIM 3中，我们检验了IGF-1的假设 受体是p44改变哺乳动物健康和寿命的主要下游效应因子。在这 目的：我们利用小鼠遗传学的力量来确定IGF-1受体是否在细胞和 P44纯合子转基因小鼠的组织是导致健康和生命损失的关键成分。 哺乳动物系统。因为这个基因是IGF-1信号转导的关键第一步，所以这个目标集中在 P53与决定人类寿命的信号通路相互作用的研究 生物体，如线虫和黑腹线虫。

We have generated a transgenic mouse in which over-expression of the short isoform of the tumor suppressor p53 (p44) results in the premature loss of health and life. We intend to delineate the molecular mechanism by which increased p44 expression accelerates the process of normal aging in this animal model. In SPECIFIC AIM 1, we test the hypothesis that a change in the p44-to-p53 ratio can modulate mammalian longevity. In this Aim, we characterize the expression of p44 during normal aging in both mouse and human (Experiment 1.1) and determine its relevance in the context of full-length p53 to the rate of aging using RNAi in cells (Experiments 1.2) and a p44-deficient p53 allele in animals (Experiment 1.3) to manipulate p44 levels. In SPECIFIC AIM 2, we test the hypothesis thatp53 modulates life-span in the mouse by coupling the Sir2 and IGF-1 pathways. In this Aim, we propose a series of biochemical experiments to elucidate the molecular mechanism by which p44 might interfere with the interaction between p53 and Sir2, including altering the interaction of p53 with its known binding partners (Experiment 2.1); altering p53 post- translational modifications, including phosphorylation and acetylation/deacetylation (Experiment 2.2); altering p53 sub-cellular localization (Experiment 2.3); and, interfering with the assembly or function of p53- dependent transcription complexes (Experiment 2.4). If p53 is the link between Sir2a and IGF signaling in the mouse, then p44 uncouples the link, and the clues to how this link functions normally lie in why it is non- functional when p44 is in excess. Finally, in SPECIFIC AIM 3, we test the hypothesis that the Igf-1 receptor is theprincipal downstream effector by which p44 alters mammalian health- and life-span. In this aim, we exploit the power of mouse genetics to determine if the Igf-1 receptor, which is altered in cells and tissues ofp44 homozygous transgenic mice, is the key component mediating the loss of health and life in this mammalian system. Because this gene is the critical first step in IGF-1 signal transduction, this Aim focuses on the interactionof p53 and the signalingpathway that plays a major role in life-span determination in lower organisms, such as C. elegans and D. melanogaster.