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

描述（由申请人提供）：我们已经产生了一种转基因小鼠，在这种小鼠中，肿瘤抑制因子p53 （p44）短异构体的过度表达会导致健康和生命的过早丧失。我们打算在这种动物模型中描述p44表达增加加速正常衰老过程的分子机制。在SPECIFIC AIM 1中，我们验证了p44- p53比值的变化可以调节哺乳动物寿命的假设。在本研究中，我们对小鼠和人类正常衰老过程中p44的表达进行了表征（实验1.1），并利用细胞中的RNAi（实验1.2）和动物中的p44缺陷p53等位基因（实验1.3）来确定其在全长p53与衰老速度的相关性。在SPECIFIC AIM 2中，我们验证了p53通过Sir2和IGF-1通路偶联来调节小鼠寿命的假设。在本研究中，我们提出了一系列生化实验来阐明p44可能干扰p53和Sir2之间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的翻译后修饰，包括磷酸化和乙酰化/去乙酰化（实验2.2）；改变p53亚细胞定位（实验2.3）；干扰p53依赖转录复合物的组装或功能（实验2.4）。如果p53是小鼠体内Sir2a和IGF信号之间的纽带，那么p44就会断开这种纽带，而这种纽带如何正常发挥作用的线索就在于为什么当p44过量时它不起作用。最后，在SPECIFIC AIM 3中，我们验证了Igf-1受体是p44改变哺乳动物健康和寿命的主要下游效应物的假设。为此，我们利用小鼠遗传学的力量来确定在p44纯合子转基因小鼠的细胞和组织中发生改变的Igf-1受体是否是介导这种哺乳动物系统中健康和生命丧失的关键成分。由于该基因是IGF-1信号转导的关键第一步，因此本目的重点关注p53与在低等生物（如秀丽隐杆线虫和黑胃线虫）的寿命决定中起主要作用的信号通路的相互作用。

DESCRIPTION (provided by applicant): 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 that p53 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 the principal 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 of p44 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 interaction of p53 and the signaling pathway that plays a major role in life-span determination in lower organisms, such as C. elegans and D. melanogaster.