EFFECTS OF AGING ON P38 SIGNALING PATHWAY IN MOUSE LIVER

衰老细胞由于活性氧（ROS）而积累氧化剂损伤的线粒体DNA（mtDNA）。这导致ROS产生增加，导致更多的DNA损伤和线粒体功能障碍。我们认为ROS影响p38 MAPK应激反应通路的功能。为了支持这一假设，我们已经显示了C57 BL/6小鼠肝脏中p38蛋白的年龄相关修饰，例如，磷酸化蛋白水平增加;它们对3- NPA（复合物II（琥珀酸脱氢酶）的抑制剂）产生的ROS的去磷酸化反应;以及老年肝脏中缺乏这种反应。我们提出：（a）老年组织中功能障碍的线粒体产生的RQS导致p38信号通路的基础活性增加;（B）老年肝脏中p38通路蛋白对3-NPA应答的失败表明信号功能受损。我们将在以下5个目标中检验这一假设： 目的1将确定3-NPA抑制复合体II（SDH）产生活性氧的位点和机制，以及3-NPA、鱼藤酮和抗霉素A产生的活性氧在p38激活中的作用。目的2将表征老年小鼠（24个月）p38活性的基础水平增加; 3- NPA在年轻（3个月）和中年（12个月）小鼠中激活p38通路的机制;以及老年肝脏中这种反应的失败。目的3将表征年龄特异性3-NPA刺激的p38 MAPK激活剂的去磷酸化。目的4研究转录因子ATF-2和CEBP β活性随年龄增加而增加的机制，以及3-NPA不能激活这些蛋白的机制，并表征p38在激活细胞凋亡中的作用。 线粒体伴侣蛋白目的5将研究衰老和氧化应激对线粒体维持的影响，使用96个线粒体蛋白基因的微阵列来确定衰老对其在老年肝脏中表达的影响以及对3-NPA的响应。我们将确定羰基化和硝化线粒体4-羟基壬烯修饰的p38 MAPK蛋白的水平，并将这种氧化损伤与线粒体功能障碍相关联。在这个项目中，我们开始深入分析对细胞生存至关重要的分子信号相互作用;我们使用基因阵列和蛋白质组学技术来帮助理解与年龄相关的关键生物学功能改变的全球影响。因此，我们的项目将确定与年龄相关的组织功能下降的重要因果因素。

Aging cells accumulate oxidant-damaged mitochondriat DNA (mtDNA) due to reactive oxygen species (ROS). This leads to increased ROS production, to more DNA damage and mitochondrial dysfunction. We propose that ROS affects the function of the p38 MAPK stress response pathway. To support this hypothesis we have shown age-associated modifications to p38 proteins in C57BL/6 mouse livers, e.g., increased levels of phosphorylated protein ; their dephosphorylation in response to ROS generated by 3- NPA, an inhibitor of Complex II (succinic dehydrogense); and a lack of this response in aged liver. We propose that (a) RQS generated by dysfunctional mitochondria in aged tissue cause an increased basal activity of the p38 signaling pathway; (b) failure of the p38 pathway proteins to respond to 3-NPA in aged livers suggests signaling functions are compromised. We will test this hypothesis in the following 5 Aims: Aim 1 will identify the site and mechanisms of ROS generation by 3-NPA's inhibition of complex II (SDH) and the role of ROS generated by 3-NPA, rotenone and Antimycin A in p38 activation. Aim 2 will characterize the increased basal levels of p38 activity in aged mice (24 mo); the mechanism of p38 pathway activation by 3- NPA in young (3 mo) and middle-aged (12 mo) mice; and the failure of this response in aged livers. Aim 3 will characterize the age-specific 3-NPA-stimulated dephosphorylation of p38 MAPK activators. Aim 4 will study the mechanism of age-associated increase in activity of the transcription factors ATF-2 and CEBPbeta, and the failure of 3-NPA to activate these proteins, and characterize the role of p38 in activation of mitochondrial chaperones. Aim 5 will examine the effect of aging and oxidative stress on mitochondrial maintenance, using a microarray of 96 mitochondrial protein genes to determine the effects of aging on their expression in aged livers and in response to 3-NPA. We will determine the levels of carbonylated and nitrated mitochondrial 4-hydroxynonena-modified p38 MAPK proteins and correlate this oxidative damage to mitochondrial dysfunction. In this project we initiate an in-depth analysis of the molecular signaling interactions critical to cellular survival; we use gene array and proteomics technology to assist in understanding global effects of age-associated alteration of critical biological functions. Our Project wilt thus identify important causal factors in the age-associated decline in tissue function.