EFFECTS OF AGING ON P38 SIGNALING PATHWAY IN MOUSE LIVER

衰老细胞由于活性氧(ROS)积累氧化损伤的线粒体DNA(MtDNA)。这会导致ROS的产生增加，导致更多的DNA损伤和线粒体功能障碍。我们认为ROS影响p38MAPK应激反应通路的功能。为了支持这一假设，我们展示了C57BL/6小鼠肝脏中p38蛋白的年龄相关修饰，例如，磷酸化蛋白水平的增加；它们对3-NPA(复合体II(琥珀酸脱氢酶)的抑制剂)产生的ROS的反应中的去磷酸化；以及在衰老的肝脏中缺乏这种反应。我们认为：(A)老年组织中功能失调的线粒体产生的RQS导致p38信号通路的基础活性增加；(B)老年肝脏中的p38通路蛋白不能对3-NPA做出反应，表明信号功能受到损害。我们将在以下5个目标中检验这一假设： 目的1确定3-NPA抑制复合体II(SDH)产生ROS的部位和机制，以及3-NPA、鱼藤酮和抗霉素A产生的ROS在p38激活中的作用。目的2研究老年小鼠(24个月)p38活性的基础水平升高，青年(3个月)和中年(12个月)小鼠3-NPA激活p38通路的机制，以及这种反应在老年小鼠肝脏中的失败。目的3将表征AGE特异性的3-NPA刺激的p38 MAPK激活剂的去磷酸化。目的4研究增龄相关转录因子ATF-2和CEBPβ活性增加的机制，以及3-NPA不能激活这些蛋白的机制，并表征p38在激活这些蛋白中的作用。 线粒体伴侣。目的5研究衰老和氧化应激对线粒体维持的影响，使用96个线粒体蛋白基因芯片来确定衰老对其在衰老肝脏中表达的影响以及对3-NPA的响应。我们将测定线粒体4-羟基二十二醇修饰的p38MAPK蛋白的羧化和硝化水平，并将这种氧化损伤与线粒体功能障碍联系起来。在这个项目中，我们启动了对细胞生存至关重要的分子信号相互作用的深入分析；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功能变化的全球影响。因此，我们的WILT项目确定了与年龄相关的组织功能下降的重要原因。

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.