EFFECTS OF AGING ON P38 SIGNALING PATHWAY IN MOUSE LIVER

衰老对小鼠肝脏 P38 信号通路的影响

• 批准号: 6814766
• 负责人: JOHN NMN PAPACONSTANTINOU
• 金额: $ 33.42万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6814766
• 关键词: age difference; aging; antifungal antibiotics; biological signal transduction; cell senescence; enzyme activity; free radical oxygen; gel electrophoresis; laboratory mouse; liver; liver cells; matrix assisted laser desorption ionization; microarray technology; mitochondrial DNA; mitochondrial disease /disorder; mitogen activated protein kinase; molecular chaperones; oxidative stress; phosphorylation; plant insecticide; propionates; protein localization; protein protein interaction; proteomics; stress; tissue /cell culture; transcription factor

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.

由于活性氧 (ROS)，衰老细胞会积累氧化损伤的线粒体 DNA (mtDNA)。这会导致 ROS 产生增加，导致更多 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，我们展示了 C57BL/6 小鼠肝脏中 p38 蛋白的与年龄相关的修饰，例如磷酸化蛋白水平增加；它们响应 3-NPA（复合物 II（琥珀酸脱氢）抑制剂）产生的 ROS 而去磷酸化；而衰老的肝脏则缺乏这种反应。我们认为 (a) 衰老组织中功能失调的线粒体产生的 RQS 会导致 p38 信号通路基础活性增加； (b) 老化肝脏中 p38 途径蛋白无法对 3-NPA 做出反应，表明信号传导功能受到损害。我们将在以下 5 个目标中检验这一假设： 目标 1 将确定 3-NPA 抑制复合物 II (SDH) 产生 ROS 的位点和机制，以及 3-NPA、鱼藤酮和抗霉素 A 产生的 ROS 在 p38 激活中的作用。目标 2 将表征老年小鼠（24 个月）p38 活性基础水平的增加； 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；以及衰老肝脏中这种反应的失败。目标 3 将表征年龄特异性 3-NPA 刺激的 p38 MAPK 激活剂去磷酸化。目标 4 将研究转录因子 ATF-2 和 CEBPbeta 活性与年龄相关增加的机制，以及 3-NPA 未能激活这些蛋白质的机制，并表征 p38 在激活 线粒体伴侣。目标 5 将检查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衰老肝脏中的表达以及对 3-NPA 的反应的影响。我们将测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联系起来。在这个项目中，我们对对细胞生存至关重要的分子信号相互作用进行了深入分析；我们使用基因阵列和蛋白质组学技术来帮助了解与年龄相关的关键生物功能改变的整体影响。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原因。