AGE-DEPENDENT MODULATION OF AP-ENDONUCELEASE FUNCTIONS

衰老综合征可以说是由活性氧物种(ROS)引起的慢性氧化应激累积效应的结果，ROS主要产生于线粒体(MT)。核基因组和线粒体基因组都是ROS的靶点，损伤的修复主要通过DNA碱基切除修复途径进行，其中AP-内切酶(APE)起核心作用。Mt猿参与线粒体DNA损伤的修复，由主要哺乳动物核猿(APE1)的N端裂解产生。APE1也是一种转录调节因子，既是转录因子(TF)的还原激活因子，如P53和低氧 诱导因子(HIF)，并通过与负的钙反应元件(NCaRE)和剪应力反应元件(SSRE)结合本身作为转铁蛋白。这种结合活性通过其乙酰化而增强。许多包含这些顺式元件的基因的表达随着年龄的增长而下降，乙酰化APE1(AcAPE1)水平的下降表明与这些元件结合的TF的活性，包括APE1，受到年龄的影响。 为了与计划项目阐明线粒体产生的氧化应激在衰老中的作用这一广泛目标保持一致，项目2的目标是使用来自野生型和转基因年轻、中年和老年小鼠的肝脏和分离的肝细胞。野生型和转基因动物表现出核和mtAPE水平的变化。剪切力敏感的原代内皮细胞经历复制衰老，也将被用于各种分子和细胞生物学方法，以检测APE在修复核和线粒体DNA损伤以及调节年龄调节的SSRE-1和nCaRE依赖基因表达方面的双向功能。我们将与项目1和项目3合作：(1)检测acape1和ape1的水平是否受到衰老的影响，导致细胞对氧化应激的反应减弱；(2)测试ape1和mtAPE是否限制了ROS诱导的DNA损伤的修复，导致这种损伤的衰老积累，并导致NAT功能障碍；(3)检测依赖衰老的SSRE、nCaRE激活小鼠和水平改变了APE或mtAPE的孤立肝细胞中基因的激活，以及对这些动物寿命和认知功能的影响；以及(4)识别转基因小鼠中受APE水平变化影响的基因，通过基因组和 蛋白质组学分析。这些研究将为核和线粒体DNA损伤在维持氧化应激中的作用以及APE 1在依赖年龄的信号通路调节中的作用提供重要的新见解。

The aging syndrome arguably results from cumulative effects of chronic oxidative stress caused by reactive oxygen species (ROS), generated mostly in the mitochondria (mt). Both the nuclear and mitochondrial genomes are among the targets of ROS, in which the damage is repaired primarily via the DNA base excision repair pathway with AP-endonuclease (APE) playing a central role. The mt APE, involved in the repair of mtDNA damage, is generated by N-terminal cleavage of the major mammalian nuclear APE (APE1). APE1 is also a transcriptional regulator, both as a reductive activator of transcription factors (TF), such as p53 and hypoxia inducible factor (HIF), and as a TF in its own right, by binding to negative Ca 2+ response elements (nCaRE) and shear stress response elements (SSRE). This binding activity is enhanced by its acetylation. An age-dependent decline in the expression of many genes containing these cis elements, and decreased in the acetylated APE1 (AcAPE1) level suggest that activities of the TFs binding to these elements, including APE1, are affected by aging. In keeping with the broad objective of the Program Project to elucidate the role ofmt-generated oxidative stress in aging, the goal of Project 2 is to use livers and isolated hepatocytes from wild-type and transgenic young, middle aged and old mice. The wild type and transgenic animals express altered levels of nuclear and mtAPE. Shear stress-sensitive primary endothelial cells which undergo replicative senescence will also be used in a variety of molecular and cell biological approaches, to examine APE's bipartite functions in repairing nuclear and mtDNA damage, and in regulating age-modulated expression of SSRE-, and nCaRE-dependent genes. We will collaborate with Projects 1 and 3 to: (1) examine whether AcAPE1 and APE1 levels are affected by aging, leading to diminished cellular responses to oxidative stress; (2) test whether APE1 and mtAPE are limiting in repair ofROS-induced DNA damage, leading to aging-dependent accumulation of such damage, and to nat dysfunction; (3) examine aging-dependent modulation of SSRE-, nCaRE-dependent activation of genes in mice and isolated hepatocytes with altered APE or mtAPE levels, and the effect on the life span and cognitive functions of these animals; and (4) identify genes affected by altered APE levels in transgenic mice, by genomic and proteomic analysis. These studies should provide important new insights into the role of nuclear and mt DNA damage in sustaining oxidative stress, and of APE 1 in age-dependent modulation of signaling pathways.