AGE-DEPENDENT MODULATION OF AP-ENDONUCELEASE FUNCTIONS

衰老综合征可以说是由主要在线粒体（mt）中产生的活性氧（ROS）引起的慢性氧化应激的累积效应引起的。细胞核和线粒体基因组都是ROS的靶点，其中损伤主要通过DNA碱基切除修复途径修复，AP-内切核酸酶（APE）发挥核心作用。线粒体APE是哺乳动物细胞核APE（APE 1）的N端裂解产物，参与线粒体DNA损伤的修复。APE 1也是一种转录调节因子，既作为转录因子（TF）的还原激活因子，如p53和缺氧 诱导因子（HIF），并作为TF本身，通过结合负Ca 2+反应元件（nCaRE）和剪切应力反应元件（SSRE）。这种结合活性通过其乙酰化而增强。许多含有这些顺式元件的基因的表达呈年龄依赖性下降，乙酰化APE 1（AcAPE 1）水平下降，表明TF与这些元件（包括APE 1）结合的活性受到衰老的影响。 为了与阐明线粒体产生的氧化应激在衰老中的作用的计划项目的广泛目标保持一致，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达改变水平的核和mtAPE。剪切应力敏感的原代内皮细胞进行复制性衰老也将用于各种分子和细胞生物学方法，检查APE的二分功能，在修复核和mtDNA损伤，并在调节SSRE和nCaRE依赖基因的年龄调节表达。我们将与项目1和3合作：（1）检查AcAPE 1和APE 1水平是否受衰老影响，导致细胞对氧化应激的反应减弱;（2）测试APE 1和mtAPE是否限制ROS诱导的DNA损伤的修复，导致这种损伤的衰老依赖性积累，并导致nat功能障碍;（3）检查具有改变的APE或mtAPE水平的小鼠和分离的肝细胞中SSRE-、nCaRE-依赖性基因活化的衰老依赖性调节，以及对这些动物的寿命和认知功能的影响;和（4）通过基因组和 蛋白质组学分析这些研究应该提供重要的新的见解，核和线粒体DNA损伤的作用，在维持氧化应激，APE 1的年龄依赖性调制的信号通路。

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.