Regulatory Functions of APE1 - An Essential Repair Protein

描述（由申请人提供）：AP-内切核酸酶（APE）在修复内源性诱导的和诱导氧化应激的环境因子诱导的大多数基因组损伤中起核心作用。哺乳动物APE（APE 1/Ref-1）还在转录调节中发挥作用：既作为涉及Cys 65（或可能另一个Cys残基）的转录因子的还原激活因子，又直接作为抑制负钙反应元件（nCaRE）依赖性甲状旁腺、肾素和可能的其他基因的反式作用辅因子（乙酰化形式）。乙酰化的APE 1还与内皮细胞中存在于PDGF、eNOS和其他剪切激活基因中的剪切应力反应元件（SSRE）结合。APE 1不直接与顺式元件结合，而是与反式作用复合物中存在的其他蛋白质结合。此外，APE 1与Y盒特异性反式作用因子YB-1的稳定结合表明其在调节甚至包括p53在内的其他基因中的作用。我们已经产生了条件小鼠胚胎成纤维细胞（MEF）缺乏内源性APE 1等位基因，但表达人类APE 1转基因;删除转基因诱导MEF的凋亡，这表明APE 1是必不可少的体细胞以及小鼠胚胎。通过提供外源性APE 1来防止细胞凋亡，突变以破坏其修复功能或其乙酰化位点表明这两种功能的重要作用。APE 1对细胞存活的调节活性也从其在癌细胞中的频繁过表达中得到提示。此外，APE 1和p53在小鼠自发肿瘤诱导中的协同作用暗示APE 1在癌症预防中。尽管大量文献记载了APE 1的多种调控功能，但其在各种系统中的调控功能所需的特定序列基序、结构域和活性位点尚未明确定义。我们的中心假设是，APE 1，除了进行内源性基因组损伤的必要修复，调节作为一个共激活剂或共阻遏基因，这是必不可少的生存或维持稳态后暴露于压力。为了验证这一假设并阐明APE 1的结构-功能要求，我们将追求三个目标：（i）确定APE 1的特定残基和基序在MEF突变体中通过互补作用防止凋亡的要求;（2）表征APE 1与作为原型系统的nCaRE-、SSRE-和Y-结合盒复合物中存在的其他反式作用因子的相互作用;（3）通过基因芯片初步筛选，证实APE 1对部分糖尿病相关基因的调控作用。从这些研究中获得的结果将提供一个全面的图片APE 1的不同的调节活动的分子基础。此外，我们的研究将有助于确定APE 1调节的肿瘤诱导和促进的关键信号通路，这可能是癌症治疗的潜在靶点。

DESCRIPTION (provided by applicant): AP-endonuclease (APE) plays a central role in repair of most genomic damage induced endogenously and by environmental agents that induce oxidative stress. The mammalian APE, (APE1/Ref-1) additionally functions in transcriptional regulation: both as a reductive activator of transcription factors involving Cys 65 (or possibly another Cys residue) and directly, as a trans-acting co-factor (in acetylated form) in repressing negative Ca2+ response element (nCaRE)-dependent parathyroid, renin, and possibly other genes. Acetylated APE1 also binds to shear stress response element (SSRE), present in PDGF, eNOS and other shear-activated genes in endothelial cells. APE1 does not directly bind to a cis element, but to other proteins present in trans-acting complexes. Furthermore, APE1's stable binding to the Y-box specific trans-acting factor YB-1 suggests its role in regulation of even other genes including p53. We have generated conditional mouse embryo fibroblasts (MEF) lacking endogenous APE1 alleles but expressing human APE1 transgene; deletion of the transgene induces apoptosis of MEF, which showed that APE1 is essential for somatic cells as well as for the mouse embryo. Prevention of apoptosis by providing exogenous APE1, mutated to inactivate its repair function or its acetylation sites indicates essential roles of both functions. Regulatory activity of APE1 for cell survival is also suggested from its frequent overexpression in cancer cells. Furthermore, synergy between APE1 and p53 in spontaneous tumor induction in mice implicates APE1 in cancer prevention. In spite of extensive documentation of APE1's diverse regulatory functions, the requirement of its specific sequence motifs, domains and active sites for regulatory functions in various systems has not been clearly defined. Our central hypothesis is that APE1, in addition to carrying out essential repair of endogenous genome damage, regulates as a co-activator or co-repressor of genes which are essential for survival or for maintaining homeostasis after exposure to stress. To test this hypothesis and to elucidate APE1's structure- function requirements for the regulatory activities we will pursue three aims: (i) to define the requirements of APE1's specific residues and motifs for preventing apoptosis in MEF mutants by complementation; (2) to characterize APE1's interaction with other trans-acting factors present in nCaRE-, SSRE-, and Y-bound-box complexes chosen as prototype systems; and (3) to confirm the regulatory functions of APE1 for a few apoptosis-linked genes identified by preliminary gene chip screening. The results obtained from these studies will provide a comprehensive picture of the molecular bases for APE1's diverse regulatory activities. Furthermore, our studies should help identify APE1-regulated key signaling pathways in tumor induction and promotion which could be potential targets for cancer therapy.