Regulatory Functions of APE1 - An Essential Repair Protein

描述（由申请人提供）：ap -内切酶（APE）在大多数内源性和由诱导氧化应激的环境因子引起的基因组损伤的修复中起核心作用。哺乳动物的APE （APE1/Ref-1）在转录调控中也起作用：既作为涉及Cys 65（或可能是另一种Cys残基）的转录因子的还原激活因子，又直接作为反式作用辅助因子（以乙酰化形式）抑制负Ca2+反应元件（nCaRE）依赖的甲状旁腺、肾素和可能的其他基因。乙酰化的APE1还与剪切应力反应元件（SSRE）结合，存在于内皮细胞中的PDGF、eNOS和其他剪切激活基因中。APE1不直接与顺式元件结合，而是与反式作用复合物中的其他蛋白质结合。此外，APE1与Y-box特异性反式作用因子YB-1的稳定结合表明其在调节包括p53在内的其他基因中的作用。我们产生了缺乏内源性APE1等位基因但表达人APE1转基因的条件小鼠胚胎成纤维细胞（MEF）；转基因基因的缺失诱导MEF凋亡，这表明APE1对体细胞和小鼠胚胎都是必需的。通过提供外源性APE1来预防细胞凋亡，突变使其修复功能或乙酰化位点失活，表明这两种功能都具有重要作用。APE1在癌细胞中的频繁过表达也提示了其对细胞存活的调节作用。此外，APE1和p53在小鼠自发性肿瘤诱导中的协同作用暗示了APE1在癌症预防中的作用。尽管有大量关于APE1多种调控功能的文献记载，但在各种系统中调控功能对其特定序列基序、结构域和活性位点的要求还没有明确定义。我们的中心假设是，APE1除了对内源性基因组损伤进行必要的修复外，还作为对生存或在暴露于压力后维持体内平衡所必需的基因的共同激活或共同抑制因子进行调节。为了验证这一假设并阐明APE1对调控活动的结构-功能要求，我们将追求三个目标：(i)确定APE1的特定残基和基序的要求，以通过互补防止MEF突变体的凋亡；(2)表征APE1与作为原型系统的nCaRE-、SSRE-和y - binding -box复合物中存在的其他反式作用因子的相互作用；(3)确认APE1对初步基因芯片筛选发现的少数凋亡相关基因的调控功能。从这些研究中获得的结果将为APE1的各种调控活动提供一个全面的分子基础。此外，我们的研究应该有助于确定ape1调控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.