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The Ape1-NPM1 Axis and Telomere Maintenance

Ape1-NPM1 轴和端粒维护

基本信息

批准号：
9196330
负责人：
Bruce F. Demple
金额：
$ 16.97万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-16 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9196330
关键词：
Acetylation Acute Myelocytic Leukemia Address Alanine Alkylating Agents Arginine Base Excision Repairs Binding Blast Cell Cell Line Cell Nucleolus Cell Nucleus Cells Charge Chromosomal Instability Chromosomal Stability Chromosome abnormality Chromosomes Complex Cytoplasm DNA DNA Damage DNA Maintenance DNA Repair DNA Repair Endonuclease DNA-(apurinic or apyrimidinic site) lyase Defect Dependence Development Down-Regulation Ectopic Expression Embryo Employee Strikes Etiology Failure Fibroblasts Frequencies Genetic Recombination Human Human Cell Line Lead Length Lesion Lysine Maintenance Malignant Neoplasms Mammalian Cell Measures Methods Monitor Mus Mutagens Mutation N-terminal NPM1 gene Occupations Oxidation-Reduction Pathway interactions Patients Play Principal Investigator Process Proteins Protocols documentation RNA Interference Role Structure Telomerase Telomere Maintenance Telomeric Repeat Binding Protein 2 Testing Work cancer therapy cell injury genotoxicity inhibitor/antagonist mutant nucleophosmin oxidative DNA damage prevent programs public health relevance telomere tool tumor

项目摘要

DESCRIPTION (provided by applicant): Telomeres require special mechanisms for their maintenance, and disruption of these mechanisms, or of the telomeric structures themselves, occur in nearly all cancers. Damage to telomeric DNA disrupts telomere maintenance and can result in chromosome rearrangements. We found recently that Ape1 protein, the central player of base excision DNA repair, is required for proper telomere maintenance in both normal mammalian cells and in tumor lines. Ape1 is required for normal binding of the protective protein TRF2, and Ape1 deficiency leads to increased telomeric binding of POT1. The AP endonuclease activity (DNA repair function) of Ape1 is required for telomere maintenance. Although Ape1 is found throughout the nucleus, it accumulates preferentially in the nucleolus via interaction with nucleophosmin (NPM1), from which it is released in DNA-damaged cells through acetylation of lysines near the Ape1 N- terminus. While DNA damage and telomere disruption are mechanistically related by Ape1, it is unknown how the modulation of Ape1 by NPM1 plays a role in this process. Moreover, the NPM1c+ mutations that occur in about 1/3 of acute myelogenous leukemias (AML) lead to relocation of NPM1 to the cytoplasm, carrying Ape1 with it. In this revised application, we will examine the effects of the NPM1-Ape1 interaction on telomere maintenance, and its possible role in the etiology of AML and other cancers. Although Ape1 cannot be eliminated, we have established RNAi protocols for its efficient down-regulation. The interaction with NPM1 can be controlled by replacement of the key N-terminal lysines (27, 31, 32, 35) of Ape1 with alanines (to mimic the uncharged acetylated state) or arginines (to prevent actylation and retain charge). These tools, and cells expressing the NPM1c+ protein, will be used to determine whether the Ape1-NPM1 interaction is important for telomere maintenance under both normal conditions, and in the face of DNA damage by oxidative or alkylating agents, which generate lesions processed by base excision repair. Telomere length, and the occupation by TRF2 protein, will be monitored by methods we have already established.

描述（由申请人提供）：端粒需要特殊的机制来维持，而这些机制或端粒结构本身的破坏几乎发生在所有癌症中。端粒 DNA 的损伤会破坏端粒的维持，并可能导致染色体重排。我们最近发现，Ape1 蛋白是碱基切除 DNA 修复的核心参与者，是正常哺乳动物细胞和肿瘤细胞系中正确维持端粒所必需的。 Ape1 是保护蛋白 TRF2 正常结合所必需的，Ape1 缺陷会导致 POT1 端粒结合增加。 Ape1 的 AP 核酸内切酶活性（DNA 修复功能）是端粒维持所必需的。尽管 Ape1 存在于整个细胞核中，但它通过与核磷蛋白 (NPM1) 相互作用优先在核仁中积累，并通过 Ape1 N 末端附近的赖氨酸的乙酰化将其释放到 DNA 损伤的细胞中。虽然 DNA 损伤和端粒破坏在机制上与 Ape1 相关，但 NPM1 对 Ape1 的调节如何在此过程中发挥作用尚不清楚。此外，大约 1/3 的急性髓性白血病 (AML) 中发生的 NPM1c+ 突变导致 NPM1 重新定位到细胞质，并携带 Ape1。在此修订后的申请中，我们将研究 NPM1-Ape1 相互作用对端粒维持的影响，及其在 AML 和其他癌症病因学中的可能作用。尽管 Ape1 无法被消除，但我们已经建立了 RNAi 方案来有效下调 Ape1。与 NPM1 的相互作用可以通过用丙氨酸（以模拟不带电的乙酰化状态）或精氨酸（以防止乙酰化并保留电荷）替换 Ape1 的关键 N 端赖氨酸（27、31、32、35）来控制。这些工具和表达 NPM1c+ 蛋白的细胞将用于确定 Ape1-NPM1 相互作用对于正常条件下以及面对氧化剂或烷化剂造成的 DNA 损伤（产生通过碱基切除修复处理的损伤）的端粒维持是否重要。端粒长度和 TRF2 蛋白的占据将通过我们已经建立的方法进行监测。