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（由适用提供）：端粒需要特殊的机制来维护，这些机制的破坏或远程建筑本身几乎都发生在几乎所有的癌症中。远程仪DNA的损害会破坏端粒维护，并可能导致染色体重排。我们最近发现，在正常哺乳动物细胞和肿瘤系中，APE1蛋白是基本惊喜DNA修复的中心作用，需要适当的端粒维持。 APE1是受保护蛋白TRF2的正常结合所必需的，并且APE1缺乏会导致POT1的远程仪结合增加。 APE1的AP核酸内切酶活性（DNA修复功能）是端粒维护所必需的。尽管在整个核中都发现了APE1，但它通过与核素蛋白（NPM1）的相互作用在核仁中优先积累，从而通过APE1 N末端附近的赖氨酸的乙酰化在DNA损伤细胞中释放出来。尽管DNA损伤和端粒破坏是通过APE1机械相关的，但尚不清楚NPM1对APE1的调制如何在此过程中起作用。此外，在约1/3的急性粒细胞性白血病（AML）中发生的NPM1C+突变导致NPM1迁移到细胞质，并随身携带APE1。在此修订后的应用中，我们将研究NPM1-APE1相互作用对端粒维护的影响，以及其在AML和其他癌症病因中的可能作用。尽管无法消除APE1，但我们已经建立了RNAi协议以有效下调。可以通过替换APE1的关键N末端赖氨酸（27、31、32、35）与丙氨酸（模拟未加成的乙酰化状态）或精氨酸（以防止诱导并保留诱导和保留电荷）来控制与NPM1的相互作用。这些工具以及表达NPM1C+蛋白的细胞将用于确定APE1-NPM1相互作用对于在正常条件下以及面对氧化或烷基化剂DNA损伤的端粒维持是否重要，这会产生基本惊喜修复处理的水平。端粒长度和TRF2蛋白的启动将通过我们已经建立的方法来监测。