Regulatory Functions of APE1 acetylation

APE1 乙酰化的调节功能

基本信息

批准号：
8504783
负责人：
Kishor K Bhakat
金额：
$ 26.42万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8504783
关键词：
ABCB1 gene Acetylation Affect Affinity Alkylating Agents Antibodies Antineoplastic Agents Apoptosis Base Excision Repairs Basic Science Binding Biological Assay Carrier Proteins Cell Line Cell Survival Cells Cessation of life ChIP-on-chip Charge Clinical Complex DNA DNA Damage DNA Microarray Chip DNA Repair DNA glycosylase DNA-(apurinic or apyrimidinic site) lyase Data Down-Regulation Drug Efflux Drug resistance EP300 gene Embryo Environment Excision Fibroblasts Gene Chips Gene Expression Gene Expression Profiling Gene Targeting Genes Genetic Transcription Genome Genomics Goals Human In Vitro Ionizing radiation Lead Microarray Analysis Molecular Mus N-terminal Oxidation-Reduction Pathway interactions Pharmaceutical Preparations Physiological Play Proteins Reactive Oxygen Species Resistance Resources Role Signal Pathway Site Testing Therapeutic Toxic effect Transcriptional Regulation Tumor Cell Line base chromatin immunoprecipitation endonuclease experience histone acetyltransferase human APEX1 protein in vivo inhibitor/antagonist innovation mutant neoplastic cell novel novel therapeutic intervention overexpression prevent promoter repaired small molecule transcription factor tumor

项目摘要

DESCRIPTION (provided by applicant): DNA base excision repair (BER) pathway repairs damage caused by reactive oxygen species, alkylating agents and ionizing radiation, and so protects cells against the toxic effects of various endogenous and exogenous agents. The mammalian AP-endonuclease (APE1) plays a central role in the BER pathway by repairing abasic (AP) sites generated in DNA spontaneously or after removal of damaged bases by DNA glycosylases. Besides this key role in DNA repair, APE1 acts as a transcriptional regulator that binds to several transcription factors and regulates their function via both redox-dependent and -independent mechanisms; this requires its non-conserved N-terminal domain. We have shown that APE1 can be acetylated at Lys 6 and 7, which modulates its transcriptional regulatory functions. Later we showed that APE1 inactivation induces apoptosis in conditionally APE1-nullizygous cells; both its DNA repair function and acetylation sites are needed to reverse this effect. Our preliminary data show that the positive charges of these Lys are essential for APE1's interaction with all its binding partners in cells, and neutralizing the positive charges of these Lys residues by acetylation abolishes these interactions. APE1 acetylation also enhances its endonuclease activity and affects total AP-site repair in an in vitro BER assay. Our studies will thus test the hypothesis that the positive charges of the acetylable Lys residues in APE1's N-terminal domain, and their neutralization by acetylation, regulate both its essential DNA repair and transcriptional regulatory functions, and thus play a critical role in cell survival and drug resistance of tumor cells. This hypothesis will be tested in three specific aims. Aim1 will ask how APE1's acetylation affects its interaction with other proteins or DNA to modulate repair of damaged bases or AP sites, both in vitro and in cells. Using cells stably expressing WT, nonacetylable K6R/K7R or acetylation-mimic K6Q/K7Q APE1, we will test the hypothesis that in the absence of APE1's acetylable Lys residues cells accumulate AP sites or single-strand breaks in the genome, which triggers apoptosis. Aim2 will ask how AcAPE1 levels are increased in tumor cells after genotoxic drug treatment to alter its complex formation with partner proteins and modulate its DNA repair activity and expression of the drug-efflux transporter protein MDR1 via its transcriptional regulatory function. Using a panel of tumor cell lines we will test for a correlation between AcAPE1 levels and their resistance to chemotherapeutics dugs. In Aim3, gene expression analysis and ChIP-on-Chip assays, which combine chromatin immunoprecipitation with DNA microarray analysis in cells stably expressing WT or nonacetylable K6R/K7R APE1, will be used to identify target genes that are directly regulated by APE1 acetylation, and the signaling pathways responsible for inducing apoptosis in the absence of APE1 acetylation. Together, our results should advance our understanding of how APE1's acetylation plays a critical role in both DNA damage repair in vivo and in regulation of transcription, which have a profound consequence for cell survival.

描述（由申请人提供）：DNA碱基切除修复（BER）途径由活性氧，烷基化剂和电离辐射造成的损害，从而保护细胞免受各种内源性和外源性剂的毒性作用。哺乳动物AP-核酸酶（APE1）通过自发或通过DNA糖基化酶去除受损碱后在DNA中产生的Abasic（AP）位点在BER途径中起着核心作用。除了在DNA修复中的关键作用外，APE1还充当转录调节剂，它与几个转录因子结合并通过依赖氧化还原依赖性和非依赖性机制来调节其功能。这需要其非保存的N末端结构域。我们已经表明，在Lys 6和7处可以对APE1进行乙酰基，从而调节其转录调节函数。后来，我们表明APE1失活诱导有条件的APE1无脂细胞凋亡。其DNA修复功能和乙酰化位点都需要扭转这种效果。我们的初步数据表明，这些LYS的正电荷对于APE1与细胞中所有结合伴侣的相互作用至关重要，并且通过乙酰化来消除这些相互作用的这些LYS残基的正电荷。 APE1乙酰化还增强了其核酸内切酶的活性，并影响体外BER测定中的总AP位点修复。因此，我们的研究将检验以下假设：APE1的N末端结构域中乙酰基裂基残基的阳性电荷及其通过乙酰化的中和，调节其必不可少的DNA修复和转录调节功能，从而在细胞存活和细胞存活中起关键作用肿瘤细胞的耐药性。该假设将以三个特定目的进行检验。 AIM1将询问APE1的乙酰化如何影响其与其他蛋白质或DNA的相互作用，以调节受损碱或AP位点的修复，无论是在体外还是在细胞中。使用稳定表达WT，不可乙酰基K6R/K7R或乙酰化模拟K6Q/K7Q APE1的细胞，我们将测试以下假设：在没有APE1的乙酰基乳糖基裂解的情况下，在基因组中积累了AP位点或单链破裂的基因组，这触发了凋亡。 AIM2将询问遗传毒性药物治疗后肿瘤细胞中ACAPE1水平如何增加，以改变其与伴侣蛋白的复合形成，并通过其转录调节功能调节其DNA修复活性和药物效能转运蛋白MDR1的表达。使用一系列肿瘤细胞系，我们将测试ACAPE1水平及其对化学疗法挖掘的耐药性之间的相关性。在AIM3中，将染色质免疫沉淀与DNA微阵列分析结合在稳定表达WT或无乙酰基K6R/K7R APE1的细胞中的基因表达分析和芯片芯片分析中，将用于识别受APE1乙酰化和APE1乙酰化和直接调节的靶基因在没有APE1乙酰化的情况下，负责诱导凋亡的信号通路。总之，我们的结果应提高我们对APE1乙酰化如何在体内DNA损伤修复和转录调节中起关键作用的理解，这对细胞存活产生了深远的影响。