Alteration of NAD Metabolism by Chemical Carcinogens

化学致癌物改变 NAD 代谢

• 批准号: 6783168
• 负责人: MYRON K JACOBSON
• 金额: $ 35.59万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6783168
• 关键词: ADP ribosylation; DNA damage; DNA repair; N methyl N' nitro N nitrosoguanidine; NAD nucleosidase; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; apoptosis; carcinogen testing; cell line; chemical carcinogen; chemical carcinogenesis; confocal scanning microscopy; enzyme activity; green fluorescent proteins; high performance liquid chromatography; immunocytochemistry; mitochondrial membrane; neoplasm /cancer genetics; nicotinamide adenine dinucleotide; nucleotide metabolism; polymerase chain reaction; protein structure; western blottings

DESCRIPTION (provided by applicant): NAD metabolism is rapidly altered following genotoxic stress by the activation of ADP-ribose (ADPR) polymer cycles. These cycles are mediated by poly(ADP-ribose) polymerases 1 and 2 (PARP-1, PARP-2) and poly ADP-ribose glycohydrolase (PARG) and they play a major role in the maintenance of genomic integrity by mediating cell recovery or cell death following genotoxic stress. PARG functions in the nucleus where ADPR polymers are synthesized but we have discovered a mitochonddal isoform of PARG. The hypothesis to be tested is that nuclear and mitochondrial PARG are key components of a signaling and sensing mechanism reporting the amount of DNA damage and integrating this information with key mitochondrial regulators of cell death. The presence of PARG in mitochondria is intriguing as PARP-1 initiated ADPR polymer cycles are linked to release of apoptosis inducing factor (AIF) from mitochondria following genotoxic stress but PARP-1 itself is not present in mitochondria. Our hypothesis predicts that nuclear PARG promotes cell recovery and prevents cell death by preventing formation of free ADPR polymers at low levels of genotoxic stress but generates free ADPR polymers that can be released from the nucleus following high levels of genotoxic stress to participate in AIF release from mitochondria. The mitochondrial PARG isoform prevents inappropriate AIF release by ADPR polymers and must be saturated with ADPR polymers before AIF can be released from mitochondda. The specific aims by which this hypothesis will be tested include defining the role of nuclear and mitochonddal PARG isoforms in DNA damage response signaling leading to cell recovery and cell death (Aim 1), defining the regulation of PARG gene expression and the relationship between the expression of the PARG gene and the mitochondrial import protein gene TIM23 with which it shares a common promoter (Aim 2), and defining key structural features of the PARG protein (Aim 3). We will modulate PARG content and activity using PARG gene disrupted mouse cells, novel cell permeable PARG inhibitors, and by overexpression PARG isoforms and site-directed mutants. The innovation of the application is that is represents a new paradigm for cross talk between nucleus and mitochondria for regulation of cell death and that powerful genetic and chemical tools have been developed to test the central hypothesis. The proposed research also provides the opportunity to evaluate PARG as a therapeutic target.

描述（由申请人提供）：通过激活ADP-核糖（ADPR）聚合物周期，NAD代谢在遗传毒性应激后迅速改变。这些循环是由聚（ADP-核糖）聚合酶1和2（PARP-1，PARP-2）和多ADP-核糖糖糖酶（PARG）介导的，它们通过介导基因组完整性而在遗传毒性应激后介导细胞恢复或细胞死亡中起着重要作用。 PARG在合成ADPR聚合物的核中功能，但我们发现了PARG的线粒体同工型。要测试的假设是，核和线粒体PARG是报告和感测机制的关键组成部分，该机制报告了DNA损伤的量，并将这些信息与细胞死亡的关键线粒体调节剂相结合。线粒体中PARG的存在引人入胜，因为PARP-1启动的ADPR聚合物周期与遗传毒性应激后线粒体诱导凋亡因子（AIF）的释放有关，但在线粒体中不存在PARP-1本身。我们的假设预测，核PARG会促进细胞的恢复，并通过防止在低水平的遗传毒性应激下形成游离ADPR聚合物，从而防止细胞死亡，但会产生游离ADPR聚合物，这些聚合物可以在高水平的遗传毒性应激之后从核中释放出来，从而参与Mitochonochria的AIF释放。线粒体PARG同工型可防止ADPR聚合物释放不适当的AIF AIF释放，并且必须在可以从Mitochondda释放AIF之前饱和ADPR聚合物。 The specific aims by which this hypothesis will be tested include defining the role of nuclear and mitochonddal PARG isoforms in DNA damage response signaling leading to cell recovery and cell death (Aim 1), defining the regulation of PARG gene expression and the relationship between the expression of the PARG gene and the mitochondrial import protein gene TIM23 with which it shares a common promoter (Aim 2), and defining key structural features of the PARG蛋白质（目标3）。我们将使用PARG基因中断的小鼠细胞，可渗透的PARG抑制剂以及过表达的PARG同工型和定位突变体来调节PARG含量和活性。该应用的创新是代表了细胞核与线粒体之间的跨交谈的新范式，以调节细胞死亡，并且已经开发出强大的遗传和化学工具来检验中心假设。拟议的研究还提供了将PARG评估为治疗靶点的机会。