Alteration of NAD Metabolism by Chemical Carcinogens

化学致癌物改变 NAD 代谢

• 批准号: 6891465
• 负责人: MYRON K JACOBSON
• 金额: $ 36.62万
• 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/6891465
• 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）：NAD 代谢在基因毒性应激后通过 ADP-核糖（ADPR）聚合物循环的激活而迅速改变。这些循环由聚（ADP-核糖）聚合酶 1 和 2（PARP-1、PARP-2）和聚 ADP-核糖糖水解酶 (PARG) 介导，它们通过介导细胞恢复或恢复，在维持基因组完整性方面发挥着重要作用。遗传毒性应激后的细胞死亡。 PARG 在合成 ADPR 聚合物的细胞核中发挥作用，但我们发现了 PARG 的线粒体亚型。待测试的假设是，核和线粒体 PARG 是信号传导和传感机制的关键组成部分，报告 DNA 损伤量并将此信息与细胞死亡的关键线粒体调节因子整合。线粒体中 PARG 的存在很有趣，因为 PARP-1 启动的 ADPR 聚合物循环与遗传毒性应激后线粒体释放凋亡诱导因子 (AIF) 有关，但 PARP-1 本身并不存在于线粒体中。我们的假设预测，核 PARG 通过在低水平的基因毒性应激下阻止游离 ADPR 聚合物的形成来促进细胞恢复并防止细胞死亡，但在高水平的基因毒性应激后生成游离 ADPR 聚合物，这些聚合物可以从细胞核中释放，参与 AIF 的释放。线粒体。线粒体 PARG 亚型可防止 ADPR 聚合物不当释放 AIF，并且必须先用 ADPR 聚合物饱和，然后才能从线粒体中释放 AIF。检验这一假设的具体目标包括确定核和线粒体 PARG 亚型在导致细胞恢复和细胞死亡的 DNA 损伤反应信号传导中的作用（目标 1）、确定 PARG 基因表达的调节以及表达之间的关系。描述了 PARG 基因和线粒体输入蛋白基因 TIM23（目标 2），并定义了 PARG 蛋白的关键结构特征（目标 3）。我们将使用 PARG 基因破坏的小鼠细胞、新型细胞渗透性 PARG 抑制剂以及过表达 PARG 亚型和定点突变体来调节 PARG 含量和活性。该应用的创新之处在于它代表了细胞核和线粒体之间用于调节细胞死亡的串扰的新范例，并且已经开发出强大的遗传和化学工具来测试中心假设。拟议的研究还提供了评估 PARG 作为治疗靶点的机会。