Alteration of NAD Metabolism by Chemical Carcinogens

描述（由申请人提供）：基因毒性应激后，通过激活adp核糖（ADPR）聚合物循环，NAD代谢迅速改变。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，它们通过介导基因毒性应激后的细胞恢复或细胞死亡，在维持基因组完整性方面发挥重要作用。PARG在合成ADPR聚合物的细胞核中起作用，但我们已经发现了PARG的线粒体异构体。待验证的假设是，核和线粒体PARG是信号和传感机制的关键组成部分，报告DNA损伤的数量，并将这些信息与细胞死亡的关键线粒体调节因子整合在一起。PARG在线粒体中的存在是有趣的，因为PARP-1启动的ADPR聚合物循环与基因毒性应激后线粒体中凋亡诱导因子（AIF）的释放有关，但PARP-1本身不存在于线粒体中。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和防止细胞死亡，但在高水平的基因毒性应激下产生可从细胞核释放的游离ADPR聚合物，参与线粒体的AIF释放。线粒体PARG异构体阻止了ADPR聚合物释放不适当的AIF，并且必须在AIF从线粒体释放之前被ADPR聚合物饱和。验证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损伤反应信号中的作用（目标1），确定PARG基因表达的调控以及PARG基因与线粒体进口蛋白基因TIM23的表达之间的关系（目标2），并确定PARG蛋白的关键结构特征（目标3）。我们将使用PARG基因被破坏的小鼠细胞、新型细胞渗透性PARG抑制剂、过表达PARG异构体和位点定向突变体来调节PARG的含量和活性。该应用程序的创新之处在于，它代表了细胞核和线粒体之间的串扰调节细胞死亡的新范式，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。

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.