Alteration of NAD Metabolism by Chemical Carcinogens

描述（由申请方提供）：遗传毒性应激后，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，它们通过介导遗传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ADPR聚合物合成的细胞核中发挥作用，但我们已经发现了PARG的线粒体亚型。待检验的假设是，核和线粒体PARG是信号传导和传感机制的关键组分，该机制报告DNA损伤的量并将该信息与细胞死亡的关键线粒体调节因子整合。PARG在线粒体中的存在是令人感兴趣的，因为PARP-1启动的ADPR聚合物循环与遗传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通过在低水平的遗传毒性应激下防止游离ADPR聚合物的形成来促进细胞恢复并防止细胞死亡，但产生游离ADPR聚合物，其可以在高水平的遗传毒性应激后从核中释放以参与从线粒体释放AIF。线粒体PARG同种型防止ADPR聚合物不适当地释放AIF，并且在AIF可以从线粒体释放之前必须被ADPR聚合物饱和。检验这一假设的具体目的包括确定核和线粒体PARG同种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（Aim 1），确定PARG基因表达的调节和PARG基因表达与线粒体输入蛋白基因TIM 23之间的关系（Aim 2），并定义PARG蛋白的关键结构特征（Aim 3）。我们将使用PARG基因破坏的小鼠细胞、新型细胞渗透性PARG抑制剂以及通过过表达PARG同种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.