A novel role for PARP1 in regulation of autophagy

描述(由申请人提供)：广泛的文献记载了聚(ADP-核糖)聚合酶1(PARP1)在DNA修复中的作用。PARP1识别并被单链DNA断裂激活。激活后，它聚合(ADP-核糖)，参与染色质组织和DNA修复的各种蛋白质。最近的研究表明，PARP1的生存效应除了在DNA修复中发挥作用外，可能还反映了它对诱导自噬的贡献，自噬是一种过程，胞浆和受损的细胞器被包裹在称为自噬小体的空泡中，自噬小体与溶酶体融合，导致被隔离的货物降解。PARP1可能通过利用NAD进行多聚(ADP-核糖基)来介导这种诱导，从而导致代谢缺陷。因此，它激活AMPK，进而抑制mTOR活性，从而否定后者对自噬的主动抑制。相反，我们有数据表明，PARP1可能在自噬中发挥作用，与其最终抑制mTOR的条件的建立无关。具体地说，我们发现，与野生型MEF相比，PARP1基因敲除(PARP1-/-)的小鼠胚胎成纤维细胞(MEF)在mTOR抑制剂雷帕霉素诱导自噬后，自噬形成减少。此外，对积极参与自噬的人卵巢癌细胞株的免疫沉淀(IP)研究表明，PARP1和ATG12-ATG5以复合体的形式存在。由于ATG12-ATG5是位于自噬小体上的ATG12-ATG5/ATG16复合体的一个组成部分，在自噬体膜的合成和延伸中起着关键作用，因此这种复合体从未被报道过，而且意义重大。我们假设PARP1池的一部分存在于与自噬合成机械成员的复合体中，并且是最佳自噬小体合成所必需的。本申请的总体目的是确定：1)在接收到自噬诱导信号后，是否需要PARP1来合成自噬小体，以及2)PARP1是否存在于自噬小体上与ATG12-ATG5或ATG12-ATG5/ATG16的复合体中。这两个目标的另一个组成部分将是确定其中一种特性是否需要PARP1的聚(ADP-核糖基)活性。为了实现这些目标，将使用共聚焦荧光显微镜和免疫生物化学分析来监测雷帕霉素和饥饿诱导的人卵巢癌细胞株在载体或PARP1 shRNA、野生型MEF和PARP1-/-MEF的培养中的自噬小体合成。这些细胞将用于IP和共聚焦荧光共定位研究，以确定PARP1是否定位于ATG12-ATG5、ATG12-ATG5/ATG16复合体和自噬小体。表达多聚(ADP-核糖基)死亡PARP1的工程细胞系和PARP1的药理抑制剂将被用于检测PARP1是否必须对复合体的形成和自噬小体的合成具有活性。正在做出重大努力，使用PARP1和自噬的抑制剂来增强细胞毒药物的治疗效果。我们的研究调查了PARP1的一个潜在的新功能，它与自噬有关。

DESCRIPTION (provided by applicant): An extensive literature documents the role of poly(ADP-ribose)polymerase 1 (PARP1) in DNA repair. PARP1 recognizes and is activated by single strand DNA breaks. Upon activation, it poly(ADP-ribosyl)ates a variety of proteins involved in chromatin organization and DNA repair. Recent studies suggest that the prosurvival effects of PARP1 may, in addition to its role in DNA repair, reflect its contributions to the induction of autophagy, a process whereby cytosol and damaged organelles are encapsulated in vacuoles termed autophagosomes that fuse with lysosomes, leading to degradation of the sequestered cargo. PARP1 presumably mediates this induction via its utilization of NAD for poly(ADP-ribosyl)ation, which results in a metabolic deficiency. Consequently, it activates AMPK and in turn inhibits mTOR activity, thus negating the latter's active suppression of autophagy. In contrast, we have data suggesting that PARP1 may play a role in autophagy unrelated to its establishment of conditions that ultimately inhibit mTOR. Specifically, we found that PARP1 knockout (PARP1-/-) murine embryonic fibroblasts (MEFs), relative to wild type MEFs, exhibit reduced autophagosome formation following induction of autophagy with the mTOR inhibitor rapamycin. Furthermore, immunoprecipitation (IP) studies with human ovarian cancer cell lines actively engaged in autophagy suggested that PARP1 and ATG12-ATG5 exist as a complex. Such complexes have never been reported and are significant since ATG12-ATG5 is a component of the ATG12-ATG5/ATG16 complex that resides on the autophagosome, which plays a key role in the synthesis and elongation of the autophagosome membrane. We hypothesize that a portion of the PARP1 pool exists in a complex with members of the autophagic synthetic machinery and is required for optimal autophagosome synthesis. The overall aims of this application are to determine: 1) if PARP1 is required for the synthesis of autophagosomes following receipt of an autophagy-inducing signal, and 2) if PARP1 exists in a complex with ATG12-ATG5 or ATG12- ATG5/ATG16 on the autophagosomes. An additional component of both aims will be to determine if either property requires the poly(ADP-ribosyl)ating activity of PARP1. To achieve these goals confocal fluorescence microscopy and immunobiochemical assays will be used to monitor rapamycin- and starvation-induced autophagosome synthesis in cultures of human ovarian cancer cell lines transfected with vector or PARP1 shRNA, wild type MEFs and PARP1-/- MEFs. The cells will be used in IP and confocal fluorescence colocalization studies to determine if PARP1 localizes to ATG12-ATG5, ATG12-ATG5/ATG16 complexes and autophagosomes. Engineered cell lines expressing poly(ADP-ribosyl)ation dead PARP1 and pharmacological inhibitors of PARP1 will be used to examine if PARP1 must be active for complex formation and autophagosome synthesis. Significant efforts are being made to use inhibitors of PARP1 and autophagy to potentiate the therapeutic effectiveness of cytotoxic drugs. Our studies investigate a potentially novel function for PARP1 that links it to autophagy.