A novel role for PARP1 in regulation of autophagy

描述（由申请人提供）：大量文献记录了聚（ADP-核糖）聚合酶1（PARP 1）在DNA修复中的作用。PARP 1识别单链DNA断裂并被其激活。在激活后，它聚（ADP-核糖基）与参与染色质组织和DNA修复的各种蛋白质作用。最近的研究表明，PARP 1的促生存作用可能，除了其在DNA修复中的作用，反映了其对诱导自噬的贡献，自噬是一个过程，其中胞质溶胶和受损的细胞器被封装在称为自噬体的空泡中，自噬体与溶酶体融合，导致被隔离的货物降解。PARP 1可能通过其利用NAD进行聚（ADP-核糖基）化来介导这种诱导，这导致代谢缺陷。因此，它激活AMPK并反过来抑制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PARP 1可能在自噬中发挥作用，与其建立最终抑制mTOR的条件无关。具体而言，我们发现PARP 1敲除（PARP 1-/-）鼠胚胎成纤维细胞（MEF），相对于野生型MEF，表现出减少的自噬体形成后，诱导自噬与mTOR抑制剂雷帕霉素。此外，对积极参与自噬的人卵巢癌细胞系的免疫沉淀（IP）研究表明，PARP 1和ATG 12-ATG 5作为复合物存在。这种复合物从未被报道过，并且是重要的，因为ATG 12-ATG 5是存在于自噬体上的ATG 12-ATG 5/ATG 16复合物的组分，其在自噬体膜的合成和延伸中起关键作用。我们假设PARP 1库的一部分存在于与自噬合成机制成员的复合物中，并且是最佳自噬体合成所需的。本申请的总体目的是确定：1）在接收到自噬诱导信号后，自噬体的合成是否需要PARP 1，以及2）PARP 1是否存在于自噬体上与ATG 12-ATG 5或ATG 12-ATG 5/ATG 16的复合物中。这两个目标的另一个组成部分将是确定是否有任何一种性质需要PARP 1的聚（ADP-核糖基）化活性。为了实现这些目标，共聚焦荧光显微镜和免疫生物化学测定将用于监测雷帕霉素和饥饿诱导的自噬体合成的人卵巢癌细胞系转染载体或PARP 1 shRNA，野生型MEFs和PARP 1-/- MEFs的文化。这些细胞将用于IP和共聚焦荧光共定位研究，以确定PARP 1是否定位于ATG 12-ATG 5、ATG 12-ATG 5/ATG 16复合物和自噬体。表达聚（ADP-核糖基）化死亡PARP 1和PARP 1的药理学抑制剂的工程化细胞系将用于检查PARP 1是否必须具有复合物形成和自噬体合成的活性。正在做出重大努力来使用PARP 1和自噬的抑制剂来增强细胞毒性药物的治疗效果。我们的研究调查了PARP 1的一种潜在的新功能，将其与自噬联系起来。

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.