A novel role for PARP1 in regulation of autophagy

PARP1 在自噬调节中的新作用

• 批准号: 8565852
• 负责人: GEN SHENG WU
• 金额: $ 16.53万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8565852
• 关键词: Antineoplastic Agents; Antithymoglobulin; Autophagocytosis; Autophagosome; Biological Assay; Cancer cell line; Cell Line; Cell Survival; Cell physiology; Cells; Chromatin; Chromatin Structure; Clinical Trials; Complex; Cytosol; Cytotoxic agent; DNA Damage; DNA Repair; DNA Repair Pathway; DNA Single Strand Break; DNA biosynthesis; Data; Embryo; Encapsulated; Engineering; Excision; Exhibits; Exposure to; Family; Fibroblasts; Fluorescence; Fluorescence Microscopy; Genome Stability; Goals; Human; Immunoprecipitation; Knock-out; Link; Literature; Lysosomes; Maintenance; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Membrane; Metabolic; Monitor; Mus; Nutritional; Organelles; Pathway interactions; Pharmaceutical Preparations; Play; Poly(ADP-ribose) Polymerases; Process; Property; Proteins; Public Health; Recruitment Activity; Regulation; Relative (related person); Reporting; Research; Role; Signal Transduction; Sirolimus; Starvation; Testing; Transcriptional Regulation; Vacuole; base; cytotoxicity; design; effective therapy; human FRAP1 protein; inhibitor/antagonist; mTOR Inhibitor; member; membrane synthesis; novel; operation; public health relevance; small hairpin RNA; therapeutic effectiveness; vector

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.

描述（由申请人提供）：大量文献记录了聚（adp -核糖）聚合酶1 （PARP1）在DNA修复中的作用。PARP1识别单链DNA断裂并被激活。一旦被激活，它的聚（adp -核糖基）会产生多种参与染色质组织和DNA修复的蛋白质。最近的研究表明，除了在DNA修复中的作用外，PARP1的促生存作用可能反映了它对诱导自噬的贡献，这一过程中，细胞质和受损的细胞器被包裹在称为自噬体的液泡中，与溶酶体融合，导致被隔离的货物降解。PARP1可能通过利用NAD进行聚（adp -核糖基）化介导这种诱导，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，与最终抑制mTOR的条件建立无关。具体来说，我们发现PARP1敲除（PARP1-/-）小鼠胚胎成纤维细胞（mef），相对于野生型mef，在mTOR抑制剂雷帕霉素诱导自噬后，自噬体的形成减少。此外，免疫沉淀（IP）对活跃自噬的人卵巢癌细胞系的研究表明，PARP1和ATG12-ATG5作为复合物存在。此类复合物从未被报道过，但其意义重大，因为ATG12-ATG5是位于自噬体上的ATG12-ATG5/ATG16复合物的一个组分，在自噬体膜的合成和延伸中起关键作用。我们假设PARP1库的一部分存在于自噬合成机制成员的复合体中，并且是最佳自噬体合成所必需的。本应用程序的总体目的是确定：1)PARP1在收到自噬诱导信号后是否需要自噬体的合成，以及2)PARP1是否存在于自噬体上的ATG12-ATG5或ATG12-ATG5 /ATG16复合物中。这两个目标的另一个组成部分将是确定是否任何性质都需要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化分析来监测转染载体或PARP1 shRNA、野生型mef和PARP1-/- mef的人卵巢癌细胞系培养物中雷帕霉素和饥饿诱导的自噬体合成。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复合物和自噬体。通过表达聚（adp -核糖基）化死亡的PARP1和PARP1的药理学抑制剂的工程细胞系，将检测PARP1是否必须在复合物形成和自噬体合成中具有活性。人们正在努力使用PARP1和自噬抑制剂来增强细胞毒性药物的治疗效果。我们的研究探讨了PARP1与自噬相关的一种潜在的新功能。