Role of PARP-1 in Mediating Inflammatory Gene Transcription

描述(申请人提供)：炎性介质产生过多可导致主要器官衰竭和损伤。一组明确的炎症基因的转录需要在被称为增强体的高度有序的复合体中同时进行特定的DNA-蛋白质和蛋白质-蛋白质相互作用。多聚(ADP-核糖)聚合酶-1(PARP-1)调节炎症基因表达的转录因子，重要的是PARP-1缺陷(PARP-1-/-)小鼠被证明对炎症性疾病具有抵抗力。总体假设是PARP-1被招募到启动子上，用聚ADP-核糖修饰转录调节因子，并介导招募转录调节因子组装增强体进行协同基因转录。我们的具体目标是：(1)确定PARP-1是否以序列特异性的方式与诱导型一氧化氮合酶启动子结合；(2)研究PARP-1是否通过与序列特异的转录因子(S)相互作用并增加其DNA结合活性而被招募到诱导型一氧化氮合酶启动子；(3)研究p42/44MAPK是否直接或间接地磷酸化并诱导PARP-1对内毒素的催化活性；(4)确定PARP-1是否通过聚(腺苷二磷酸-核糖)靶向转录调节器，以实现对内毒素的有效基因转录；(5)通过比较野生型和PARP-1-/-细胞，(Ii)同时分析PARP-1介导的招募，以及(Iii)iNOS、TNFpha、IL-1β和IL-6的招募，鉴定PARP-1介导转录调节因子对启动子招募的特征；以及(6)通过在PARP-1-/-细胞中表达野生型PARP-1和催化失活的PARP-1突变体，明确确定PARP-1的特异性及其对炎症基因调控的催化活性。本研究的意义在于为PARP-1调控炎症基因协同转录的机制(S)提供新的见解。炎症基因转录对免疫反应和确定炎症性疾病的发病至关重要，如感染性休克、急性肺部炎症、阿尔茨海默病、多发性硬化症和艾滋病毒相关痴呆。

DESCRIPTION (provided by applicant): Overproduction of inflammatory mediators can lead to major organ failure and damage. The transcription of a defined set of inflammatory genes requires the simultaneous specific DNA-protein and protein-protein interactions in highly ordered complexes called enhanceosome. Poly(ADP-ribose) polymerase-1 (PARP-1) regulates transcription factors responsible for inflammatory gene expression, and importantly PARP-1-deficient (PARP-1-/-) mice were shown to be resistant to inflammatory diseases. The overall HYPOTHESES are that PARP-1 is recruited to the promoter, modifies transcription regulators with poly(ADP-ribose), and mediates recruiting transcription regulators to assemble the enhanceosome for synergistic gene transcription. Our SPECIFIC AIMS are to (1) determine whether PARP- 1 binds the iNOS promoter in a sequence-specific manner; (2) investigate if PARP-1 is recruited to the iNOS promoter by interacting with a sequence-specific transcription factor(s) and increasing its DNA binding activity; (3) investigate whether p42/44MAPK directly or indirectly phosphorylates and induces PARP-1 catalytic activity in response to LPS; (4) determine whether PARP-1 targets transcription regulators with poly(ADP-ribose) for the efficient gene transcription in response to LPS; (5) characterize PARP-1 mediating the recruitment of transcription regulators to the promoters (i) by comparing wild-type and PARP-1-/- cells, (ii) by simultaneously analyzing PARP-1-mediated recruitments, and (iii) of iNOS, TNFalpha, IL-1beta, and IL-6; and (6) definitively determine the specificity of PARP-1 and its catalytic activity on inflammatory gene regulation by expressing wild-type PARP-1 and catalytically inactive PARP-1 mutant in PARP-1-/- cells. The SIGNIFICANCE of the proposed studies is to offer new insights into the mechanism(s) by which PARP-1 may regulate and be regulated to achieve synergistic inflammatory gene transcription. The inflammatory gene transcription is critical for immune responses and for determining the onset of inflammatory diseases such as septic shock, acute lung inflammation, Alzheimer's disease, multiple sclerosis, and HIV-associated dementia.