Role of PARP-1 in Mediating Inflammatory Gene Transcription

描述（由申请人提供）：炎症介质的过量产生可导致主要器官衰竭和损伤。一组特定炎症基因的转录需要在称为增强体的高度有序复合物中同时进行特异性dna -蛋白质和蛋白质-蛋白质相互作用。聚（adp -核糖）聚合酶-1 （PARP-1）调节负责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抗性。总的假设是，PARP-1被招募到启动子上，用聚（adp -核糖）修饰转录调控因子，并介导招募转录调控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结合iNOS启动子；(2)研究PARP-1是否通过与序列特异性转录因子相互作用并增加其DNA结合活性而被招募到iNOS启动子；(3)研究p42/44MAPK是否直接或间接磷酸化并诱导PARP-1对LPS的催化活性；(4)确定PARP-1是否通过多聚（adp -核糖）靶向转录调节因子，以响应LPS有效地进行基因转录；(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、TNFalpha、il -1 β和IL-6，表征PARP-1介导的转录调节因子募集到启动子的过程；(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。所提出的研究的意义在于为PARP-1调节和被调节以实现协同炎症基因转录的机制提供了新的见解。炎症基因转录对于免疫反应和确定感染性休克、急性肺部炎症、阿尔茨海默病、多发性硬化症和艾滋病相关痴呆等炎症性疾病的发病至关重要。

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.