Role of PARP-1 in Mediating Inflammatory Gene Transcription

描述（由申请人提供）：炎症介质的过度产生可导致主要器官衰竭和损伤。一组确定的炎症基因的转录需要在称为增强体的高度有序的复合物中同时发生特异性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）调节负责炎性基因表达的转录因子，重要的是，PARP-1缺陷（PARP-1-/-）小鼠显示出对炎性疾病的抗性。总的假设是PARP-1被募集到启动子，用聚（ADP-核糖）修饰转录调节子，并介导募集转录调节子以组装增强子用于协同基因转录。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结合活性而被募集到iNOS启动子;（3）研究p42/44 MAPK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是否靶向具有多聚腺苷酸的转录调节子。（5）通过比较野生型和PARP-1-/-细胞，（ii）通过同时分析PARP-1介导的募集，和（iii）iNOS、TNF α、IL-1 β和IL-6，表征PARP-1介导的转录调节因子向启动子的募集;和（6）通过在PARP-1-/-细胞中表达野生型PARP-1和无催化活性的PARP-1突变体，确定PARP-1的特异性及其对炎症基因调节的催化活性。拟议研究的意义是为PARP-1可能调节和被调节以实现协同炎症基因转录的机制提供新的见解。炎性基因转录对于免疫应答和确定炎性疾病如感染性休克、急性肺部炎症、阿尔茨海默病、多发性硬化和HIV相关痴呆的发作至关重要。

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.