Regulation of TNF Receptor-Mediated Apoptosis

描述（由申请人提供）：TNF特异性拮抗剂治疗类风湿关节炎（RA）的临床疗效表明TNF在RA发病机制中的关键作用。TNF受体1 （TNF receptor 1, TNFR1）与其受体之一结合，可激活NF-kB转录因子，导致基因表达或caspase途径导致细胞凋亡。最近的研究表明，NF-kB诱导抗凋亡基因的表达，从而拮抗同时激活的凋亡途径。因此，NF-kB调控的抗凋亡基因是什么以及它们如何抑制细胞凋亡是一个重要的问题。一个Jurkat T细胞突变体缺乏IKK /NEMO （NF-kB信号通路的重要组成部分），由于这种缺陷，该突变体对tnf诱导的细胞凋亡的敏感性显著增加。A20是一种NP- b调控基因，通过阻断TNF通路中的死亡结构域激酶RIP或RIP上游分子，有效阻断突变细胞中TNF诱导的凋亡。在Specific Aim 1中，A20的精确分子靶点将通过上位分期研究来确定，并结合基于我们目前对近端TNFR1信号转导的理解而制定的假设的检验。在Specific Aim 2中，将通过鉴定与A20相互作用的蛋白来研究A20利用的机制。所使用的方法将基于A20与其合作伙伴之间的从头相互作用，这种相互作用可以通过抗A20抗体进行分离。随后将使用若干方案来确定相关的合作伙伴。在Specific aim中，我们将在该突变细胞系中寻找不再被TNF上调的基因，然后对其破坏凋亡途径的能力进行功能测试。该项目的最终结果将是对TNF调控的途径和下游基因的更多了解，这可能为RA的药理调节提供额外的途径。

DESCRIPTION (provided by applicant): The clinical efficacy of TNF-specific antagonists in the treatment of rheumatoid arthritis (RA) points to the critical role of TNF in RA pathogenesis. Binding of TNF to one of its receptors, TNF receptor 1 (TNFR1) can activate NF-kB transcription factors leading to gene expression or the caspase pathway leading to apoptosis. Recent studies have shown that NF-kB induces the expression of anti-apoptotic genes, which then antagonize the apoptosis pathway that is concurrently activated. Therefore, an important question is what are the anti-apoptotic genes regulated by NF-kB and how do they inhibit apoptosis. A Jurkat T cell mutant that is deficient in IKK /NEMO, an essential component of the NF-kB signaling pathway, has been isolated and as a result of this deficiency, this mutant exhibits a profound increase in sensitivity to TNF-induced apoptosis. A20, a NP- B-regulated gene, effectively blocks TNF-induced apoptosis in the mutant cell by blocking either RIP, a death domain kinase in the TNF pathway, or a molecule upstream of RIP. In Specific Aim 1, the precise molecular target of A20 will be defined by epistasis staging studies, combined with the testing of hypothesis that were formulated based on our current understanding of proximal TNFR1 signal transduction. In Specific Aim 2, the mechanism utilized by A20 will be examined by identifying proteins that interact with A20. The approaches used will be based on the de novo interaction between A20 with its partners, which can be isolated by anti-A20 antibodies. A number of schemes will be used subsequently to identify the associated partners. In Specific Aim3, a search for genes that are no longer upregulated by TNF in this mutant cell line will be conducted and then functionally tested for their ability to disrupt the apoptosis pathway. The end result of this project will be a greater knowledge of the pathways and downstream genes regulated by TNF, which may suggest additional avenues for pharmacological modulation in RA.