Regulation of TNF Receptor-Mediated Apoptosis

描述(由申请人提供)：肿瘤坏死因子特异性拮抗剂治疗类风湿性关节炎(RA)的临床疗效表明，肿瘤坏死因子在类风湿关节炎发病机制中起着关键作用。肿瘤坏死因子受体1(TNFR1)是肿瘤坏死因子与其受体的结合，可激活核因子-kB转录因子，导致基因表达或半胱氨酸天冬氨酸氨基转移酶途径，导致细胞凋亡。最近的研究表明，核因子-kB可诱导抗凋亡基因的表达，从而拮抗同时激活的细胞凋亡途径。因此，一个重要的问题是，受核因子-kB调控的抗凋亡基因是什么，它们是如何抑制细胞凋亡的。一个Jurkat T细胞突变体已经被分离到，该突变体缺乏作为核因子-kB信号通路的重要组成部分的ikk/Nemo，并且由于这一缺陷，该突变体对肿瘤坏死因子诱导的细胞凋亡的敏感性显著增加。A20是一个受NP-B调节的基因，通过阻断RIP(肿瘤坏死因子途径中的死亡区域激酶)或RIP上游的分子，有效地阻断了肿瘤坏死因子诱导的突变细胞的凋亡。在具体目标1中，A20的确切分子靶点将通过上位性分期研究，结合基于我们目前对近端TNFR1信号转导的理解而形成的假设检验来定义。在具体目标2中，将通过识别与A20相互作用的蛋白质来研究A20所利用的机制。所使用的方法将基于A20与其伙伴之间的从头相互作用，这种相互作用可以通过抗A20抗体分离出来。随后将使用一些方案来确定相关的合作伙伴。在特定的Aim3中，将在这个突变的细胞系中搜索不再被肿瘤坏死因子上调的基因，然后进行功能测试，以确定它们扰乱细胞凋亡途径的能力。该项目的最终结果将是对肿瘤坏死因子调节的通路和下游基因有更多的了解，这可能为类风湿关节炎的药理调节提供更多的途径。

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.