Cloning Negative Regulators of TNF Signaling

描述（由申请人提供）：肿瘤坏死因子- α (TNF)现在已知是炎症过程的关键组成部分，过量的TNF水平在包括类风湿关节炎在内的严重炎症性疾病中起病理作用。因此，对TNF受体1 （TNFR1）信号如何调控的更深入了解可能为治疗干预提供新的靶点。TNFR1的连接导致NF-kappaB通路的激活或凋亡通路的激活。在另一项niaid资助项目中，我们提出研究NF-kappaB通路关闭凋亡通路TNFR1信号通路的机制，重点研究A20介导的机制。此外，我们的目标之一是鉴定其他可以抑制TNF信号传导至凋亡途径的新基因。在本应用的目的1中，我们建议开发一种表达克隆策略，以功能性地分离抑制TNFR1凋亡信号的cdna。该策略将基于我们之前在研究中描述的Jurkat T细胞突变体，该突变体缺乏IKK活性，因此对tnf介导的细胞凋亡高度敏感。在这些细胞中，编码抑制凋亡途径的蛋白质的cDNA的存在将赋予tnf处理的细胞生存优势，并且可以从存活的细胞中克隆出来。除了抑制凋亡通路的分子外，发现抑制NF-kappaB通路的分子也将促进我们对TNF信号传导的理解。为了在Aim 2中做到这一点，我们已经产生了一种不同的Jurkat细胞系，每当NF-kappaB被激活时，它就会经历自杀。因此，该细胞系可以作为筛选NF-kappaB通路抑制基因的平台，再次使用死亡作为选择工具。含有抑制NF-kappaB通路的cDNA的细胞将在TNF或PMA处理中存活，然后可以分离cDNA。这两种克隆策略都基于类似的原则，需要类似的方法。这些方法的发展将加速鉴定在TNF信号传导中具有特定调节功能的基因。

DESCRIPTION (provided by applicant): Tumor necrosis factor- alpha (TNF) is now known to be a critical component of the inflammatory process and excess TNF levels plays a pathological role in severe inflammatory diseases including rheumatoid arthritis. Therefore a greater understanding of how TNF receptor 1 (TNFR1) signaling is regulated may suggest new targets for therapeutic interventions. Ligation of TNFR1 leads either to the activation of the NF-kappaB pathway or to the activation of the apoptosis pathway. In another NIAID-funded grant, we proposed to study the mechanism by which TNFR1 signaling to the apoptosis pathway can be shut down by the NF-kappaB pathway, focusing on that mediated by A20. In addition, one of our aims was to identify other novel genes that can inhibit TNF signaling to the apoptosis pathway. In Aim 1 of this application, we propose to develop an expression cloning strategy to functionally isolate cDNAs that inhibit TNFR1 signaling to apoptosis. This strategy will be based on using a Jurkat T cell mutant we described previously in our grant that is deficient in IKK activity and thus highly sensitive to TNF-mediated apoptosis. The presence of a cDNA encoding a protein that inhibits the apoptosis pathway in these cells will confer a survival advantage to TNF-treated cells and can be cloned from the surviving cells. In addition to molecules that inhibit the apoptosis pathway, the identification of molecules that inhibit the NF-kappaB pathway would also advance our understanding of TNF signaling. In order to do so in Aim 2, we have generated a different Jurkat cell line that undergoes suicide whenever NF-kappaB is activated. Therefore, this cell line can be used as a platform to screen for genes that inhibit the NF-kappaB pathway, again using death as a selection tool. Cells harboring cDNAs that inhibit the NF-kappaB pathway will survive TNF or PMA treatment and the cDNA can then be isolated. Both cloning strategies are based on similar principles and required similar methodologies. The development of these approaches will accelerate the identification of genes with specific regulatory functions in TNF signaling.