Cloning Negative Regulators of TNF Signaling

描述（由申请人提供）：目前已知肿瘤坏死因子-α（TNF）是炎症过程的关键组分，过量的TNF水平在包括类风湿性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（TNFR 1）信号传导如何调节的更深入了解可能为治疗干预提供新的靶点。TNFR 1的连接导致NF-κ B通路的激活或凋亡通路的激活。在另一个NIAID资助的基金中，我们提议研究TNFR 1信号转导到凋亡通路的机制，该机制可以通过NF-κ B通路关闭，重点是A20介导的机制。此外，我们的目标之一是确定其他新的基因，可以抑制肿瘤坏死因子信号的凋亡途径。在本申请的目的1中，我们提出开发表达克隆策略以功能性分离抑制TNFR 1信号传导至细胞凋亡的cDNA。这一策略将基于使用Jurkat T细胞突变体，我们先前在我们的资助中描述了该突变体缺乏IKK活性，因此对TNF介导的细胞凋亡高度敏感。编码抑制这些细胞中细胞凋亡途径的蛋白质的cDNA的存在将赋予TNF处理的细胞存活优势，并且可以从存活的细胞克隆。除了抑制细胞凋亡途径的分子外，抑制NF-κ B途径的分子的鉴定也将促进我们对TNF信号传导的理解。为了在目标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-κ B被激活时，该细胞系就会发生自杀。因此，该细胞系可用作筛选抑制NF-κ B通路的基因的平台，再次使用死亡作为选择工具。携带抑制NF-κ B途径的cDNA的细胞将在TNF或PMA处理后存活，然后可以分离cDNA。两种克隆战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识别在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.