Cloning Negative Regulators of TNF Signaling

描述(申请人提供)：肿瘤坏死因子-α(肿瘤坏死因子-α)现在被认为是炎症过程中的一个关键成分，而过量的肿瘤坏死因子水平在包括类风湿性关节炎在内的严重炎症性疾病中起着病理作用。因此，进一步了解肿瘤坏死因子受体1(TNFR1)信号是如何调控的，可能会为治疗干预提供新的靶点。结扎TNFR1可以激活核因子-kappaB途径，也可以激活细胞凋亡途径。在NIAID资助的另一项拨款中，我们建议研究核因子-kappaB途径关闭TNFR1信号到细胞凋亡途径的机制，重点是A20介导的机制。此外，我们的目标之一是确定其他可以抑制肿瘤坏死因子信号传导至细胞凋亡途径的新基因。在本申请的目的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信号转导细胞凋亡的cDNA。这一策略将基于使用Jurkat T细胞突变体，我们在之前的研究中描述了这种突变体，它缺乏IKK活性，因此对肿瘤坏死因子介导的细胞凋亡高度敏感。在这些细胞中，编码一种抑制细胞凋亡途径的蛋白质的cDNA的存在将为经肿瘤坏死因子处理的细胞提供生存优势，并可以从存活的细胞中克隆出来。除了抑制细胞凋亡途径的分子外，识别抑制核因子-kappaB途径的分子也将促进我们对肿瘤坏死因子信号转导的理解。为了在目标2中做到这一点，我们创造了一个不同的Jurkat细胞系，每当核因子-kappaB被激活时，该细胞系就会经历自杀。因此，这个细胞系可以作为一个平台来筛选抑制核因子-kappaB途径的基因，再次使用死亡作为选择工具。携带抑制核因子-kappaB途径的cDNA的细胞将在肿瘤坏死因子或PMA处理后存活，然后可以分离出cDNAs。这两种克隆战略都基于相似的原则和所需的相似方法。这些方法的发展将加速寻找在肿瘤坏死因子信号转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.