Negative Regulators of NFkappaB

描述（由申请人提供）： 我们已经使用遗传方法分离突变细胞系，其中NF κ B的激活是组成性的。通过过表达抑制NF κ B组成性激活的蛋白质来补充这些细胞，已经导致了几种新的候选负调节因子的功能鉴定。NF κ B的组成性激活在许多不同的癌症中是常见的，其中NF κ B调节的基因产物抑制细胞凋亡的能力是重要的。我们将研究如何不同的负调节控制途径，可以导致激活NF κ B在响应许多不同的细胞应激，通过追求三个具体目标：具体目标1A：验证候选人的NF κ B负调节和识别新的候选人。我们将消除正常细胞中候选负调节因子的表达，并测试随后NF κ B的组成性激活。我们还将测试每种候选负调节因子在代表不同互补组和具有生物化学上不同表型的组成型突变细胞中的作用。此外，我们将测试这些蛋白质是否影响NF κ B的诱导响应IL-1和TNF。为了鉴定其他候选负调控因子，我们将通过使用逆转录病毒cDNA文库以及随机激活基因表达（一种可驱动任何内源基因高表达的新技术）补充其他组成型突变体。具体目标1B：NF κ B和癌症的负调节因子。我们将确定新的负调节剂的表达水平已经在手，和额外的被隔离，在各种癌症中，NFKappaB是组成型活性，以确定肿瘤类型中的特定调节剂的损失是频繁的。然后，我们将开始探索使用负调节因子作为诊断辅助手段和治疗靶点的潜力。具体目标2：分析每种经验证的负调节剂。将采用各种测定来确定每种经验证的负调节剂的生理作用。我们将分析它们对IKappaB α磷酸化和已知参与组成性或诱导性NF κ B活化的激酶活化的影响。我们将通过免疫共沉淀、GST下拉和酵母双杂交分析来鉴定与负调控相关的蛋白质。我们还将进行详细的结构功能分析的负调节，以帮助阐明其作用机制。

DESCRIPTION (provided by applicant): We have used genetic methods to isolate mutant cell lines in which the activation of NFKappaB is constitutive. Complementation of these cells by overexpressing proteins that suppress the constitutive activation of NFKappaB has led to the functional identification of several novel candidate negative regulators. Constitutive activation of NFKappaB is common in many different cancers, where the ability of NFKappaB-regulated gene products to suppress apoptosis is important. We will investigate how different negative regulators control pathways that can lead to the activation of NFKappaB in response to many different cellular stresses by pursuing three specific aims: Specific Aim 1A: Validation of candidate negative regulators of NFKB and identification of new candidates. We will ablate the expression of candidate negative regulators in normal cells and test for consequent constitutive activation of NFKB. We will also test the effect of each candidate negative regulator in constitutive mutant cells that represent different complementation groups and that have biochemically distinct phenotypes. Also, we will test whether these proteins affect the induction of NFKappaB in response to IL-1 and TNF. To identify additional candidate negative regulators, we will complement additional constitutive mutants by using retroviral cDNA libraries and also by using Random Activation of Gene Expression, a novel technology that can drive high expression of any endogenous gene. Specific Aim 1B: Negative regulators of NFKappaB and cancer. We will determine the levels of expression of the novel negative regulators already in hand, and of additional ones to be isolated, in a variety of cancers in which NFKappaB is constitutively active, to identity tumor types in which loss of a particular regulator is frequent. We will then begin to explore the potential of using negative regulators as diagnostic aids and as possible targets for therapy. Specific Aim 2: Analysis of each validated negative regulator. A variety of assays will be employed to ascertain the physiological role of each validated negative regulator. We will analyze their effects on IKappaBalpha phosphorylation and on the activation of kinases already known to be involved in constitutive or induced NFKappaB activation. We will identify proteins associated with negative regulators by using co-immunoprecipitations, GST pull-downs and yeast two-hybrid analyses. We will also perform detailed structure-function analyses of negative regulators to help elucidate their mechanisms of action.