Negative Regulators of NFkappaB

描述(申请人提供)：我们已经使用遗传方法分离了NFKappaB激活是结构性的突变细胞系。通过过度表达抑制NFKappaB结构性激活的蛋白质来补充这些细胞，已经导致了几个新的候选负调控因子的功能鉴定。NFKappaB的结构性激活在许多不同的癌症中很常见，其中NFKappaB调节的基因产物抑制细胞凋亡的能力是重要的。我们将通过追求三个具体目标来研究不同的负调控因子如何控制导致NFKappaB激活的途径，以响应许多不同的细胞压力：具体目标1A：验证NFKB的候选负调控因子并确定新的候选候选因子。我们将去除正常细胞中候选负调控因子的表达，并测试NFKB随后的结构性激活。我们还将测试每个候选负调控因子在代表不同互补基团和具有不同生化表型的结构性突变细胞中的效果。此外，我们还将测试这些蛋白是否会影响IL-1和TNF对NFKappaB的诱导。为了确定更多的候选负调控因子，我们将通过使用逆转录病毒cDNA文库和随机激活基因表达(一种可以驱动任何内源基因高表达的新技术)来补充更多的构成突变体。具体目标1B：NFKappaB的负调控与癌症。我们将确定NFKappaB具有结构性活性的各种癌症中已有的新的负调控因子的表达水平，以及有待分离的其他负调控因子的表达水平，以确定经常丢失特定调控因子的肿瘤类型。然后，我们将开始探索使用负性调节剂作为诊断辅助工具和可能的治疗靶点的可能性。具体目标2：分析每个经过验证的负调节因子。将使用各种分析来确定每个有效的负调节因子的生理作用。我们将分析它们对IKappaBalpha磷酸化的影响，以及对已知参与结构性或诱导性NFKappaB激活的激酶的激活的影响。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。我们还将对负调控因子进行详细的结构-功能分析，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.