Activators and repressors of NFkappaB and IRF3/7 in innate immunity

描述(申请人提供)：先天免疫中NFkappaB和IRF3/7的激活物和抑制物摘要：关于激活天然和获得性免疫反应的中央协调器--核因子-kB的途径已知很多，但关于核因子-kB如何在非应激细胞中受到控制仍有许多需要了解的。我们将使用插入突变来确定新的核因子-kB激活抑制物。我们还将与李晓霞和甘斯森合作，利用他们实验室已经建立的遗传选择，发现和研究激活核因子-kB、IRF7和IRF3对Toll样受体8(TLR8)和TLR3做出反应的途径的新的正负调控因子。我们开发了一种新的病毒载体，将新的启动子引入基因组的许多基因位点。在产生的显性突变中，驱动下游DNA编码的蛋白质过度表达的启动子可以随意移除，证明突变是由插入引起的。我们用这种方法鉴定了赖氨酸去甲基酶FBXL11是一种新的核因子-kB负调控因子，其表达受核因子-kB的调控。具体目标1A：我们将确定FBXL11的作用机制。具体目标1B：我们将研究其他突变体，在这些突变体中，不同负调控因子的表达是由插入的启动子驱动的。具体目标2：我们将研究响应TLR8的信号通路，TLR8通常由单链RNA激活。具体目标2A：我们将分离TLR8依赖启动子的激活被负调控子抑制的显性突变体。具体目标2B：我们将分离显性突变体，在这些突变体中，依赖于IRF7的启动子的结构性激活受到正调控因子的刺激。具体目标2C：我们将使用基因陷阱载体和促进目标基因剩余未突变等位基因丢失或失活的策略相结合，分离信号所需蛋白质表达已被消融的隐性突变体。具体目标2D：我们将分离核因子-kB被结构性激活的隐性突变体。具体目标2E：我们将研究我们新发现的TLR8通路的新成分或负调控因子如何在信号转导中发挥作用。具体目标3：我们将研究响应双链RNA(DsRNA)的信号通路，双链RNA通过TLR3激活NF-kB和IRF3，也通过涉及RIG-I和同源蛋白的内部途径。具体目标3A：我们将确定作为TLR3反应启动子对dsRNA反应的显性负调节因子的细胞蛋白。具体目标3B：我们将分离IRF3反应启动子被结构性激活的显性突变体，以鉴定正作用蛋白。具体目标3C：我们将研究新发现的响应dsRNA的通路的新成分或负调控因子如何在信号传递中发挥作用。我们强大的选择系统和强大的新基因方法的结合，可以极大地加深目前对驱动和调节重要的先天免疫介体的途径的理解。失控的炎症是一个主要的健康问题，影响到许多不同的疾病，包括癌症。我们使用新的遗传学方法来研究正常的、积极的和消极的对照，这些对照在不需要的时候控制炎症途径，在需要的时候激活它们。我们的发现有可能在几种疾病环境中导致新的治疗方法。

DESCRIPTION (provided by applicant): Activators and repressors of NFkappaB and IRF3/7 in innate immunity Abstract: Much is known about pathways that activate NF-kB, a central coordinator of innate and adaptive immune responses, but there is still much to learn about how NF-kB is kept in check in unstressed cells. We will use insertional mutagenesis to identify novel inhibitors of NF-kB activation. We will also collaborate with Xiaoxia Li and Ganes Sen to uncover and study novel positive and negative regulators of pathways that activate NF-kB, IRF7 and IRF3 in response to Toll-like receptor 8 (TLR8) and TLR3, using genetic selections that their labs have already established. We have developed a novel viral vector that introduces new promoters into many loci in the genome. In the resulting dominant mutants, the promoter, which drives the over-expression of a protein encoded by downstream DNA, can be removed at will, proving that the mutation was caused by the insertion. We have used this method to identify the lysine demethylase FBXL11 as a novel negative regulator of NF-kB whose expression is regulated by NF-kB. Specific Aim 1A: we will determine the mechanism of action of FBXL11. Specific Aim 1B: we will study additional mutants in which the expression of different negative regulators has been driven by inserted promoters. Specific Aim 2: we will study signaling pathways that respond to TLR8, which is normally activated by single-stranded RNA. Specific Aim 2A: we will isolate dominant mutants in which the activation of TLR8-dependent promoters is suppressed by negative regulators. Specific Aim 2B: we will isolate dominant mutants in which the constitutive activation of an IRF7-dependent promoter is stimulated by positive regulators. Specific Aim 2C: we will isolate recessive mutants in which the expression of a protein required for signaling has been ablated, using a gene-trap vector in combination with a strategy to promote loss or inactivation of the remaining unmutated allele of the targeted gene. Specific Aim 2D: we will isolate recessive mutants in which NF-kB has been activated constitutively. Specific Aim 2E: we will investigate how the novel components or negative regulators of the TLR8 pathway that we have newly identified function in signaling. Specific Aim 3: we will study signaling pathways that respond to double-stranded RNA (dsRNA), which activates NF-kB and IRF3 through TLR3 and also through an internal pathway involving RIG-I and homologous proteins. Specific Aim 3A: we will identify cellular proteins that function as dominant negative regulators of the response of TLR3-responsive promoters to dsRNA. Specific Aim 3B: we will isolate dominant mutants in which an IRF3-responsive promoter has been activated constitutively, to identify positively acting proteins. Specific Aim 3C: we will investigate how newly identified novel components or negative regulators of pathways that respond to dsRNA function in signaling. Our combination of robust selective systems and powerful new genetic methods can add much to the current understanding of pathways that drive and regulate important mediators of innate immunity. Uncontrolled inflammation is a major health problem that impacts many different diseases, including cancer. We use novel genetic methods to study the normal positive and negative controls that keep inflammatory pathways in check when they are not needed and activate them when they are. Our findings have the potential to lead to new therapeutic approaches in several disease settings.