Control of NF-kappaB and Inflammation by COMMD proteins

描述(申请人提供)：核因子-βB是一种保守的转录因子，在宿主抵抗感染中发挥重要作用。虽然我们对核因子-βB是如何被激活的理解已经取得了相当大的进展，但对其活性是如何被终止的知之甚少。然而，就疾病机制而言，负责终止核因子-βB的机制的改变很可能在持续性炎症中发挥核心作用。这项拨款支持的研究表明，DNA结合的核转录因子B亚基的降解是转录终止的关键机制。我们确定了一种对这些效应负责的泛素连接酶，它包含一种名为COMMD1的蛋白质作为必要的辅因子。这个途径主要限制促炎基因的表达，蛋白质降解需要对核因子？B亚单位relA进行翻译后修饰(PTM)。关键悬而未决的问题包括COMMD1是否可以控制体内的炎症，这一途径是如何具体调控促炎基因的，以及触发Rela泛素化的确切PTM是什么。我们的目标是研究COMMD1在体内炎症中的作用，并揭示控制其在先天性免疫系统中活动的关键机制。根据初步数据，我们假设COMMD1是一种抗炎因子，它与可诱导的PTM协同作用，如苏莫化和磷酸化，以调节促炎基因。为了实现我们的目标，我们提出了这些特定的目标：(1)Commd1在体内炎症反应中的作用：我们发现COMMD1的表达在炎症性疾病的背景下受到抑制，这表明这种现象可能有助于持续和/或更严重的炎症。为了测试这一概念，我们培育了髓系特异性Commd1-/-小鼠，这些小鼠对脂多糖注射的敏感性增加。在这里，我们将利用这个独特的小鼠模型来评估终止机制在脓毒症期间炎症控制中的作用。(2)COMMD1基因特异性染色质募集的基础：COMMD1基因特异性募集是通过一种未知的机制发生的。初步研究表明，COMMD1与相扑的结合是染色质结合的主要原因。在这里，我们将研究相扑结合和染色质募集到促炎基因启动子之间的联系。通过将相扑结合缺陷突变体引入Commd1/-髓系细胞，将评估这些事件在免疫调节中的相关性。(2)控制PTMS终止NF-B/relA：除了COMMD1募集到染色质外，RelA的PTMS还需要蛋白质降解。在这个目标中，我们将定义靶向RELA进行降解所需的PTM，并将通过将抗降解形式的RELA重新引入RelA/-髓系细胞来评估这些PTM的生物学意义。总之，这项建议将解决一个极其重要的问题，通过提供对防止持续炎症的体内平衡机制的重要见解，推动核因子-B领域向前发展。

DESCRIPTION (provided by applicant): NF-?B is a conserved transcription factor that plays an essential role in the host defense to infection. While considerable advances have been made in our understanding of how NF-?B is activated, lesser is known about how its activity is terminated. Nevertheless, in terms of disease mechanism, it is likely that alterations in the mechanisms responsible for terminating NF-?B play a central role in persistent inflammation. Studies supported by this grant identified that the degradation of DNA-bound NF-?B subunits is a critical mechanism for transcriptional termination. We identified a ubiquitin ligase responsible for these effects, which contains as an essential cofactor the protein known as COMMD1. This pathway primarily limits the expression of pro-inflammatory genes and post-translational modifications (PTMs) of the NF-?B subunit RelA are required for protein degradation. Key outstanding questions include whether COMMD1 can control inflammation in vivo, how is this pathway specifically regulating pro-inflammatory genes, and what are the precise PTMs that trigger RelA ubiquitination. Our goal is to investigate the role of COMMD1 in inflammation in vivo and to uncover key mechanisms that control its activity in the innate immune system. Based on preliminary data we hypothesize that COMMD1 is an anti-inflammatory factor that cooperates with inducible PTMs such as sumoylation and phosphorylation in order to regulate pro-inflammatory genes. In pursuit of our goal, we propose these Specific Aims: (1) Role of Commd1 in inflammatory responses in vivo: We find that COMMD1 expression is repressed in the setting of inflammatory diseases, suggesting that this phenomenon may contribute to persistent and/or more severe inflammation. To test this notion, we have generated myeloid specific Commd1-/- mice, which display increased susceptibility to LPS administration. Here we will utilize this unique mouse model to evaluate the role of termination mechanisms in the control of inflammation during sepsis. (2) Basis for gene-specific chromatin recruitment of COMMD1: Gene-specific recruitment of COMMD1 occurs through a yet unknown mechanism. Preliminary studies suggest that SUMO binding by COMMD1 is responsible for chromatin binding. Here we will examine the link between SUMO binding and chromatin recruitment to pro-inflammatory gene promoters. The relevance of these events in immune regulation will be assessed by introducing SUMO binding defective mutants into Commd1-/- myeloid cells. (2) Control of termination by PTMs of NF-?B /RelA: In addition to COMMD1 recruitment to chromatin, PTMs of RelA are required for protein degradation. In this Aim we will define the PTMs required to target RelA for degradation and will assess the biological significance of these PTMs by reintroducing degradation-resistant forms of RelA into rela-/- myeloid cells. Altogether, this proposal will tackle a critically significant problem that will propel the NF-?B field forward by providing important insights into homeostatic mechanisms that prevent persistent inflammation.