Control of NF-kappaB and Inflammation by COMMD proteins

描述（由申请人提供）：NF-？B是一种保守的转录因子，在宿主防御感染中起重要作用。虽然取得了相当大的进展，我们的理解如何NF-？当B被激活时，关于其活性如何被终止的了解较少。然而，在疾病机制方面，它很可能是在负责终止NF-？B在持续性炎症中起核心作用。这项资助支持的研究表明，DNA结合NF-？B亚基是转录终止的关键机制。我们确定了一个泛素连接酶负责这些影响，其中包含作为一个重要的辅因子的蛋白质称为COMMD 1。这一途径主要限制表达的促炎基因和翻译后修饰（PTM）的NF-？B亚基RelA是蛋白质降解所必需的。关键的悬而未决的问题包括COMMD 1是否可以控制体内炎症，该途径如何特异性调节促炎基因，以及触发RelA泛素化的精确PTM是什么。我们的目标是研究COMMD 1在体内炎症中的作用，并揭示控制其在先天免疫系统中活性的关键机制。基于初步数据，我们假设COMMD 1是一种抗炎因子，与诱导型PTM如类小泛素化和磷酸化合作，以调节促炎基因。为了实现我们的目标，我们提出了以下具体目标：（1）COMMD 1在体内炎症反应中的作用：我们发现COMMD 1的表达在炎症性疾病中受到抑制，这表明这种现象可能导致持续和/或更严重的炎症。为了测试这一概念，我们已经产生了骨髓特异性Commd 1-/-小鼠，其显示出对LPS施用的易感性增加。在这里，我们将利用这种独特的小鼠模型，以评估在败血症的炎症控制的终止机制的作用。(2)COMMD 1基因特异性染色质募集的基础：COMMD 1基因特异性募集通过一种未知的机制发生。初步研究表明，COMMD 1与SUMO的结合是染色质结合的原因。在这里，我们将研究SUMO结合和染色质募集促炎基因启动子之间的联系。将通过将SUMO结合缺陷突变体引入Commd 1-/-骨髓细胞来评估这些事件在免疫调节中的相关性。(2)通过NF-？B/RelA：除了COMMD 1募集到染色质外，RelA的PTM也是蛋白质降解所必需的。在这个目标中，我们将定义靶向RelA降解所需的PTM，并将通过将RelA的抗降解形式重新引入Rela-/-骨髓细胞来评估这些PTM的生物学意义。总之，这项建议将解决一个至关重要的问题，将推动NF-？B领域的发展提供了重要的见解，稳态机制，防止持续性炎症。 公共卫生相关性：持续性炎症是人类疾病的核心机制，导致组织破坏和癌症风险增加。很可能终止炎症反应的机制的改变在持续性炎症中起重要作用。然而，这方面的炎症研究较少。该提议特别关注控制转录因子NF-B的终止途径，NF-B是炎症反应中的关键参与者。

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. PUBLIC HEALTH RELEVANCE: Persistent inflammation is a central mechanism in human disease, leading to tissue destruction and an increased risk for cancer. It is likely that alterations in the mechanisms that terminate inflammatory responses play a significant role in persistent inflammation. Nevertheless, this aspect of inflammation is less studied. This proposal is specifically focused on the termination pathways that control the transcription factor NF- B, a critical player in the inflammatory response.