Control of NF-kappaB and Inflammation by COMMD proteins

描述（由申请人提供）：NF-κB是一种保守的转录因子，在宿主防御感染中发挥重要作用。虽然我们对 NF-κB 如何激活的理解已经取得了相当大的进展，但对其活性如何终止却知之甚少。然而，就疾病机制而言，终止 NF-κB 的机制的改变很可能在持续性炎症中发挥着核心作用。这项资助支持的研究发现，DNA 结合的 NF-κB 亚基的降解是转录终止的关键机制。我们发现了一种负责这些作用的泛素连接酶，它含有一种重要的辅助因子 COMMD1 蛋白。该途径主要限制促炎基因的表达，并且蛋白质降解需要 NF-κB 亚基 RelA 的翻译后修饰 (PTM)。关键的悬而未决的问题包括 COMMD1 是否可以控制体内炎症、该通路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 COMMD1 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。根据初步数据，我们假设 COMMD1 是一种抗炎因子，可与可诱导的 PTM（如苏酰化和磷酸化）配合来调节促​​炎基因。为了实现我们的目标，我们提出以下具体目标：（1）Commd1 在体内炎症反应中的作用：我们发现 COMMD1 表达在炎症性疾病中受到抑制，表明这种现象可能导致持续和/或更严重的炎症。为了测试这个概念，我们培育了骨髓特异性 Commd1-/- 小鼠，它们对 LPS 给药的敏感性增加。在这里，我们将利用这种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 基因特异性染色质招募的基础：COMMD1 基因特异性招募是通过一种尚不清楚的机制发生的。初步研究表明 COMMD1 的 SUMO 结合负责染色质结合。在这里，我们将检查 SUMO 结合和染色质募集到促炎基因启动子之间的联系。这些事件在免疫调节中的相关性将通过将 SUMO 结合缺陷突变体引入 Commd1-/- 骨髓细胞来评估。 (2) NF-κB/RelA 的 PTM 控制终止：除了 COMMD1 募集到染色质之外，RelA 的 PTM 也是蛋白质降解所必需的。在此目标中，我们将定义以 RelA 为目标进行降解所需的 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。总而言之，该提案将解决一个至关重要的问题，通过提供对预防持续性炎症的稳态机制的重要见解，推动 NF-κB 领域的发展。

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.