The role of MCPIP1 in regulating NF-kB signaling and macrophage activation

描述（由申请人提供）：活化的巨噬细胞在许多炎性疾病中起重要作用，包括败血性休克和动脉粥样硬化。然而，限制巨噬细胞炎症的分子机制尚未完全了解。MCP诱导蛋白1（MCP-induced protein 1，MCPIP 1）是近年来发现的一种含有CCCH锌指的蛋白质，可被单核细胞趋化蛋白1（MCP-1）诱导表达，因此命名为MCPIP 1。在我们以前的工作中，我们已经确定了MCPIP 1作为一种新的负调节巨噬细胞炎症激活。在我们最近的研究中，我们进一步发现MCPIP 1作为一种去泛素化酶，可以通过从关键蛋白质如TNF受体衔接因子（TRAFs）、RIP和I：B1中去除泛素部分来负调节NF-：B信号传导。因此，MCPIP 1缺陷小鼠自发地发展为炎症综合征并过早死亡。来自MCPIP 1-/-小鼠的巨噬细胞显示出炎症基因表达的高度上调，以及大大增加的NF-kB活化，以及TRAF 2和TRAF 6的多聚泛素化增加。此外，体外试验直接证明了纯化的MCPIP 1的去泛素化活性。基于这些有趣的发现，我们推测MCPIP 1主要通过TRAF家族的去泛素化来抑制NF-κ B信号传导和微噬细胞活化。本提案的总体目标是通过使用生物化学和遗传学相结合的方法来检验中心假设。特别地，我们将建立MCPIP 1介导的TRAFs去泛素化作为NF-：B信号转导调节以及巨噬细胞活化的重要机制，在体外和体内。我们将通过以下方法达到这一目的：1）体外确定MCPIP 1去泛素化酶的直接分子靶点; 2）利用MCPIP 1基因敲除小鼠的原代细胞和组织确定MCPIP 1去泛素化酶的体内靶点; 3）通过序列突变分析定位MCPIP 1去泛素化酶的活性结构域; 4）确定去泛素化酶结构域和RNase结构域之间的功能关系：5）确定MCPIP 1的去泛素化酶活性对NF-：B信号传导抑制以及巨噬细胞活化的相对贡献; 6）确定MCPIP 1去泛素化酶在人巨噬细胞中的作用。完成这些研究不仅有助于理解巨噬细胞活化的分子基础，而且还有助于开发新的药物治疗炎症性疾病，如动脉粥样硬化。 公共卫生相关性：本研究的目的是探讨新发现的锌指蛋白MCPIP 1在调节NF-κ B信号转导和巨噬细胞活化中的作用及其分子机制。这项研究计划的完成不仅有助于我们了解MCPIP 1在巨噬细胞活化中的功能和分子机制，而且还为包括动脉粥样硬化在内的炎症性疾病的潜在新疗法提供了关键信息。

DESCRIPTION (provided by applicant): Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. However, the molecular mechanisms limiting macrophage inflammation are not completely understood. MCP-induced protein 1 (MCPIP1) is a recently identified CCCH-zinc finger containing protein, which was significantly induced by monocyte chemotactic protein 1 (MCP-1) and thus designated as MCPIP1. In our previous works, we have identified MCPIP1 as a novel negative regulator of macrophage inflammatory activation. In our recent studies, we have further found that MCPIP1 acts as a deubiquitinating enzyme that may negatively regulates NF-:B signaling by removing ubiquitin moieties from critical proteins, such as TNF receptor adaptor factors (TRAFs), RIP and I:B1. Consistently, MCPIP1- deficient mice spontaneously developed inflammatory syndrome and died prematurely. Macrophages from MCPIP1-/- mice showed high up-regulation of inflammatory gene expression, together with a greatly increased NF-kB activation, as well as increased polyubiquitination of TRAF2 and TRAF6. Furthermore, in vitro assay directly demonstrated the deubiquitinating activity of purified MCPIP1. Based on these intriguing findings, we hypothesize that MCPIP1 represses NF-kB signaling and microphage activation mainly through deubiquitination of TRAF family. The overall objective of this proposal is to test the central hypothesis by using combined biochemical and genetic approaches. Specially, we will establish MCPIP1- mediated deubiquitination of TRAFs as an essential mechanism in the regulation of NF-:B signaling as well as macrophage activation both in vitro and in vivo. We will attain the objective of this aim by using following approaches: 1) determine the direct molecular targets of MCPIP1 deubiquitinase in vitro; 2) determine the in vivo targets of MCPIP1 deubiquitinase using the primary cells and tissues from MCPIP1 knockout mouse; 3) map the active domain of MCPIP1 deubiquitinase through serial mutagenesis analysis; 4) define the functional relationship between deubiquitinase domain and RNase domain; 5) determine the relative contribution of the deubiquitinase activity of MCPIP1 to the suppression of NF-:B signaling as well as macrophage activation; 6) determine the role of MCPIP1 deubiquitinase in human macrophages. Completion of the proposed studies will not only help to understand the molecular basis of macrophage activation, but also implicate in the development of novel drug therapies against inflammatory diseases such as atherosclerosis. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to explore the role and molecular mechanisms of a newly identified zinc finger protein MCPIP1 in regulating NF-KB signaling and macrophage activation. Completion of this research program will not only help us understand the function and molecular mechanisms of MCPIP1 in macrophage activation, but also provide critical information for potential new therapies for inflammatory diseases including atherosclerosis.