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

描述(申请人提供)：激活的巨噬细胞在许多炎症性疾病中发挥重要作用，包括感染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不完全清楚。单核细胞趋化蛋白1(MCP-1)是新近发现的一种含CCCH-锌指的蛋白，由单核细胞趋化蛋白1(MCP-1)诱导，命名为MCPIP1。在我们以前的工作中，我们已经确定MCPIP1是一种新的巨噬细胞炎症激活的负调控因子。在我们最近的研究中，我们进一步发现，MCPIP1作为一种去泛素酶，可能通过去除关键蛋白质中的泛素部分，如肿瘤坏死因子受体适配器因子(TRAF)、RIP和I：B1来负向调节NF-：B信号转导。一直以来，MCPIP1基因缺陷的小鼠会自发发展成炎症综合征并过早死亡。MCPIP1-/-小鼠的巨噬细胞表现出炎症基因表达的高度上调，同时显著增加了NF-kB的激活，以及TRAF2和TRAF6的多泛素化增加。此外，体外实验直接证明了纯化的MCPIP1具有去泛素化活性。基于这些有趣的发现，我们推测MCPIP1主要通过TRAF家族的去泛素化来抑制NF-kB信号和巨噬细胞的激活。这项提议的总体目标是通过结合使用生化和遗传方法来检验中心假说。特别是，我们将建立MCPIP1介导的TRAF去泛素化作为在体内外调节NF-：B信号和巨噬细胞激活的一个重要机制。我们将通过以下方法达到这一目标：1)确定MCPIP1去泛素酶的体外直接分子靶点；2)利用MCPIP1基因敲除小鼠的原代细胞和组织来确定MCPIP1去泛素酶的体内靶点；3)通过连续突变分析定位MCPIP1去泛素酶的活性区域；4)确定去泛素酶结构域和RNase域之间的功能关系；5)确定MCPIP1去泛素酶活性在抑制NF-：B信号转导和巨噬细胞激活中的相对贡献；6)确定MCPIP1去泛素酶在人巨噬细胞中的作用。这些研究的完成不仅将有助于了解巨噬细胞活化的分子基础，而且将有助于开发针对动脉粥样硬化等炎症性疾病的新型药物治疗方法。 公共卫生相关性：这项建议的目标是探索一种新发现的锌指蛋白MCPIP1在调节NF-KB信号和巨噬细胞激活方面的作用和分子机制。这项研究的完成不仅有助于我们了解MCPIP1在巨噬细胞激活中的功能和分子机制，而且还将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.