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

项目摘要 激活的巨噬细胞在包括感染性休克在内的许多炎症性疾病中发挥重要作用。 和动脉粥样硬化。然而，限制巨噬细胞炎症的分子机制并不是 完全理解。MCP诱导蛋白1(MCPIP1)是新近发现的一种CCCH-锌指 含有由单核细胞趋化蛋白1(MCP-1)显著诱导的蛋白质，因此 指定为MCPIP1。在我们以前的工作中，我们已经确定MCPIP1是一种新的负性调节因子 巨噬细胞炎症激活。在我们最近的研究中，我们进一步发现MCPIP1作为一种 一种脱泛素酶，可能通过去除泛素部分来负面调节核因子-B信号 关键蛋白，如肿瘤坏死因子受体适配器因子(TRAF)、RIP和I？B？一如既往，MCPIP1- 基因缺陷的小鼠会自发地患上炎症综合症并过早死亡。巨噬细胞来自 MCPIP1-/-小鼠表现出炎症基因表达的高度上调，以及显著的 增加了核因子-B的激活，以及增加了TRAF2和TRAF6的多泛素化。此外，在 体外实验直接证明了纯化的MCPIP1具有去泛素化活性。基于这些耐人寻味 结果，我们推测MCPIP1主要抑制核因子-kB信号和巨噬细胞的激活 通过TRAF家族的去泛素化。这项提议的总体目标是测试中央 使用生化和遗传学相结合的方法进行假说。特别是，我们将建立MCPIP1- 介导的TRAF去泛素化也是调节核因子-βB信号的重要机制 作为巨噬细胞在体外和体内的激活。我们将通过以下方式实现这一目标： 方法如下：1)体外测定MCPIP1脱泛素酶的直接分子靶点； 利用MCPIP1原代细胞和组织确定MCPIP1脱泛素酶的体内靶点 3)通过系列突变分析定位MCPIP1脱泛素酶的活性区域； 4)定义去泛素酶结构域和核糖核酸酶结构域之间的功能关系；5)确定 MCPIP1脱泛素酶活性在抑制核因子B信号转导中的相对作用 6)测定MCPIP1脱泛素酶在人巨噬细胞中的作用。 完成拟议的研究不仅有助于了解巨噬细胞的分子基础 激活，但也牵涉到抗炎症性疾病新药疗法的发展 比如动脉粥样硬化。

Project Summary 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¿B¿. 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-¿B 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.