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

项目摘要 活化的巨噬细胞在包括感染性休克在内的许多炎症性疾病中起重要作用 和动脉粥样硬化。然而，限制巨噬细胞炎症的分子机制并不 完全理解MCP诱导蛋白1（MCP-induced protein 1，MCPIP 1）是最近发现的CCCH-锌指蛋白 含蛋白，这是显着诱导单核细胞趋化蛋白1（MCP-1），因此 命名为MCPIP1。在我们以前的工作中，我们已经确定MCPIP1作为一种新的负调节因子， 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 一种去泛素化酶，可通过去除NF-κ B的泛素部分来负调节NF-κ B B信号传导。 关键蛋白质，如TNF受体衔接因子（TRAF）、RIP和I B。一致性，MCPIP 1- 缺陷小鼠自发地发展出炎性综合征并过早死亡。巨噬细胞 MCPIP 1-/-小鼠表现出炎症基因表达的高度上调，同时也表现出极大的免疫抑制作用。 增加NF-B活化，以及增加TRAF 2和TRAF 6的多聚泛素化。更以 体外实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 研究结果，我们假设MCPIP1主要抑制NF-κ B信号传导和微噬细胞活化， 通过TRAF家族的去泛素化。本提案的总体目标是测试中央 生物化学和遗传学方法相结合的假设。特别地，我们将建立MCPIP 1- TRAFs介导的去泛素化也是NF-B信号调节的重要机制 在体外和体内都表现为巨噬细胞活化。我们将通过以下方式实现这一目标： 以下方法：1）确定体外MCPIP 1去泛素化酶的直接分子靶点; 2） 使用来自MCPIP 1的原代细胞和组织确定MCPIP 1去泛素化酶的体内靶点 3）通过连续突变分析定位MCPIP 1去泛素化酶的活性结构域; 4)确定去泛素化酶结构域与RNase结构域之间的功能关系; 5）确定去泛素化酶结构域与RNase结构域之间的功能关系。 MCPIP 1的去泛素化酶活性对NF-B信号转导抑制的相对贡献 作为巨噬细胞活化; 6）确定MCPIP 1去泛素化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，而且还涉及针对炎症性疾病的新型药物疗法的开发 例如动脉粥样硬化。

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.