Posttranscriptional regulation of TNFa by Carm1 in Macrophages

描述（由申请人提供）：巨噬细胞是先天免疫反应的核心组成部分，通过产生炎症介质，如细胞因子和趋化因子，以及吞噬作用。巨噬细胞产生炎性细胞因子与炎性、自身免疫性甚至代谢性疾病有关。我们发现蛋白精氨酸甲基转移酶（PRMT）家族成员Carm1是巨噬细胞中TLR4诱导的TNF¿生物合成的负调节因子。PRMTs催化从S-腺苷蛋氨酸到精氨酸残基上的胍基氮原子的甲基加成。Carm1通常被描述为一种转录辅激活因子。我们发现，在Carm1缺失的巨噬细胞中，lps诱导的TNF -蛋白水平升高，而TNF - mRNA水平相同，这表明Carm1在转录后调节TNF -的产生。此外，巨噬细胞和中性粒细胞谱系中缺失Carm1的小鼠对lps诱导的脓毒性休克模型的易感性增强，这与血清TNF¿水平升高有关。Carm1通过转录后机制负向调节TNF¿代表了精氨酸甲基转移酶调节先天免疫的新机制。我们研究的目的是确定car1调节TNF -生物合成的机制。我们假设Carm1负调控TNF¿的转录后加工。我们的建议有两个具体目标。具体目标1。定义Carm1与TNF¿产生的交集。我们将研究Carm1对TNF - mRNA剪接、TNF - mRNA的核输出、TNF - mRNA信息稳定性、TNF - mRNA翻译和TNF -蛋白加工的影响。在本研究中，我们将明确指出与Carm1交叉的TNF转录后调控阶段。该Aim的结果将使我们能够专注于来自Aim 2的Carm1底物，这些底物最有可能调节TNF的产生。我们的TSRI同事，RNA调控专家Jamie Williamson将为这次Aim提供指导（见附信）。具体目标2。构建巨噬细胞中carm1介导的蛋白质组学格局。为了更好地了解Carm1在分子水平上调节TNF -表达的抑制作用，我们将使用质谱分析Carm1 WT和Carm1 KO巨噬细胞的精氨酸甲基化蛋白质组。这一目标将与TSRI生理蛋白质组学中心和Ben Cravatt博士的实验室合作进行（见附信）。该Aim对于确定Carm1调节TNF -加工的机制至关重要，Aim 1的后续研究。

DESCRIPTION (provided by applicant): Macrophages are a central component of the innate immune response by the production of inflammatory mediators, such as cytokines and chemokines, and by phagocytosis. The production of inflammatory cytokines by macrophages has been linked to inflammatory, autoimmune, and even metabolic diseases. We find that the protein arginine methyltransferase (PRMT) family member Carm1 is a negative regulator of TLR4 induced TNF¿ biosynthesis in macrophages. PRMTs catalyze the addition of a methyl group from S- adenosylmethionine to guanidino nitrogen atoms on arginine residues. Carm1 has classically been described as a transcriptional coactivator. We find that LPS-induced TNF¿ protein levels are increased in Carm1 deficient macrophages, whereas TNF¿ mRNA levels are equivalent, suggesting that Carm1 regulates TNF¿ production post-transcriptionally. In addition, mice bearing a deletion of Carm1 within the macrophage and neutrophil lineages show enhanced susceptibility to the LPS-induced septic shock model, which correlates with increased serum levels of TNF¿. Negative regulation of TNF¿ by Carm1 through posttranscriptional mechanisms represents a novel mechanism for arginine methyltransferases in regulating innate immunity. The goals of our studies are to identify the mechanism behind Carm1's regulation of TNF¿ biosynthesis. We hypothesize that Carm1 negatively regulates the post-transcriptional processing of TNF¿. Our proposal has two specific aims. SPECIFIC AIM 1. To define the intersection of Carm1 with TNF¿ production. We will examine the impact of Carm1 on TNF¿ mRNA splicing, nuclear export of TNF¿ mRNA, TNF¿ message stability, TNF¿ mRNA translation, and TNF¿ protein processing. In this Aim, we will pinpoint the stage(s) of TNF¿ posttranscriptional regulation that intersects with Carm1. The results from this Aim will allow us to focus in on Carm1 substrates from Aim 2 that most likely regulate TNF¿ production. Our TSRI colleague and RNA regulation expert Jamie Williamson will provide guidance for this Aim (see attached letter). SPECIFIC AIM 2. To build the CARM1-mediated proteomic landscape in macrophages. To better understand the inhibitory role for Carm1 in regulating TNF¿ expression on a molecular level, we will profile the arginine methylation proteome of Carm1 WT and Carm1 KO macrophages using mass spectrometry. This Aim will be performed in collaboration with the TSRI Center for Physiological Proteomics and Dr. Ben Cravatt's laboratory (see attached letter). This Aim will be essential to identify the mechanism by which Carm1 regulates TNF¿ processing, follow-up studies for Aim 1.