Posttranscriptional regulation of TNFa by Carm1 in Macrophages

巨噬细胞是先天免疫应答的中心成分，通过产生炎性 介质，如细胞因子和趋化因子，以及通过吞噬作用。炎性细胞因子的产生 被巨噬细胞感染与炎症，自身免疫，甚至代谢性疾病有关。我们发现 蛋白质精氨酸甲基转移酶（PRMT）家族成员Carm 1是TLR 4诱导的负调节因子， 巨噬细胞中的TNF合成PRMT催化S-甲基的加成， 腺苷甲硫氨酸与精氨酸残基上的胍基氮原子连接。卡尔1号被经典地描述为 作为转录辅激活因子。我们发现LPS诱导的TNF？蛋白水平在Carm 1中增加， 缺陷的巨噬细胞，而TNF？mRNA水平是相等的，这表明Carm 1调节TNF？ 转录后的生产。此外，巨噬细胞内Carm 1缺失的小鼠， 中性粒细胞谱系对LPS诱导的脓毒性休克模型显示出增强的易感性，这与 血清TNF水平升高。Carm 1通过转录后途径对TNF？的负调控 机制代表了精氨酸甲基转移酶调节先天免疫的新机制。 我们研究的目标是确定Carm 1调节TNF生物合成的机制。 我们假设Carm 1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.确定Carm 1与TNF产生的交叉点。 我们将研究Carm 1对TNF <$mRNA剪接、TNF <$mRNA核输出、TNF <$mRNA核转录和TNF <$mRNA核转录的影响。 信息稳定性、TNF？mRNA翻译和TNF？蛋白质加工。在本目标中，我们将指出 与Carm 1交叉的TNF？转录后调节阶段。这一目标的结果将允许 我们将重点放在来自Aim 2的Carm 1底物上，该底物最有可能调节TNF的产生。我们的TSRI同事 和RNA调控专家杰米威廉姆森将提供这一目标的指导（见所附的信）。 具体目标2.在巨噬细胞中构建CARM 1介导的蛋白质组景观。 为了更好地了解Carm 1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Carm 1 WT和Carm 1 KO巨噬细胞的精氨酸甲基化蛋白质组 光谱法该目标将与TSRI生理蛋白质组学中心合作执行 和本·克拉瓦特博士的实验室（见所附信件）。这一目标对于确定机制至关重要， Carm 1调节TNF的加工，Aim 1的后续研究。 !

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. !