Posttranscriptional regulation of TNFa by Carm1 in Macrophages

描述(申请人提供)：巨噬细胞是先天免疫反应的中心成分，通过产生炎症介质，如细胞因子和趋化因子，以及吞噬作用。巨噬细胞产生的炎性细胞因子与炎症、自身免疫、甚至代谢性疾病有关。我们发现精氨酸甲基转移酶(PRMT)家族成员Carm1是TLR4诱导巨噬细胞合成肿瘤坏死因子的负性调节因子。PRMTs催化S-腺苷甲硫氨酸上的甲基加成到精氨酸残基上的胍基氮原子上。Carm1被经典地描述为转录辅助激活因子。我们发现，在Carm1缺乏的巨噬细胞中，内毒素诱导的肿瘤坏死因子？蛋白水平增加，而肿瘤坏死因子？的mRNA水平相同，这表明Carm1在转录后调节肿瘤坏死因子的产生。此外，巨噬细胞和中性粒细胞系中Carm1基因缺失的小鼠表现出对内毒素诱导的感染性休克模型的敏感性增加，这与血清中肿瘤坏死因子水平的增加有关。Carm1通过转录后机制负调控肿瘤坏死因子，这是精氨酸甲基转移酶调节天然免疫的一种新机制。我们研究的目的是确定CAM1‘S调节肿瘤坏死因子生物合成的机制。我们假设Carm1负向调节肿瘤坏死因子的转录后处理。我们的建议有两个具体目标。具体目的1.明确Carm1与肿瘤坏死因子产生的交叉点。我们将研究Carm1对肿瘤坏死因子mRNA的剪接、核输出、信息稳定性、翻译和蛋白质加工的影响。在这个目标中，我们将准确地定位与Carm1交叉的肿瘤坏死因子转录后调控的阶段(S)。这一目标的结果将使我们能够专注于目标2中最有可能调节肿瘤坏死因子产生的Carm1底物。我们的TSRI同事兼RNA调控专家杰米·威廉姆森将为这一目标提供指导(见所附信件)。特异性目的2.构建CARM1介导的巨噬细胞蛋白质组学图谱。为了在分子水平上更好地了解Carm1在调节肿瘤坏死因子表达中的抑制作用，我们将利用质谱仪对Carm1 WT和Carm1 KO巨噬细胞的精氨酸甲基化蛋白质组进行分析。这一目标将与TSRI生理蛋白质组学中心和Ben Cravatt博士的实验室合作实现(见附信)。这一目标对于确定Carm1调节肿瘤坏死因子加工的机制至关重要，目标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.