Posttranscriptional regulation of TNFa by Carm1 in Macrophages

巨噬细胞中 Carm1 对 TNFa 的转录后调节

• 批准号: 8786493
• 负责人: KERRI A MOWEN
• 金额: $ 23.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-20 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8786493
• 关键词: Anabolism; Applications Grants; Arginine; Autoimmune Process; Biogenesis; Biological Assay; Bone Marrow; Cells; Collaborations; Data; Drug Industry; Enzymes; Equipment and supply inventories; Event; Family member; Gene Expression; Gene Targeting; Genetic Transcription; Genetic Translation; Goals; Health; Histones; Immune; Immune response; Inflammation; Inflammation Mediators; Inflammatory; Inhibitory Concentration 50; Laboratories; Letters; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Messenger RNA; Metabolic Diseases; Methylation; Modeling; Modification; Molecular; Mus; Natural Immunity; Nitrogen; Nuclear Export; Nuclear Hormone Receptors; Phagocytosis; Physiological; Predisposition; Process; Production; Protein-Arginine N-Methyltransferase; Proteins; Proteome; Proteomics; Publishing; RNA; RNA Splicing; Regulation; Reporting; Research Proposals; Role; S-Adenosylmethionine; Sepsis; Septic Shock; Serum; Staging; TLR4 gene; TNF gene; Therapeutic; Transcription Coactivator; Vaccination; arginine methyltransferase; cell type; chemokine; coactivator-associated arginine methyltransferase 1; cytokine; follow-up; high risk; histone methylation; inhibitor/antagonist; innovation; interest; macrophage; methyl group; mutant; neutrophil; novel; research study

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.

描述（由申请人提供）：巨噬细胞是通过产生炎性介质（如细胞因子和趋化因子）和通过吞噬作用产生的先天免疫应答的中心组分。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。我们发现蛋白质精氨酸甲基转移酶（PRMT）家族成员Carm 1是巨噬细胞中TLR 4诱导的TNF？生物合成的负调节剂。PRMT催化甲基从S-腺苷甲硫氨酸加成到精氨酸残基上的胍基氮原子上。Carm 1被经典地描述为转录辅激活因子。我们发现，在Carm 1缺陷型巨噬细胞中，LPS诱导的TNF <$蛋白水平增加，而TNF <$mRNA水平相当，这表明Carm 1在转录后调节TNF <$的产生。此外，在巨噬细胞和中性粒细胞谱系中携带Carm 1缺失的小鼠显示出对LPS诱导的脓毒性休克模型的易感性增强，这与血清TNF水平升高相关。 TNF的负调节Carm 1通过转录后机制的作用代表了精氨酸甲基转移酶调节先天免疫的一种新机制。我们研究的目标是确定Carm 1调节TNF生物合成的机制。我们假设Carm 1负调控TNF？的转录后加工。我们的建议有两个具体目标。具体目标1.确定Carm 1与TNF产生的交叉点。我们将研究Carm 1对TNF <$mRNA剪接、TNF <$mRNA核输出、TNF <$mRNA信息稳定性、TNF <$mRNA翻译和TNF <$蛋白加工的影响。在这个目标中，我们将查明与Carm 1交叉的TNF？转录后调节的阶段。来自该目标的结果将使我们能够专注于来自目标2的Carm 1底物，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Jamie威廉姆森将为这个目标提供指导（见所附信件）。具体目标2.在巨噬细胞中构建CARM 1介导的蛋白质组景观。为了更好地理解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。该目标将与TSRI生理蛋白质组学中心和Ben Cravatt博士的实验室合作进行（见随附信函）。这一目标将是必不可少的，以确定的机制，其中Carm 1调节肿瘤坏死因子处理，后续研究的目标1。