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: 8636332
• 负责人: KERRI A MOWEN
• 金额: $ 28.43万
• 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/8636332
• 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; 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; inhibitor/antagonist; innovation; interest; macrophage; methyl group; mutant; neutrophil; novel; public health relevance; research study

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. !

巨噬细胞是通过产生炎症的先天免疫反应的核心组成部分 介质，例如细胞因子和趋化因子，以及吞噬作用。炎症细胞因子的产生 巨噬细胞与炎症，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TLR4诱导的负调节剂 巨噬细胞中的生物合成。 PRMTS催化从S-添加甲基 在精氨酸残留物上，腺苷硫氨氨酸向鸟养界的氮原子。 Carm1经典描述 作为转录共激活因子。我们发现CARM1中LPS诱导的TNF蛋白水平升高 不足的巨噬细胞，而TNF？mRNA水平等效，表明CARM1调节了TNF¿ 转录后生产。另外，在巨噬细胞中删除Carm1的小鼠， 中性粒细胞谱系显示出对LPS诱导的败血性休克模型的敏感性增强，该模型与 Tnf¿的血清水平升高。 CARM1通过转录后对TNF的负调节 机理代表了精氨酸甲基转移酶在调节先天免疫中的新机制。 我们研究的目标是确定CARM1调节TNF研讨会的机制。 我们假设CARM1对TNF？的转录后处理进行负调节。我们的建议 有两个具体的目标。 特定目的1。定义Carm1与TNF€的交集。 我们将检查Carm1对TNF¿MRNA剪接的影响，TNF¿MRNA的核输出，TNF¿ 消息稳定性，tnf¿mRNA翻译和TNF？蛋白处理。在此目标中，我们将确定 与CARM1相交的转录后调节阶段。这个目标的结果将允许 我们将重点放在AIM 2中的CARM1底物上，最有可能调节TNF的生产。我们的TSRI同事 RNA监管专家杰米·威廉姆森（Jamie Williamson）将为此目标提供指导（请参阅附件）。 具体目标2。在巨噬细胞中构建CARM1介导的蛋白质组学景观。 为了更好地了解Carm1在调节TNF表达上的抑制作用，我们将 使用质量介绍Carm1 WT和Carm1 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（Ben Cravatt）博士的实验室（请参阅附件）。这个目标对于确定机制至关重要 CARM1调节TNF处理，AIM 1的后续研究。 呢