Identification of Novel Nuclear RGS10 Binding Partners

新型核 RGS10 结合伴侣的鉴定

• 批准号: 9296748
• 负责人: Shelley B Hooks
• 金额: $ 22.5万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9296748
• 关键词: Amino Acid Sequence; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Binding; Binding Proteins; Biochemical; Biological Models; Brain; Cell Nucleus; Cell membrane; Cell physiology; Cells; Chromatin; Co-Immunoprecipitations; Cytokine Gene; DNA; DNA Binding; DNA Sequence; DNA-Binding Proteins; Data; Disease; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Gene Targeting; Genes; Genetic Transcription; Immune; Inflammation; Inflammatory; Knowledge; Mediating; Microglia; Multiple Sclerosis; Multiprotein Complexes; NF-kappa B; Neuraxis; Neurodegenerative Disorders; Nuclear; Nuclear Protein; Nuclear Proteins; Nuclear Translocation; Outcome; Parkinson Disease; Pathogenesis; Physiological; Play; Point Mutation; Production; Promoter Regions; Proteins; Proteomics; RGS Domain; RGS Proteins; Regulation; Reporting; Rest; Role; Series; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; TNF gene; Testing; Time; Transcriptional Activation; Transcriptional Regulation; Work; cell type; chromatin immunoprecipitation; chromatin protein; cytokine; experimental study; factor A; gene interaction; insight; knock-down; liquid chromatography mass spectrometry; macrophage; nervous system disorder; neuroinflammation; new therapeutic target; next generation sequencing; novel; novel therapeutic intervention; promoter; transcription factor

Regulator of G-protein Signaling 10 (RGS10) regulates inflammatory signaling pathways in microglia and is implicated in multiple inflammatory disease states. However, the mechanism by which RGS10 controls microglial cell physiology and pathogenesis are undefined. The known biochemical function of RGS10 is to regulate G-protein signaling downstream of G-protein coupled receptors, but this mechanism does not account for RGS10's effects in microglia. RGS10 is enriched in the nucleus of microglia, and it regulates transcription of pro-inflammatory cytokines. Preliminary data suggest that RGS10 functions to regulate transcription by a novel, unknown nuclear mechanism involving indirect chromatin interaction. This represents an unexpected new paradigm in RGS-domain function. However, the specificity of RGS10-DNA interactions and identity of nuclear proteins that mediate the interaction with DNA are unknown. We hypothesize that RGS10 regulates proinflammatory cytokine production by interactions with transcription factors or transcription factor binding proteins in promoter regions of proinflammatory cytokine genes. The objectives of this study are to test this hypothesis, and to generate new testable hypotheses about the physiologic role of RGS10 by fully defining RGS10 protein and DNA interactions in the nucleus of microglia. Specifically, nuclear RGS10 interactions with protein and DNA will be analyzed using unbiased proteomics and next generation sequencing approaches in microglia in the following aims: Aim 1. Define specific DNA sequences associated with nuclear RGS10 in resting and activated microglia. Chromatin Immunoprecipitation followed by next generation sequencing will be used to define specific target sequences associated with nuclear RGS10 and determine whether the profile of target sites is altered following microglial activation. Sequences implicated by unbiased approaches will be confirmed by direct ChIP-PCR. Aim 2. Define RGS10 interactions with nuclear proteins in resting and activated microglia, and determine whether these interactions mediate RGS10 association with DNA. RGS10 co-immunoprecipitation will be performed in nuclear protein extracts from resting and activated microglia, and proteomics analysis of the enriched proteins using LC/MS will identify the nuclear RGS10 interactome. Protein sequences identified in the proteomics study will be confirmed by a series of direct biochemical analyses. Finally, the role of specific RGS10 protein binding partners in mediating the RGS10- DNA interactions defined in Aim 1 will be determined by performing ChIP-PCR following knock-down of specific protein binding partners. The outcome of the proposed project will be to define the first nuclear interactome for an RGS protein and identify for the first time specific sites of RGS protein-chromatin interaction.

G蛋白信号调节因子10（RGS 10）调节小胶质细胞中的炎症信号通路， 与多种炎性疾病状态有关。然而，RGS 10控制的机制 小神经胶质细胞的生理学和发病机制尚未明确。RGS 10的已知生化功能是 调节G-蛋白偶联受体下游的G-蛋白信号传导，但这种机制并不能解释 RGS 10在小胶质细胞中的作用RGS 10在小胶质细胞的细胞核中富集，并且它调节 促炎细胞因子。初步数据表明，RGS 10的功能是通过调节转录。 涉及间接染色质相互作用的新的、未知的核机制。这代表了一个意想不到的 RGS域功能的新范例。然而，RGS 10-DNA相互作用的特异性和 介导与DNA相互作用的核蛋白是未知的。我们假设RGS 10调节 通过与转录因子相互作用或转录因子结合产生促炎细胞因子 促炎细胞因子基因启动子区的蛋白质。本研究的目的是验证这一点。 假设，并通过充分定义RGS 10的生理作用来产生新的可检验的假设 RGS 10蛋白和DNA在小胶质细胞核中的相互作用。具体而言，核RGS 10与 蛋白质和DNA将使用无偏蛋白质组学和下一代测序方法进行分析， 小胶质细胞在以下目标：目标1。定义与细胞核RGS 10相关的特定DNA序列， 休眠和激活的小胶质细胞。染色质免疫沉淀，然后进行下一代测序， 用于定义与核RGS 10相关的特异性靶序列，并确定该谱是否 在小胶质细胞激活后，靶位点的变化。无偏方法所涉及的序列将被 通过直接ChIP-PCR确认。目标二。定义RGS 10与静息和静息细胞核蛋白的相互作用， 激活的小胶质细胞，并确定这些相互作用是否介导RGS 10与DNA的关联。 RGS 10共免疫沉淀将在来自静息和活化细胞的核蛋白提取物中进行。 使用LC/MS对富集的蛋白质进行蛋白质组学分析将鉴定核RGS 10 相互作用体在蛋白质组学研究中鉴定的蛋白质序列将通过一系列直接测序来证实。 生化分析最后，研究了特异性RGS 10蛋白结合伴侣在介导RGS 10- 目标1中定义的DNA相互作用将通过在敲低以下基因后进行ChIP-PCR来确定： 特异性蛋白质结合伴侣。拟议项目的结果将是确定第一个核 RGS蛋白的相互作用基因组，并首次鉴定RGS蛋白-染色质的特异性位点 互动