Identification of Novel Nuclear RGS10 Binding Partners

G蛋白信号转导调节因子10(RGS10)对小胶质细胞炎症信号通路的调节作用 与多种炎症性疾病有关。然而，RGS10控制的机制 小胶质细胞的生理和发病机制尚不清楚。已知的RGS10的生化功能是 调节G蛋白偶联受体下游的G蛋白信号，但这一机制不能解释 RGS10‘S对小胶质细胞的影响。RGS10富含在小胶质细胞的胞核中，它调节 促炎细胞因子。初步数据表明，RGS10通过一种 涉及间接染色质相互作用的新的、未知的核机制。这代表了一种意想不到的 RGS领域功能的新范式。然而，RGS10-DNA相互作用的特异性和RGS10的同源性 介导与DNA相互作用的核蛋白尚不清楚。我们假设RGS10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 促炎症细胞因子基因启动子区域的蛋白质。这项研究的目的就是验证这一点 假说，并通过充分定义RGS10的生理作用产生新的可检验的假说 RGS10蛋白和DNA在小胶质细胞胞核内的相互作用。具体地说，核RGS10与 蛋白质和DNA将使用无偏见的蛋白质组学和下一代测序方法进行分析 小胶质细胞在以下方面的目标：目的1.定义与核RGS10相关的特定DNA序列 静息并激活小胶质细胞。染色质免疫沉淀和下一代测序将 用来定义与核RGS10相关的特定靶序列，并确定是否 在小胶质细胞激活后，靶点的数量发生了变化。不偏不倚的方法所涉及的序列将是 经直接芯片聚合酶链式反应证实。目的2.确定RGS10与静息状态下核蛋白的相互作用 激活小胶质细胞，并确定这些相互作用是否介导了RGS10与DNA的结合。 RGS10免疫共沉淀将在静息和活化的核蛋白提取液中进行 小胶质细胞，用LC/MS对浓缩的蛋白质进行蛋白质组学分析将鉴定核RGS10 互动组。蛋白质组学研究中确定的蛋白质序列将通过一系列直接的 生化分析。最后，特定的RGS10蛋白结合伙伴在介导RGS10- 在AIM 1中定义的DNA相互作用将通过在击倒 特定的蛋白质结合伙伴。拟议项目的结果将是定义第一个核项目 RGS蛋白的互作组学研究及首次鉴定RGS蛋白的特异部位--染色质 互动。

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.