MECHANISMS BEHIND CX2CL1-DRIVEN MONOCYTE RECRUITMENT DURING PERIODONTITIS

牙周炎期间 CX2CL1 驱动的单核细胞募集背后的机制

• 批准号: 7720562
• 负责人: Rebecca Worthylake
• 金额: $ 22.58万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720562
• 关键词: Binding; Biological; C-terminal; CX3CL1 gene; Cell Fractionation; Co-Immunoprecipitations; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytoskeletal Modeling; Data; Dynamin; Elements; Endosomes; Endothelium; Event; Funding; Grant; Indium; Institution; Lead; Leukocytes; Literature; Location; Membrane; Membrane Protein Traffic; Numbers; PH Domain; Periodontitis; Proteins; Proteomics; Regulation; Research; Research Personnel; Resources; Signal Transduction; Source; Specific qualifier value; Specificity; Stimulus; United States National Institutes of Health; base; chemokine; design; extracellular; follow-up; genetic regulatory protein; monocyte; novel; research study; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background: This project is designed to investigate signaling from the chemokine, CX3CL1, through ROCK, to its effects on cytoskeletal elements in the context of leukocyte recruitment across the endothelium. Many extracellular stimuli use the RhoA/ROCK signaling module to phosphorylate a subset of a large number of potential substrates, and lead to selective reorganization of cytoskeletal elements, to elicit the appropriate biological response. Two potential mechanisms for determining the specific ROCK signaling events is by the formation of multi-protein complexes, and restricting signaling to particular subcellular locations. Objective: The first objective is to clearly identify mechanisms for specifying ROCK signaling, such as the composition of multi-protein complexes containing ROCK. Previously, we used a proteomics based screen to identify 38 potential binding partners. This year, we have followed up on these initial results to verify and characterize these interactions to be able to build more specific hypotheses for the regulation of ROCK activity. Results: Careful analysis of the proteomics results showed that many of the potential ROCK binding partners are unexpectedly associated with membrane dynamics. We have followed this up in two ways. First, we have been able to confirm with co-immunoprecipitation experiments that ROCK interacts with Dynamin, an important protein for regulating membrane trafficking, and a novel interaction for ROCK. Second, we have performed subcellular fractionation studies that show ROCK resides in multiple membrane compartment, and identified endosomes as a novel location for ROCK. Finally, we have initiated studies to understand what sequences within ROCK regulate its localization. We found that the C-terminal PH domain contains information to target proteins to internal membrane compartments. This is different from what was predicted in the literature, but consistent with both our proteomics and subcellular fractionation data. Discussion: Our proteomics studies were designed to identify components of multi-protein complexes containing ROCK, as a mechanism for determining signaling specificity. Interestingly, we found membrane regulatory proteins, such as dynamin, in complex with ROCK. This led us to follow the hypothesis that ROCK subcellular localization is an important mechanism for determining context specific signaling. Thus, while we began with the idea that there were two potential mechanisms for regulating ROCK signaling  the formation of mulit-protein complexes and subcellular localization, our data now indicate that the binding partners are likely important for regulating its subcellular localization, combining the two regulatory mechanisms into one.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 背景资料：该项目旨在研究趋化因子CX3CL1通过ROCK对细胞骨架元素的影响，以及白细胞在内皮细胞中的募集。 许多细胞外刺激使用RhoA/ROCK信号传导模块磷酸化大量潜在底物的子集，并导致细胞骨架元件的选择性重组，以引发适当的生物反应。 确定特异性ROCK信号传导事件的两种潜在机制是通过形成多蛋白复合物，并将信号传导限制在特定的亚细胞位置。 目的：第一个目标是明确识别指定ROCK信号传导的机制，例如包含ROCK的多蛋白复合物的组成。 在此之前，我们使用基于蛋白质组学的筛选来鉴定38种潜在的结合伴侣。 今年，我们对这些初步结果进行了跟踪，以验证和表征这些相互作用，从而能够为ROCK活性的调节建立更具体的假设。 结果如下：对蛋白质组学结果的仔细分析表明，许多潜在的ROCK结合伴侣出乎意料地与膜动力学相关。 我们从两个方面跟进此事。 首先，我们已经能够证实与免疫共沉淀实验，ROCK相互作用与发动蛋白，一个重要的蛋白质调节膜运输，和一种新的相互作用ROCK。 其次，我们进行了亚细胞分级分离研究，表明ROCK存在于多个膜室中，并确定了内体作为ROCK的新位置。 最后，我们已经启动了研究，以了解ROCK内的哪些序列调节其定位。 我们发现C-末端PH结构域包含将蛋白质靶向到内部膜隔室的信息。 这与文献中的预测不同，但与我们的蛋白质组学和亚细胞分级数据一致。 讨论内容：我们的蛋白质组学研究旨在鉴定含有ROCK的多蛋白质复合物的组分，作为确定信号特异性的机制。 有趣的是，我们发现膜调节蛋白，如发动蛋白，与ROCK复合。 这使我们遵循这样的假设，即ROCK亚细胞定位是决定环境特异性信号传导的重要机制。 因此，虽然我们开始的想法，有两个潜在的机制，调节ROCK信号  多蛋白复合物的形成和亚细胞定位，我们的数据现在表明，结合伙伴可能是重要的调节其亚细胞定位，结合成一个两种调节机制。