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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 背景：该项目旨在研究趋化因子CX3CL1通过ROCK在白细胞跨内皮细胞募集的背景下对细胞骨架元素的影响。许多细胞外刺激使用RhoA/ROCK信号模块来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。确定特定岩石信号事件的两种可能机制是通过形成多蛋白复合体和将信号限制在特定的亚细胞位置。 目的：第一个目标是清楚地确定指定ROCK信号的机制，例如包含ROCK的多蛋白复合体的组成。在此之前，我们使用了基于蛋白质组学的筛选来确定38个潜在的结合伙伴。今年，我们跟进了这些初步结果，以验证和描述这些相互作用，以便能够为调节岩石活动建立更具体的假说。 结果：对蛋白质组学结果的仔细分析表明，许多潜在的岩石结合伙伴出人意料地与膜动力学有关。我们已经通过两种方式跟进了这一点。首先，我们已经能够用免疫共沉淀实验证实岩石与动力蛋白相互作用，动力蛋白是一种调节膜运输的重要蛋白质，也是一种新的岩石相互作用。其次，我们进行了亚细胞分离研究，表明岩石驻留在多个膜室中，并确定内体是岩石的新位置。最后，我们已经开始了研究，以了解岩石中的哪些序列控制其定位。我们发现，C-末端的PH结构域包含靶向内膜室的蛋白质的信息。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：我们的蛋白质组学研究旨在确定含有ROCK的多蛋白复合体的成分，作为确定信号特异性的机制。有趣的是，我们在与ROCK的复合体中发现了膜调节蛋白，如Dynamin。这导致我们遵循这样的假设，即ROCK的亚细胞定位是确定上下文特定信号的重要机制。因此，虽然我们一开始认为有两种潜在的机制来调控岩石，发出多蛋白复合体的形成和亚细胞定位的信号，但我们的数据现在表明，结合伙伴可能对调控其亚细胞定位很重要，将这两种调控机制结合在一起。