Identifying the membrane proteins of the LBRC, a key regulator of inflammation

鉴定 LBRC 的膜蛋白（炎症的关键调节因子）

• 批准号: 8072018
• 负责人: William A Muller
• 金额: $ 19.06万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-13 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072018
• 关键词: Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Applications Grants; Area; Asthma; Atherosclerosis; Autoimmune Diseases; Biochemical; Biological Assay; Blood Vessels; CD31 Antigens; Cell Adhesion Molecules; Cell Fractionation; Cell Line; Cell membrane; Cells; Databases; Density Gradient Centrifugation; Development; Disease; Endothelial Cells; Ensure; Fluorescence Microscopy; Funding Mechanisms; Future; Gel; Goals; Immunoelectron Microscopy; Immunofluorescence Immunologic; Inflammation; Inflammatory Response; Intercellular Junctions; Kinesin; Knowledge; Lateral; Leukocytes; Life; Lymphocyte; Mass Spectrum Analysis; Membrane; Membrane Proteins; Methods; Microtubules; Molecular Motors; Names; Organelles; PECAM1 gene; Peptide Hydrolases; Peptide Mapping; Physiological; Preparation; Procedures; Process; Proteins; Proteomics; Recycling; Regulation; Research Personnel; Rest; Reticulum; Role; Side; Site; Spottings; Staging; Structure; Surface; Techniques; Testing; Time; Two-Dimensional Gel Electrophoresis; Vascular Endothelial Cell; base; cadherin 5; design; insight; junctional adhesion molecule; migration; monocyte; neutrophil; new therapeutic target; novel; protein profiling; public health relevance; scale up; two-dimensional

DESCRIPTION (provided by applicant): The lateral border recycling compartment (LBRC) is a recently-discovered membrane compartment located along the borders of vascular endothelial cells. Membrane from the LBRC recycles constitutively and rapidly between the compartment and the plasma membrane at borders between endothelial cells. The physiologic function of this constitutive recycling is not known. However, during leukocyte transmigration, recycling membrane from the LBRC is redirected-targeted to the site at which the leukocyte engages the endothelial cell junctions. It surrounds the leukocyte during the transmigration process providing increased membrane surface area in the junction and several key adhesion molecules that regulate transmigration. Blocking targeted recycling blocks leukocyte transmigration. Hence, the LBRC and its targeted recycling are critical factors regulating leukocyte transmigration in inflammation. Understanding how targeted recycling is regulated would provide insights into the control of the inflammatory response and potentially identify new therapeutic targets. Knowing the composition of the LBRC would allow us to formulate testable hypotheses about the function(s) of constitutive recycling and the regulation of targeted recycling. However, only three components of the LBRC are known: Platelet/endothelial cell adhesion molecule-1 (PECAM, CD31), CD99, and Junctional Adhesion Molecule A (JAM-A). In order to obtain an unbiased insight into the composition of the LBRC, we will isolate LBRC membrane from endothelial cell homogenates by density gradient centrifugation. We will analyze the protein composition of this membrane by two dimensional gel electrophoresis and identify membrane proteins unique to or enriched in the LBRC by comparison to the protein profiles of plasma membrane fractionated in parallel. Spots on the gels representing proteins that appear to be unique or markedly enriched in LBRC membrane will be excised, protease digested, and identified by mass spectrometry. As a complementary approach, a total proteomic comparison of LBRC and plasma membrane fractions will be performed. Candidate LBRC proteins will be validated in several ways. In the first step, we will localize these proteins in intact endothelial cells to be certain they have the correct distribution. They should be concentrated at the endothelial cell borders. Immunolocalization by confocal fluorescence microscopy will be performed. We expect most of the proteins will be well known and previously described. We will use existing antibodies against the candidate proteins where they are available and generate FLAG-tagged constructs of the candidate proteins, based on known sequences, where they are not. Those proteins that co-localize with authentic LBRC in intact cells will be further tested to determine whether they are truly components of the LBRC in functional assays. We will determine, using well-established assays in our lab, whether they recycle constitutively in the same manner as LBRC and participate in targeted recycling during leukocyte transmigration. Public Health Relevance: Most diseases (including atherosclerosis, asthma, and autoimmune diseases) involve an inflammatory response that is uncontrolled or misdirected. We have discovered a novel membrane compartment in endothelial cells (the cells that line blood vessels) that is critical for the inflammatory response. We will isolate this membrane compartment and identify its component proteins in order to understand how it functions and design better anti-inflammatory therapies.

描述（由申请人提供）：侧边界再循环室（LBRC）是最近发现的位于血管内皮细胞边界的膜室。来自LBRC的膜在腔室和内皮细胞边界的质膜之间组成性地快速循环。这种本构循环的生理功能尚不清楚。然而，在白细胞转运过程中，来自LBRC的再循环膜被重定向到白细胞与内皮细胞连接处接合的位置。在转运过程中，它包围着白细胞，在连接处提供增加的膜表面积和调节转运的几个关键粘附分子。阻断靶向再循环会阻断白细胞的转运。因此，LBRC及其靶向再循环是炎症中调节白细胞迁移的关键因素。了解靶向再循环是如何被调节的，将有助于了解炎症反应的控制，并有可能确定新的治疗靶点。了解LBRC的组成将使我们能够制定关于本构回收和目标回收调节功能的可测试假设。然而，LBRC只有三种成分是已知的：血小板/内皮细胞粘附分子-1 (PECAM, CD31)， CD99和连接粘附分子A （JAM-A）。为了获得对LBRC组成的公正见解，我们将通过密度梯度离心从内皮细胞匀浆中分离LBRC膜。我们将通过二维凝胶电泳分析该膜的蛋白质组成，并通过与平行分离的质膜蛋白质谱进行比较，确定LBRC中特有或富集的膜蛋白。凝胶上代表LBRC膜中独特或显著富集的蛋白质的斑点将被切除，蛋白酶消化，并通过质谱法鉴定。作为补充方法，将进行LBRC和质膜组分的总蛋白质组学比较。候选LBRC蛋白将通过几种方式进行验证。在第一步，我们将把这些蛋白定位在完整的内皮细胞中，以确保它们有正确的分布。它们应集中在内皮细胞边界。通过共聚焦荧光显微镜进行免疫定位。我们希望大多数蛋白质都是已知的，并且以前已经描述过了。我们将使用现有的针对候选蛋白的抗体，并根据已知的序列生成候选蛋白的flag标记结构，在它们不存在的地方。那些在完整细胞中与真正的LBRC共定位的蛋白质将在功能分析中进一步测试，以确定它们是否真正是LBRC的组成部分。我们将在实验室中使用成熟的检测方法来确定它们是否以与LBRC相同的方式进行结构性回收，并在白细胞转运过程中参与靶向回收。