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: 7872115
• 负责人: William A Muller
• 金额: $ 22.88万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7872115
• 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; Data; 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）：侧缘再循环室（LBRC）是最近发现的位于血管内皮细胞边缘沿着的膜室。来自LBRC的膜在隔室和内皮细胞之间边界处的质膜之间组成性地且快速地粘附。这种组成性再循环的生理功能尚不清楚。然而，在白细胞迁移过程中，来自LBRC的再循环膜被重定向-靶向到白细胞接合内皮细胞连接的位点。它在迁移过程中包围白细胞，增加连接处的膜表面积，并提供几种调节迁移的关键粘附分子。阻断靶向再循环可阻断白细胞迁移。因此，LBRC及其靶向再循环是调节炎症中白细胞迁移的关键因素。了解靶向再循环是如何调节的，将为炎症反应的控制提供见解，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组成性回收功能和目标回收调节的可验证假设。然而，已知LBRC仅有三种组分：血小板/内皮细胞粘附分子-1（PECAM，CD 31）、CD 99和连接粘附分子A（JAM-A）。为了获得对LBRC的组成的无偏见的了解，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来鉴定LBRC中特有或富集的膜蛋白。将切下凝胶上代表在LBRC膜中似乎独特或显著富集的蛋白质的点，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蛋白将以几种方式进行验证。在第一步中，我们将在完整的内皮细胞中定位这些蛋白质，以确保它们具有正确的分布。它们应该集中在内皮细胞边缘。将通过共聚焦荧光显微镜进行免疫定位。我们预计大多数蛋白质将是众所周知的和以前描述的。我们将使用现有的针对候选蛋白质的抗体，在它们可用的地方，并基于已知序列生成候选蛋白质的FLAG标记的构建体，在它们不存在的地方。将进一步测试与完整细胞中的真实LBRC共定位的那些蛋白质，以确定它们在功能测定中是否是LBRC的真正组分。我们将使用我们实验室中成熟的检测方法，确定它们是否以与LBRC相同的方式组成性再循环，并在白细胞迁移过程中参与靶向再循环。 公共卫生相关性：大多数疾病（包括动脉粥样硬化、哮喘和自身免疫性疾病）都涉及不受控制或误导的炎症反应。我们已经发现了一种新的膜室在内皮细胞（细胞内衬血管），这是至关重要的炎症反应。我们将分离这种膜室并鉴定其组成蛋白，以了解其功能并设计更好的抗炎疗法。