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

描述(申请人提供)：侧缘回收室(LBRC)是最近发现的一种位于血管内皮细胞边界的膜室。来自LBRC的膜在隔室和内皮细胞交界处的质膜之间结构性地、快速地循环。这种结构性循环的生理功能尚不清楚。然而，在白细胞迁移期间，来自LBRC的循环膜被重定向到白细胞与内皮细胞连接的位置。在转运过程中，它包围着白细胞，在交界处提供更大的膜表面积和几个调节转运的关键黏附分子。阻止有针对性的循环会阻止白细胞的迁移。因此，LBRC及其靶向循环是调节炎症中白细胞迁移的关键因素。了解靶向回收是如何受到监管的，将为控制炎症反应提供见解，并有可能确定新的治疗靶点。了解LBRC的组成将使我们能够就结构性回收的功能(S)和定向回收的监管提出可检验的假设。然而，LBRC只有三个已知成分：血小板/内皮细胞黏附分子-1(PECAM，CD31)、CD99和连接黏附分子A(JAM-A)。为了公正地了解LBRC的组成，我们将通过密度梯度离心法将LBRC膜从内皮细胞匀浆中分离出来。我们将通过双向凝胶电泳法分析该膜的蛋白质组成，并通过与平行分离的质膜的蛋白质谱进行比较来鉴定LBRC所特有的或富含的膜蛋白。凝胶上的代表蛋白质的斑点似乎是唯一的或明显浓缩在LBRC膜上，将被切除，蛋白酶消化，并通过质谱仪鉴定。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，以确保它们有正确的分布。它们应该集中在内皮细胞边界。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，并在此之前得到了描述。我们将在候选蛋白质可用的地方使用现有的抗体，并根据已知序列生成候选蛋白质的标记构建体，在已知序列的基础上，在未知序列的情况下生成候选蛋白质的标记构建体。那些与完整细胞中的LBRC共定位的蛋白质将进行进一步的测试，以确定它们在功能分析中是否真的是LBRC的组成部分。我们将使用我们实验室中成熟的分析方法，确定它们是否以与LBRC相同的方式构成循环，并参与白细胞迁移过程中的靶向循环。 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)涉及失控或误导的炎症反应。我们在内皮细胞(排列血管的细胞)中发现了一种新的膜室，它对炎症反应至关重要。我们将分离这个膜室并鉴定它的组成蛋白，以便了解它的功能并设计更好的抗炎疗法。

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.