Proteomic Evaluation of the Blood-Brain Barrier Receptor-Mediated Transportome

脑微血管由一类特殊的内皮细胞组成，它在血流和脑间隙之间形成细胞屏障。这个所谓的血脑屏障（BBB）将大脑微血管系统与周围血管床区分开来，因为它构成了一个物理和代谢屏障，严格调节大脑对离子、小分子、蛋白质和循环细胞的摄取。由于脑内皮细胞浆膜与血液和脑间质液接触，它们被理想地定位为信号、免疫调节和血脑间运输的控制界面。因此，血脑屏障内皮的许多独特特征可能归因于其质膜的蛋白质组成。特别是，血脑屏障上的受体介导转运（RMT）系统通过控制大分子进出大脑的转运，在健康的脑功能中发挥重要的功能作用。此外，RMT系统还可以通过其功能障碍或直接参与运输与疾病进展有关的物质（例如β -淀粉样蛋白、HIV病毒、麻疹病毒）来影响疾病过程。最后，考虑到RMT系统运输大分子的能力，它们已经越来越多地被用作将药物货物无创地运送到大脑中的手段。虽然RMT系统是一类非常重要的蛋白质，但由于技术缺陷，对它们的研究不足。RMT受体是一种膜蛋白，由于其低丰度和疏水性，使用传统的二维凝胶或串联质谱方法无法很好地描述。此外，由于血脑屏障仅占脑总体积的1/1000，因此使用整个脑组织的蛋白质组学研究可能会遗漏在血脑屏障中发挥作用的许多重要的RMT系统。因此，在这项EUREKA提案中，我们将使用我们实验室开发的一种新的、最先进的技术平台，将抗体库的力量与洗涤剂溶解血脑屏障质膜裂解物结合起来，同时识别抗体和同源血脑屏障RMT蛋白。由于可以使用洗涤剂，膜蛋白的疏水性通常会阻碍其他蛋白质组学技术，因此可以克服，从而有望更广泛地覆盖BBB RMT蛋白质组（RMT-ome）。此外，该方法使这种亚细胞血脑屏障膜蛋白质组学具有高度的体内相关性，因为新鲜分离和分离的血脑屏障被用作源组织。最后，我们的新方法允许功能膜蛋白质组学，鉴定的膜蛋白可以很容易地通过它们与RMT系统的一般成分的功能关联进行分类。利用这些新工具，将确定参与血脑屏障受体介导转运（RMT）功能的受体和机制。鉴定出的靶向RMT机制的抗体将用于评估RMT表达水平和组织定位，以及验证RMT机制的运输功能。预计这项研究的成功完成不仅会对神经科学家和细胞生物学家产生重大影响，而且最终会对数百万神经疾病患者的治疗产生重大影响。

The brain microvasculature is comprised of a specialized class of endothelium that forms a cellular barrier between the bloodstream and the interstices of the brain. This so-called blood-brain barrier (BBB) distinguishes the brain microvasculature from peripheral vascular beds because it constitutes a physical and metabolic barrier that tightly regulates brain uptake of ions, small molecules, proteins, and circulating cells. Since brain endothelial cell plasma membranes contact both the bloodstream and brain interstitial fluid, they are ideally positioned to act as the controlling interfaces for signaling, immune regulation, and transport between the blood and brain. Therefore, many of the unique characteristics of the BBB endothelium can likely be attributed to the protein composition of its plasma membranes. In particular, the receptor-mediated transport (RMT) systems at the BBB play major functional roles in healthy brain function by controlling the transport of large molecules into and out of the brain. Moreover, RMT systems can also affect disease processes by either their dysfunction or by their direct participation in transporting substances involved in disease progression (e.g. beta-amyloid, HIV virus, measles virus). Finally, given the capability for RMT systems to transport large molecules, they have been increasingly used as a means for shuttling drug cargo into the brain noninvasively. Although RMT systems are a highly significant class of proteins, they have been understudied as a consequence of technological shortcomings. RMT receptors are membrane proteins that because of their low abundance and hydrophobicity are not well profiled using traditional two dimensional gel or tandem mass spectrometry methods. In addition, since the BBB comprises only 1/1000 of the brain total volume, proteomics studies using total brain tissue likely miss many of the important RMT systems in play at the BBB. Therefore, in this EUREKA proposal we will use a new, state-of-the-art technological platform developed in our laboratory that combines the power of antibody libraries with detergent-solubilized BBB plasma membrane lysates to simultaneously identify antibodies and cognate BBB RMT proteins. Because detergents can be employed, the hydrophobic nature of membrane proteins that often hampers other proteomics technologies can be overcome, thereby promising a broader coverage of the BBB RMT proteome (RMT-ome). Moreover, the approach enables such subcellular BBB membrane proteomics with high in vivo relevance because freshly isolated and subfractionated BBB is used as source tissue. Finally, our new approaches allow functional membrane proteomics in that identified membrane proteins can be readily categorized by their functional association with a generalized component of RMT systems. Taking advantage of these new tools, receptors and machinery that are involved in receptor-mediated transport (RMT) function at the BBB will be identified. Identified antibodies targeting RMT machinery will then used to assess RMT expression levels and tissue localization as well as to validate the transport function of the RMT machinery. Successful completion of the proposed research is predicted to have substantial impact not only for neuroscientists and cell biologists alike, but ultimately for the treatment of millions suffering from neurological disease.