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

脑微血管系统由一类特殊的内皮细胞组成，它在血流和大脑间隙之间形成细胞屏障。这种所谓的血脑屏障(BBB)将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。由于脑内皮细胞质膜同时接触血液和脑间质液体，它们被理想地定位为控制信号、免疫调节和血液和大脑之间传输的接口。因此，血脑屏障内皮细胞的许多独特特征很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的运输(RMT)系统通过控制大分子的进出大脑，在健康的大脑功能中发挥着重要的功能作用。此外，RMT系统还可以通过其功能障碍或通过其直接参与运输参与疾病进展的物质(例如，β-淀粉样蛋白、艾滋病毒、麻疹病毒)来影响疾病进程。最后，考虑到RMT系统传输大分子的能力，它们越来越多地被用作将药物货物非侵入性地运送到大脑中的一种手段。尽管RMT系统是一类非常重要的蛋白质，但由于技术上的缺陷，对它们的研究一直不足。RMT受体是一种膜蛋白，由于其丰度低和疏水性，不能用传统的二维凝胶或串联质谱学方法很好地描述。此外，由于血脑屏障仅占大脑总体积的1/1000，使用总脑组织的蛋白质组学研究可能会错过血脑屏障发挥作用的许多重要的RMT系统。因此，在这份尤里卡提案中，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，该平台将抗体库的能力与洗涤剂溶解的BBB质膜裂解物相结合，以同时识别抗体和同源BBB RMT蛋白。由于可以使用洗涤剂，因此可以克服膜蛋白的疏水性，这往往会阻碍其他蛋白质组技术的发展，从而有望扩大BBB RMT蛋白质组(RMT-OME)的覆盖范围。此外，这种方法使得这种亚细胞BBB膜蛋白质组学具有很高的体内相关性，因为新分离的和亚分离的BBB被用作来源组织。最后，我们的新方法允许功能膜蛋白质组学，因为所识别的膜蛋白可以很容易地通过它们与RMT系统的广义组件的功能关联来分类。利用这些新的工具，参与受体介导的转运(RMT)功能的受体和机制将被识别出来。识别的针对RMT机制的抗体将用于评估RMT的表达水平和组织定位，以及验证RMT机制的运输功能。这项拟议研究的成功完成预计将不仅对神经科学家和细胞生物学家产生重大影响，而且最终将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.