Development of a virion display (VirD) array to profile human GPCR interactions

 描述（由申请人提供）：约三分之一的人类蛋白质组由膜蛋白组成，这些膜蛋白属于具有多种生物化学活性的蛋白质家族，如转运蛋白、通道、受体、识别分子和粘附分子。然而，膜蛋白，特别是那些含有多通道跨膜结构域的膜蛋白，是众所周知的难以研究，因为它们必须嵌入膜中以保持天然构象，并且许多需要适当的翻译后修饰（PTM），如糖基化。使用去污剂的膜蛋白的生物化学纯化限制了通量，并且不可避免地破坏了膜蛋白的天然环境，在大多数情况下导致功能损失。为了促进膜蛋白的高通量生物化学分析，我们最近报道了VirD阵列技术，通过该技术在单纯疱疹的膜包膜中展示多次通过的人跨膜蛋白（即，HSV-1）病毒体以其天然构象。在这个探索性的R33应用中，我们建议采用这种新技术来构建一个由人类所有G蛋白偶联受体（GPCR）家族组成的高含量VirD阵列。VirD阵列方法具有几个明显的优点：1）显示的人膜蛋白包埋在宿主细胞膜中，这是一个更生理相关的环境，可以帮助维持它们的活性; 2）GPCR蛋白可能在病毒体包膜中正确折叠; 3）由于病毒利用人类分泌途径，所展示的人蛋白质在通过分泌途径转运时可能保持其典型的PTM。GPCR VirD阵列的应用将通过配体分析来证明，重点是癌症相关的GPCR。我们预计，开发一个高通量的平台，使其生化活性的功能构象的膜蛋白的轮廓将有一个重要的影响，通过简化小分子筛选方法的药物发现。我们设想，一旦建立了这样一个高含量、高通量的VirD阵列平台，它将使各种实验室能够针对膜蛋白进行高通量筛选，以识别各种类型受体的配体，系统地分析膜蛋白-蛋白相互作用，并分析膜蛋白的PTM。

 DESCRIPTION (provided by applicant): Approximately one-third of the human proteome is comprised of membrane proteins that belong to protein families with a wide variety of biochemical activities, such as transporters, channels, receptors, recognition molecules, and adhesion molecules. However, membrane proteins, especially those containing multi-pass transmembrane domains, are notoriously difficult to study because they have to be embedded in a membrane to maintain a native conformation and many require proper posttranslational modifications (PTMs), such as glycosylation. Biochemical purification of membrane proteins using detergents limits the throughput and unavoidably disrupts the native environment of membrane proteins, resulting in loss of functionality in most cases. To facilitate high-throughput biochemical analyses of membrane proteins, we have recently reported a VirD array technology by which multi-pass human transmembrane proteins were displayed in the membrane envelop of herpes simplex (i.e., HSV-1) virions in their native conformations. In this explorative R33 application, we propose to employ this new technology to build a high-content VirD array comprised of all of the G protein coupled receptor (GPCR) family in humans. The VirD array approach has several obvious advantages: 1) Displayed human membrane proteins are embedded in host cell membranes, a more physiologically relevant environment that can help maintain their activity; 2) The GPCR proteins are likely to be folded correctly in the virion envelopes; 3) Since the virus exploits the human secretory pathways, the displayed human proteins are likely to maintain their canonical PTMs as they are transported through the secretory pathways. Application of the GPCR VirD arrays will be demonstrated via ligand profiling with a focus on cancer-related GPCRs. We expect that development of a high-throughput platform that enables profiling membrane proteins in a functional conformation for their biochemical activities will have an important impact on drug discovery by streamlining small molecule screening methods. We envision that once such a high-content, high-throughput VirD array platform is established, it will enable a variety of laboratories to perform high-throughput screens for novel drug target identification against membrane proteins, to identify ligands of various types of receptors, to systematically profile membrane protein-protein interactions, and to profile PTMs of membrane proteins.