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

开发病毒粒子展示 (VirD) 阵列来分析人类 GPCR 相互作用

基本信息

批准号：
9247705
负责人：
PRASHANT J DESAI
金额：
$ 38.55万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9247705
关键词：
Address Affinity Biochemical Biological C5AR2 gene Calcium Cell Adhesion Molecules Cell Proliferation Cell Survival Cell membrane Cells Chemistry Cloning Collection Detergents Development Drug Targeting Engineering Environment Escherichia coli F2R gene Family G-Protein-Coupled Receptors Genes Genetic Recombination Genome Glass Herpes Simplex Infections Herpesvirus 1 Hormones Human IL8RB gene Image Immunology procedure Incidence Insecta Integral Membrane Protein Knowledge Label Laboratories Ligand Binding Ligands Lipids Malignant Neoplasms Measures Membrane Membrane Proteins Methods Mission Modification Molecular Molecular Conformation Neoplasm Metastasis Pathway interactions Peptides Pharmaceutical Preparations Physiological Plasmids Post-Translational Protein Processing Production Protein Family Protein Microchips Proteins Proteome Public Health Receptor Activation Receptor Signaling Reporting Research Slide Specificity Standardization Structure Surface System Technology Therapeutic Time Transmembrane Domain United States National Institutes of Health Validation Virion Virus Yeasts angiogenesis base cell growth chemokine density drug discovery glycosylation high throughput screening human disease improved insight loss of function member new technology new therapeutic target protein protein interaction public health relevance receptor screening small molecule success therapeutic target tumorigenesis

项目摘要

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.

描述（由申请人提供）：大约三分之一的人类蛋白质组由属于具有多种生化活性的蛋白质家族的膜蛋白组成，例如转运蛋白、通道、受体、识别分子和粘附分子。然而，膜蛋白，尤其是那些含有多次跨膜结构域的膜蛋白，非常难以研究，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），例如糖基化。使用去污剂对膜蛋白进行生化纯化会限制通量，并且不可避免地破坏膜蛋白的天然环境，导致大多数情况下功能丧失。为了促进膜蛋白的高通量生化分析，我们最近报道了一种 VirD 阵列技术，通过该技术，多次通过的人类跨膜蛋白以其天然构象展示在单纯疱疹（即 HSV-1）病毒粒子的膜包膜中。在这个探索性的 R33 应用中，我们建议采用这项新技术来构建由人类所有 G 蛋白偶联受体 (GPCR) 家族组成的高内涵 VirD 阵列。 VirD阵列方法有几个明显的优势：1）展示的人膜蛋白嵌入宿主细胞膜中，这是一个与生理学更相关的环境，可以帮助维持其活性； 2）GPCR蛋白很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，因此展示的人类蛋白质在通过分泌途径运输时可能会维持其规范的 PTM。 GPCR VirD 阵列的应用将通过配体分析来证明，重点是与癌症相关的 GPCR。我们预计，开发一个高通量平台，能够分析功能构象的膜蛋白的生化活性，将通过简化小分子筛选方法对药物发现产生重要影响。我们设想，一旦建立了这样一个高内涵、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新药物靶点识别的高通量筛选，识别各种类型受体的配体，系统地分析膜蛋白-蛋白质相互作用，以及分析膜蛋白的PTM。