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.

Public Health Relevance

In this application, we will develop a VirD array, comprised of all the members of the human G-protein-coupled receptor (GPCR) family, and apply it to profile ligand-receptor interactions. This project is relevant to public health because the success of this project is expected to provide insights into the molecular mechanisms by which GPCRs, as the major family of drug targets, specifically and quantitatively recognize different ligands and small molecules. The proposed research is thus relevant to part of the NIH's mission, as this advance in fundamental biological knowledge will help identify better therapeutics for a broad range of human diseases.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (ZCA1)
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Dickherber, Anthony J
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Johns Hopkins University
Schools of Medicine
United States
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Ma, Guangzhong; Syu, Guan-Da; Shan, Xiaonan et al. (2018) Measuring Ligand Binding Kinetics to Membrane Proteins Using Virion Nano-oscillators. J Am Chem Soc 140:11495-11501
Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A et al. (2017) Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods. Proc Natl Acad Sci U S A 114:E8885-E8894
Wan, Jun; Su, Yijing; Song, Qifeng et al. (2017) Methylated cis-regulatory elements mediate KLF4-dependent gene transactivation and cell migration. Elife 6:
Huang, Yi; Zhu, Heng (2017) Protein Array-based Approaches for Biomarker Discovery in Cancer. Genomics Proteomics Bioinformatics 15:73-81
Li, Renfeng; Wang, Leyao; Liao, Gangling et al. (2012) SUMO binding by the Epstein-Barr virus protein kinase BGLF4 is crucial for BGLF4 function. J Virol 86:5412-21
Zheng, Dasheng; Wan, Jun; Cho, Yong Gu et al. (2011) Comparison of humoral immune responses to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus using a viral proteome microarray. J Infect Dis 204:1683-91
Zhu, Jian; Liao, Gangling; Shan, Liang et al. (2009) Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4. J Virol 83:5219-31