Cytomegalovirus Deubiquitinase pUL48: Identifying Substrate and Inhibitors

巨细胞病毒去泛素酶 pUL48：识别底物和抑制剂

基本信息

批准号：
8105826
负责人：
D Wade Gibson
金额：
$ 20.3万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-19 至 2012-07-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8105826
关键词：
Acquired Immunodeficiency Syndrome Affinity Amino Acid Sequence Antiviral Agents Attention Baculoviruses Biochemical Biological Biological Assay Biological Process Catalytic Domain Cells Chemicals Chemotherapy-Oncologic Procedure Congenital Abnormality Cysteine Protease Cytomegalovirus Deoxyribonucleases Environment Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Funding Genetic Genome Herpesviridae Human Herpesvirus 5 species Immune system Immunoassay In Situ Infection Insecta Length Libraries Mass Spectrum Analysis Medical Methods Molecular Weight Open Reading Frames Organ Transplantation Peptide Hydrolases Pilot Projects Preparation Process Proteins Protocols documentation Publications Publishing Recombinants Reporting Request for Applications Research Resources Rest Role Site Source Testing Ubiquitin Viral Virion Virus Virus Inhibitors Virus Replication Work base design enzyme activity enzyme substrate high throughput screening inhibitor/antagonist member monomer multidisciplinary mutant parent grant prototype public health relevance research study success therapy design thioester

项目摘要

DESCRIPTION (provided by applicant): Herpes-group viruses have recently been discovered to encode a ubiquitin-specific cysteine protease, whose biological function is unknown. Its catalytic domain lies within a 300 to 400 amino-acid sequence that is the amino terminal end of the largest protein encoded by each herpesvirus genome. Although its activity is not absolutely essential for virus replication, it appears to enhance the efficiency of the process. Our discovery that this deubiquitinylating enzyme (DUB) is active in the context of the full-length protein led to two key findings on which the work proposed here is based. First, the DUB is an integral constituent of infectious virions and second, it is the predominant if not sole DUB activity present in purified virions. We are studying the herpesvirus DUB in human cytomegalovirus (HCMV, HHV5) because this sexually transmissible agent is of increasing medical relevance in association with AIDS, organ transplantation, cancer chemotherapy, and birth defects resulting from transplacental infections. It is also the prototypic 2-herpesvirus and there is an underlying need to identify and understand differences between it and members of the 2- and 3- herpesviruses. In human cytomegalovirus the protein hosting the DUB activity is encoded by open reading frame UL48, is 253 kDa, and is called the high-molecular-weight protein (HMWP) or pUL48. The specific aims we propose follow directly from results of published and pilot studies done to define the biological purpose of this enzyme and determine its potential as an antiviral target. Our immediate aims are to substantiate the hypothesis that HCMV virions contain a substrate of the viral DUB (critical to understanding function and mechanism, but so far unidentified), and to develop an assay that can be adapted to high-throughput format to screen for inhibitors of the HCMV DUB. We will apply a combination of genetic, physical, biochemical, enzymological, and biological approaches to accomplish these aims in a multidisciplinary environment. PUBLIC HEALTH RELEVANCE: Cytomegalovirus is a herpes-group virus that is a threat to people with weakened immune systems, including the very young and the very old. This research is directed at a recently discovered protease that removes ubiquitin from proteins, but whose function and potential as an antiviral target are unknown. We propose to identify the substrate(s) of this enzyme and identify inhibitors of its activity in high-throughput assays.

描述（由申请人提供）：最近发现疱疹病毒编码泛素特异性半胱氨酸蛋白酶，其生物学功能尚不清楚。其催化结构域位于 300 至 400 个氨基酸序列内，该序列是每个疱疹病毒基因组编码的最大蛋白质的氨基末端。尽管它的活性对于病毒复制并不是绝对必要的，但它似乎提高了该过程的效率。我们发现这种去泛素化酶 (DUB) 在全长蛋白质的背景下具有活性，这导致了本文提出的工作所基于的两个关键发现。首先，DUB 是感染性病毒粒子的组成部分；其次，它是纯化病毒粒子中存在的主要 DUB 活性（如果不是唯一的话）。我们正在研究人类巨细胞病毒（HCMV、HHV5）中的疱疹病毒 DUB，因为这种性传播媒介与艾滋病、器官移植、癌症化疗和经胎盘感染引起的出生缺陷的相关性越来越大。它也是原型2-疱疹病毒，并且存在识别和理解它与2-和3-疱疹病毒成员之间的差异的潜在需要。在人巨细胞病毒中，具有 DUB 活性的蛋白质由开放阅读框 UL48 编码，大小为 253 kDa，被称为高分子量蛋白 (HMWP) 或 pUL48。我们提出的具体目标直接遵循已发表的和试点研究的结果，以确定这种酶的生物学目的并确定其作为抗病毒靶点的潜力。我们的近期目标是证实 HCMV 病毒粒子含有病毒 DUB 底物的假设（对于理解功能和机制至关重要，但迄今为止尚未确定），并开发一种可适应高通量格式的检测方法来筛选抑制剂HCMV DUB。我们将结合遗传、物理、生化、酶学和生物学方法在多学科环境中实现这些目标。公共卫生相关性：巨细胞病毒是一种疱疹病毒，对免疫系统较弱的人（包括幼儿和老年人）构成威胁。这项研究针对的是最近发现的一种蛋白酶，它可以从蛋白质中去除泛素，但其作为抗病毒靶点的功能和潜力尚不清楚。我们建议在高通量测定中鉴定该酶的底物并鉴定其活性的抑制剂。