Impact of herpesvirus protein kinases on host protein modification

疱疹病毒蛋白激酶对宿主蛋白修饰的影响

• 批准号: 8888392
• 负责人: Renfeng Li
• 金额: $ 24.9万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8888392
• 关键词: Binding; Bioinformatics; Biology; Cells; Cellular biology; Complex; Cytomegalovirus; DNA Damage; Ensure; Environment; Enzymes; Event; Fostering; Future; HTATIP gene; Herpesviridae; Herpesvirus 1; Human; Human Herpesvirus 4; Human Herpesvirus 8; In Vitro; Infection; Infectious Mononucleosis; Knowledge; Lead; Life Cycle Stages; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mentors; Modification; Nuclear; Pathway interactions; Phase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Protein Processing; Protein Kinase; Protein Microchips; Proteins; Proteomics; Role; Scientist; Signal Transduction; Site; Training; Ubiquitin; Validation; Viral; Viral Proteins; Virus Replication; Work; base; cellular targeting; drug development; experience; high throughput screening; in vivo; insight; lytic replication; member; multidisciplinary; novel; novel strategies; pathogen; response; success; virology

The Epstein-Barr virus (EBV) protein kinase BGLF4 is a member of the conserved herpesvirus protein kinases, a group of enzymes conserved throughout all subfamilies of Herpesviridae. Knowledge of the cellular protein substrates of BGLF4 is essential for understanding BGLF4 function; however, only a small number of host substrates have been characterized to date. In addition to inducing protein phosphorylation, BGLF4 also regulates global host protein SUMOylation in a kinase activity dependent manner; however, no specific examples of a BGLF4 regulated host protein have been described. To understand the changes brought about in the cellular environment by EBV infeciton, it is not only critical to identify the BGLF4 in vivo targets and their precise phosphorylation sites, but also to have an approach that can unambiguously dissect the complex signaling networks regulated by BGLF4. We previously identified several hundred host substrates phosphorylated in vitro by EBV BGLF4 and by three other orthologous kinases. Bioinformatic analysis of their shared substrates revealed that proteins involved in the DNA damage response (DDR) pathway were statistically enriched. Further studies demonstrated that BGLF4 actively induces a host DDR via TIP60 phosphorylation to foster viral replication. Interestingly, my recent work demonstrated that binding of the small ubiquitin-like modifier (SUMO) is critical for the BGLF4-induced protein phosphorylation involved in the DDR. BGLF4 also inhibits host protein SUMOylation in both a SUMO binding and kinase activity dependent manner. Based on these observations, I hypothesize that EBV BGLF4 induces a dynamic alteration of cellular protein phosphorylation and SUMOylation to create a suitable environment for efficient virus replication. In order to precisely identify the signaling events downstream of BGLF4, I will work with my co-mentor Dr. Akhilesh Pandey, who is a leading scientist in mass spectrometry, proteomics and bioinformatics. My primary mentor Dr. Diane Hayward, who has over 30 years experience with EBV, will guide my validation of the host substrates and demonstration of their role in EBV biology. Specifically, during the K99 phase, I will globally identify BGLF4-regulated phosphorylation and SUMOylation targets in EBV-infected cells, pinpoint the key host pathways targeted by BGLF4, and validate the identified substrates. In the R00 phase, I will elucidate the mechanisms by which BGLF4 mediated changes in phosphorylation and SUMOylation of host proteins facilitate virus replication. My proposed studies during the mentored phase of this application are an ideal vehicle for multidisciplinary training in virology, proteomics and bioinformatics, all of which will ensure my future success in the subsequent independent phase. The proposed studies are novel in that they explore the role of a viral protein BGLF4 as an active player in regulating the crosstalk between protein phosphorylation and SUMOylation. They will also significantly increase our understanding of viral manipulation of host phosphoryation and SUMOylation, which should lead to novel insights into pathogen-host interactions.

Epstein-Barr 病毒 (EBV) 蛋白激酶 BGLF4 是保守疱疹病毒蛋白的成员 激酶，一组在疱疹病毒科所有亚科中保守的酶。细胞知识 BGLF4 的蛋白质底物对于理解 BGLF4 功能至关重要；然而，只有一小部分 迄今为止，宿主底物已被表征。除了诱导蛋白质磷酸化外，BGLF4 还 以激酶活性依赖性方式调节全局宿主蛋白 SUMOylation；但没有具体 已经描述了 BGLF4 调节的宿主蛋白的例子。来了解所带来的变化 由于 EBV 感染的细胞环境，识别 BGLF4 体内靶点及其作用不仅至关重要 精确的磷酸化位点，而且还要有一种可以明确剖析复合物的方法 信号网络受 BGLF4 调节。我们之前鉴定了数百种宿主底物 在体外被 EBV BGLF4 和其他三种直系同源激酶磷酸化。对其进行生物信息学分析 共享底物揭示了参与 DNA 损伤反应 (DDR) 途径的蛋白质 统计丰富。进一步的研究表明 BGLF4 通过 TIP60 主动诱导宿主 DDR 磷酸化以促进病毒复制。有趣的是，我最近的工作表明，小 泛素样修饰剂 (SUMO) 对于 DDR 中 BGLF4 诱导的蛋白质磷酸化至关重要。 BGLF4 还以 SUMO 结合和激酶活性依赖性方式抑制宿主蛋白 SUMO 化。 基于这些观察，我假设 EBV BGLF4 诱导细胞的动态改变 蛋白质磷酸化和SUMO化为病毒高效复制创造合适的环境。 为了精确识别 BGLF4 下游的信号事件，我将与我的共同导师 Dr. Akhilesh Pandey 是质谱、蛋白质组学和生物信息学领域的领先科学家。我的小学 拥有 30 多年 EBV 经验的导师 Diane Hayward 博士将指导我对宿主的验证 底物及其在 EBV 生物学中的作用的证明。具体来说，在K99阶段，我会在全球范围内 识别 EBV 感染细胞中 BGLF4 调节的磷酸化和 SUMO 化靶点，查明关键宿主 BGLF4 靶向的途径，并验证已识别的底物。在R00阶段，我将阐明 BGLF4 介导宿主蛋白磷酸化和 SUMOylation 变化的机制 病毒复制。我在本申请的指导阶段提出的研究是一个理想的工具 病毒学、蛋白质组学和生物信息学的多学科培训，所有这些都将确保我未来在 随后的独立阶段。拟议的研究是新颖的，因为它们探讨了病毒的作用 蛋白质 BGLF4 作为调节蛋白质磷酸化和蛋白质之间串扰的积极参与者 SUMO化。它们还将显着增加我们对病毒操纵宿主的理解 磷酸化和SUMO化，这应该会导致对病原体-宿主相互作用的新见解。