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: 8488143
• 负责人: Renfeng Li
• 金额: $ 8.46万
• 依托单位: JOHNS HOPKINS 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488143
• 关键词: Binding; Bioinformatics; Biology; Cells; Cellular biology; Complex; Cytomegalovirus; DNA Damage; Ensure; Environment; Enzymes; Epstein-Barr Virus Infections; 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

DESCRIPTION (provided by applicant): 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 infection, it is not only critical to identify the BGLF4 in vivo targets and their precise phosphorylation sites, but alo 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 phosphorylation and SUMOylation, which should lead to novel insights into pathogen-host interactions. Epstein-Barr virus (EBV) causes infectious mononucleosis and is implicated in several human cancers. EBV expresses a protein kinase, BGLF4, that controls multiple critical steps in the viral life cycle and is a target for anti-viral drug development. I propose a novel strategy using high throughput mass spectrometry to dissect the complex signaling networks regulated by BGLF4.

描述（由申请人提供）：Epstein-Barr病毒（EBV）蛋白激酶BGLF4是保守的疱疹病毒蛋白激酶的成员，这是一组保存在所有疱疹性疱疹的亚家族中的酶。对BGLF4的细胞蛋白底物的了解对于理解BGLF4功能至关重要。但是，迄今为止，只有少量的主机基材被表征。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球宿主蛋白sumoylation。但是，尚未描述过BGLF4调控宿主蛋白的具体例子。为了了解EBV感染在细胞环境中带来的变化，不仅要在体内靶标及其精确的磷酸化位点识别BGLF4至关重要，而且ALO的方法可以明确剖析由BGLF4调节的复杂信号网络。我们先前鉴定出数百个宿主底物在体外磷酸化，而EBV BGLF4和其他三个直系同源激酶。对其共享底物的生物信息学分析表明，参与DNA损伤反应（DDR）途径的蛋白质在统计上富集。进一步的研究表明，BGLF4通过TIP60磷酸化积极诱导宿主DDR以促进病毒复制。有趣的是，我最近的工作表明，小型泛素样修饰剂（SUMO）的结合对于DDR涉及的BGLF4诱导的蛋白磷酸化至关重要。 BGLF4还以Sumo结合和激酶活性依赖性方式抑制宿主蛋白Sumoylation。基于这些观察结果，我假设EBV BGLF4诱导了细胞蛋白磷酸化和sumoylation的动态改变，以创建适合有效病毒复制的环境。为了准确识别BGLF4下游的信号事件，我将与我的同事Akhilesh Pandey博士一起工作，他是质谱，蛋白质组学和生物信息学领域的领先科学家。我的主要导师戴安娜·海沃德（Diane Hayward）博士在EBV方面拥有30多年的经验，他将指导我对宿主基板的验证，并证明其在EBV生物学中的作用。具体而言，在K99阶段，我将在EBV感染的细胞中全局识别BGLF4调节的磷酸化和Sumoylation靶标，请查明由BGLF4靶向的关键主机途径，并验证已识别的底物。在R00阶段，我将阐明BGLF4介导的宿主蛋白磷酸化和Sumoylation的变化的机制，促进病毒复制。我在该应用的指导阶段提出的研究是对病毒学，蛋白质组学和生物信息学培训多学科培训的理想工具，所有这些都将确保我在随后的独立阶段的未来成功。拟议的研究是新颖的，因为它们探讨了病毒蛋白BGLF4作为调节蛋白质磷酸化和sumoylation之间串扰的活性参与者的作用。它们还将显着提高我们对宿主磷酸化和Sumoylation的病毒操纵的理解，这应该导致对病原体宿主相互作用的新见解。爱泼斯坦 - 巴尔病毒（EBV）引起感染性单核细胞增多症，并与几种人类癌症有关。 EBV表达蛋白激酶BGLF4，该蛋白激酶控制病毒生命周期中的多个关键步骤，并且是抗病毒药物发育的靶标。我提出了一种使用高吞吐量质谱法的新型策略，以剖析由BGLF4调节的复杂信号网络。