Impact of herpesvirus protein kinases on host protein modification

描述（由申请人提供）：EB病毒（EBV）蛋白激酶BGLF 4是保守疱疹病毒蛋白激酶的成员，保守疱疹病毒蛋白激酶是一组在疱疹病毒科所有亚科中保守的酶。了解BGLF 4的细胞蛋白底物对于理解BGLF 4功能至关重要;然而，迄今为止仅对少数宿主底物进行了表征。除了诱导蛋白磷酸化，BGLF 4还以激酶活性依赖性方式调节整体宿主蛋白SUMO化;然而，尚未描述BGLF 4调节的宿主蛋白的具体实例。为了了解EBV感染引起的细胞环境变化，不仅要确定BGLF 4的体内靶点及其精确的磷酸化位点，而且还必须有一种方法可以明确地剖析BGLF 4调控的复杂信号网络。我们以前确定了数百个主机基板在体外磷酸化的EBV BGLF 4和其他三个orthoglycerin激酶。对它们共享底物的生物信息学分析显示，参与DNA损伤反应（DDR）途径的蛋白质在统计学上富集。进一步的研究表明，BGLF 4通过TIP 60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，小泛素样修饰物（SUMO）的结合对于参与DDR的BGLF 4诱导的蛋白磷酸化至关重要。BGLF 4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 BGLF 4诱导细胞蛋白磷酸化和SUMO化的动态改变，以创造一个合适的环境，有效的病毒复制。为了精确识别BGLF 4下游的信号事件，我将与我的共同导师Akhilesh Pandey博士合作，他是质谱，蛋白质组学和生物信息学的领先科学家。我的主要导师Diane海沃德博士拥有30多年的EBV经验，她将指导我验证宿主底物并证明它们在EBV生物学中的作用。具体而言，在K99阶段，我将在EBV感染的细胞中全面鉴定BGLF 4调节的磷酸化和SUMO化靶点，查明BGLF 4靶向的关键宿主途径，并验证鉴定的底物。在R 00阶段，我将阐明BGLF 4介导的宿主蛋白磷酸化和SUMO化变化促进病毒复制的机制。我在本申请的指导阶段提出的研究是病毒学，蛋白质组学和生物信息学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取得成功。所提出的研究是新颖的，因为它们探索了病毒蛋白BGLF 4作为调节蛋白磷酸化和SUMO化之间的串扰的活性参与者的作用。它们还将显著增加我们对宿主磷酸化和SUMO化的病毒操纵的理解，这将导致对病原体-宿主相互作用的新见解。EB病毒（EBV）引起传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BGLF 4，其控制病毒生命周期中的多个关键步骤，并且是抗病毒药物开发的靶点。我提出了一种新的策略，使用高通量质谱法来剖析由BGLF 4调控的复杂信号网络。

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.