Abstract：
Protein phosphorylation and dephosphorylation are the most common and important regulatory mechanisms in signal transduction, which play a vital role in immune defense response. Our previous study has found the level of tyrosine phosphorylation was significantly changed in the hemocytes of Fenneropenaeus chinensis upon white spot syndrome virus (WSSV) infection. In order to explore the relationship between protein phosphorylation and WSSV infection, the quantitative phosphoproteomics was employed to identify differential phosphorylated proteins in hemocytes of F. chinensis before and after WSSV infection, and elucidate the role of key differential phosphorylated proteins in WSSV infection process. The results showed that a total of 147 differential phosphorylated proteins were identified in the hemocytes, including 64 phosphorylated proteins and 83 dephosphorylated proteins, which were mostly enriched in pyruvate metabolism, TCA cycle, glycolysis, and ribosomal biosynthesis. Functional analysis of differential phosphorylated proteins showed that they were involved in cell apoptosis, cell phagocytosis, cell metabolism and antiviral infection. A total of 236 differential phosphorylation sites were found, including 91 modified sites in the phosphorylation proteins and 145 modified sites in the dephosphorylation proteins. Motif analysis showed that these phosphorylation sites could activate mitogen-activated protein kinase, P70 S6 kinase and other kinases in hemocytes. Moveover, the phosphorylation levels of eukaryotic protein initiation factor 4E binding proteins and histone H3 were further determined by ELISA and Western blotting, which both exhibited a significant increase post WSSV infection and reach their peak levels at 6 and 12 h, respectively. Moreover, we found that lactate, a metabolite closely related to pyruvate metabolism, TCA cycle and glycolysis, was significantly increased in the hemocytes after WSSV infection. This study revealed the protein phosphorylation response in hemocytes of F. chinensis to WSSV infection, which help to clarify the response characteristics and virus resistance mechanism of hemocytes in F. chinensis, and also facilitate further understanding of the interaction between WSSV and shrimp hemocytes.
摘要：
蛋白质磷酸化和去磷酸化是信号转导中最常见和最重要的调控机制,在免疫防御反应中起着至关重要的作用。我们先前的研究发现，白斑综合症病毒（WSSV）感染后，中国明对虾血细胞中酪氨酸磷酸化水平发生了显着变化。为了探讨蛋白磷酸化与WSSV感染的关系,定量磷酸化蛋白质组学用于鉴定WSSV感染前后华氏杆菌血细胞中的差异磷酸化蛋白，并阐明关键差异磷酸化蛋白在WSSV感染过程中的作用。结果表明，在血细胞中总共鉴定出147种差异磷酸化蛋白，包括64个磷酸化蛋白和83个去磷酸化蛋白，主要富含丙酮酸代谢，TCA循环,糖酵解,和核糖体生物合成。差异磷酸化蛋白的功能分析显示它们参与细胞凋亡,细胞吞噬，细胞代谢和抗病毒感染。共发现236个差异磷酸化位点,包括磷酸化蛋白中的91个修饰位点和去磷酸化蛋白中的145个修饰位点。基序分析表明,这些磷酸化位点可以激活丝裂原活化蛋白激酶,血细胞中的P70S6激酶和其他激酶。Moveover,通过ELISA和Westernblotting进一步测定真核蛋白起始因子4E结合蛋白和组蛋白H3的磷酸化水平,两者在WSSV感染后均表现出显着增加，并在6和12小时达到峰值水平，分别。此外，我们发现乳酸，与丙酮酸代谢密切相关的代谢产物，TCA循环和糖酵解，WSSV感染后血细胞显著增加。本研究揭示了华氏杆菌血细胞对WSSV感染的蛋白磷酸化反应,有助于阐明黄连血细胞的应答特性和抗病毒机制,并有助于进一步了解WSSV与对虾血细胞之间的相互作用。
