PDF(Pubmed)
Abstract：
Cryphonectria parasitica, the chestnut blight fungus, and hypoviruses are excellent models for examining fungal pathogenesis and virus-host interactions. Increasing evidence suggests that lysine acetylation plays a regulatory role in cell processes and signalling. To understand protein regulation in C. parasitica by hypoviruses at the level of posttranslational modification, a label-free comparative acetylome analysis was performed in the fungus with or without Cryphonectria hypovirus 1 (CHV1) infection. Using enrichment of acetyl-peptides with a specific anti-acetyl-lysine antibody, followed by high accuracy liquid chromatography-tandem mass spectrometry analysis, 638 lysine acetylation sites were identified on 616 peptides, corresponding to 325 unique proteins. Further analysis revealed that 80 of 325 proteins were differentially acetylated between C. parasitica strain EP155 and EP155/CHV1-EP713, with 43 and 37 characterized as up- and down-regulated, respectively. Moreover, 75 and 65 distinct acetylated proteins were found in EP155 and EP155/CHV1-EP713, respectively. Bioinformatics analysis revealed that the differentially acetylated proteins were involved in various biological processes and were particularly enriched in metabolic processes. Differences in acetylation in C. parasitica citrate synthase, a key enzyme in the tricarboxylic acid cycle, were further validated by immunoprecipitation and western blotting. Site-specific mutagenesis and biochemical studies demonstrated that the acetylation of lysine-55 plays a vital role in the regulation of the enzymatic activity of C. parasitica citrate synthase in vitro and in vivo. These findings provide a valuable resource for the functional analysis of lysine acetylation in C. parasitica, as well as improving our understanding of fungal protein regulation by hypoviruses from a protein acetylation perspective.
摘要：
寄生虫，栗病真菌,和低病毒是检查真菌发病机理和病毒-宿主相互作用的极好模型。越来越多的证据表明赖氨酸乙酰化在细胞过程和信号传导中起调节作用。为了在翻译后修饰的水平上了解低病毒在寄生虫中的蛋白质调节，在有或没有低毒病毒1(CHV1)感染的真菌中进行了无标记的比较乙酰基组分析.使用特定的抗乙酰赖氨酸抗体富集乙酰肽，其次是高精度液相色谱-串联质谱分析，在616个肽上鉴定出638个赖氨酸乙酰化位点，对应于325种独特的蛋白质。进一步分析显示，在寄生虫梭菌菌株EP155和EP155/CHV1-EP713之间，325种蛋白质中有80种差异乙酰化，其中43种和37种特征为上调和下调，分别。此外，在EP155和EP155/CHV1-EP713中分别发现了75和65种不同的乙酰化蛋白。生物信息学分析显示，差异乙酰化的蛋白质参与各种生物过程，特别是在代谢过程中富集。寄生虫柠檬酸合酶的乙酰化差异，三羧酸循环中的关键酶，通过免疫沉淀和蛋白质印迹进一步验证。定点诱变和生化研究表明，赖氨酸-55的乙酰化在体外和体内对寄生虫柠檬酸合酶酶活性的调节中起着至关重要的作用。这些发现为寄生虫中赖氨酸乙酰化的功能分析提供了宝贵的资源，以及从蛋白质乙酰化的角度提高我们对低病毒真菌蛋白质调节的理解。
