巴西弧菌(Vibrio brasiliensis)磷酸酶RsbU的结构特性与去磷酸化活性研究  

Structure properties and dephosphorylation activity of phosphatase RsbU from Vibrio brasiliensis

作　　者：王馨远 张如梦 王勃然 贾鑫 WANG Xinyuan;ZHANG Rumeng;WANG Boran;JIA Xin(College of Food Science&Nutritional Engineering,China Agricultural University,Beijing 100083,China)

机构地区：[1]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《中国酿造》2024年第12期55-63,共9页China Brewing

基　　金：国家重点研发计划(2021YFD2100102);国家自然科学基金(31901790)。

摘　　要：应激信号传导通路中激酶B因子调控蛋白T(RsbT)在受环境胁迫后会被激活并磷酸化,但其如何将信号传导给下游信号传导蛋白B因子调控蛋白U(Rsb U)仍然未知。为了防治弧菌感染,明确弧菌在环境胁迫下的耐受机制及应激信号传导,该研究以巴西弧菌(Vibrio brasiliensis)为模式菌株,利用生物信息学软件分析其理化性质、功能位点和互作机制,探讨了其RsbU与RsbT的互作模式,并对RsbU的酶学性质研究。结果表明,成功构建表达载体并纯化RsbU蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,蛋白分子质量均与预期大小相符。Rsb U本质上是一种2C类蛋白磷酸酶(PP2C),蛋白互作网络分析数据库Sring得到的结果显示,RsbU与RsbT之间的互作分数超过0.98。酶学性质结果表明,RsbU是Ser/Thr磷酸酶,去磷酸化能力较弱且主要在前期作用,自身不携带磷酸基团。因此推断Rsb U的N端主要与RsbT结合来接收信号,而C端与RsbT的结合属于弱结合,用于去磷酸化,从而完成上下游之间应激信号传导。In the stress signal transduction pathway,the regulator of sigma B protein T(RsbT)can be activated and phosphorylated under environmental stress.However,how it transmits its signal to the downstream signaling protein regulator of sigma B protein U(RsbU)remains unknown.To prevent and control Vibrio infection and clarify the tolerance mechanism and stress signal transduction of Vibrio under environmental stress,using Vibrio brasiliensis as a model strain,its physicochemical properties,functional sites and interaction mechanism were analyzed using bioinformatics software,the interaction mode of RsbU and RsbT was explored,and the enzymatic properties of RsbU were investigated.The results showed that the expression vectors were successfully constructed,and the RsbU proteins were purified.The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the molecular weight of these proteins was in accordance with the expected size.RsbU was essentially a 2C protein phosphatase(PP2C),and the results obtained from Sring,a database for protein interactions network analysis,showed that the interaction scores between RsbU and RsbT were more than 0.98.Enzymatic properties results showed that the RsbU was a Ser/Thr phosphatase with a weak dephosphorylation ability that mainly acted in the prephase stage,and did not carry a phosphate group itself.Therefore,it was inferred that the N-terminal of RsbU mainly bound to RsbT to receive signals,while the C-terminal of RsbU bound to RsbT weakly for dephosphorylation,thus completing the upstream and downstream stress signaling.

关 键 词：关键调控蛋白RsbU 磷酸酶 应激信号传导通路 去磷酸化活性 

分 类 号：TS201.3[轻工技术与工程—食品科学]