START家族Rv0164蛋白在耻垢分枝杆菌中的表达及垂钓互作蛋白

Expression and protein interaction analysis of the START family protein Rv0164 in Mycobacterium smegmatis

【目的】START家族蛋白的突变或者错误表达使哺乳动物产生肾上腺皮质增生、乳腺癌和结肠癌等疾病;START家族蛋白是植物发育过程中重要的调节因子;尚未阐明START家族蛋白作为细菌必需基因的作用机制。结核分枝杆菌必需基因Rv0164属于START家族,功能未知,研究Rv0164作用机制将为START家族分子机制增添新理论。【方法】生物信息学方法分析Rv0164序列特征;模式菌耻垢分枝杆菌中表达Rv0164并分析蛋白的细胞定位;Co-immunoprecipitation(Co-IP)方法垂钓Rv0164的相互作用蛋白,质谱鉴定互作蛋白,酵母双杂交和Pull down验证蛋白相互作用。【结果】Rv0164的N端17个氨基酸在分枝杆菌中不保守;Rv0164无信号肽;Rv0164定位在细胞质中,受蛋白降解机制调控,该机制在细菌生长平台期比对数期活性弱;N端缺失使Rv0164在平台期和对数期均不稳定;Rv0164结合多个胞内蛋白。【结论】Rv0164的N端肽段增加了蛋白的稳定性;Rv0164是一个胞内蛋白;Rv0164能够结合细菌生存必需蛋白。

[Objective]Mutations or abnormal expressions of START family proteins in mammals are the basis for diseases such as adrenal hyperplasia,breast cancer and colon cancer.START family proteins are key regulators of plant development.The mechanism of the essential START proteins in prokaryote is unclear.Rv0164 belongs to the START family and is an essential gene with unknown function in Mycobacterium tuberculosis.Therefore,exploring the function of Rv0164 will add a new theory to the molecular mechanisms of START family.[Methods]Bioinformatics method was used to analyze the sequence of Rv0164.Rv0164 was expressed in the model strain Mycobacterium smegmatis and analyzed the protein localization.The interaction proteins of Rv0164 were identified by Co-IP and mass spectrometry,then verified by yeast two hybrid and pull down.[Results]The N-terminal 17 residues of Rv0164 were not conserved in Mycobacterium.Rv0164 had no signal peptide.Rv0164 was localized in cytoplasm and regulated by some proteostasis mechanisms which were weaker in stationary phase than in logarithmic phase.Deletion of the N-terminus of Rv0164 produced protein instability in both stationary phase and logarithmic phase.Rv0164 interacted with several cytosolic proteins.[Conclusion]The N-terminus of Rv0164 enhances protein stability.Rv0164 localizes in cytoplasm and can bind essential proteins.