Functional Characterization of Sirtuin-like Protein in Mycobacterium smegmatis
Functional Characterization of Sirtuin-like Protein in Mycobacterium smegmatis
复制标题
耻垢分枝杆菌中 Sirtuin 样蛋白的功能表征
DOI：
10.1021/acs.jproteome.5b00359
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发表时间：
2015-11-01
影响因子：
4.4
通讯作者：
Deng, Haiteng
中科院分区：
文献类型：
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作者：
Gu, Lixiao;Chen, Yuling;Deng, Haiteng
烟酰胺腺嘌呤二核苷酸（NAD）依赖的去乙酰化酶（sirtuins）在原核生物和真核生物中都是保守的。哺乳动物sirtuins的功能和调控已被广泛研究，并表明sirtuins在调节生物过程中发挥重要作用，而分枝杆菌sirtuins的功能则较少探索。为了研究sirtuin样蛋白在分枝杆菌中的功能，本研究将耻垢分枝杆菌的sirtuin MSMEG_5175在耻垢分枝杆菌菌株mc(2)155中过表达，从而产生MSMEG_5175过表达菌株mc(2)155- ms5175。对mc(2)155- ms5175菌株的生理特性进行了分析，结果表明，与含空pMV261质粒的mc(2)155 （mc(2)155-pMV261）相比，其胞内NAD水平较低，对异烟肼（INH）的抗性较高。定量蛋白质组学分析mc(2)155-pMV261和mc(2)155-MS5175之间的差异表达蛋白。在3032个鉴定的蛋白中，MSMEG_5175过表达导致34个蛋白上调，72个蛋白下调，这些蛋白涉及代谢激活、转录和翻译、抗氧化和DNA修复等多种细胞过程。mc(2)155-MS5175菌株过氧化氢酶（catalase peroxidase, KatG） mRNA和蛋白表达水平均下调，表明mc(2)155-MS5175细胞NAD含量降低和KatG表达下调是其抗INH能力增强的原因之一。结合免疫沉淀和蛋白质组学分析，研究人员发现，与mc(2)155-pMV261相比，mc(2)155-MSS175中27个蛋白的乙酰化程度降低，这表明这些蛋白包括RNA聚合酶（rpoC）的β引物亚基、核糖体蛋白和代谢酶是MSMEG_5175的底物。rpoC的乙酰化变化可能会影响其功能并引起全局基因转录的改变。综上所述，这些结果表明MSMEG_5175调节多种细胞过程，导致分枝杆菌对INH的抗性增加，并为进一步探索分枝杆菌中蛋白质乙酰化的功能提供了有用的生物学资源。
Nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (sirtuins) are well conserved from prokaryotes to eukaryotes. Functions and regulations of mammalian sirtuins have been extensively studied and indicate that sirtuins play an important role in regulation of biological processes, whereas functions of mycobacterial sirtuins were less explored. To examine functions of the sirtuin-like protein in mycobacteria, a Mycobacterium smegmatis sirtuin, MSMEG_5175, was overexpressed in a M. smegmatis strain mc(2)155 to generate an MSMEG_5175-overexpression strain (mc(2)155-MS5175) in the present study. The physiological aspects of mc(2)155-MS5175 strain were characterized showing that they had a lower intracellular NAD level and a higher resistance to isoniazid (INH) as compared to mc(2)155 containing empty pMV261 plasmid (mc(2)155-pMV261). Quantitative proteomic analysis was carried out to determine differentially expressed proteins between mc(2)155-pMV261 and mc(2)155-MS5175. Among 3032 identified proteins, overexpression of MSMEG_5175 results in up-regulation of 34 proteins and down-regulation of 72 proteins, which involve in diverse cellular processes including metabolic activation, transcription and translation, antioxidant, and DNA repair. Down-regulation of catalase peroxidase (KatG) expression in both mRNA and protein levels were observed in mc(2)155-MS5175 strain, suggesting that a decrease in cellular NAD content and down-regulation of KatG expression contribute to the higher resistance to INH in mc(2)155-MS5175. Using a combination of immunoprecipitation and proteomic analysis, we found that acetylation in 27 proteins was decreased in mc(2)155-MSS175 as compared to those in mc(2)155-pMV261, suggesting that these proteins including the beta prime subunit of RNA polymerase (rpoC), ribosomal proteins, and metabolic enzymes were substrates of MSMEG_5175. Acetylation changes in rpoC may affect its function and cause changes in global gene transcription. Taken together, these results suggest that MSMEG_5175 regulates diverse cellular processes resulting in an increase in INH resistance in mycobacteria, and provide a useful resource to further biological exploration into functions of protein acetylation in mycobacteria.