乙酰化修饰调控结核杆菌异柠檬酸裂合酶的研究

doi:10.13523/j.cb.20150602

中国生物工程杂志

2015, Vol. 35

Issue (6): 8-13 DOI: 10.13523/j.cb.20150602

研究报告

乙酰化修饰调控结核杆菌异柠檬酸裂合酶的研究

李瑶瑶, 毕静, 王艺红, 秦云贺, 张雪莲

复旦大学生命科学学院遗传工程国家重点实验室上海 200433

Lysine-322 Acetylation Negatively Regulates Isocitrate Lyase of Mycobacterium tuberculosis

LI Yao-yao, BI Jing, WANG Yi-hong, QIN Yun-he, ZHANG Xue-lian

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China

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摘要：

目的:探索结核杆菌异柠檬酸裂合酶(ICL)蛋白322位点赖氨酸(Lys322)的乙酰化修饰对蛋白功能的调控作用。方法:构建结核杆菌ICL蛋白原核表达载体pET28a-icl,并对Lys322位点进行定点突变为精氨酸(Arg,R)和谷氨酰胺(Glu,Q),体外表达纯化获得重组蛋白ICL^WT、ICL^322R和ICL^322Q。通过Western blotting和酶活性测定来揭示Lys322位点突变前后对蛋白的乙酰化修饰水平及蛋白功能的影响。结果:Western blotting检测发现大肠杆菌表达体系获得的ICL^WT、ICL^322R和ICL^322Q蛋白均有较高水平的蛋白赖氨酸乙酰化修饰信号,较ICL^WT,ICL^322R和ICL^322Q突变蛋白的酶活性分别下降了大约50%和70%。结论:在大肠杆菌的表达体系中,ICL蛋白可以获得乙酰化修饰。ICL^322Q突变蛋白酶活性的显著下降,揭示Lys322位点乙酰化修饰对ICL蛋白的功能存在负向调控。为未来深入探索赖氨酸乙酰化修饰对结核杆菌代谢,潜伏感染的调控作用奠定了基础。

关键词： 异柠檬酸裂合酶; 乙酰化修饰; 定点突变

Abstract:

Objective: Isocitrate lyase (ICL) of Mycobacterium tuberculosis (Mtb) was acetylated at lysine 322. The aim was to investigate ICL Lys322 acetylation and its regulatory function in activity of ICL. Methods: The prokaryotic expression plasmids pET28a-icl was constructed and Lys322 of ICL was site-directed mutated into arginine (Arg, R) and glutamine (Gln, Q), respectively. Then recombinant proteins ICL^WT,ICL^322R and ICL^322Qwere expressed and purified in vitro. Acetylation of wild type ICL and its mutants were examined by Western blotting with anti-acetyllysine antibody, and enzyme activities of proteins were evaluated. Results: Western blotting showed that ICL^WT,ICL^322R and ICL^322Q were indeed acetylated proteins. Compared with activity of ICL^WT, mutation of Lys322 to arginine decreased the enzyme activity by approximately 50%, and glutamine substitution dramatically decreased the activity more than 70%. Conclusion: The results of Western blotting showed that the ICL and two mutant proteins can be acetylated effectively in E. coli expression system. Decreasing in ICL^322Q activity suggested that Lys322 acetylation could negatively regulate isocitrate lyase of Mycobacterium tuberculosis. This provided a foundation to study an important aspect of Mtb, the metabolic regulation of Mtb, which may lead to new knowledge on latent infection.

Key words: Isocitrate lyase acetylation site-directed mutagenesis

收稿日期: 2015-03-13 出版日期: 2015-06-25

ZTFLH:

Q81

基金资助:

国家自然科学基金资助项目(81261120558、30901828)

通讯作者: 张雪莲 E-mail: xuelianzhang@fudan.edu.cn

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引用本文:

李瑶瑶, 毕静, 王艺红, 秦云贺, 张雪莲. 乙酰化修饰调控结核杆菌异柠檬酸裂合酶的研究[J]. 中国生物工程杂志, 2015, 35(6): 8-13.

LI Yao-yao, BI Jing, WANG Yi-hong, QIN Yun-he, ZHANG Xue-lian. Lysine-322 Acetylation Negatively Regulates Isocitrate Lyase of Mycobacterium tuberculosis. China Biotechnology, 2015, 35(6): 8-13.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150602 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I6/8

[1] 毕静, 张雪莲. 原核生物中蛋白质乙酰化修饰的研究进展. 中国生物工程杂志, 2013, 33(9): 79-84. Bi J, Zhang X L.Protein acetylation in prokaryotes. China Biotechnology, 2013,33(9):79-84.

[2] Yu B J, Kim J A, Moon J H, et al. The diversity of lysine-acetylated proteins in Escherichia coli. Journal of Microbiology and Biotechnology, 2008, 18(9): 1529-1536.

[3] Wang Q, Zhang Y, Yang C, et al. Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science, 2010, 327(5968): 1004-1007.

[4] Liu F, Yang M, Wang X, et al. Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis. Molecular & Cellular Proteomics, 2014, 13(12): 3352-336

[5] Xie L, Wang X, Zeng J, et al. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis. The International Journal of Biochemistry & Cell Biology, 2015, 59(2): 193-202.

[6] Murthy PS, Sirsi M, Ramakrishnan T. Effect of age on the enzymes of tricarboxylic acid and related cycles in Mycobacterium tuberculosis H37Rv. Am Rev Respir Dis, 1973, 108(3): 689-690.

[7] Zu Bentrup K H, Miczak A, Swenson D L, et al. Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis. Journal of Bacteriology, 1999, 181(23): 7161-7167.

[8] Wayne L G, Lin K Y. Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions. Infect Immun, 1982, 37 (3): 1042-1049.

[9] Sturgill-Koszycki S, Haddix PL, Russell D G. The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis, 1997, 18(14): 2558-2565.

[10] Xiong Y, Guan K L. Mechanistic insights into the regulation of metabolic enzymes by acetylation. The Journal of Cell Biology, 2012, 198(2): 155-164.

[11] Yu W, Lin Y, Yao J, et al. Lysine 88 acetylation negatively regulates ornithine carbamoyltransferase activity in response to nutrient signals. Journal of Biological Chemistry, 2009, 284(20): 13669-13675.

[12] Zhao D, Zou S W, Liu Y, et al. Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer. Cancer Cell, 2013, 23(4): 464-476.

[13] Kim E Y, Kim W K, Kang H J, et al. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity. Journal of Lipid Research, 2012, 53(9): 1864-1876.

[14] Muñoz-Elías E J, Upton A M, Cherian J, et al. Role of the methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular growth, and virulence. Molecular Microbiology, 2006, 60(5): 1109-1122.

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