二硫键氧化还原异构酶(DsbM)与转录调控因子(PA0056)的相互作用
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摘要
二硫键氧化还原异构酶(disulfide oxidoreductase,DsbM)分布于原核细胞中,主要参与周质空间中膜蛋白的折叠.转录调控因子(lysR type transcriptional regulator,PA0056)是一类包含4个半胱氨酸的蛋白,当以氧化态形式存在时,可调控下游基因的转录,参与调节抗氧化或者抗药性.本实验室前期研究证明DsbM参与氨基糖苷类耐药性的调节.关注铜绿假单胞菌中DsbM和PA0056之间的相互作用,分别构建了酵母双杂交(yeast two-hybrid test)和双分子荧光互补系统(bi-molecular fluorescence complementation, BiFC)的载体.通过酵母双杂交实验,发现DsbM和PA0056之间存在较强的蛋白质相互作用.通过BiFC实验,发现共同转化表达DsbM和PA0056载体的酵母中可观察到强烈的黄色荧光,表明DsbM和PA0056可以发生相互作用.本研究为深入研究DsbM在铜绿假单胞菌耐氨基糖苷类抗生素耐药机制奠定基础.
Disulfide oxidoreductase(DsbM) exists widely in prokaryotic cells. DsbM was involved in the protein folding pathway of many cell envelope proteins in the periplasmic space. LysR type transcriptional regulator(PA0056), has four cysteines. Oxidized PA0056 can activate the transcription of genes which regulate antioxidant process or antibiotic resistance. Previous work in this laboratory has demonstrated that DsbM was involved in the regulation of aminoglycoside resistance. This study focused on the interaction between DsbM and PA0056. Vectors were constructed for yeast two-hybrid and Bi-molecular fluorescence complementation(BiFC), respectively. Through yeast two-hybrid test, it was found that there is a physical interaction between DsbM and PA0056. Through the BiFC test, it was found that a strong yellow fluorescence signal could be observed in yeast when co-transforming yeast with DsbM and PA0056. These results indicated that DsbM and PA0056 can interact with each other. This study lay a foundation for further exploring the function and mechanism of DsbM regulated resistance to aminoglycoside in Pseudomonas aeruginosa infection.
Disulfide oxidoreductase(DsbM) exists widely in prokaryotic cells. DsbM was involved in the protein folding pathway of many cell envelope proteins in the periplasmic space. LysR type transcriptional regulator(PA0056), has four cysteines. Oxidized PA0056 can activate the transcription of genes which regulate antioxidant process or antibiotic resistance. Previous work in this laboratory has demonstrated that DsbM was involved in the regulation of aminoglycoside resistance. This study focused on the interaction between DsbM and PA0056. Vectors were constructed for yeast two-hybrid and Bi-molecular fluorescence complementation(BiFC), respectively. Through yeast two-hybrid test, it was found that there is a physical interaction between DsbM and PA0056. Through the BiFC test, it was found that a strong yellow fluorescence signal could be observed in yeast when co-transforming yeast with DsbM and PA0056. These results indicated that DsbM and PA0056 can interact with each other. This study lay a foundation for further exploring the function and mechanism of DsbM regulated resistance to aminoglycoside in Pseudomonas aeruginosa infection.
引文
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16 Li M,Guan X,Wang X,et al.DsbM affects aminoglycoside resistance in Pseudomonas aeruginosa by the reduction of OxyR[J].FEMS Microbiol Lett,2014,352(2):184-189.
17白芳,殷腾飞,徐海津,等.用酵母双杂交系统研究铜绿假单胞菌EtfA和EtfB蛋白的相互作用[J].南开大学学报:自然科学版,2008,41(2):1-6.
18刘盈盈,刘宇,黄奎,等.拟南芥钙依赖蛋白激酶CPK12与bZIP转录因子ABF4相互作用研究[J].四川大学学报,2018,55(6):1 301-1 306.
19袁敏,邢继红,王莉,等.拟南芥开花抑制因子TFL1与GRFs蛋白的相互作用[J].中国农业科学,2017,50(10):1772-1780.
20麻楠,乔金柱,孙天杰,等.小麦翻译控制肿瘤蛋白(TCTP)与索马甜类蛋白(TLP)的相互作用[J].农业生物技术学报,2018,26(6):911-919.
21 Martin J L.Thioredoxina fold for all reasons[J].Structure,1995,3(3):245-250.
2 Carmel H O,Storz G.Roles of the glutathione-and thioredoxin-dependent reduction systems in the Escherichia coli and saccharomyces cerevisiae responses to oxidative stress[J].Annu Rev Microbiol,2000,54(1):439-461.
3 Lee D G,Urbach J M,Wu G,et al.Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[J].Genome Biology,2006,7(10):R90-R98.
4 Wook C,Small D A,Freshteh T,et al.Microarray analysis of toxicogenomic effects of peracetic acid on Pseudomonas aeruginosa[J].Environ Sci Technol,2005,39(15):5 893-5 899.
5 Lau G W,Britigan B E,Hassett D J.Pseudomonas aeruginosa Oxy R is required for full virulence in rodent and insect models of infection and for resistance to human neutrophils[J].Infection&Immunity,2005,73(4):2 550-2 553.
6 Wang X,Li M,Liu L,et al.DsbM,a novel disulfide oxidoreductase affects aminoglycoside resistance in Pseudomonas aeruginosa by OxyR-regulated response[J].J Microbiol,2012,50(6):932-938.
7 Kalai M,Giri N,Camilo V,et al.Dynamics of Pseudomonas aeruginosa genome evolution[J].Proc Natl Acad Sci USA,2008,105(8):3 100-3 105.
8 Kurokawa Y,Yanagi H,Yura T.Overexpression of protein disulfide isomerase DsbC stabilizes multiple-disulfide-bonded recombinant protein produced and transported to the periplasm in Escherichia coli[J].Applied&Environmental Microbiology,2000,66(9):3 960-3 965.
9 Winsor G L V R,Thea,Lo R,Khaira B,et al.Pseudomonas genome database:facilitating user-friendly,comprehensive comparisons of microbial genomes[J].Nucleic Acids Res,2009,37(1):D483.
10 Pérez R E,Collado V J.Common history at the origin of the position-function correlation in transcriptional regulators in archaea and bacteria[J].J Mol Evol,2001,53(3):172-179.
11 Junsong S,Albrecht K.A lysR-type regulator is involved in the negative regulation of genes encoding seleniumfree hydrogenases in the archaeon Methanococcus voltae[J].Mol Microbiol,2010,52(2):563-571.
12 Stec E,Witkowska Z M,Hryniewicz M,et al.Structural basis of the sulphate starvation response in E-coli:Crystal structure and mutational analysis of the cofactor-binding domain of the cbl transcriptional regulator[J].JMol Biol,2006,364(3):309-322.
13 Kullik I,Stevens J,Toledano M B,et al.Mutational analysis of the redox-sensitive transcriptional regulator OxyR:regions important for DNA binding and multimerization[J].J Bacteriol,1995,177(5):1 285-1 291.
14 Christman M F,Storz G,Ames B N.OxyR,a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium,is homologous to a family of bacterial regulatory proteins[J].Proc Natl Acad Sci U S A,1989,86(10):3 484-3 488.
15 Jolanta Z,Kierzek A M.The structure of full-length LysR-type transcriptional regulators.Modeling of the fulllength OxyR transcription factor dimer[J].Nucleic Acids Res,2003,31(5):1 444-1 454.
16 Li M,Guan X,Wang X,et al.DsbM affects aminoglycoside resistance in Pseudomonas aeruginosa by the reduction of OxyR[J].FEMS Microbiol Lett,2014,352(2):184-189.
17白芳,殷腾飞,徐海津,等.用酵母双杂交系统研究铜绿假单胞菌EtfA和EtfB蛋白的相互作用[J].南开大学学报:自然科学版,2008,41(2):1-6.
18刘盈盈,刘宇,黄奎,等.拟南芥钙依赖蛋白激酶CPK12与bZIP转录因子ABF4相互作用研究[J].四川大学学报,2018,55(6):1 301-1 306.
19袁敏,邢继红,王莉,等.拟南芥开花抑制因子TFL1与GRFs蛋白的相互作用[J].中国农业科学,2017,50(10):1772-1780.
20麻楠,乔金柱,孙天杰,等.小麦翻译控制肿瘤蛋白(TCTP)与索马甜类蛋白(TLP)的相互作用[J].农业生物技术学报,2018,26(6):911-919.
21 Martin J L.Thioredoxina fold for all reasons[J].Structure,1995,3(3):245-250.