极长链脂肪酸的合成缺陷对酵母细胞膜的稳定性和多烯类药物敏感性的影响
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摘要
【背景】脂肪酸延长酶家族参与脂肪酸代谢具有真核生物的高度保守性,且与膜脂的代谢密切相关。但细胞极长链脂肪酸(Very long-chain fatty acid,VLCFA)的合成缺陷对膜的稳定性及多烯类药物的敏感性影响并不完全明晰。【目的】探究细胞VLCFA延长酶ELO1、ELO2和ELO3的作用及功能。【方法】研究脂肪酸延长酶缺陷型elo1?、elo2?和elo3?对多烯类药物两性霉素B (Amphotericin B,AmB)、制霉菌素(Nystatin,Ny)及唑类硝酸益康唑(Econazolenitrate,Eco)的响应,检测不同酵母细胞的麦角固醇,检测其对Na+的响应及胞内钠钾离子水平。【结果】发现细胞VLCFA延长酶ELO2和ELO3缺陷后对AmB高度敏感;VLCFA延长酶缺陷突变株elo2?和elo3?对其它多烯类药物Ny及唑类药物Eco也十分敏感;细胞膜不饱和脂肪酸增加也会改变膜的稳定性,实验结果表明外源油酸(Oleic acid,OLA)增加了elo2?和elo3?突变体的AmB敏感性;相对野生型BY4741和elo1?,缺陷菌株elo2?和elo3?中麦角固醇的含量有显著下降;钠钾离子平衡是维护细胞正常生理的必要条件,也是检测细胞膜稳定性的重要参数,发现VLCFA的合成缺陷菌株对高浓度的NaCl比野生型菌株更敏感,使用ICP-AES检测不同浓度AmB胁迫下细胞内钠钾离子水平,也显示VLCFA延长酶缺陷菌株中,钠水平表现出上升趋势,并且细胞内钾含量明显降低。【结论】细胞VLCFA的合成缺陷会导致细胞膜更脆弱、稳定性下降,从而提高真菌对多烯类药物的敏感性,也表明脂肪酸延长酶是潜在的抗真菌治疗靶点。
[Background] Very long-chain fatty acids(VLCFA) are involved in the synthesis of membrane lipid sphingolipids and closely related to the metabolism of membrane lipid ergosterol and phospholipids.Defects in the synthesis of VLCFA in cells can damage the stability of cell membranes and thus enhance the fungal susceptibility to polyene-based drugs. However, the effect of the synthesis defects of VLCFA on the membrane stability and the sensitivity to polyene drugs is not entirely clear. [Objective] The role and function of the cell long-chain fatty acid elongases ELO1, ELO2 and ELO3. [Methods] The response of mutants with the synthesis defects of VLCFA, elo1?, elo2? and elo3?, to amphotericin B(AmB), one of the polyene drugs, and other drugs, such as nystatin(Ny) and econazole nitrate(Eco) was investigated.The ergosterol contents of different yeast cells were detected, and its response to Na+ and intracellular sodium and potassium levels was examined. [Results] We found that elo2? and elo3? mutants were highly sensitive to AmB. In addition, these two mutants were also sensitive to other drugs, such as Ny and Eco.Generally increased unsaturated fatty acids affect membrane stability. Our results showed that exogenous oleic acid(OLA) increased the sensitivity of elo2? and elo3? mutants to AmB. Compared with those of the wild-type and elo1? mutant, the ergosterol contents of the defective strains elo2? and elo3? decreased significantly. Sodium-potassium ion balance was a necessary condition for maintaining the normal physiology of cells, and was also an important parameter for measuring cell membrane stability. We found that defective strains were more sensitive to high concentrations of NaCl than wild-type strains. The levels of sodium and potassium in cells under different concentrations of AmB were detected by ICP-AES. The data showed that sodium levels were elevated in VLCFA synthesis-deficient strains compared with the wild-type strain. The potassium content in mutants was decreased significantly. [Conclusion] The synthetic defects of VLCFA could lead to the more fragile cell membrane and lower stability, thus increasing the sensitivity of fungi to polyenes. It suggests that fatty acid elongase could be the target of potential antifungal therapy.
[Background] Very long-chain fatty acids(VLCFA) are involved in the synthesis of membrane lipid sphingolipids and closely related to the metabolism of membrane lipid ergosterol and phospholipids.Defects in the synthesis of VLCFA in cells can damage the stability of cell membranes and thus enhance the fungal susceptibility to polyene-based drugs. However, the effect of the synthesis defects of VLCFA on the membrane stability and the sensitivity to polyene drugs is not entirely clear. [Objective] The role and function of the cell long-chain fatty acid elongases ELO1, ELO2 and ELO3. [Methods] The response of mutants with the synthesis defects of VLCFA, elo1?, elo2? and elo3?, to amphotericin B(AmB), one of the polyene drugs, and other drugs, such as nystatin(Ny) and econazole nitrate(Eco) was investigated.The ergosterol contents of different yeast cells were detected, and its response to Na+ and intracellular sodium and potassium levels was examined. [Results] We found that elo2? and elo3? mutants were highly sensitive to AmB. In addition, these two mutants were also sensitive to other drugs, such as Ny and Eco.Generally increased unsaturated fatty acids affect membrane stability. Our results showed that exogenous oleic acid(OLA) increased the sensitivity of elo2? and elo3? mutants to AmB. Compared with those of the wild-type and elo1? mutant, the ergosterol contents of the defective strains elo2? and elo3? decreased significantly. Sodium-potassium ion balance was a necessary condition for maintaining the normal physiology of cells, and was also an important parameter for measuring cell membrane stability. We found that defective strains were more sensitive to high concentrations of NaCl than wild-type strains. The levels of sodium and potassium in cells under different concentrations of AmB were detected by ICP-AES. The data showed that sodium levels were elevated in VLCFA synthesis-deficient strains compared with the wild-type strain. The potassium content in mutants was decreased significantly. [Conclusion] The synthetic defects of VLCFA could lead to the more fragile cell membrane and lower stability, thus increasing the sensitivity of fungi to polyenes. It suggests that fatty acid elongase could be the target of potential antifungal therapy.
引文
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[14]Wang XY.Effect of salt stress and aluminum stress on the composition and content of membrane lipids in rice[D].Beijing:Master’s Thesis of University of Chinese Academy of Sciences,2016(in Chinese)王鑫月.盐胁迫和铝胁迫对水稻膜脂组分和含量的影响[D].北京:中国科学院大学硕士学位论文,2016
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[22]Hama K,Fujiwara Y,Morita M,et al.Profiling and imaging of phospholipids in brains of Abcd1-deficient mice[J].Lipids,2018,53(1):85-102
[23]Cheng X,Du XX,Wang HD,et al.Study on the response of fatty acid elongase defects to lipid metabolism and oleic acid stress in Saccharomyces cerevisiae[J].Chinese Journal of Cell Biology,2018,40(9):1494-1503(in Chinese)程迅,杜秀秀,王寒冬,等.脂肪酸延长酶缺陷对酵母细胞脂质代谢及油酸胁迫响应的影响[J].中国细胞生物学报,2018,40(9):1494-1503
[24]Peter A,Weigert C,Staiger H,et al.Induction of stearoyl-CoAdesaturase protects human arterial endothelial cells against lipotoxicity[J].American Journal of Physiology-Endocrinology and Metabolism,2008,295(2):E339-E349
[25]Nguyen LN,Nosanchuk JD.Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis:an essential role of fatty acid desaturase Ole1[J].Cell Cycle,2011,10(18):3159-3167
[26]Lockshon D,Surface LE,Kerr EO,et al.The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function[J].Genetics,2007,175(1):77-91
[27]Zhai ZH,Wang XZ,Ding MX.Cell Biology[M].4th ed.Beijing:Higher Education Press,2011:62(in Chinese)翟中和,王喜忠,丁明孝.细胞生物学[M].4版.北京:高等教育出版社,2011:62
[28]Sun TH,Zhao CX,Jin FX.Effect of several kinds of inorganic ions on yeasts metabolism and mechanisms of acid producing in fermentation[J].Journal of Dalian Institute of Light Industry,2002,21(1):29-32(in Chinese)孙廷宏,赵长新,金凤燮.几种无机离子对啤酒酵母生理代谢的影响及发酵过程的产酸机制[J].大连轻工业学院学报,2002,21(1):29-32
[29]Quist TM,Sokolchik I,Shi HZ,et al.HOS3,an ELO-like gene,inhibits effects of ABA and implicates a S-1-P/ceramide control system for abiotic stress responses in Arabidopsis thaliana[J].Molecular Plant,2009,2(1):138-151
[2]Wang Q,Du XX,Ma K,et al.A critical role for very long-chain fatty acid elongases in oleic acid-mediated Saccharomyces cerevisiae cytotoxicity[J].Microbiological Research,2018,207:1-7
[3]Toke DA,Martin CE.Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,1996,271:18413-18422
[4]Tehlivets O,Scheuringer K,Kohlwein SD.Fatty acid synthesis and elongation in yeast[J].Biochimica Et Biophysica Acta,2007,1771(3):255-270
[5]Ohanian J,Ohanian V.Sphingolipids in mammalian cell signalling[J].Cellular&Molecular Life Science,2001,58(14):2053-2068
[6]Schneiter R,Brügger B,Amann CM,et al.Identification and biophysical characterization of a very-long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes[J].Biochemical Journal,2004,381(3):941-949
[7]Lorenz RT,Parks LW.Physiological effects of fenpropimorph on wild-type Saccharomyces cerevisiae and fenpropimorph-resistant mutants[J].Antimicrobial Agents&Chemotherapy,1991,35(8):1532-1537
[8]David D,Sundarababu S,Gerst JE.Involvement of long chain fatty acid elongation in the trafficking of secretory vesicles in yeast[J].Journal of Cell Biology,1998,143(5):1167-1182
[9]Eisenkolb M,Zenzmaier C,Leitner E,et al.A specific structural requirement for ergosterol in long-chain fatty acid synthesis mutants important for maintaining raft domains in yeast[J].Molecular Biology of the Cell,2002,13(12):4414-4428
[10]Jiang ZL,Lin JQ.New progress in research on polyene antifungal drugs[J].China Pharmaceuticals,2006,15(7):62-63(in Chinese)蒋正立,林建群.多烯类抗真菌药物的研究新进展[J].中国药业,2006,15(7):62-63
[11]Kaiser C,Michaelis S,Mitchell A.Methods in Yeast Genetics:ACold Spring Harbor Laboratory Course Manual[M].New York:Cold Spring Harbor Laboratory Press,1994:232
[12]Kurat CF,Natter K,Petschnigg J,et al.Obese yeast:triglyceride lipolysis is functionally conserved from mammals to yeast[J].Journal of Biological Chemistry,2006,281(1):491-500
[13]Arthington-Skaggs BA,Jradi H,Desai T,et al.Quantitation of ergosterol content:novel method for determination of fluconazole susceptibility of Candida albicans[J].Journal of Clinical Microbiology,1999,37(10):3332-3337
[14]Wang XY.Effect of salt stress and aluminum stress on the composition and content of membrane lipids in rice[D].Beijing:Master’s Thesis of University of Chinese Academy of Sciences,2016(in Chinese)王鑫月.盐胁迫和铝胁迫对水稻膜脂组分和含量的影响[D].北京:中国科学院大学硕士学位论文,2016
[15]Shreaz S,Shiekh RA,Raja V,et al.Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II)complex with ligand derived from cinnamaldehyde[J].Chemico-Biological Interactions,2016,247:64-74
[16]Gruszecki WI,Gago?M,Here?M,et al.Organization of antibiotic amphotericin B in model lipid membranes.A mini review[J].Cellular&Molecular Biology Letters,2003,8:161-170
[17]Ostrosky-Zeichner L,Casadevall A,Galgiani JN,et al.An insight into the antifungal pipeline:selected new molecules and beyond[J].Nature Reviews Drug Discovery,2010,9(9):719-727
[18]Fanos V,Cataldi L.Amphotericin B-induced nephrotoxicity:a review[J].Journal of Chemotherapy,2000,12(6):463-470
[19]Mansfield BE,Oltean HN,Oliver BG,et al.Azole drugs are imported by facilitated diffusion in Candida albicans and other pathogenic fungi[J].PLoS Pathogens,2010,6(9):e1001126
[20]White TC,Marr KA,Bowden RA.Clinical,cellular,and molecular factors that contribute to antifungal drug resistance[J].Clinical Microbiology Reviews,1998,11(2):382-402
[21]Lupetti A,Danesi R,Campa M,et al.Molecular basis of resistance to azole antifungals[J].Trends in Molecular Medicine,2002,8(2):76-81
[22]Hama K,Fujiwara Y,Morita M,et al.Profiling and imaging of phospholipids in brains of Abcd1-deficient mice[J].Lipids,2018,53(1):85-102
[23]Cheng X,Du XX,Wang HD,et al.Study on the response of fatty acid elongase defects to lipid metabolism and oleic acid stress in Saccharomyces cerevisiae[J].Chinese Journal of Cell Biology,2018,40(9):1494-1503(in Chinese)程迅,杜秀秀,王寒冬,等.脂肪酸延长酶缺陷对酵母细胞脂质代谢及油酸胁迫响应的影响[J].中国细胞生物学报,2018,40(9):1494-1503
[24]Peter A,Weigert C,Staiger H,et al.Induction of stearoyl-CoAdesaturase protects human arterial endothelial cells against lipotoxicity[J].American Journal of Physiology-Endocrinology and Metabolism,2008,295(2):E339-E349
[25]Nguyen LN,Nosanchuk JD.Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis:an essential role of fatty acid desaturase Ole1[J].Cell Cycle,2011,10(18):3159-3167
[26]Lockshon D,Surface LE,Kerr EO,et al.The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function[J].Genetics,2007,175(1):77-91
[27]Zhai ZH,Wang XZ,Ding MX.Cell Biology[M].4th ed.Beijing:Higher Education Press,2011:62(in Chinese)翟中和,王喜忠,丁明孝.细胞生物学[M].4版.北京:高等教育出版社,2011:62
[28]Sun TH,Zhao CX,Jin FX.Effect of several kinds of inorganic ions on yeasts metabolism and mechanisms of acid producing in fermentation[J].Journal of Dalian Institute of Light Industry,2002,21(1):29-32(in Chinese)孙廷宏,赵长新,金凤燮.几种无机离子对啤酒酵母生理代谢的影响及发酵过程的产酸机制[J].大连轻工业学院学报,2002,21(1):29-32
[29]Quist TM,Sokolchik I,Shi HZ,et al.HOS3,an ELO-like gene,inhibits effects of ABA and implicates a S-1-P/ceramide control system for abiotic stress responses in Arabidopsis thaliana[J].Molecular Plant,2009,2(1):138-151