巴斯德毕赤酵母Orm1蛋白在细胞生长和内质网功能维持方面的功能
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摘要
【目的】鉴定巴斯德毕赤酵母ORM1基因;研究ORM1基因缺失对毕赤酵母生长、内质网压力应答、细胞钙稳态调节和活性氧水平等方面的影响。【方法】利用生物信息学软件对毕赤酵母Orm1蛋白进行序列比对和分析;利用PCR介导的同源重组法构建orm1Δ缺失菌株,将回补质粒p IB1-ORM1转入orm1Δ菌株构建回补菌株;研究ORM1基因缺失对毕赤酵母生长的影响;以Fluo-3 AM染色法测定胞质钙含量;以DCFH-DA染色法分析胞内活性氧水平;以实时荧光定量PCR技术研究ORM1基因缺失对毕赤酵母非折叠蛋白应答、钙稳态和抗氧化系统基因表达的影响;使用试剂盒分析毕赤酵母抗氧化系统过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性及谷胱甘肽(GSH)的含量。【结果】在毕赤酵母基因组数据库中比对出酿酒酵母Orm1和Orm2的同源蛋白,并将该蛋白编码基因命名为ORM1;毕赤酵母ORM1基因缺失导致细胞生长受到明显抑制,对衣霉素引起的内质网压力敏感性增强,非折叠蛋白应答激活,细胞钙稳态紊乱,活性氧积累,抗氧化系统激活。【结论】由于非折叠蛋白应答、钙稳态调节、活性氧积累等均与内质网功能息息相关,因此,巴斯德毕赤酵母ORM1基因编码的Orm1蛋白在细胞生长及内质网正常功能的维持过程中发挥重要作用。
[Objective] This study aimed to identify the ORM1 gene in Pichia pastoris, and to illustrate the effect of its deletion on cell growth, endoplasmic reticulum stress response, cell calcium homeostasis and reactive oxygen species accumulate on. [Methods] Sequence alignment and analysis were done by the relevant bioinformatic software. The orm1Δ mutant was constructed by PCR-mediated gene disruption, and the reconstituted strain orm1Δ+ORM1 was constructed by transforming orm1Δ with the p IB1-ORM1 plasmid. The growth rates of the strains were detected using both liquid and the solid media. Gene expression related to the unfolded protein response, calcium homeostasis and antioxidant system was detected via real-time PCR. The activity of antioxidant enzymes, including catalase and superoxide dismutase, and the content of reduced glutathione were measured by relevant kits. [Results] The P. pastoris ORM1 gene sharing high homology with S. cerevisiae ORM1 and ORM2, was identified in P. pastoris genome database. Deletion of ORM1 caused growth defect, increased sensitivity to endoplasmic reticulum stress caused by tunicamycin, activation of unfolded protein response, disturbance of calcium homeostasis, reactive oxygen species accumulation, and activation of the antioxidant system. [Conclusion] Because unfolded protein response, calcium homeostasis, and production of reactive oxygen species are all correlated with ER function, P. pastoris Orm1 protein plays critical roles in cell growth and maintenance of ER functions.
[Objective] This study aimed to identify the ORM1 gene in Pichia pastoris, and to illustrate the effect of its deletion on cell growth, endoplasmic reticulum stress response, cell calcium homeostasis and reactive oxygen species accumulate on. [Methods] Sequence alignment and analysis were done by the relevant bioinformatic software. The orm1Δ mutant was constructed by PCR-mediated gene disruption, and the reconstituted strain orm1Δ+ORM1 was constructed by transforming orm1Δ with the p IB1-ORM1 plasmid. The growth rates of the strains were detected using both liquid and the solid media. Gene expression related to the unfolded protein response, calcium homeostasis and antioxidant system was detected via real-time PCR. The activity of antioxidant enzymes, including catalase and superoxide dismutase, and the content of reduced glutathione were measured by relevant kits. [Results] The P. pastoris ORM1 gene sharing high homology with S. cerevisiae ORM1 and ORM2, was identified in P. pastoris genome database. Deletion of ORM1 caused growth defect, increased sensitivity to endoplasmic reticulum stress caused by tunicamycin, activation of unfolded protein response, disturbance of calcium homeostasis, reactive oxygen species accumulation, and activation of the antioxidant system. [Conclusion] Because unfolded protein response, calcium homeostasis, and production of reactive oxygen species are all correlated with ER function, P. pastoris Orm1 protein plays critical roles in cell growth and maintenance of ER functions.
引文
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[18]Volmer R,Ron D.Lipid-dependent regulation of the unfolded protein response[J].Current Opinion in Cell Biology,2015,33:67-73
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[20]Jadhav S,Russo S,Cottier S,et al.Valproate induces the unfolded protein response by increasing ceramide levels[J].Journal of Biological Chemistry,2016,291(42):22253-22261
[21]Gaspar ML,Aregullin MA,Jesch SA,et al.Inositol induces a profound alteration in the pattern and rate of synthesis and turnover of membrane lipids in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2006,281(32):22773-22785
[22]Chang HJ,Jones EW,Henry SA.Role of the unfolded protein response pathway in regulation of INO1 and in the sec14 bypass mechanism in Saccharomyces cerevisiae[J].Genetics,2002,162(1):29-43
[23]Brice SE,Alford CW,Cowart LA.Modulation of sphingolipid metabolism by the phosphatidylinositol-4-phosphate phosphatase Saclp through regulation of phosphatidylinositol in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2009,284(12):7588-7596
[24]Cyert MS.Calcineurin signaling in Saccharomyces cerevisiae:how yeast go crazy in response to stress[J].Biochemical and Biophysical Research Communications,2003,311(4):1143-1150
[25]Niles BJ,Joslin AC,Fresques T,et al.TOR Complex 2-Ypk1signaling maintains sphingolipid homeostasis by sensing and regulating ROS accumulation[J].Cell Reports,2014,6(3):541-552
[2]Hjelmqvist L,Tuson M,Marfany G,et al.ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins[J].Genome Biology,2002,3(6):research0027.1-0027.16
[3]Breslow DK,Collins SR,Bodenmiller B,et al.Orm family proteins mediate sphingolipid homeostasis[J].Nature,2010,463(7284):1048-1053
[4]Gururaj C,Federman RS,Chang A.Orm proteins integrate multiple signals to maintain sphingolipid homeostasis[J].Journal of Biological Chemistry,2013,288(28):20453-20463
[5]Hou NS,Gutschmidt A,Choi DY,et al.Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(22):E2271-E2280
[6]Wu HX,Ng BSH,Thibault G.Endoplasmic reticulum stress response in yeast and humans[J].Bioscience Reports,2014,34(4):e00118
[7]Pineau L,Colas J,Dupont S,et al.Lipid-induced ER stress:synergistic effects of sterols and saturated fatty acids[J].Traffic,2009,10(6):673-690
[8]Cox JS,Chapman RE,Walter P.The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane[J].Molecular and Cellular Biology,1997,8(9):1805-1814
[9]Bonilla M,Nastase KK,Cunningham KW.Essential role of calcineurin in response to endoplasmic reticulum stress[J].The EMBO Journal,2002,21(10):2343-2353
[10]Haynes CM,Titus EA,Cooper AA.Degradation of misfolded protein prevents ER-Derived oxidative stress and cell death[J].Molecular Cell,2004,15(5):767-776
[11]Tu BP,Weissman JS.Oxidative protein folding in eukaryotes:mechanisms and consequences[J].Journal of Cell Biology,2004,164(3):341-346
[12]Ternes P,Wobbe T,Schwarz M,et al.Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris[J].Journal of Biological Chemistry,2011,286(13):11401-11414
[13]Miyake Y,Kozutsumi Y,Nakamura S,et al.Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant,ISP-1/myriocin[J].Biochemical and Biophysical Research Communications,1995,211(2):396-403
[14]Klose C,Surma MA,Gerl MJ,et al.Flexibility of a eukaryotic lipidome-insights from yeast lipidomics[J].PLo S One,2012,7(4):e35063
[15]Mahto KK,Singh A,Khandelwal NK,et al.An assessment of growth media enrichment on lipid metabolome and the concurrent phenotypic properties of Candida albicans[J].PLo S One,2014,9(11):e113664
[16]Liu M,Huang CJ,Polu SR,et al.Regulation of sphingolipid synthesis through Orm1 and Orm2 in yeast[J].Journal of Cell Science,2012,125(10):2428-2435
[17]Han S,Lone MA,Schneiter R,et al.Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(13):5851-5856
[18]Volmer R,Ron D.Lipid-dependent regulation of the unfolded protein response[J].Current Opinion in Cell Biology,2015,33:67-73
[19]Epstein S,Kirkpatrick CL,Castillon GA,et al.Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells[J].The Journal of Lipid Research,2012,53(3):412-420
[20]Jadhav S,Russo S,Cottier S,et al.Valproate induces the unfolded protein response by increasing ceramide levels[J].Journal of Biological Chemistry,2016,291(42):22253-22261
[21]Gaspar ML,Aregullin MA,Jesch SA,et al.Inositol induces a profound alteration in the pattern and rate of synthesis and turnover of membrane lipids in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2006,281(32):22773-22785
[22]Chang HJ,Jones EW,Henry SA.Role of the unfolded protein response pathway in regulation of INO1 and in the sec14 bypass mechanism in Saccharomyces cerevisiae[J].Genetics,2002,162(1):29-43
[23]Brice SE,Alford CW,Cowart LA.Modulation of sphingolipid metabolism by the phosphatidylinositol-4-phosphate phosphatase Saclp through regulation of phosphatidylinositol in Saccharomyces cerevisiae[J].Journal of Biological Chemistry,2009,284(12):7588-7596
[24]Cyert MS.Calcineurin signaling in Saccharomyces cerevisiae:how yeast go crazy in response to stress[J].Biochemical and Biophysical Research Communications,2003,311(4):1143-1150
[25]Niles BJ,Joslin AC,Fresques T,et al.TOR Complex 2-Ypk1signaling maintains sphingolipid homeostasis by sensing and regulating ROS accumulation[J].Cell Reports,2014,6(3):541-552