嗜热四膜虫胱硫醚γ-裂解酶Cgl1的表达、定位及功能分析
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摘要
含硫氨基酸在不同的生物体中具有重要调节功能,转硫途径相关酶促进半胱氨酸的生成和硫化氢产生。本研究从嗜热四膜虫中鉴定一种胱硫醚γ-裂解酶(cystathionineγ-lyase 1,CGL1,TTHERM_00052400)基因。CGL1在营养生长期高水平表达,而在饥饿阶段和有性生殖期,维持在较低的表达水平。通过密码子优化,人工合成CGL1基因,构建重组表达质粒pGEX-CGL1,转化大肠杆菌BL21(DE3)。E.coli/pGEX-CGL1表达重组蛋白质GST-Cgl1,并通过亲和层析获得纯化。GST-Cgl1裂解胱硫醚产生半胱氨酸,也具有裂解半胱氨酸和同型半胱氨酸产生H_2S的活性。进一步构建重组质粒pNEO4-3HA-CGL1和pSMC1hpNEO-CGL1,转化四膜虫细胞,获得带有HA标签和干扰CGL1的突变体细胞株。免疫荧光定位表明,HA-Cgl1生长期定位在亲本大核,饥饿期定位在细胞质,有性生殖前期定位在亲本大核,而在后期定位在胞质中。CGL1干扰的突变体细胞株在有性生殖过程中不能形成合子核,发育中的小核异常降解,产生仅有大核的异常单细胞。结果表明,嗜热四膜虫含有进化中保守的胱硫醚γ-裂解酶Cgl1。Cgl1具有产生和裂解半胱氨酸的活性。Cgl1定位在细胞质和细胞核中,参与了有性生殖过程细胞核的发育。
Sulfur-containing amino acids have important regulatory functions in different organisms, and transsulfuration pathway-related enzymes promote the production of cysteine and hydrogen sulfide. In this study, a cystathionine γ-lyase(CGL1, TTHERM_00052400) gene was identified from T. thermophila. CGL1 was expressed at a high level in the vegetative growth stage, while maintaining a low expression level during the starvation and the sexual reproduction stage. The CGL1 was synthesized by codon optimization, and the recombinant expression plasmid pGEX-CGL1 was constructed and transformed into E. coli BL21(DE3). GST-Cgl1 was expressed in the E.coli BL21(DE3)/pGEX-CGL1 and purified by affinity chromatography. GST-Cgl1 cleaved cystathionine to produce cysteine, and simultaneously cleaved cysteine and homocysteine to produce H_2S. Furthermore, the plasmids pNEO4-3 HA-CGL1 and pSMC1 hpNEO-CGL1 were constructed and the mutant strains were created, respectively. Immunofluorescence localization showed that the HA-Cgl1 localized in the parental macronucleus at the vegetative growth stage, localized in the cytoplasm at the starvation stage,and localized in the parental macronucleus in the early stage and cytoplasm during the late conjugation stage. CGL1 knockdown cells failed to produce the zygotic nuclei and formed micronucleus defective cells. The results indicate that cystathionine γ-lyase is an evolutionary conservative enzyme in T. thermophile. Cgl1 could produce cysteine and cleavage of cysteine. Cgl1 localizes in the cytoplasm and macronuclei and is also involved in formation of new nuclei during the sexual development stage.
Sulfur-containing amino acids have important regulatory functions in different organisms, and transsulfuration pathway-related enzymes promote the production of cysteine and hydrogen sulfide. In this study, a cystathionine γ-lyase(CGL1, TTHERM_00052400) gene was identified from T. thermophila. CGL1 was expressed at a high level in the vegetative growth stage, while maintaining a low expression level during the starvation and the sexual reproduction stage. The CGL1 was synthesized by codon optimization, and the recombinant expression plasmid pGEX-CGL1 was constructed and transformed into E. coli BL21(DE3). GST-Cgl1 was expressed in the E.coli BL21(DE3)/pGEX-CGL1 and purified by affinity chromatography. GST-Cgl1 cleaved cystathionine to produce cysteine, and simultaneously cleaved cysteine and homocysteine to produce H_2S. Furthermore, the plasmids pNEO4-3 HA-CGL1 and pSMC1 hpNEO-CGL1 were constructed and the mutant strains were created, respectively. Immunofluorescence localization showed that the HA-Cgl1 localized in the parental macronucleus at the vegetative growth stage, localized in the cytoplasm at the starvation stage,and localized in the parental macronucleus in the early stage and cytoplasm during the late conjugation stage. CGL1 knockdown cells failed to produce the zygotic nuclei and formed micronucleus defective cells. The results indicate that cystathionine γ-lyase is an evolutionary conservative enzyme in T. thermophile. Cgl1 could produce cysteine and cleavage of cysteine. Cgl1 localizes in the cytoplasm and macronuclei and is also involved in formation of new nuclei during the sexual development stage.
引文
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[3] Mozzarelli A, Bettati S, Campanini B, et al. The multifaceted pyridoxal 5′-phosphate-dependent O- acetylserine sulfhydrylase[J]. Biochim Biophys Acta, 2011, 1814(11): 1497-1510
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[7] Nozaki T, Shigeta Y, Saito-Nakano Y, et al. Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma[J]. J Biol Chem, 2001, 276(9): 6516-6523
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[9] Maresi E, Janson G, Fruncillo S, et al. Functional characterization and structure-guided mutational analysis of the transsulfuration enzyme cystathionine gamma-lyase from Toxoplasma gondii[J]. Int J Mol Sci, 2018, 19(7)
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[11] Chen X, Jhee K H, Kruger W D. Production of the neuromodulator H2S by cystathionine beta-synthase via the condensation of cysteine and homocysteine[J]. J Biol Chem, 2004, 279(50): 52082-52086
[12] 许静, 王伟, 梁爱华. 嗜热四膜虫δ微管蛋白基因的克隆、鉴定及细胞定位[J]. 中国生物化学与分子生物学报(Xu J, Wang W, Liang AH. Characterization and cellular localization of δ-tubulin from Tetrahymena thermophila[J]. Chin J Biochem Mol Biol), 2013, 29(5): 430-436
[13] Giordana L, Mantilla B S, Santana M, et al. Cystathionine gamma-lyase, an enzyme related to the reverse transsulfuration pathway, is functional in Leishmania spp[J]. J Eukaryot Microbiol, 2014, 61(2): 204-213
[14] 梅玉东, 金欣欣, 黄丽琴. 硫巯基化:一种新的蛋白质翻译后修饰[J]. 中国生物化学与分子生物学报(Mei YD, Jin XX, Huang LQ. Thiothiolation: a novel post-translational modification of proteins[J]. Chin J Biochem Mol Biol), 2018, 34(9): 911-920
[15] Possenti A, Fratini F, Fantozzi L, et al. Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle[J]. BMC Genomics, 2013, 14: 183
[16] Wang R. Two′s company, three′s a crowd: can H2S be the third endogenous gaseous transmitter?[J]. FASEB J, 2002, 16(13): 1792-1798
[17] Khersonsky O, Tawfik D S. Enzyme promiscuity: a mechanistic and evolutionary perspective[J]. Annu Rev Biochem, 2010, 79: 471-505
[18] Jeffery C J. Moonlighting proteins[J]. Trends Biochem Sci, 1999, 24(1): 8-11
[19] Bruinenberg PG, De Roo G, Limsowtin G. Purification and characterization of cystathionine (gamma)- lyase from Lactococcus lactis subsp. cremoris SK11: possible role in flavor compound formation during cheese maturation[J]. Appl Environ Microbiol, 1997, 63(2): 561-566
[20] Ferla M P, Brewster J L, Hall K R, et al. Primordial-like enzymes from bacteria with reduced genomes[J]. Mol Microbiol, 2017, 105(4): 508-524
[21] Alting AC, Engels W, van Schalkwijk S, et al. Purification and characterization of cystathionine (beta)- lyase from Lactococcus lactis subsp. cremoris B78 and its possible role in flavor development in cheese[J]. Appl Environ Microbiol, 1995, 61(11): 4037-4042
[22] Williams RA, Westrop GD, Coombs GH. Two pathways for cysteine biosynthesis in Leishmania major[J]. Biochem J, 2009, 420(3): 451-462
[23] Paape D, Barrios-Llerena M E, Le Bihan T, et al. Gel free analysis of the proteome of intracellular Leishmania mexicana[J]. Mol Biochem Parasitol, 2010, 169(2): 108-114
[24] Zheng Y, Liao F, Lin X, et al. Cystathionine gamma-lyase-hydrogen sulfide Induces Runt-related transcription factor 2 sulfhydration, thereby increasing osteoblast activity to promote bone fracture healing[J]. Antioxid Redox Signal, 2017, 27(11): 742-753
[25] Wang L, Shi H, Zhang X, et al. I157172, a novel inhibitor of cystathionine gamma-lyase, inhibits growth and migration of breast cancer cells via SIRT1-mediated deacetylation of STAT3[J]. Oncol Rep, 2019, 41 (1): 427-436
[2] Brosnan J T, Brosnan M E. The sulfur-containing amino acids: an overview[J]. J Nutr, 2006, 136(6 Suppl): 1636s-1640s
[3] Mozzarelli A, Bettati S, Campanini B, et al. The multifaceted pyridoxal 5′-phosphate-dependent O- acetylserine sulfhydrylase[J]. Biochim Biophys Acta, 2011, 1814(11): 1497-1510
[4] Hannibal L, Blom H J. Homocysteine and disease: Causal associations or epiphenomenons?[J]. Mol Aspects Med, 2017, 53: 36-42
[5] Szabo C, Papapetropoulos A. International union of basic and clinical pharmacology. CII: Pharmacological modulation of H2S levels: H2S donors and H2S biosynthesis inhibitors[J]. Pharmacol Rev, 2017, 69(4): 497-564
[6] Tokoro M, Asai T, Kobayashi S, et al. Identification and characterization of two isoenzymes of methionine gamma-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways[J]. J Biol Chem, 2003, 278(43): 42717-42727
[7] Nozaki T, Shigeta Y, Saito-Nakano Y, et al. Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma[J]. J Biol Chem, 2001, 276(9): 6516-6523
[8] Marciano D, Santana M, Nowicki C. Functional characterization of enzymes involved in cysteine biosynthesis and H2S production in Trypanosoma cruzi[J]. Mol Biochem Parasitol, 2012, 185(2): 114-120
[9] Maresi E, Janson G, Fruncillo S, et al. Functional characterization and structure-guided mutational analysis of the transsulfuration enzyme cystathionine gamma-lyase from Toxoplasma gondii[J]. Int J Mol Sci, 2018, 19(7)
[10] 曹珊珊, 牛卫宁, 羊梦林等. 胱硫醚β-裂解酶的表达纯化及性质研究[J]. 化学与生物工程(Cao SS, Niu WN, Yang ML, et al. Expression, purification and properties of cystathionine β-lyase [J]. Chem Bioeng), 2011, 28(9): 27-31
[11] Chen X, Jhee K H, Kruger W D. Production of the neuromodulator H2S by cystathionine beta-synthase via the condensation of cysteine and homocysteine[J]. J Biol Chem, 2004, 279(50): 52082-52086
[12] 许静, 王伟, 梁爱华. 嗜热四膜虫δ微管蛋白基因的克隆、鉴定及细胞定位[J]. 中国生物化学与分子生物学报(Xu J, Wang W, Liang AH. Characterization and cellular localization of δ-tubulin from Tetrahymena thermophila[J]. Chin J Biochem Mol Biol), 2013, 29(5): 430-436
[13] Giordana L, Mantilla B S, Santana M, et al. Cystathionine gamma-lyase, an enzyme related to the reverse transsulfuration pathway, is functional in Leishmania spp[J]. J Eukaryot Microbiol, 2014, 61(2): 204-213
[14] 梅玉东, 金欣欣, 黄丽琴. 硫巯基化:一种新的蛋白质翻译后修饰[J]. 中国生物化学与分子生物学报(Mei YD, Jin XX, Huang LQ. Thiothiolation: a novel post-translational modification of proteins[J]. Chin J Biochem Mol Biol), 2018, 34(9): 911-920
[15] Possenti A, Fratini F, Fantozzi L, et al. Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle[J]. BMC Genomics, 2013, 14: 183
[16] Wang R. Two′s company, three′s a crowd: can H2S be the third endogenous gaseous transmitter?[J]. FASEB J, 2002, 16(13): 1792-1798
[17] Khersonsky O, Tawfik D S. Enzyme promiscuity: a mechanistic and evolutionary perspective[J]. Annu Rev Biochem, 2010, 79: 471-505
[18] Jeffery C J. Moonlighting proteins[J]. Trends Biochem Sci, 1999, 24(1): 8-11
[19] Bruinenberg PG, De Roo G, Limsowtin G. Purification and characterization of cystathionine (gamma)- lyase from Lactococcus lactis subsp. cremoris SK11: possible role in flavor compound formation during cheese maturation[J]. Appl Environ Microbiol, 1997, 63(2): 561-566
[20] Ferla M P, Brewster J L, Hall K R, et al. Primordial-like enzymes from bacteria with reduced genomes[J]. Mol Microbiol, 2017, 105(4): 508-524
[21] Alting AC, Engels W, van Schalkwijk S, et al. Purification and characterization of cystathionine (beta)- lyase from Lactococcus lactis subsp. cremoris B78 and its possible role in flavor development in cheese[J]. Appl Environ Microbiol, 1995, 61(11): 4037-4042
[22] Williams RA, Westrop GD, Coombs GH. Two pathways for cysteine biosynthesis in Leishmania major[J]. Biochem J, 2009, 420(3): 451-462
[23] Paape D, Barrios-Llerena M E, Le Bihan T, et al. Gel free analysis of the proteome of intracellular Leishmania mexicana[J]. Mol Biochem Parasitol, 2010, 169(2): 108-114
[24] Zheng Y, Liao F, Lin X, et al. Cystathionine gamma-lyase-hydrogen sulfide Induces Runt-related transcription factor 2 sulfhydration, thereby increasing osteoblast activity to promote bone fracture healing[J]. Antioxid Redox Signal, 2017, 27(11): 742-753
[25] Wang L, Shi H, Zhang X, et al. I157172, a novel inhibitor of cystathionine gamma-lyase, inhibits growth and migration of breast cancer cells via SIRT1-mediated deacetylation of STAT3[J]. Oncol Rep, 2019, 41 (1): 427-436