牙鲆甲状腺素受体β互作蛋白的鉴定及其受甲状腺素调控的分析
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摘要
甲状腺素(TH)在牙鲆(Paralichthys olivaceus)变态发育过程中具有重要的调控作用,其通过与甲状腺素受体结合并在甲状腺素受体结合蛋白的共同作用下发挥生物学功能。利用免疫沉淀和质谱分析法鉴定了牙鲆甲状腺素受体β(TRβ)的结合蛋白,并采用实时荧光定量PCR技术检测了结合蛋白在牙鲆变态过程中受外源甲状腺素调控的变化。从TRβ多克隆抗体与牙鲆胚胎细胞总蛋白的免疫沉淀复合物中鉴定得到了21个牙鲆蛋白,生物信息学分析显示它们参与到多种生命活动中。对其中7个代表性蛋白的基因在出膜后第21天(21 dph)和第28天(28 dph)的牙鲆仔鱼体内的转录水平检测发现,它们的表达在外源甲状腺素处理后均表现出显著变化,其中beta-F1-ATPase、Hsp90β、Cct6A、RPS15A、Slc25a5和EF1a的表达受外源甲状腺素处理显著下调,CK1截然相反,它的表达受甲状腺素处理显著上调;硫脲处理对所分析的基因则几乎都没有明显影响。本研究为进一步探究甲状腺素受体介导甲状腺素调控牙鲆变态提供了新的见解。
Thyroid hormone( TH) plays an important role in the metamorphosis of Paralichthys olivaceus. It exerts biological functions through the combination of thyroid hormone receptors together with thyroid hormone receptor interacting proteins. In this study,immunoprecipitation and mass spectrometry were used to identify the interacting proteins of Paralichthys olivaceus thyroid hormone receptor β,and real-time fluorescence quantitative PCR was used to detect the changes of the interacting proteins in the metamorphosis of Paralichthys olivaceus affected by exogenous thyroid hormone at 21 dph and 28 dph. We identified 21 Paralichthys olivaceus proteins from immunoprecipitated complexes of TRβ polyclonal antibodies and total Paralichthys olivaceus embryonic cell proteins. Bioinformatics analysis revealed that they participated in various life activities. The mRNA expression levels of seven representative proteins were detected and their expressions showed significant changes after exogenous thyroid hormone treatment. The expression of beta-F1-ATPase, Hsp90β, Cct6 A, RPS15 A, Slc25 a5, and EF1 a was significantly downregulated by exogenous thyroid hormone treatment. In contrast,CK1 was significantly up-regulated by thyroid hormone treatment,and thiourea treatment had almost no significant effect on the analyzed genes. This study provides new insights into the role of thyroid hormone receptor-mediated regulation of thyroid hormone in Paralichthys olivaceus metamorphosis.
Thyroid hormone( TH) plays an important role in the metamorphosis of Paralichthys olivaceus. It exerts biological functions through the combination of thyroid hormone receptors together with thyroid hormone receptor interacting proteins. In this study,immunoprecipitation and mass spectrometry were used to identify the interacting proteins of Paralichthys olivaceus thyroid hormone receptor β,and real-time fluorescence quantitative PCR was used to detect the changes of the interacting proteins in the metamorphosis of Paralichthys olivaceus affected by exogenous thyroid hormone at 21 dph and 28 dph. We identified 21 Paralichthys olivaceus proteins from immunoprecipitated complexes of TRβ polyclonal antibodies and total Paralichthys olivaceus embryonic cell proteins. Bioinformatics analysis revealed that they participated in various life activities. The mRNA expression levels of seven representative proteins were detected and their expressions showed significant changes after exogenous thyroid hormone treatment. The expression of beta-F1-ATPase, Hsp90β, Cct6 A, RPS15 A, Slc25 a5, and EF1 a was significantly downregulated by exogenous thyroid hormone treatment. In contrast,CK1 was significantly up-regulated by thyroid hormone treatment,and thiourea treatment had almost no significant effect on the analyzed genes. This study provides new insights into the role of thyroid hormone receptor-mediated regulation of thyroid hormone in Paralichthys olivaceus metamorphosis.
引文
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[18] BARKER R N. Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells[J]. Clinical&Experimental Immunology,2002,127(2):220-225.
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[21] YAMASHITA M,YABU T,OJIMA N. Stress protein HSP70 in fish[J]. Aqua,2010,3(4):111-141.
[22]王鹏飞.鳜热休克蛋白和低氧反应基因的克隆和表达研究[D].广州:中山大学,2014.WANG P F. Cloning and expression analysis of heat shock protein and hypoxia responsive genes in mandarin fish(Siniperca chuatsi)[D]. Guangzhou:Sun Yat-sen University,2014.
[23] NIE Z Q,WU Y G,MENG J Z. The function and application of molecular chaperone[J]. Chinese Bulletin of Life Sciences,2006,18(1):84-89.
[24] HORWICH A L,FENTON W A,CHAPMAN E.Two families of chaperonin:Physiology and mechanism[J]. Annual Review of Cell&Developmental Biology,2007,23(1):115.
[25]李光耀.核糖体蛋白RPS15A促进白血病U937细胞恶性增殖的分子机制研究[D].济南:山东大学,2016.LI G Y. Regulation mechanism of RPS15A in promoting leukemia U937 cell proliferation[D].Jinan:Shandong University,2016.
[26] JACOBUS W E,LEHNINGER A L. Creatine kinase of rat heart mitochondria[J]. Journal of Biological Chemistry,1973,248(13):4803-4810.
[27] SCHLATTNER U,TOKARSKASCHLATTNER M,WALLIMANN T. Mitochondrial creatine kinase in human health and disease[J]. BBA-Molecular Basis of Disease,2006,1762(2):164-180.
[28]周多奇,龚莉,钱振宇.肌酸激酶的生理功能与运动[J].安庆师范学院学报(自科版),2016,22(3):113-118.ZHOU D Q,GONG L,QIAN Z Y. Physiological function of creatine kinase and sport[J]. Journal of Anqing Teachers College(Natural Science Edition),2016,22(3):113-118.
[29] SEPPET E K,SAKS V A. Thyroid hormones and the creatine kinase system in cardiac cells[J].Molecular&Cellular Biochemistry,1994,133-134(1):299-309.
[30] SMITH D P. The relationship between serum creatine kinase and thyroid hormones:In vivo and in vitro studies[J]. Clinica Chimica Acta,1976,68(3):333-338.
[31] YANG L,HE Y,KONG Q,et al. Isolation,nucleotide identification and tissue expression of three novel ovine genes—SLC25A4,SLC25A5,and SLC25A6[J]. Molecular Biology Reports,2010,37(6):2743-2748.
[32] JANG J Y,YUN C,JEON Y K,et al. Suppression of adenine nucleotide translocase-2 by vector-based siRNA in human breast cancer cells induces apoptosis and inhibits tumor growth in vitro and in vivo[J]. Breast Cancer Research,2008,10(1):R11.
[33]张力维,李勇鹏,姚瑶,等.香樟延伸因子EF1a基因片段的克隆及表达分析[J].中南林业科技大学学报,2015(5):122-128.ZHANG L W,LI Y P,YAO Y,et al. Cloning and expression analysis of EF1a gene fragment of elongation factor from Cinnamomum camphora[J].Journal of Central South University of Forestry&Technology,2015(5):122-128.
[34] FU J,MOMCILOVIC I,PRASAD P V V. Roles of protein synthesis elongation factor EF-Tu in heat tolerance in plants[J]. Journal of Botany,2012(2012-6-3).
[35] GOULART-SILVA F,TEIXEIRA S S,LUCHESSI A D, et al. Potential contribution of translational factors on T3-induced insulin synthesis by pancreatic beta cells[J]. Thyroid Official Journal of the American Thyroid Association,2012,22(6):637.
[2] WU Y,KOENIG R J. Gene regulation by thyroid hormone[J]. Trends in Endocrinology&Metabolism,2000,11(6):207-211.
[3] FURUYA F, HANOVER J A, CHENG S Y.Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormoneβreceptor[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(6):1780-1785.
[4]郭纯.免疫共沉淀技术的研究进展[J].中医药导报,2007(12):86-89.GUO C. Study progress of co-immunoprecipitation technology[J]. Guiding Journal of TCM, 2007(12):86-89.
[5]施杨,居慧,喻杰,等.牙鲆NR4A1基因在甲状腺素诱导仔鱼变态中的表达调控分析[J].海洋渔业,2017,39(4):433-442.SHI Y,JU H,YU J,et al. Expression regulation of Paralichthys olivaceus NR4A1 gene in the metamorphic larvae induced by thyroid hormone[J].Marine Fisheries,2017,39(4):433-442.
[6] YU J,FU Y,SHI Z. Coordinated expression and regulation of deiodinases and thyroid hormone receptors during metamorphosis in the Japanese flounder(Paralichthys olivaceus)[J]. Fish Physiology and Biochemistry,2017,43(2):321-336.
[7] SU Y,FU Y,ZHANG H,et al. Identification and expression of SRF targeted by miR-133a during early development of Paralichthys olivaceus[J]. Fish Physiology and Biochemistry,2015,41(5):1093-1104.
[8] CHEN S L,REN G C,SHA Z X,et al. Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation[J]. Diseases of Aquatic Organisms,2004,60(3):241-6.
[9] INUI Y, MIWA S. Thyroid hormone induces metamorphosis of flounder larvae[J]. General and Comparative Endocrinology,1985,60(3):450-454.
[10] LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J]. Methods,2001,25(4):402-408.
[11]翟曜耀,刘晓霞,魏秀芳.免疫沉淀技术的最新进展[J].现代生物医学进展,2010(10):1997-2000.ZHAI Y Y, LIU X X, WEI X F. The latest development of immunoprecipitation[J]. Progress in Modern Biomedicine,2010(10):1997-2000.
[12] REN X M,SHAND S H,TAKIMOTO K. Effective association of Kv channel-interacting proteins with Kv4 channel is mediated with their unique core peptide[J]. Journal of Biological Chemistry,2003,278(44):43564-43570.
[13] WEI Z,THOMAS D,KARLE C A,et al. Protein kinase A-mediated phosphorylation of HERG potassium channels in a human cell line[J].Chinese Medical Journal,2002,115(5):668-676.
[14] HOTCHKISS K,HARVEY M,PACHECO M,et al.Ion channel proteins in mouse and human vestibular tissue[J]. Otolaryngology-Head and Neck Surgery,2005,132(6):916-923.
[15]张霞,彭艳,俞丽丽,等.抗人F1-F0 ATP合成酶beta亚基单抗的制备及其抗肿瘤活性研究[J].中国免疫学杂志,2008,24(11):984-987.ZHANG X,PENG Y,YU L L,et al. Preparation and anti-tumor activity of mouse monoclonal antibody against human F1-F0 ATP synthase beta subunit[J]. Chinese Journal of Immunology,2008,24(11):984-987.
[16]崔杰,徐德昌,李滨胜,等.甜菜ATP合酶β亚基基因atp B的克隆、序列分析及进化[J].植物研究,2006(5):583-588.CUI J,XU D C,LI B S,et al. Cloning,sequence analysis and evolution of gene atp B sugar beet ATP synthase beta subunit[J]. Bulletin of Botanical Research,2006(5):583-588.
[17] KRYSKO DV,VANDENABEELE P. From regulation of dying cell engulfment to development of anti-cancer therapy[J]. Cell Death and Differentiation,2008,15(1):29-38.
[18] BARKER R N. Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells[J]. Clinical&Experimental Immunology,2002,127(2):220-225.
[19] CHAMPAGNE E,MARTINEZ L O,COLLET X,et al. Ecto-F1Fo ATP synthase/F1 ATPase:metabolic and immunological functions.[J]. Current Opinion in Lipidology,2006,17(3):279-284.
[20] ROBERTS R J,AGIUS C,SALIBA C,et al. Heat shock proteins(chaperones)in fish and shellfish and their potential role in relation to fish health:a review[J]. Journal of Fish Diseases,2010,33(10):789-801.
[21] YAMASHITA M,YABU T,OJIMA N. Stress protein HSP70 in fish[J]. Aqua,2010,3(4):111-141.
[22]王鹏飞.鳜热休克蛋白和低氧反应基因的克隆和表达研究[D].广州:中山大学,2014.WANG P F. Cloning and expression analysis of heat shock protein and hypoxia responsive genes in mandarin fish(Siniperca chuatsi)[D]. Guangzhou:Sun Yat-sen University,2014.
[23] NIE Z Q,WU Y G,MENG J Z. The function and application of molecular chaperone[J]. Chinese Bulletin of Life Sciences,2006,18(1):84-89.
[24] HORWICH A L,FENTON W A,CHAPMAN E.Two families of chaperonin:Physiology and mechanism[J]. Annual Review of Cell&Developmental Biology,2007,23(1):115.
[25]李光耀.核糖体蛋白RPS15A促进白血病U937细胞恶性增殖的分子机制研究[D].济南:山东大学,2016.LI G Y. Regulation mechanism of RPS15A in promoting leukemia U937 cell proliferation[D].Jinan:Shandong University,2016.
[26] JACOBUS W E,LEHNINGER A L. Creatine kinase of rat heart mitochondria[J]. Journal of Biological Chemistry,1973,248(13):4803-4810.
[27] SCHLATTNER U,TOKARSKASCHLATTNER M,WALLIMANN T. Mitochondrial creatine kinase in human health and disease[J]. BBA-Molecular Basis of Disease,2006,1762(2):164-180.
[28]周多奇,龚莉,钱振宇.肌酸激酶的生理功能与运动[J].安庆师范学院学报(自科版),2016,22(3):113-118.ZHOU D Q,GONG L,QIAN Z Y. Physiological function of creatine kinase and sport[J]. Journal of Anqing Teachers College(Natural Science Edition),2016,22(3):113-118.
[29] SEPPET E K,SAKS V A. Thyroid hormones and the creatine kinase system in cardiac cells[J].Molecular&Cellular Biochemistry,1994,133-134(1):299-309.
[30] SMITH D P. The relationship between serum creatine kinase and thyroid hormones:In vivo and in vitro studies[J]. Clinica Chimica Acta,1976,68(3):333-338.
[31] YANG L,HE Y,KONG Q,et al. Isolation,nucleotide identification and tissue expression of three novel ovine genes—SLC25A4,SLC25A5,and SLC25A6[J]. Molecular Biology Reports,2010,37(6):2743-2748.
[32] JANG J Y,YUN C,JEON Y K,et al. Suppression of adenine nucleotide translocase-2 by vector-based siRNA in human breast cancer cells induces apoptosis and inhibits tumor growth in vitro and in vivo[J]. Breast Cancer Research,2008,10(1):R11.
[33]张力维,李勇鹏,姚瑶,等.香樟延伸因子EF1a基因片段的克隆及表达分析[J].中南林业科技大学学报,2015(5):122-128.ZHANG L W,LI Y P,YAO Y,et al. Cloning and expression analysis of EF1a gene fragment of elongation factor from Cinnamomum camphora[J].Journal of Central South University of Forestry&Technology,2015(5):122-128.
[34] FU J,MOMCILOVIC I,PRASAD P V V. Roles of protein synthesis elongation factor EF-Tu in heat tolerance in plants[J]. Journal of Botany,2012(2012-6-3).
[35] GOULART-SILVA F,TEIXEIRA S S,LUCHESSI A D, et al. Potential contribution of translational factors on T3-induced insulin synthesis by pancreatic beta cells[J]. Thyroid Official Journal of the American Thyroid Association,2012,22(6):637.