小鼠肌肉生长抑制素Y309和C310位点缺失突变对蛋白结构的影响
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摘要
肌肉生长抑制素(myostatin, MSTN)又名生长分化因子8 (growth-differentiation factor 8, GDF-8),是转化生长因子-β(transforming growth factor-β, TGF-β)家族成员之一,广泛参与机体生长调控过程。作为肌肉生长的负调控因子,MSTN基因突变会造成肌细胞过度增殖、肌纤维数量增加和肌纤维肥大。本研究通过测序技术及生物信息学方法,对小鼠(Mus musculus) MSTN基因Y309和C310位点缺失突变前后编码蛋白的多级结构及其功能进行预测分析。结果表明,MSTN基因突变小鼠第3外显子nt 175～180发生缺失突变,肌肉组织中MSTN基因mRNA表达量减少,MSTN蛋白表达量也减少,MSTN蛋白受体激活素Ⅱ型受体基因(activin typeⅡreceptor, ActR2b)表达量减少,与肌肉发育相关的成肌决定因子基因(myogenin determination, MyoD),生肌决定因子5基因(myogenic factor 5, Myf5)以及肌细胞生成素基因(myogenin, Myog)的表达量发生显著上调,表现出肌肉肥大的表型。分析突变后的MSTN蛋白结构发现,缺失Y(309)和C(310)位点两个氨基酸破坏了MSTN成熟肽的二硫键,β延伸链延长,蛋白结构改变。蛋白功能预测表明,在MSTN突变蛋白内缺失了一个蛋白结合位点,从而影响了其与受体的结合。本研究表明,小鼠MSTN第3外显子nt 175～180是影响MSTN蛋白功能的核心序列,缺失突变后会产生肌肉肥大的表型,为研究MSTN基因功能提供了良好的小鼠模型。
Myostatin(MSTN), also known as GDF-8(growth-differentiation factor 8), is a member of the transforming growth factor-beta(TGF-β) super-family, which participates in various process of body growth regulation. As a negative regulatory of muscle growth, its mutation will lead to the excessive proliferation of muscle cells and muscle hypertrophy. To study how MSTN mutation affect its protein structure and biology functions, the multi-level structure and function changes of MSTN were predicted and analyzed by bioinformatics methods, based on our MSTN gene knock-out mice(Mus musculus). The results showed that the nt 175～180 sequence in the third exon of the MSTN gene was missing, the expression level of the MSTN decreased which led to deprived the MSTN protein in muscle, the expression level of myogenin determination gene(Myod), myogenic factor 5 gene(Myf5) and myogenin gene(Myog) increased significantly, activin typeⅡreceptor b3 gene(ActR2 b3), the receptor protein of the MSTN decreased and the muscle hypertrophy. Analysis of the deletion mutation MSTN protein structure, it lost the Y(309) and C(310) amino acids, which elongated the beta pleated sheet and changed the protein structure. The functional prediction result showed that, the mutant MTSN protein of these mice lost a protein bonding site, which reduced its bonding capability to the receptor and impaired its biological functions. This study showed that the third exon nt 175～180 of MSTN in mice was the core sequence that affected the function of MSTN protein, and resulted in the muscle hypertrophy phenotype, which would provide a good mouse model for studying the function of MSTN gene.
Myostatin(MSTN), also known as GDF-8(growth-differentiation factor 8), is a member of the transforming growth factor-beta(TGF-β) super-family, which participates in various process of body growth regulation. As a negative regulatory of muscle growth, its mutation will lead to the excessive proliferation of muscle cells and muscle hypertrophy. To study how MSTN mutation affect its protein structure and biology functions, the multi-level structure and function changes of MSTN were predicted and analyzed by bioinformatics methods, based on our MSTN gene knock-out mice(Mus musculus). The results showed that the nt 175～180 sequence in the third exon of the MSTN gene was missing, the expression level of the MSTN decreased which led to deprived the MSTN protein in muscle, the expression level of myogenin determination gene(Myod), myogenic factor 5 gene(Myf5) and myogenin gene(Myog) increased significantly, activin typeⅡreceptor b3 gene(ActR2 b3), the receptor protein of the MSTN decreased and the muscle hypertrophy. Analysis of the deletion mutation MSTN protein structure, it lost the Y(309) and C(310) amino acids, which elongated the beta pleated sheet and changed the protein structure. The functional prediction result showed that, the mutant MTSN protein of these mice lost a protein bonding site, which reduced its bonding capability to the receptor and impaired its biological functions. This study showed that the third exon nt 175～180 of MSTN in mice was the core sequence that affected the function of MSTN protein, and resulted in the muscle hypertrophy phenotype, which would provide a good mouse model for studying the function of MSTN gene.
引文
曹婷,周汉林,荀文娟,等.2017.MSTN基因对猪骨骼肌发育调控的作用及其研究进展[J].基因组学与应用生物学,36(4):1511-1517.(Cao T,Zhou H,Xun W,et al.2017.The effect of MSTN gene on the regulation of skeletal muscle development of pig and its research progress[J].Genomics and Applied Biology,36(4):1511-1517).
高峰,邢沈阳,于海滨,等.2014.牛MSTN基因克隆及编码区E1-224->C定点突变前后生物信息学对比分析[J].中国兽医学报,34(5):825-830.(Gao F,Xing S Y,Yu HB,et al.2014.Cloning of bovine MSTN gene and bioinformatics comparative analysis between coding region with E1-224-A>C mutation site and normal type[J].Chinese Journal of Veterinary Science,34(5):825-830.)
魏著英,白春玲,李光鹏.2018.牛肌肉生长抑制素基因突变的遗传效应与育种应用[J].生物技术进展,45(03):19-27;99.(Wei Z Y,Bai C L,Li G P.2018.Genetic effect and breeding application of myostatin gene mutation in beef cattle[J].Current Biotechnology,45(03):19-27;99.)
佟彬,张立,李光鹏.2017.中国肉牛分子与基因修饰育种研究进展[J].遗传,39(11):984-101.(Tong B,Zhang L,Li G P.2017.Progress in the molecular and genetic modification breeding of beef cattle in China[J].Hereditas,39(11):984-1015.)
张丽,刘丽霞,李强子,等.2017.天祝白牦牛MSTN基因编码区克隆及生物信息学分析[J].浙江农业学报.29(4):618-624.(Zhang L,Liu L X,Li Q Z,et al.2017.Cloning and bioinformatics analysis of MSTN gene of Tianzhu white yak[J].Acta Agriculturae Zhejiangensis,29(4):618-624.)
Allen D L,Hittel D S,Mcpherron A C.2011.Expression and function of myostatin in obesity,diabetes,and exercise adaptation[J].Medicine&Science in Sports&Exercise,43(10):1828-1835.
Arnold K,Bordoli L,Kopp J,et al.2006.The SWISS-MOD-EL Workspace:A web-based environment for protein structure homology modeling[J].Bioinformatics,22(2):195-201.
Bellinge R H,Liberles D A,Iaschi S P,et al.2005.Myostatin and its implications on animal breeding:A review[J].Animal Genetics,36(1):1-6.
Biasini M,Bienert S,Waterhouse A,et al.2014.SWISS-MODEL:Modelling protein tertiary and quaternary structure using evolutionary information[J].Nucleic Acids Research,42(W1):252-258.
Bordoli L,Kiefer F,Arnold K,et al.2008.Protein structure homology modeling using SWISS-MODEL workspace[J].Nature Protocols,4(1):1-13.
Buel D R,Dilip K G.2008.Clinical,agricultural,and evolutionary biology of myostatin:A comparative review[J].Endocrine Reviews,29(5):513-514.
Cash J N,Angerman E B,Kattamuri C,et al.2012.Structure of myostatin follistatin-like 3:N-terminal domains of follistatin-type molecules exhibit alternate modes of binding[J].Journal of Biological Chemistry,287(2):1043-1053.
Chen Y,Ye J,Cao L,et al.2010.Myostatin regulates glucose metabolism via the AMP-activated protein kinase pathway in skeletal muscle cells[J].International Journal of Biochemistry&Cell Biology,42(12):2072-2081.
Chillemi G,Fiorani P,Castelli S,et al.2005.Effect on DNArelaxation of the single Thr718Ala mutation in human topoisomerase I:A functional and molecular dynamics study[J].Nucleic Acids Research,33(10):3339-3350.
Chillemi G,D'Annessa I,Fiorani P,et al.2008.Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations[J].Nucleic Acids Research,36(17):5645-5651.
Combet C,Blanchet C,Geourjon C,et al.2000.NPS@:Network protein sequence analysis[J].Trends in Biochemical Sciences,25(3):147-150.
Cotton T R,Fischer G,Wang X,et al.2018.Structure of the human pro-myostatin precursor and determinants of growth factor latency[J].Embo Journal,37(3):e97883.
Georges M.2010.When less means more:Impact of myostatin in animal breeding[J].Immunology Endocrine&Metabolic Agents in Medicinal Chemistry,10(4):240-248.
Haidet A M,Rizo L,Handy C,et al.2008.Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors[J].Proceeding of the National Academy Sciences of the USA,105(11):4318-4322.
Kambadur R,Sharma M,Smith T,et al.1997.Mutations in myostatin(GDF8)in double-muscled Belgian Blue and Piedmontese cattle[J].Genome Research,7(9):910-916.
Kotzsch A,Nickel J,Seher A,et al.2009.Crystal structure analysis reveals a spring-loaded latch as molecular mechanism for GDF-5-type I receptor specificity[J].Embo Journal,28(7):937-947.
Li N,Yang Q,Walker R G,et al.2015.Myostatin attenuation in vivo reduces adiposity,but activates adipogenesis[J].Endocrinology,157(1):282-291.
Lee S J.2007.Quadrupling muscle mass in mice by targeting TGF-βsignaling pathways[J].PLoS ONE,2(8):e789.
Manfredi L H,Paula-Gomes S,Zanon N M,et al.2017.Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents[J].Brazilizan Journal of Medical and Biological Research,50(12):e6733.
Mcpherron A C,Lawler A M,Lee S J.1997.Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[J].Nature,387(6628):83-90.
Mcpherron A C,Lee S J.2002.Suppression of body fat accumulation in myostatin-deficient mice[J].Journal of Clinical Investigation,109(5):595-601.
Mosher D S,Quiqnon P,Bustamante C D,et al.2007.A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs[J].PLoS Genetics,3(5):e79.
Oldham J M,Martyn J A,Sharma M,et al.2001.Molecular expression of myostatin and MyoD is greater in doublemuscled than normal-muscled cattle fetuses[J].American Journal of Physiology-Regulatory,Integrative and Comparative Physiology,280(5):1488-1493.
Petersen T N,Brunak S,von Heijne G,et al.2011.SignalP4.0:Discriminating signal peptides from transmembrane regions[J].Nature Methods,8:785-786
Pirruccello-Straub M,Jackson J,Wawersik S,et al.2018.Blocking extracellular bactivation of myostatin as a strategy for treating muscle wasting[J].Scientific Reports,8(1):2292.
Rebbapragada A,Enchabane H,Wrana J L,et al.2003.Myostatin signals through a transforming growth factor like signaling pathway to block adipogenesis[J].Molecular and Celluar Biology,23(20):7230-7242.
Sabrout E I,Aqqaq S.2018.Association of melanocortin(MC4R)and myostatin(MSTN)genes with carcass quality in rabbit[J].Meat Science,137:67-70
Silvia B,Alessio V,Giovanni C.2016.Structural and dynamic characterization of the C313Y mutation in myostatin dimeric protein,responsible for the“double muscle”phenotype in piedmontese cattle[J].Frontiers in Genetics,7:14
Wang K,Ouyang H,Xie Z,et al.2015.Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system[J].Scientific Reports,5:16623.
Wiener P1,Smith J A,Lewis A M,et al.2002.Muscle-related traits in cattle:The role of the myostatin gene in the South Devon breed[J].Genetics selection Evolution,34(2):221-32.
Xue Y,Ren J,Gao X,et al.2008.GPS 2.0,a tool to predict kinase-specific phosphorylation sites in hierarchy[J].Molecular and Cellular Proteomics,7(9):1598-1608.
高峰,邢沈阳,于海滨,等.2014.牛MSTN基因克隆及编码区E1-224->C定点突变前后生物信息学对比分析[J].中国兽医学报,34(5):825-830.(Gao F,Xing S Y,Yu HB,et al.2014.Cloning of bovine MSTN gene and bioinformatics comparative analysis between coding region with E1-224-A>C mutation site and normal type[J].Chinese Journal of Veterinary Science,34(5):825-830.)
魏著英,白春玲,李光鹏.2018.牛肌肉生长抑制素基因突变的遗传效应与育种应用[J].生物技术进展,45(03):19-27;99.(Wei Z Y,Bai C L,Li G P.2018.Genetic effect and breeding application of myostatin gene mutation in beef cattle[J].Current Biotechnology,45(03):19-27;99.)
佟彬,张立,李光鹏.2017.中国肉牛分子与基因修饰育种研究进展[J].遗传,39(11):984-101.(Tong B,Zhang L,Li G P.2017.Progress in the molecular and genetic modification breeding of beef cattle in China[J].Hereditas,39(11):984-1015.)
张丽,刘丽霞,李强子,等.2017.天祝白牦牛MSTN基因编码区克隆及生物信息学分析[J].浙江农业学报.29(4):618-624.(Zhang L,Liu L X,Li Q Z,et al.2017.Cloning and bioinformatics analysis of MSTN gene of Tianzhu white yak[J].Acta Agriculturae Zhejiangensis,29(4):618-624.)
Allen D L,Hittel D S,Mcpherron A C.2011.Expression and function of myostatin in obesity,diabetes,and exercise adaptation[J].Medicine&Science in Sports&Exercise,43(10):1828-1835.
Arnold K,Bordoli L,Kopp J,et al.2006.The SWISS-MOD-EL Workspace:A web-based environment for protein structure homology modeling[J].Bioinformatics,22(2):195-201.
Bellinge R H,Liberles D A,Iaschi S P,et al.2005.Myostatin and its implications on animal breeding:A review[J].Animal Genetics,36(1):1-6.
Biasini M,Bienert S,Waterhouse A,et al.2014.SWISS-MODEL:Modelling protein tertiary and quaternary structure using evolutionary information[J].Nucleic Acids Research,42(W1):252-258.
Bordoli L,Kiefer F,Arnold K,et al.2008.Protein structure homology modeling using SWISS-MODEL workspace[J].Nature Protocols,4(1):1-13.
Buel D R,Dilip K G.2008.Clinical,agricultural,and evolutionary biology of myostatin:A comparative review[J].Endocrine Reviews,29(5):513-514.
Cash J N,Angerman E B,Kattamuri C,et al.2012.Structure of myostatin follistatin-like 3:N-terminal domains of follistatin-type molecules exhibit alternate modes of binding[J].Journal of Biological Chemistry,287(2):1043-1053.
Chen Y,Ye J,Cao L,et al.2010.Myostatin regulates glucose metabolism via the AMP-activated protein kinase pathway in skeletal muscle cells[J].International Journal of Biochemistry&Cell Biology,42(12):2072-2081.
Chillemi G,Fiorani P,Castelli S,et al.2005.Effect on DNArelaxation of the single Thr718Ala mutation in human topoisomerase I:A functional and molecular dynamics study[J].Nucleic Acids Research,33(10):3339-3350.
Chillemi G,D'Annessa I,Fiorani P,et al.2008.Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations[J].Nucleic Acids Research,36(17):5645-5651.
Combet C,Blanchet C,Geourjon C,et al.2000.NPS@:Network protein sequence analysis[J].Trends in Biochemical Sciences,25(3):147-150.
Cotton T R,Fischer G,Wang X,et al.2018.Structure of the human pro-myostatin precursor and determinants of growth factor latency[J].Embo Journal,37(3):e97883.
Georges M.2010.When less means more:Impact of myostatin in animal breeding[J].Immunology Endocrine&Metabolic Agents in Medicinal Chemistry,10(4):240-248.
Haidet A M,Rizo L,Handy C,et al.2008.Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors[J].Proceeding of the National Academy Sciences of the USA,105(11):4318-4322.
Kambadur R,Sharma M,Smith T,et al.1997.Mutations in myostatin(GDF8)in double-muscled Belgian Blue and Piedmontese cattle[J].Genome Research,7(9):910-916.
Kotzsch A,Nickel J,Seher A,et al.2009.Crystal structure analysis reveals a spring-loaded latch as molecular mechanism for GDF-5-type I receptor specificity[J].Embo Journal,28(7):937-947.
Li N,Yang Q,Walker R G,et al.2015.Myostatin attenuation in vivo reduces adiposity,but activates adipogenesis[J].Endocrinology,157(1):282-291.
Lee S J.2007.Quadrupling muscle mass in mice by targeting TGF-βsignaling pathways[J].PLoS ONE,2(8):e789.
Manfredi L H,Paula-Gomes S,Zanon N M,et al.2017.Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents[J].Brazilizan Journal of Medical and Biological Research,50(12):e6733.
Mcpherron A C,Lawler A M,Lee S J.1997.Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[J].Nature,387(6628):83-90.
Mcpherron A C,Lee S J.2002.Suppression of body fat accumulation in myostatin-deficient mice[J].Journal of Clinical Investigation,109(5):595-601.
Mosher D S,Quiqnon P,Bustamante C D,et al.2007.A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs[J].PLoS Genetics,3(5):e79.
Oldham J M,Martyn J A,Sharma M,et al.2001.Molecular expression of myostatin and MyoD is greater in doublemuscled than normal-muscled cattle fetuses[J].American Journal of Physiology-Regulatory,Integrative and Comparative Physiology,280(5):1488-1493.
Petersen T N,Brunak S,von Heijne G,et al.2011.SignalP4.0:Discriminating signal peptides from transmembrane regions[J].Nature Methods,8:785-786
Pirruccello-Straub M,Jackson J,Wawersik S,et al.2018.Blocking extracellular bactivation of myostatin as a strategy for treating muscle wasting[J].Scientific Reports,8(1):2292.
Rebbapragada A,Enchabane H,Wrana J L,et al.2003.Myostatin signals through a transforming growth factor like signaling pathway to block adipogenesis[J].Molecular and Celluar Biology,23(20):7230-7242.
Sabrout E I,Aqqaq S.2018.Association of melanocortin(MC4R)and myostatin(MSTN)genes with carcass quality in rabbit[J].Meat Science,137:67-70
Silvia B,Alessio V,Giovanni C.2016.Structural and dynamic characterization of the C313Y mutation in myostatin dimeric protein,responsible for the“double muscle”phenotype in piedmontese cattle[J].Frontiers in Genetics,7:14
Wang K,Ouyang H,Xie Z,et al.2015.Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system[J].Scientific Reports,5:16623.
Wiener P1,Smith J A,Lewis A M,et al.2002.Muscle-related traits in cattle:The role of the myostatin gene in the South Devon breed[J].Genetics selection Evolution,34(2):221-32.
Xue Y,Ren J,Gao X,et al.2008.GPS 2.0,a tool to predict kinase-specific phosphorylation sites in hierarchy[J].Molecular and Cellular Proteomics,7(9):1598-1608.