猪PKM2基因的序列分析与组织表达及亚细胞定位
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摘要
运用RT–PCR技术克隆猪PKM2基因的编码区序列,对该序列进行生物信息学分析,研究PKM2基因的组织表达及亚细胞定位。结果显示:猪PKM2基因CDS全长1 596 bp,编码531个氨基酸;预测其蛋白相对分子质量为5.79×104,等电点为7.96,含有多个磷酸化位点,为稳定蛋白;PKM2蛋白含有丙酮酸激酶家族的barreldomain和alpha/beta domain 2个结构域,其蛋白二级结构中α–螺旋和无规则卷曲占主要类型;猪PKM2蛋白序列在物种间非常保守,系统进化树分析表明,该蛋白与兔的亲缘关系最近;组织表达显示,猪PKM2基因在肾、小肠中相对高表达,而在肝脏与肌肉中相对低表达;亚细胞定位显示,PKM2蛋白在猪3D4/21和PK15细胞中表达于细胞质,而不表达于细胞核。
The coding region of PKM2 was cloned by RT-PCR, and was subjected to sequence bioinformatics analysis, tissue expression profiling and subcellular localization. The results showed that the CDS of porcine PKM2 gene was 1 596 bp in length, which encodes 531 amino acids with the molecular weight 5.79×104, the isoelectric point of 7.96, and multiple phosphorylation sites. PKM2 protein had two domains, pyruvate kinase, barrel domain and pyruvate kinase, alpha/beta domain. Regarding to secondary structure, the α-helices and random coil accounted for the main types in the PKM2 protein. The PKM2 protein sequence was very conserved among species, and phylogenetic tree analysis showed that the procine PKM2 protein is closely related to rabbits. The tissue expression profiling revealed that PKM2 gene was highly expressed in kidney and small intestine, and was relatively low expressed in liver and muscle. Subcellular localization showed that the PKM2 protein is expressed in the cytoplasm in porcine 3 D4/21 and PK15 cells but not in the nucleus.
The coding region of PKM2 was cloned by RT-PCR, and was subjected to sequence bioinformatics analysis, tissue expression profiling and subcellular localization. The results showed that the CDS of porcine PKM2 gene was 1 596 bp in length, which encodes 531 amino acids with the molecular weight 5.79×104, the isoelectric point of 7.96, and multiple phosphorylation sites. PKM2 protein had two domains, pyruvate kinase, barrel domain and pyruvate kinase, alpha/beta domain. Regarding to secondary structure, the α-helices and random coil accounted for the main types in the PKM2 protein. The PKM2 protein sequence was very conserved among species, and phylogenetic tree analysis showed that the procine PKM2 protein is closely related to rabbits. The tissue expression profiling revealed that PKM2 gene was highly expressed in kidney and small intestine, and was relatively low expressed in liver and muscle. Subcellular localization showed that the PKM2 protein is expressed in the cytoplasm in porcine 3 D4/21 and PK15 cells but not in the nucleus.
引文
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[5]WONG N,OJO D,YAN J,et al.PKM2 contributes to cancer metabolism[J].Cancer Lett,2015,356(2):184-191.
[6]HSU M C,HUNG W C.Pyruvate kinase M2 fuels multiple aspects of cancer cells:from cellular metabolism,transcriptional regulation to extracellular signaling[J].Mol Cancer,2018,17:35.
[7]PALSSON-MCDERMOTT E M,CURTIS A M,GOELG,et al.Pyruvate kinase M2 regulates Hif-1αactivity and IL-1βinduction and is a critical determinant of the warburg effect in LPS-activated macrophages[J].Cell Metabolism,2015,21(1):65-80.
[8]XIE M,YU Y,KANG R,et al.PKM2-dependent glycolysis promotes NLRP3 and AIM2 inflammasome activation[J].Nat Commun,2016,7:13280.
[9]SHIRAI T,NAZAREWICZ R R,WALLIS B B,et al.The glycolytic enzyme PKM2 bridges metabolic and inflammatory dysfunction in coronary artery disease[J].JExp Med,2016,213(3):337-354.
[10]方晓敏,赵为民,付言峰,等.猪支原体肺炎发生的品种敏感差异及分子基础[J].中国农业科学,2015,48(14):2839-2847.
[11]LEBIODA L,STEC B.Crystal structure of enolase indicates that enolase and pyruvate kinase evolved from a common ancestor[J].Nature,1988,333:683-686.
[12]YAMADA K,NOGUCHI T.Alteration of isozyme gene expression during cell differentiation and oncogenesis[J].Nippon Rinsho,1995,53:1112-1118.
[13]MAZUREK S.Pyruvate kinase type M2:a key regulator of the metabolic budget system in tumor cells[J].Int JBiochem Cell Biol,2011,4(7)3:969-980.
[14]CLOWER C V,CHATTERJEE D,WANG Z,et al.The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism[J].Proc Natl Acad Sci USA,2010,107(5):1894-1899.
[15]PANASYUK G,ESPEILLAC C,CHAUVIN C.PPARγcontributes to PKM2 and HK2 expression in fatty liver[J].Nat Commun,2012,3:672.
[16]YANG W W,XIA Y,JI H T,et al.Nuclear PKM2regulatesβ-catenin transactivation upon EGFR activation[J].Nature,2011,480:118-122.
[17]HOSHINO A,HIRST J A,FUJII H.Regulation of cell proliferation by interleukin-3-induced nuclear translocation of pyruvate kinase[J].J Biol Chem,2007,282(24):17706-17711.
[18]GAO X L,WANG H Z,YANG J J,et al.Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase[J].Mol Cell,2012,45(5):598-609.
[19]YANG W W,XIA Y,HAWKE D,et al.PKM2phosphorylates histone H3 and promotes gene transcription and tumorigenesis[J].Cell,2012,150(4):685-696.