UPF0586 Protein C9orf41 Homolog Is Anserine-producing Methyltransferase
UPF0586 Protein C9orf41 Homolog Is Anserine-producing Methyltransferase
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DOI：
10.1074/jbc.m115.640037
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发表时间：
2015-07-10
影响因子：
4.8
通讯作者：
de Heer, Emile
中科院分区：
文献类型：
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作者：
Drozak, Jakub;Piecuch, Maria;de Heer, Emile
鹅肌肽（β-丙氨酰-N(P-i)-甲基-L-组氨酸）是肌肽（β-丙氨酰-L-组氨酸）的甲基化衍生物，是脊椎动物骨骼肌的丰富成分。尽管它被建议用作质子缓冲剂和自由基清除剂，但其生理功能仍然是神秘的。鹅氨酸的形成是由肌肽 N-甲基转移酶催化的，最近在鸡中鉴定为组胺 N-甲基转移酶样（HNMT 样）蛋白。尽管哺乳动物基因组中不存在 HNMT 样基因，但大多数哺乳动物物种中都报道了肌肽 N-甲基转移酶的活性。在本研究中，我们从大鼠肌肉中纯化了约 2600 倍的肌肽 N-甲基转移酶。在制剂中鉴定出与酶活性共洗脱的三种与 45、50 和 70 kDa 相似的多肽。对这些多肽进行质谱分析，确定 UPF0586 蛋白 C9orf41 同源物是唯一有意义的候选物。大鼠 UPF0586 及其酵母、鸡和人直系同源物在 COS-7 细胞中表达并纯化至均质。尽管经色谱和质谱分析证实，所有重组蛋白均催化鹅肌肽的形成，但大鼠 UPF0586 对肌肽的活性比其他直系同源物更高。表达重组 UPF5086 蛋白的 HeLa 细胞的共聚焦显微镜显示它们存在于细胞质和细胞核中。肌肽和甘氨酸是所有研究的 UPF0586 直系同源物的最佳底物，尽管这些酶也会甲基化其他含 L-组氨酸的二肽和三肽。最后，用大鼠或人 UPF0586 和肌肽合酶共转染 COS-7 细胞，将细胞转化为高效的鹅氨酸生产者。我们得出结论，UPF0586 是哺乳动物肌肽 N-甲基转移酶，并假设它也可能在真核生物中充当肽或蛋白质甲基转移酶。
Anserine (beta-alanyl-N(P-i)-methyl-L-histidine), a methylated derivative of carnosine (beta-alanyl-L-histidine), is an abundant constituent of vertebrate skeletal muscles. Although it has been suggested to serve as a proton buffer and radical scavenger, its physiological function remains mysterious. The formation of anserine is catalyzed by carnosine N-methyltransferase, recently identified in chicken as histamine N-methyltransferaselike (HNMT-like) protein. Although the HNMT-like gene is absent in mammalian genomes, the activity of carnosine N-methyltransferase was reported in most mammalian species. In the present investigation, we purified carnosineN-methyltransferase from rat muscles about 2600-fold. Three polypeptides of similar to 45, 50, and 70 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of UPF0586 protein C9orf41 homolog as the only meaningful candidate. Rat UPF0586 and its yeast, chicken, and human orthologs were expressed in COS-7 cells and purified to homogeneity. Although all recombinant proteins catalyzed the formation of anserine, as confirmed by chromatographic and mass spectrometry analysis, rat UPF0586 was more active on carnosine than other orthologs. Confocal microscopy of HeLa cells expressing recombinant UPF5086 proteins revealed their presence in both cytosol and nucleus. Carnosine and Gly-His were the best substrates for all UPF0586 orthologs studied, although the enzymes also methylated other L-histidine-containing di- and tripeptides. Finally, cotransfection of COS-7 cells with rat or human UPF0586 and carnosine synthase transformed the cells into efficient anserine producers. We conclude that UPF0586 is mammalian carnosine N-methyl-transferase and hypothesize that it may also serve as a peptide or protein methyltransferase in eukaryotes.