Najbauer J, Johnson B A, Aswad D W
School of Biological Sciences, University of California, Irvine 92717-4550.
Arch Biochem Biophys. 1992 Feb 14;293(1):85-92. doi: 10.1016/0003-9861(92)90369-8.
It was demonstrated recently that substrates for protein N-methyltransferases (J. Najbauer and D. W. Aswad, 1990, J. Biol. Chem. 265, 12,717-12,721) and protein carboxyl methyltransferases (J. Najbauer, B. A. Johnson, and D. W. Aswad, 1991, Anal. Biochem. 197, 412-420) accumulate when rat PC12 cells are cultured in the presence of the methylation inhibitor, adenosine dialdehyde. In the present report, we have further characterized this phenomenon in PC12 cells and in two other, widely used cell types. Adenosine dialdehyde was found to increase the methyl-accepting capacity of proteins in human skin fibroblasts and mouse Sp2/0 myeloma cells. However, both the level of methyl incorporation in untreated cells and the amount of stimulation afforded by inhibitor treatment were substantially lower in these cells than in PC12 cells. All three cell lines accumulated methyl acceptor(s) at 17-21 kDa. The PC12 cells and the fibroblasts also exhibited stimulation of three apparently similar proteins in the 33- to 38-kDa region, where several arginine-methylated proteins involved in RNA processing would be expected. The optimal conditions for methylation of PC12 cell extracts with regard to pH, time of methylation, and S-[methyl-3H]adenosyl-L-methionine concentration were characterized. Increased methyl incorporation was detected after adenosine dialdehyde treatments as short as 2 h, and methylation of most substrates continued to increase as the time of treatment was extended to 72 h. The kinetics of accumulation varied from substrate to substrate. Fluorograms of two-dimensional gels of extracts from untreated PC12 cells incubated in the presence of S-[methyl-3H]adenosyl-L-methionine revealed patterns of methyl incorporation similar to those of treated cells, but longer exposure times were necessary (e.g., 35 days vs 7 days). These findings suggest that the inhibitor treatment works mainly by inhibiting the post- or cotranslational methylation of a "normal" array of cellular proteins.
最近有研究表明，当大鼠嗜铬细胞瘤（PC12）细胞在甲基化抑制剂腺苷二醛存在的情况下培养时，蛋白质N - 甲基转移酶的底物（J. Najbauer和D. W. Aswad，1990，《生物化学杂志》265卷，12717 - 12721页）以及蛋白质羧基甲基转移酶的底物（J. Najbauer，B. A. Johnson和D. W. Aswad，1991，《分析生物化学》197卷，412 - 420页）会积累。在本报告中，我们进一步对PC12细胞以及另外两种广泛使用的细胞类型中的这一现象进行了表征。发现腺苷二醛可增加人皮肤成纤维细胞和小鼠Sp2/0骨髓瘤细胞中蛋白质的甲基接受能力。然而，这些细胞中未处理细胞的甲基掺入水平以及抑制剂处理所提供的刺激量均明显低于PC12细胞。所有三种细胞系均在17 - 21 kDa处积累甲基受体。PC12细胞和成纤维细胞在33至38 kDa区域还表现出对三种明显相似蛋白质的刺激，预计该区域会有几种参与RNA加工的精氨酸甲基化蛋白质。确定了PC12细胞提取物甲基化的最佳条件，包括pH、甲基化时间以及S - [甲基 - ³H]腺苷 - L - 甲硫氨酸浓度。腺苷二醛处理仅2小时后就检测到甲基掺入增加，并且随着处理时间延长至72小时，大多数底物的甲基化持续增加。积累的动力学因底物而异。在S - [甲基 - ³H]腺苷 - L - 甲硫氨酸存在下孵育的未处理PC12细胞提取物的二维凝胶荧光图谱显示，甲基掺入模式与处理过的细胞相似，但需要更长的曝光时间（例如，35天对7天）。这些发现表明，抑制剂处理主要通过抑制细胞内“正常”蛋白质阵列的翻译后或共翻译甲基化起作用。
