Oya-Ito Tomoko, Naito Yuji, Takagi Tomohisa, Handa Osamu, Matsui Hirofumi, Yamada Masaki, Shima Keisuke, Yoshikawa Toshikazu
Department of Medical Proteomics, Kyoto Prefectural University of Medicine, 465 Kaji-i, Kyoto 602-8566, Japan.
Biochim Biophys Acta. 2011 Jul;1812(7):769-81. doi: 10.1016/j.bbadis.2011.03.017. Epub 2011 Apr 6.
The molecular mechanisms underlying the posttranslational modification of proteins in gastrointestinal cancer are still unknown. Here, we investigated the role of methylglyoxal modifications in gastrointestinal tumors. Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins. By using a monoclonal antibody to methylglyoxal-modified proteins, we found that murine heat-shock protein 25 and human heat-shock protein 27 were the major adducted proteins in rat gastric carcinoma mucosal cell line and human colon cancer cell line, respectively. Furthermore, we found that heat-shock protein 27 was modified by methylglyoxal in ascending colon and rectum of patients with cancer. However, methylglyoxal-modified heat-shock protein 25/heat-shock protein 27 was not detected in non cancerous cell lines or in normal subject. Matrix-associated laser desorption/ionization mass spectrometry/mass spectrometry analysis of peptide fragments identified Arg-75, Arg-79, Arg-89, Arg-94, Arg-127, Arg-136, Arg-140, Arg-188, and Lys-123 as methylglyoxal modification sites in heat-shock protein 27 and in phosphorylated heat-shock protein 27. The transfer of methylglyoxal-modified heat-shock protein 27 into rat intestinal epithelial cell line RIE was even more effective in preventing apoptotic cell death than that of native control heat-shock protein 27. Furthermore, methylglyoxal modification of heat-shock protein 27 protected the cells against both the hydrogen peroxide- and cytochrome c-mediated caspase activation, and the hydrogen peroxide-induced production of intracellular reactive oxygen species. The levels of lactate converted from methylglyoxal were increased in carcinoma mucosal cell lines. Our results suggest that posttranslational modification of heat-shock protein 27 by methylglyoxal may have important implications for epithelial cell injury in gastrointestinal cancer.
胃肠道癌中蛋白质翻译后修饰的分子机制仍不清楚。在此，我们研究了甲基乙二醛修饰在胃肠道肿瘤中的作用。甲基乙二醛是一种由细胞糖酵解中间产物产生的反应性二羰基化合物，它能与蛋白质发生非酶反应。通过使用针对甲基乙二醛修饰蛋白的单克隆抗体，我们发现小鼠热休克蛋白25和人类热休克蛋白27分别是大鼠胃癌黏膜细胞系和人类结肠癌细胞系中的主要加合蛋白。此外，我们发现癌症患者升结肠和直肠中的热休克蛋白27被甲基乙二醛修饰。然而，在非癌细胞系或正常受试者中未检测到甲基乙二醛修饰的热休克蛋白25/热休克蛋白27。对肽片段进行基质辅助激光解吸/电离质谱/质谱分析，确定了热休克蛋白27和磷酸化热休克蛋白27中的精氨酸-75、精氨酸-79、精氨酸-89、精氨酸-94、精氨酸-127、精氨酸-136、精氨酸-140、精氨酸-188和赖氨酸-123为甲基乙二醛修饰位点。与天然对照热休克蛋白27相比，将甲基乙二醛修饰的热休克蛋白27转入大鼠肠上皮细胞系RIE中，在预防凋亡性细胞死亡方面甚至更有效。此外，热休克蛋白27的甲基乙二醛修饰可保护细胞免受过氧化氢和细胞色素c介导的半胱天冬酶激活，以及过氧化氢诱导的细胞内活性氧生成。癌黏膜细胞系中由甲基乙二醛转化而来的乳酸水平升高。我们的结果表明，甲基乙二醛对热休克蛋白27的翻译后修饰可能对胃肠道癌中的上皮细胞损伤具有重要意义。
