Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Arch Toxicol. 2012 Nov;86(11):1693-702. doi: 10.1007/s00204-012-0893-4. Epub 2012 Jun 30.
We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42 kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42 kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.
我们之前开发了一种筛选方法，通过甲基汞（MeHg）识别通过 S-巯基化发生聚集的蛋白质，发现大鼠精氨酸酶 I 是 MeHg 的靶蛋白（Kanda 等人，在《Arch Toxicol》82:803-808, 2008）。在本研究中，我们从大鼠肝制剂中鉴定出另一种 S-汞化的蛋白质，其亚基质量为 42 kDa，从而促进其聚集。二维 SDS-聚丙烯酰胺凝胶电泳和随后使用基质辅助激光解吸电离飞行时间质谱的肽质量指纹图谱分析表明，42 kDa 蛋白是 NAD 依赖性山梨醇脱氢酶（SDH）。用重组大鼠 SDH，我们发现 MeHg 通过半胱氨酸 44、119、129 和 164 与 SDH 共价结合，从而抑制其催化活性、释放锌离子并促进蛋白质聚集。突变分析表明，与活性部位锌原子相连的半胱氨酸 44 和半胱氨酸 129 在 MeHg 介导的 SDH 聚集过程中起着至关重要的作用。辅因子 NAD 的预处理，而不是 NADP 或 FAD，显著阻止了 SDH 的聚集。讨论了 NAD 对 MeHg 引起的 SDH 聚集的这种保护作用。
