Modulation of activity and inhibitor sensitivity of rabbit aldose reductase-like protein (AKR1B19) by oxidized glutathione and SH-reagents
Modulation of activity and inhibitor sensitivity of rabbit aldose reductase-like protein (AKR1B19) by oxidized glutathione and SH-reagents
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DOI：
10.1016/j.cbi.2012.11.026
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发表时间：
2013-02-25
影响因子：
5.1
通讯作者：
Hara, Akira
中科院分区：
文献类型：
--
作者：
Endo, Satoshi;Fujimoto, Airi;Hara, Akira
兔醛酮还原酶 (AKR) 1B19 是人醛糖还原酶样蛋白 (ARLP) AKR1B10 的直向同源物，显示出 86% 的氨基酸序列同一性。与已知的醛还原酶相比，AKR1B19 对 4-oxo-2-nonenal（脂质过氧化的主要产物）表现出最高的催化效率。在本研究中，我们发现在不存在或存在 NADPH 的情况下，添加 10 μM SH 试剂（对氯汞苯磺酸和对氯汞苯甲酸）后，AKR1B19 的还原酶活性立即被激活约 5 倍。此外，与谷胱甘肽二硫化物 (GSSG) 孵育 1 小时可诱导 AKR1B19 最大 3 倍激活。通过与二硫苏糖醇和谷胱甘肽进一步孵育，活化的酶转化为天然酶。 AKR1B19 的 C299S 突变消除了激活，并通过质谱分析鉴定了谷胱甘肽化的 Cys299。 Cys299 修饰的酶根据底物和抑制剂的不同表现出不同的动力学变化。在4-氧代-2-壬烯醛的还原中，催化效率提高。因此，AKR1B10 可以通过 GSSG/谷胱甘肽的细胞比例进行调节，并在氧化应激条件下更有效地充当细胞毒性醛的解毒酶。此外，在 AKR1B10 和大鼠 ARLP 中未检测到 GSSG 的这种活性改变，表明 AKR1B19 中 Cys299 附近存在 GSSG 结合位点。 (C) 2012 Elsevier Ireland Ltd. 保留所有权利。
Rabbit aldo-keto reductase (AKR) 1B19 is an ortholog of human aldose reductase-like protein (ARLP), AKR1B10, showing 86% amino acid sequence identity. AKR1B19 exhibits the highest catalytic efficiency for 4-oxo-2-nonenal, a major product of lipid peroxidation, compared to known reductases of this aldehyde. In this study, we found that the reductase activity of AKR1B19 was activated to about 5-fold immediately after the addition of 10 mu M SH-reagents (p-chloromercuriphenylsulfonic acid and p-chloromercuribenzoic acid) in the absence or presence of NADPH. In addition, a maximum of 3-fold activation of AKR1B19 was induced by incubation with glutathione disulfide (GSSG) for 1 h. The activated enzyme was converted into the native enzyme by further incubation with dithiothreitol and glutathione. The activation was abolished by the C299S mutation of AKR1B19, and the glutathionylated Cys299 was identified by mass spectrometry analysis. The Cys299-modified enzyme displayed different kinetic alterations depending on substrates and inhibitors. In the reduction of 4-oxo-2-nonenal, the catalytic efficiency was increased. Thus, AKR1B10 may be modulated by cellular ratio of GSSG/glutathione and more efficiently act as a detoxifying enzyme for the cytotoxic aldehyde under oxidatively stressed conditions. Furthermore, such an activity alteration by GSSG was not detected in AKR1B10 and rat ARLPs, suggesting the presence of a GSSG-binding site near Cys299 in AKR1B19. (C) 2012 Elsevier Ireland Ltd. All rights reserved.