Swiss Center for Applied Human Toxicology and Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
Toxicology. 2012 Nov 15;301(1-3):72-8. doi: 10.1016/j.tox.2012.07.001. Epub 2012 Jul 11.
Dithiocarbamates and organotins can inhibit enzymes by interacting with functionally essential sulfhydryl groups. Both classes of chemicals were shown to inhibit human 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which converts active cortisol into inactive cortisone and has a role in renal and intestinal electrolyte regulation and in the feto-placental barrier to maternal glucocorticoids. In fish, 11β-HSD2 has a dual role by inactivating glucocorticoids and generating the major androgen 11-ketotestosterone. Inhibition of this enzyme may enhance glucocorticoid and diminish androgen effects in fish. Here, we characterized 11β-HSD2 activity of the model species zebrafish. A comparison with human and mouse 11β-HSD2 revealed species-specific substrate preference. Unexpectedly, assessment of the effects of thiram and several organotins on the activity of zebrafish 11β-HSD2 showed weak inhibition by thiram and no inhibition by any of the organotins tested. Sequence comparison revealed the presence of an alanine at position 253 on zebrafish 11β-HSD2, corresponding to cysteine-264 in the substrate-binding pocket of the human enzyme. Substitution of alanine-253 by cysteine resulted in a more than 10-fold increased sensitivity of zebrafish 11β-HSD2 to thiram. Mutating cysteine-264 on human 11β-HSD2 to serine resulted in 100-fold lower inhibitory activity. Our results demonstrate significant species differences in the sensitivity of human and zebrafish 11β-HSD2 to inhibition by thiram and organotins. Site-directed mutagenesis revealed a key role of cysteine-264 in the substrate-binding pocket of human 11β-HSD2 for sensitivity to sulfhydryl modifying agents.
二硫代氨基甲酸盐和有机锡可以通过与功能上必需的巯基基团相互作用来抑制酶。这两类化学物质都被证明可以抑制人类 11β-羟甾脱氢酶 2（11β-HSD2），该酶将活性皮质醇转化为非活性皮质酮，在肾脏和肠道电解质调节以及胎儿-胎盘屏障对母体糖皮质激素的作用中发挥作用。在鱼类中，11β-HSD2 具有双重作用，既能使糖皮质激素失活，又能产生主要的雄激素 11-酮睾酮。抑制这种酶可能会增强糖皮质激素的作用，并减少鱼类的雄激素作用。在这里，我们对模式物种斑马鱼的 11β-HSD2 活性进行了表征。与人类和小鼠 11β-HSD2 的比较揭示了物种特异性的底物偏好。出乎意料的是，评估噻唑烷和几种有机锡对斑马鱼 11β-HSD2 活性的影响表明，噻唑烷的抑制作用较弱，而测试的任何有机锡都没有抑制作用。序列比较显示，在斑马鱼 11β-HSD2 中，第 253 位存在丙氨酸，对应于人类酶底物结合口袋中的半胱氨酸-264。丙氨酸-253 突变为半胱氨酸导致斑马鱼 11β-HSD2 对噻唑烷的敏感性增加了 10 多倍。将人类 11β-HSD2 中的半胱氨酸-264 突变为丝氨酸导致抑制活性降低 100 倍。我们的研究结果表明，噻唑烷和有机锡对人类和斑马鱼 11β-HSD2 的抑制敏感性存在显著的种间差异。定点突变显示半胱氨酸-264 在人类 11β-HSD2 的底物结合口袋中对于巯基修饰剂的敏感性起着关键作用。
