Activation/deactivation of acetylcholinesterase by H2O2:: more evidence for oxidative stress in vitiligo
Activation/deactivation of acetylcholinesterase by H2O2:: more evidence for oxidative stress in vitiligo
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DOI：
10.1016/j.bbrc.2004.01.082
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发表时间：
2004-03-05
影响因子：
3.1
通讯作者：
Wood, JM
中科院分区：
文献类型：
--
作者：
Schallreuter, KU;Elwary, SMA;Wood, JM
此前已证明人表皮合成和降解乙酰胆碱并表达毒蕈碱和烟碱受体。这些胆碱能系统与正常健康皮肤的表皮钙梯度的发展和分化有关。在白癜风中，这些患者的表皮中会发生严重的氧化应激，H2O2 积聚在 10(-3) M 范围内，同时由于酶活性位点失活，过氧化氢酶表达/活性降低。研究还表明，必需辅因子 (6R)-L-erythro-5,6,7,8-四氢生物蝶呤通过蝶呤-4a-甲醇胺脱水酶 (PCD) 和二氢蝶啶还原酶 (DHPR) 的整个循环受到酶结构中 Trp/Met 残基的 H2O2 氧化的影响，导致这些蛋白质失活。现在，利用荧光免疫化学，我们发现白癜风患者的表皮 H2O2 几乎不产生表皮乙酰胆碱酯酶 (AchE)。使用纯重组人 AchE 的动力学分析表明，低浓度的 H2O2 (10(-6) M) 通过增加 V-max > 2 倍来激活该酶，同时高浓度的 H2O2 (10(-3) M) 抑制该酶，并显着降低 V-max。该结果通过在 lambda(max) 280nm 处的 Trp 荧光后的荧光激发光谱证实。基于已建立的人类 AchE 3D 结构的分子模型支持 H2O2 介导的 Trp(432)、Trp(435) 和 Met(436) 氧化会移动并扰乱酶的活性位点 His(440)，从而导致蛋白质失活。据我们所知，这些结果首次确定了 H2O2 对 AchE 的调节作用。此外，研究表明，H2O2 介导的 AchE 氧化对白癜风中已确定的氧化应激有显着贡献。 (C) 2004 Elsevier Inc. 保留所有权利。
Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3) M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunchistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetyleholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6) M) activate this enzyme by increasing the V-max > 2-fold, meanwhile high concentrations of H2O2 (10(-3) M) inhibit the enzyme with a significant decrease in V-max. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambda(max) 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo. (C) 2004 Elsevier Inc. All rights reserved.