Reactions of glyceraldehyde 3-phosphate dehydrogenase sulfhydryl groups with bis-electrophiles produce DNA-protein cross-links but not mutations
Reactions of glyceraldehyde 3-phosphate dehydrogenase sulfhydryl groups with bis-electrophiles produce DNA-protein cross-links but not mutations
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DOI：
10.1021/tx7003618
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发表时间：
2008-02-01
影响因子：
4.1
通讯作者：
Guengerich, F. Peter
中科院分区：
文献类型：
--
作者：
Loecken, Elisabeth M.;Guengerich, F. Peter
环境污染物1，2-二溴乙烷和二环氧丁烷是重要的工业化学品丁二烯的氧化产物，是双功能亲电体，已知具有致突变性和致癌性。双亲电试剂可发挥其毒性作用的一种机制是通过诱导遗传毒性和致突变DNA-肽交联。这种机制已经在过表达DNA修复蛋白O-6-烷基鸟嘌呤DNA-烷基转移酶（AGT）或谷胱甘肽S-转移酶的系统中得到证实，并涉及与亲核半胱氨酸残基的反应。通过筛选核蛋白与模型单功能亲电体的反应性，研究了DNA-蛋白质交联形成是双亲电体遗传毒性的更一般机制的假设。甘油醛3-磷酸脱氢酶（GAPDH）被鉴定为候选物，因为在用模型亲电体进行的反应筛选中两个半胱氨酸残基（Cys（152）和Cys（246））的亲核性（Dennehy，M. K.等人（2006）Chem.Res.Toxicol. 19、20-29）。孵育GAPDH与双亲电体导致其催化活性的抑制，但仅在高浓度的二环氧丁烷。体外试验表明，在二环氧丁烷的存在下，DNA-GAPDH交联形成，并使用质谱分析确认在Cys（246）的双亲电试剂反应性。与AGT相反，人GAPDH在大肠杆菌中的过表达没有增强二环氧丁烷的诱变。我们提出，缺乏突变增强部分是由于GAPDH对双亲电体的固有较低反应性以及相对于AGT降低的DNA结合能力，从而防止体内形成DNA-蛋白质交联和增强的诱变。
The environmental contaminant 1,2-dibromoethane and diepoxybutane, an oxidation product of the important industrial chemical butadiene, are bis-functional electrophiles and are known to be mutagenic and carcinogenic. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA-peptide cross-links. This mechanism has been shown in systems overexpressing the DNA repair protein O-6-alkylguanine DNA-alkyltransferase (AGT) or glutathione S-transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein cross-link formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate because of the nucleophilicity of two cysteine residues (Cys(152) and Cys(246)) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19, 20-29). Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity, but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH cross-link formation in the presence of diepoxybutane, and bis-electrophile reactivity at Cys(246) was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. We propose that the lack of mutational enhancement is in part due to the inherently lower reactivity of GAPDH toward bis-electrophiles as well as the reduced DNA binding ability relative to AGT, preventing the in vivo formation of DNA-protein cross-links and enhanced mutagenesis.