Fisher Ashley A, Labenski Matthew T, Malladi Srinivas, Gokhale Vijay, Bowen Martina E, Milleron Rania S, Bratton Shawn B, Monks Terrence J, Lau Serrine S
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, USA.
Biochemistry. 2007 Oct 2;46(39):11090-100. doi: 10.1021/bi700613w. Epub 2007 Sep 7.
Electrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized. Hydroquinone, and its mercapturic acid pathway metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of HQ and its thioether metabolites are in part a consequence of their oxidation to the corresponding electrophilic 1,4-benzoquinones (BQ). We now report that BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c. Furthermore, we have identified specific glutamic acid residues within cytochrome c as novel sites of NAC-BQ adduction. The microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. The solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Postadduction chemistry subsequently alters the nature of the final adduct. Using molecular modeling, the impact of BQ and NAC-BQ adduction on cytochrome c was visualized, revealing the spatial rearrangement of critical residues necessary for protein-protein interactions. Consequently, BQ-adducted cytochrome c fails to initiate caspase-3 activation in native lysates and also inhibits Apaf-1 oligomerization into an apoptosome complex in a purely reconstituted system. In summary, a combination of mass spectroscopic, molecular modeling, and biochemical approaches confirms that electrophile-protein adducts produce structural alterations that influence biological function.
内源性产生的亲电试剂，或通过药物及其他环境化学物质的代谢生物活化作用产生的亲电试剂，能够与蛋白质内的各种亲核位点结合。决定亲电试剂介导的翻译后修饰的位点选择性易感性的因素，以及此类改变的后果，在很大程度上仍不为人所知。为了鉴定和表征化学介导的蛋白质加合物，使用了具有已知毒性的亲电试剂。对苯二酚及其硫醚酸途径代谢物会导致大鼠肾近端小管细胞坏死和肾致癌性。对苯二酚及其硫醚代谢物的不良反应部分是它们氧化为相应的亲电1,4 - 苯醌（BQ）的结果。我们现在报告，BQ和2 -（N - 乙酰半胱氨酸 - S - 基）苯醌（NAC - BQ）优先结合到细胞色素c内溶剂暴露的富含赖氨酸区域。此外，我们已确定细胞色素c内的特定谷氨酸残基是NAC - BQ加合的新位点。加合位点处的微环境决定了初始特异性和最终加合物的结构。加合氨基酸和相邻氨基酸的溶剂可及性和局部pKa有助于加合的选择性。加合后化学作用随后改变了最终加合物的性质。使用分子建模，可视化了BQ和NAC - BQ加合对细胞色素c的影响，揭示了蛋白质 - 蛋白质相互作用所需关键残基的空间重排。因此，BQ加合的细胞色素c在天然裂解物中无法启动caspase - 3激活，并且在纯重组系统中也抑制Apaf - 1寡聚形成凋亡小体复合物。总之，质谱、分子建模和生化方法相结合证实亲电试剂 - 蛋白质加合物产生影响生物学功能的结构改变。
