Aristolochic acids I and II (AA-I/II) are carcinogenic principles of plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, -hydroxyaristolactam and -sulfonyloxyaristolactam metabolites are transported circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of -sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSA) and its AA complexes (HSA/AA-I and HSA/AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 10 M for HSA/AA-I 8.4 and 9.0 · 10 M for HSA/AA-II. Fluorescence quenching of the HSA Trp suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.

中文翻译：

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人血清白蛋白转运马兜铃酸及其活性代谢物的结构和机制见解


马兜铃酸 I 和 II (AA-I/II) 是植物的致癌成分，已用于传统医学实践，并被发现为食品污染物。虽然氨基酸的有害作用已得到广泛认可，但仍缺乏信息来定义其致癌机制。在肝脏中生物活化后，β-羟基马兜铃内酰胺和-磺酰氧基马兜铃内酰胺代谢物被运输至循环系统，并通过与细胞 DNA 反应而引发致癌作用。在本研究中，我们应用 DNA 加合物分析、X 射线晶体学、等温滴定量热法和荧光猝灭来研究人血清白蛋白 (HSA) 在调节 AA 致癌性中的作用。我们发现 HSA 延长了 β-磺酰氧基马兜铃内酰胺-I 与 DNA 的半衰期和反应活性，从而保护活化的 AA 免遭杂解。应用新型混合血浆 HSA 结晶方法，我们报告了富含肉豆蔻酸的 HSA (HSA) 及其 AA 复合物（HSA/AA-I 和 HSA/AA-II）的高分辨率结构，分辨率为 1.9 Å。 AA-I位于HSA子域IB内，而AA-II占据子域IIA和IB。 ITC 结合谱揭示了两个复合物中两个不同的 AA 位点，HSA/AA-I 的关联常数为 1.5 和 0.5·10 M，HSA/AA-II 的关联常数为 8.4 和 9.0·10 M。 HSA Trp 的荧光猝灭表明脂肪酸对配体结合亲和力有不同的影响。总的来说，我们的结构和热力学表征对 AA 结合、运输、毒性和潜在变构产生了重要的见解，这些是阐明 HSA 在调节 AA 致癌性中的机制作用的关键决定因素。