Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biological NO in HeLa and HuH7 cell lines. CYP2B6 is also down-regulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 down-regulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent down-regulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide releasing molecule 2 (CORM2) treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation and ubiquitination of CYP2B6 protein, but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole (4CPI) blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317 and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation.

中文翻译：

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酪氨酸硝化有助于一氧化氮刺激的 CYP2B6 降解。


在 HeLa 和 HuH7 细胞系中，人细胞色素 P450 (P450) CYP2B6 响应 NO 供体二丙三胺 NONOate (DPTA) 和生物 NO，经历一氧化氮 (NO) 依赖性蛋白酶体降解。在原代人肝细胞中，CYP2B6 也会被 NO 下调。我们假设NO或衍生的活性氮可能产生酪氨酸和/或半胱氨酸残基的加合物，导致CYP2B6下调，并根据预测的溶剂可及性选择Tyr和Cys残基进行突变。 HuH7 细胞中表达的 CYP2B6V5-Y317A、-Y380A 和 -Y190A 突变蛋白对 DPTA 引起的降解的敏感性低于野生型 (WT) 酶，表明这些酪氨酸是 NO 依赖性下调的目标。与WT酶相反，用DPTA或硼替佐米+DPTA处理后，Y317A或Y380A突变体没有表现出高分子量(HMM)种类的增加。一氧化碳释放分子 2 (CORM2) 处理导致 2B6 酶活性快速抑制、显着 HMM 物种生成和 CYP2B6 蛋白泛素化，但不刺激 CYP2B6 降解。 CYP2B6 抑制剂 4-(4-氯苯基)咪唑 (4CPI) 阻断 NO 依赖性 CYP2B6 降解，表明 NO 进入活性位点很重要。分子动力学模拟预测 CYP2B6 的酪氨酸硝化会引起二级结构的显着不稳定扰动，并消除酶功能可能所需的相关运动。我们认为，活性氮对 Y190、Y317 和 Y380 的累积硝化会导致 CYP2B6 不稳定，这可能与血红素亚硝基化协同作用，以靶向酶进行降解。