Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated 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 downregulation, 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 downregulation. 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 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 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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酪氨酸硝化促进一氧化氮刺激的CYP2B6降解

人类细胞色素P450（P450）CYP2B6受到一氧化氮（NO）依赖性蛋白酶体降解，以响应HeLa和HuH7细胞系中的NO供体二丙烯三胺NONOate（DPTA）和生物NO。CYP2B6在原代人肝细胞中也被NO下调。我们假设NO或衍生的反应性氮物种可能会生成酪氨酸和/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了Tyr和Cys残基进行突变。在HuH7细胞中表达的CYP2B6V5-Y317A，-Y380A和-Y190A突变蛋白对野生型（WT）酶对DPTA引起的降解的敏感性较低，表明这些酪氨酸是NO依赖性下调的靶标。与WT酶相反，在用DPTA或硼替佐米+ DPTA处理后，Y317A或Y380A突变体并未显示出高分子量（HMM）物种增加。一氧化碳释放分子2的处理引起2B6酶活性的快速抑制，显着的HMM物种生成和CYP2B6蛋白的泛素化，但不刺激CYP2B6降解。CYP2B6抑制剂4-（4-氯苯基）咪唑阻断了NO依赖型CYP2B6的降解，提示NO进入活性位点很重要。分子动力学模拟预测，CYP2B6的酪氨酸硝化将引起二级结构的不稳定干扰，并消除酶功能可能需要的相关运动。我们建议Y190，Y317，