Abdelmegeed Mohamed A, Moon Kwan-Hoon, Chen Chi, Gonzalez Frank J, Song Byoung-Joon
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, USA.
Biochem Pharmacol. 2010 Jan 1;79(1):57-66. doi: 10.1016/j.bcp.2009.07.016. Epub 2009 Aug 4.
It is well established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400mg/kg) for 4 and 24h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.
众所周知，在摄入毒性剂量的对乙酰氨基酚（APAP）后，过氧亚硝酸盐产生的标志——硝基酪氨酸蛋白加合物（3-NT），与细胞色素P450 2E1（CYP2E1）高度表达的肝小叶中央坏死区域共定位，这表明3-NT的形成可能在APAP介导的毒性中起关键作用。本研究旨在调查野生型和Cyp2e1基因敲除小鼠在暴露于APAP（200和400mg/kg）4小时和24小时后，CYP2E1与硝化作用（3-NT形成）以及随后泛素介导的蛋白质降解之间的关系。仅在暴露于APAP的野生型小鼠中观察到肝小叶中央坏死和3-NT形成显著增加，且呈剂量和时间依赖性，这证实了CYP2E1在APAP生物转化和毒性中的重要作用。然而，3-NT蛋白加合物的模式，在没有一氧化氮合酶（NOS）同时激活的情况下，与蛋白质泛素化相似。免疫印迹分析进一步表明，在暴露于APAP的野生型小鼠中，免疫沉淀的硝化蛋白被泛素化，这证实了硝化蛋白比天然蛋白更容易受到泛素依赖性降解的影响，导致半衰期更短。例如，胞质超氧化物歧化酶（SOD1）水平明显降低，免疫沉淀的SOD1被硝化和泛素化，这可能导致其在暴露于APAP的野生型小鼠中加速降解。这些数据表明，CYP2E1似乎在3-NT形成、蛋白质降解和肝损伤中起关键作用，这与NOS无关，并且野生型小鼠中许多蛋白质水平的降低（与Cyp2e1基因敲除小鼠相比）可能导致与APAP相关的毒性。
