Department of Pathology Microbiology and Immunology, Vanderbilt University Medical Center, 1161 21st Avenue South, Nashville, TN 37232-2561, USA.
Chem Res Toxicol. 2012 Nov 19;25(11):2310-21. doi: 10.1021/tx300198h. Epub 2012 Aug 22.
Previous studies have shown ubiquitin activating enzyme E1 to be sensitive to adduction through both Michael addition and SN(2) chemistry in vitro. E1 presents a biologically important putative protein target for adduction due to its role in initiating ubiquitin based protein processing and the involvement of impaired ubiquitin protein processing in two types of familial Parkinson's disease. We tested whether E1 is susceptible to xenobiotic-mediated electrophilic adduction in vivo and explored the potential contribution of E1 adduction to neurodegenerative events in an animal model. N,N-Diethyldithiocarbamate (DEDC) was administered to rats using a protocol that produces covalent cysteine modifications in vivo, and brain E1 protein adducts were characterized and mapped using shotgun LC-MS/MS. E1 activity, global and specific protein expression, and protein carbonyls were used to characterize cellular responses and injury in whole brain and dorsal striatal samples. The data demonstrate that DEDC treatment produced S-(ethylaminocarbonyl) adducts on Cys234 and Cys179 residues of E1 and decreased the levels of activated E1 and total ubiquitinated proteins. Proteomic analysis of whole brain samples identified expression changes for proteins involved in myelin structure, antioxidant response, and catechol metabolism, systems often disrupted in neurodegenerative disease. Our studies also delineated localized injury within the striatum as indicated by decreased levels of tyrosine hydroxylase, elevated protein carbonyl content, increased antioxidant enzyme and α-synuclein expression, and enhanced phosphorylation of tau and tyrosine hydroxylase. These data are consistent with E1 having similar susceptibility to adduction in vivo as previously reported in vitro and support further investigation into environmental agent adduction of E1 as a potential contributing factor to neurodegenerative disease. Additionally, this study supports the predictive value of in vitro screens for identifying sensitive protein targets that can be used to guide subsequent in vivo experiments.
先前的研究表明，泛素激活酶 E1 易受到迈克尔加成和 SN(2)化学体外加成。E1 由于其在启动泛素基蛋白处理中的作用以及在两种家族性帕金森病中受损的泛素蛋白处理的参与，成为生物上重要的潜在蛋白加合物靶标。我们测试了 E1 是否易受体内异生物质介导的亲电加合，并在动物模型中探索了 E1 加合对神经退行性事件的潜在贡献。使用一种在体内产生半胱氨酸共价修饰的方案，用 N,N-二乙基二硫代氨基甲酸盐（DEDC）处理大鼠，并用鸟枪法 LC-MS/MS 对脑 E1 蛋白加合物进行鉴定和定位。E1 活性、总蛋白和特异性蛋白表达以及蛋白质羰基用于表征整个大脑和背侧纹状体样本中的细胞反应和损伤。数据表明，DEDC 处理产生了 E1 上 Cys234 和 Cys179 残基的 S-(乙基氨基羰基)加合物，并降低了激活 E1 和总泛素化蛋白的水平。全脑样本的蛋白质组学分析鉴定了涉及髓鞘结构、抗氧化反应和儿茶酚代谢的蛋白质表达变化，这些系统在神经退行性疾病中经常受到破坏。我们的研究还描绘了纹状体中的局部损伤，表现为酪氨酸羟化酶水平降低、蛋白质羰基含量升高、抗氧化酶和α-突触核蛋白表达增加以及 tau 和酪氨酸羟化酶磷酸化增强。这些数据与 E1 在体内的加合易感性与先前报道的体外结果一致，并支持进一步研究环境剂对 E1 的加合作为神经退行性疾病潜在贡献因素。此外，这项研究支持了体外筛选用于识别敏感蛋白靶标以指导随后体内实验的预测价值。
