Hauser David N, Dukes April A, Mortimer Amanda D, Hastings Teresa G
Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA 15260.
Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA 15260; Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260.
Free Radic Biol Med. 2013 Dec;65:419-427. doi: 10.1016/j.freeradbiomed.2013.06.030. Epub 2013 Jun 28.
Oxidative stress and mitochondrial dysfunction are known to contribute to the pathogenesis of Parkinson's disease. Dopaminergic neurons may be more sensitive to these stressors because they contain dopamine (DA), a molecule that oxidizes to the electrophilic dopamine quinone (DAQ) which can covalently bind nucleophilic amino acid residues such as cysteine. The identification of proteins that are sensitive to covalent modification and functional alteration by DAQ is of great interest. We have hypothesized that selenoproteins, which contain a highly nucleophilic selenocysteine residue and often play vital roles in the maintenance of neuronal viability, are likely targets for the DAQ. Here we report the findings of our studies on the effect of DA oxidation and DAQ on the mitochondrial antioxidant selenoprotein glutathione peroxidase 4 (GPx4). Purified GPx4 could be covalently modified by DAQ, and the addition of DAQ to rat testes lysate resulted in dose-dependent decreases in GPx4 activity and monomeric protein levels. Exposing intact rat brain mitochondria to DAQ resulted in similar decreases in GPx4 activity and monomeric protein levels as well as detection of multiple forms of DA-conjugated GPx4 protein. Evidence of both GPx4 degradation and polymerization was observed following DAQ exposure. Finally, we observed a dose-dependent loss of mitochondrial GPx4 in differentiated PC12 cells treated with dopamine. Our findings suggest that a decrease in mitochondrial GPx4 monomer and a functional loss of activity may be a contributing factor to the vulnerability of dopaminergic neurons in Parkinson's disease.
已知氧化应激和线粒体功能障碍与帕金森病的发病机制有关。多巴胺能神经元可能对这些应激源更为敏感，因为它们含有多巴胺（DA），该分子可氧化为亲电的多巴胺醌（DAQ），后者能够与亲核氨基酸残基如半胱氨酸共价结合。鉴定对DAQ的共价修饰和功能改变敏感的蛋白质具有重要意义。我们推测，含有高度亲核的硒代半胱氨酸残基且通常在维持神经元活力中起关键作用的硒蛋白可能是DAQ的作用靶点。在此，我们报告了关于DA氧化和DAQ对线粒体抗氧化硒蛋白谷胱甘肽过氧化物酶4（GPx4）影响的研究结果。纯化的GPx4可被DAQ共价修饰，向大鼠睾丸裂解物中添加DAQ会导致GPx4活性和单体蛋白水平呈剂量依赖性降低。将完整的大鼠脑线粒体暴露于DAQ会导致GPx4活性和单体蛋白水平出现类似降低，并检测到多种形式的DA缀合GPx4蛋白。DAQ暴露后观察到了GPx4降解和聚合的证据。最后，我们在多巴胺处理的分化PC12细胞中观察到线粒体GPx4呈剂量依赖性丧失。我们的研究结果表明，线粒体GPx4单体减少和活性功能丧失可能是帕金森病中多巴胺能神经元易损性的一个促成因素。
