Pastore Anna, Tozzi Giulia, Gaeta Laura Maria, Bertini Enrico, Serafini Valentina, Di Cesare Silvia, Bonetto Valentina, Casoni Filippo, Carrozzo Rosalba, Federici Giorgio, Piemonte Fiorella
Laboratory of Biochemistry, Children's Hospital and Research Institute Bambino Gesù, Piazza S. Onofrio 4, 00165 Rome, Italy.
J Biol Chem. 2003 Oct 24;278(43):42588-95. doi: 10.1074/jbc.M301872200. Epub 2003 Aug 11.
Increasing evidence suggests that iron-mediated oxidative stress might underlie the development of neurodegeneration in Friedreich's ataxia (FRDA), an autosomal recessive ataxia caused by decreased expression of frataxin, a protein implicated in iron metabolism. In this study, we demonstrate that, in fibroblasts of patients with FRDA, the cellular redox equilibrium is shifted toward more protein-bound glutathione. Furthermore, we found that actin is glutathionylated, probably as a result of the accumulation of reactive oxygen species, generated by iron overload in the disease. Indeed, high-pressure liquid chromatography analysis of control fibroblasts in vivo treated with FeSO4 showed a significant increase in the protein-bound/free GSH ratio, and Western blot analysis indicated a relevant rise in glutathionylation. Actin glutathionylation contributes to impaired microfilament organization in FRDA fibroblasts. Rhodamine phalloidin staining revealed a disarray of actin filaments and a reduced signal of F-actin fluorescence. The same hematoxylin/eosin-stained cells showed abnormalities in size and shape. When we treated FRDA fibroblasts with reduced glutathione, we obtained a complete rescue of cytoskeletal abnormalities and cell viability. Thus, we conclude that oxidative stress may induce actin glutathionylation and impairment of cytoskeletal functions in FRDA fibroblasts.
越来越多的证据表明，铁介导的氧化应激可能是弗里德赖希共济失调（FRDA）神经退行性变发展的基础，FRDA是一种常染色体隐性共济失调，由与铁代谢有关的蛋白质——酵母辅酶A合成酶（frataxin）表达减少引起。在本研究中，我们证明，在FRDA患者的成纤维细胞中，细胞氧化还原平衡向更多与蛋白质结合的谷胱甘肽方向转变。此外，我们发现肌动蛋白被谷胱甘肽化，这可能是由于疾病中铁过载产生的活性氧积累所致。事实上，对体内用硫酸亚铁处理的对照成纤维细胞进行高压液相色谱分析显示，与蛋白质结合的/游离谷胱甘肽比率显著增加，蛋白质印迹分析表明谷胱甘肽化有相应升高。肌动蛋白谷胱甘肽化导致FRDA成纤维细胞微丝组织受损。罗丹明鬼笔环肽染色显示肌动蛋白丝紊乱，F-肌动蛋白荧光信号减弱。相同的苏木精/伊红染色细胞显示大小和形状异常。当我们用还原型谷胱甘肽处理FRDA成纤维细胞时，我们完全挽救了细胞骨架异常和细胞活力。因此，我们得出结论，氧化应激可能诱导FRDA成纤维细胞中的肌动蛋白谷胱甘肽化和细胞骨架功能受损。
