Actin Glutathionylation Increases in Fibroblasts of Patients with Friedreich's Ataxia

Abstract

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, highpressure liquid chromatography analysis of control fibroblasts in vivo treated with FeSO 4 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.Oxidative stress has been proposed to underlie neurodegeneration in Friedreich's ataxia (FRDA), 1 the most common of the hereditary ataxias, caused by severely reduced levels of frataxin, a protein implicated in iron metabolism. FRDA is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy, and increased incidence of diabetes. Most patients (95%) are homozygous for the hyperexpansion of a GAA repeat sequence in the first intron of the frataxin gene; a few are heterozygous for a GAA expansion and a point mutation (1-3). Data from yeast suggest that frataxin deficiency results in iron accumulation within mitochondria and increased sensitivity to oxidative stress (4, 5). Mouse models for FRDA exhibit cardiomyopathy, sensory nerve defects, and Fe-S enzyme deficiency followed by intramitochondrial iron deposits (6). Patients with FRDA have iron deposits in the heart, increased mitochondrial iron in fibroblasts, and greater sensitivity to oxidative stress by pro-oxidants such as FeCl 3 and hydrogen peroxide (3,7,8). Furthermore, a defective mitochondrial respiratory chain has been found in FRDA tissues, in association with iron accumulation and moderate decreases in mtDNA levels (9, 10).Iron is a crucial reagent in the Fenton reaction, as it can react with mitochondrially generated superoxide anion (O 2 . ) to produce the toxic hydroxyl radical (OH ⅐ ), and iron-mediated oxidative stress has been hypothesized to underlie the pathophysiology of the disease. Increased levels of oxidative stress markers such as plasma malondialdehyde and urine 8-hydroxy-2-deoxyguanosine have been found in patients with F...

Radical-free biology of oxidative stress

“…The mechanisms for introduction of disulfides have been studied extensively in protein processing and contribute to stability of secondary, tertiary and quaternary structure. ␤-Actin contains a conserved Cys, which results in reversible binding of proteins, S-GS-ylation, and crosslinking of actin filaments upon oxidation (33,112,140). Oxidation functions in glucocorticoid receptor translocation into nuclei (116,136), and oxidation controls export of yeast AP-1 (Yap-1) from nuclei (38,98).…”

“…Sites sensitive to changes in thiol-disulfide redox state are indicated by an "-SH/-SS-" balance. A: in early proinflammatory signaling in endothelial cells, redox-dependent signaling includes 1) extracellular Cys/CySS redox potential (55) (109); 9) processing of proteins in the secretory pathway (7,24); and 10) cytoskeletal/ surface structure (33,140,187). B: in receptormediated signaling, redox-sensitive steps include 1) metalloprotease-sensitive growth factor release (134); 2) metalloprotease-sensitive degradation of growth factor inhibitor (51); 3) redox-dependent activation of receptors (31, 94); 4) H2O2-dependent Ca 2ϩ influx and Nox-5 activation (40); 5) active-site Cys residues required for phosphatase activity [PTP1B, SHP2, PTen; (39)]; 6) Ras activity (3); 7) Src activity (48); 8) H2O2 metabolism; 9) lipoxygenase activity (42); 10) LPS activation of cytoplasmic and mitochondrial H2O2 production through Toll-like receptor 4 [TLR4 (77,139)] (42,77,139).…”

Glutathionylation of p65NF-κB correlates with proliferating/apoptotic hepatoma cells exposed to pro- and anti-oxidants

“…After sonication (Sonics Vibra Cell, Sonics & Material Inc., Newtown, CT, USA), levels of total (GSH Tot), reduced glutathione (GSH red), oxidized (GSSG) and protein-bound (ProSSG) glutathione were analyzed by HPLC. HPLC equipment and conditions to analyse various forms of glutathione have been reported (8,9).…”

“…Glutathione participates in many intracellular reactions playing different roles in cell physiology and pathophysiology (7)(8)(9)(10). Glutathione reactions include, its direct and indirect anti-oxidant action; its role as substrate to produce radicals; its conjugation with NO to form S-nitroso-glutathione adducts, its reaction with various electrophiles, physiological metabolites (e.g., estrogens, melanins, prostaglandins, and leukotrienes), and xenobiotics (e.g., bromobenzene and acetaminophen) to form mercapturates; its ability to convert formaldehyde to S-formyl-glutathione; and finally, its ability to form mixed disulfides with protein sulfhydryl groups (protein glutathionylation) (11)(12)(13)(14).…”

The yeast metacaspase is implicated in oxidative stress response in frataxin‐deficient cells

“…Iron accumulation in the mitochondria, which can participate in the Fenton reaction and increase reactive oxygen species (ROS) production, is considered an important mechanism inducing oxidative stress in frataxin-deficient cells [1,2]. However, substantial evidence shows that glutathione-dependent defenses and antioxidant enzymes are repressed or not properly regulated [6][7][8][9]. In addition, patient fibroblasts and lymphoblasts undergo apoptosis when challenged with oxidants [10][11][12].…”