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

Actin Glutathionylation Increases in Fibroblasts of Patients with Friedreich's Ataxia THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 43, Issue of October 24, pp. 42588–42595, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Actin Glutathionylation Increases in Fibroblasts of Patients with Friedreich’s Ataxia A POTENTIAL ROLE IN THE PATHOGENESIS OF THE DISEASE* Received for publication, February 21, 2003, and in revised form, August 8, 2003 Published, JBC Papers in Press, August 11, 2003, DOI 10.1074/jbc.M301872200 Anna Pastore‡, Giulia Tozzi§, Laura Maria Gaeta§, Enrico Bertini§, Valentina Serafini‡, Silvia Di Cesare¶, Valentina Bonetto**, Filippo Casoni, Rosalba Carrozzo§, Giorgio Federici‡, and Fiorella Piemonte§‡‡ From the ‡Laboratory of Biochemistry and the §Molecular Medicine and ¶Flow Cytometric Units, Children’s Hospital and Research Institute “Bambino Gesu`,” Piazza S. Onofrio, 4, 00165 Rome, Italy and the Dulbecco Telethon Institute, Pharmacological Research Institute “Mario Negri,” Via Eritrea, 62, 20157 Rome, Italy Increasing evidence suggests that iron-mediated oxi- and a point mutation (1–3). Data from yeast suggest that dative stress might underlie the development of neuro- frataxin deficiency results in iron accumulation within mito- degeneration in Friedreich’s ataxia (FRDA), an autoso- chondria and increased sensitivity to oxidative stress (4, 5). mal recessive ataxia caused by decreased expression of Mouse models for FRDA exhibit cardiomyopathy, sensory frataxin, a protein implicated in iron metabolism. In this nerve defects, and Fe-S enzyme deficiency followed by intrami- study, we demonstrate that, in fibroblasts of patients tochondrial iron deposits (6). Patients with FRDA have iron with FRDA, the cellular redox equilibrium is shifted deposits in the heart, increased mitochondrial iron in fibro- toward more protein-bound glutathione. Furthermore, blasts, and greater sensitivity to oxidative stress by pro-oxi- we found that actin is glutathionylated, probably as a dants such as FeCl and hydrogen peroxide (3, 7, 8). Further- result of the accumulation of reactive oxygen species, more, a defective mitochondrial respiratory chain has been generated by iron overload in the disease. Indeed, high- found in FRDA tissues, in association with iron accumulation pressure liquid chromatography analysis of control fi- and moderate decreases in mtDNA levels (9, 10). broblasts in vivo treated with FeSO showed a signifi- Iron is a crucial reagent in the Fenton reaction, as it can cant increase in the protein-bound/free GSH ratio, and react with mitochondrially generated superoxide anion (O )to Western blot analysis indicated a relevant rise in gluta- 2 produce the toxic hydroxyl radical (OH ), and iron-mediated thionylation. Actin glutathionylation contributes to im- oxidative stress has been hypothesized to underlie the patho- paired microfilament organization in FRDA fibroblasts. physiology of the disease. Increased levels of oxidative stress Rhodamine phalloidin staining revealed a disarray of actin filaments and a reduced signal of F-actin fluores- markers such as plasma malondialdehyde and urine 8-hy- cence. The same hematoxylin/eosin-stained cells showed droxy-2-deoxyguanosine have been found in patients with abnormalities in size and shape. When we treated FRDA FRDA, and improvement of cardiac and skeletal muscle bioen- fibroblasts with reduced glutathione, we obtained a ergetics has been observed after antioxidant treatment (11– complete rescue of cytoskeletal abnormalities and cell 15). Moreover, we found an impairment in vivo of the antioxi- viability. Thus, we conclude that oxidative stress may dant enzymes superoxide dismutase and glutathione induce actin glutathionylation and impairment of cy- peroxidase and decreased levels of free glutathione in the blood toskeletal functions in FRDA fibroblasts. of patients with FRDA (16, 17). In cells, exposure to acute oxidative stress triggers a se- quence of events characterized by depletion of antioxidant de- Oxidative stress has been proposed to underlie neurodegen- fenses and oxidative modification of proteins, lipids, and nu- eration in Friedreich’s ataxia (FRDA), the most common of the cleic acids. Protein thiols are particularly susceptible to hereditary ataxias, caused by severely reduced levels of oxidation and may represent important targets in redox signal- frataxin, a protein implicated in iron metabolism. FRDA is ing. Recently, protein glutathionylation has gained attention characterized by degeneration of the large sensory neurons and as a possible means of protein function redox regulation. One spinocerebellar tracts, cardiomyopathy, and increased inci- proposed mechanism leading to protein S-glutathionylation in dence of diabetes. Most patients (95%) are homozygous for the vivo is the thiol/disulfide exchange mechanism (18), which oc- hyperexpansion of a GAA repeat sequence in the first intron of curs when an oxidative insult changes the GSH/GSSG ratio the frataxin gene; a few are heterozygous for a GAA expansion and induces GSSG to bind to protein thiols. Several important enzymes, including phosphofructokinase (19), glycogen syn- thase (20), fructose-1,6-diphosphatase (21), 3-hydroxy-3-meth- * This work was supported in part by Grant R-03-30 from the ‘‘Fonda- zione Pierfranco e Luisa Mariani,’’ Italy. The costs of publication of this ylglutaryl-CoA reductase (22), glyceraldehyde-3-phosphate de- article were defrayed in part by the payment of page charges. This hydrogenase (23), protein kinase C (24), and guanylate cyclase article must therefore be hereby marked “advertisement” in accordance (25), and glucocorticoid receptors (26) are potentially influ- with 18 U.S.C. Section 1734 solely to indicate this fact. enced by the formation of protein adducts with glutathione. ** Assistant Telethon Scientist supported by Grant TCP.01010 from Telethon, Italy. Also transcription factors such as c-Jun appear to be redox- ‡‡ To whom correspondence should be addressed. Tel.: 390-6-6859- regulated by mechanisms that include protein S-thiolation (27, 2105; Fax: 390-6-6859-2024; E-mail: [email protected]. 28), and ubiquitin-activating enzymes become S-glutathiony- The abbreviations used are: FRDA, Friedreich’s ataxia; HPLC, lated, with a concomitant decrease in the ubiquitinylation high-pressure liquid chromatography; EE-GSH, ethyl ester-reduced glutathione. pathway, when cells are exposed to oxidants (29). A reversible 42588 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42589 FIG.2. Glutathionylated proteins in fibroblasts of FRDA pa- tients. A, glutathione bound to proteins was immunologically detected by Western blot analysis. Equal amounts of fibroblast lysates (40 g) were separated by SDS-PAGE and transferred to nitrocellulose. The glutathione conjugates were probed with monoclonal anti-GSH anti- body. Lane 1, control; lanes 2–4, FRDA fibroblasts obtained from three exemplifying patients. B, fibroblast lysates were analyzed by Western blotting using anti-actin antibody. Lane 1, control; lane 2, FRDA fibro- blasts (corresponding to the sample in lane 2 of A). C, anti-GSH anti- body was used for immunochemical detection of immunoprecipitated actin from FRDA fibroblasts. D, nitrocellulose filters were subjected to densitometric analysis and normalized by calculating the ratios be- tween mean optical density of FRDA patients (n  9) or controls (n  4) and total glutathione concentrations as obtained by HPLC analysis. glutathionylation was found to regulate actin polymerization in human epidermal carcinoma cells (30), and S-glutathionyla- tion, followed by inactivation, was reported for creatine kinase, a crucial source of ATP in myocytes, during oxidative stress (31). Furthermore, we recently found an increase in glutathio- nyl-hemoglobin in the blood of patients with FRDA, accompa- nied by a significant decrease in free glutathione (17). Free glutathione concentration, mainly represented by its reduced form, is a limiting factor in many detoxifying processes by protecting protein thiol groups from oxidation, directly as a free radical scavenger or as a cosubstrate for a number of important enzymes such as glutathione peroxidase and gluta- thione transferases (32). Under conditions of increased oxidant stress such as ischemia/reperfusion, chronic ethanol ingestion, tumor necrosis factor-induced cytotoxicity, and bile acid reten- tion in cholestasis, glutathione status is a critical factor in determining loss of mitochondrial function and cell viability as well as transcription factor activation and gene regulation (33). The relation existing among glutathione, oxidative stress, and FIG.1. HPLC analysis of glutathione status in FRDA fibro- neurodegeneration was recently reviewed by Schulz et al. (34), blasts. The GSH/GSSG ratios (A) and protein-bound (GS-Pro)/total and an important role for glutathione has been proposed for the (Tot) GSH and protein-bound/free GSH ratios (B) were determined in pathogenesis of Parkinson’s disease, where a decrease in GSH FRDA fibroblasts (n  9) and in controls (CTRL; n  4). For all concentrations in the substantia nigra was observed in preclin- experiments, p  0.05. For details, see ‘‘Experimental Procedures.’’ 42590 Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts technology. The BCA protein assay was obtained from Pierce. All ma- terials for cell cultures were from Invitrogen; all other chemicals were from Sigma. Cell Culture—Skin biopsies were taken from nine clinically affected (and genetically proven) FRDA patients (four males and five females) and four age-matched controls. Fibroblasts were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 g/ml streptomycin, 0.4% (v/v) amphotericin B (250 g/ml), and 1 mM sodium pyruvate at 37 °C in 5% CO . Fibroblasts were grown to 90% confluence. The assays were performed in triplicate, and each fibroblast strain was separately grown and processed twice. Cells were used at similar passage numbers. After washing with phos- phate-buffered saline, the cells were resuspended in 100 lofH O, sonicated three times for 2 s (Sonics Vibra Cell, Sonics & Material Inc., Newtown, CT), and subjected to biochemical analysis. HPLC Determination of Various Forms of Glutathione—The cells (treated differently) were sonicated three times for2sin0.1mlof0.1 M potassium phosphate buffer (pH 7.2). After sonication, 50 lof12% sulfosalicylic acid were added, and the GSH content in the acid-soluble fraction was determined (free GSH). The protein pellet was dissolved in 150 lof0.1 N NaOH, and protein-bound GSH was determined. To measure oxidized glutathione (GSSG), cells were sonicated three times for2sin0.1mlof0.1 M potassium phosphate buffer (pH 7.2) containing 10 mM N-ethylmaleimide. Total GSH was determined in fibroblast lysates before adding 12% sulfosalicylic acid. GSH levels were calcu- lated by subtracting GSSG concentrations from free GSH values. Pro- tein concentrations were quantified by the BCA protein assay. Derivat- ization and chromatography procedures were carried out as previously reported (38). Analysis of Glutathione Conjugates by Western Blotting—40 gof lysate sample were subjected to 12% SDS-polyacrylamide gel electro- phoresis, and the proteins were transferred to a nitrocellulose mem- brane overnight at 70 mA. The membrane was blocked with 5% nonfat dry milk in 100 mM NaCl and 10 mM Tris-HCl (pH 7.8) containing 0.1% Tween 20 for2hat room temperature and probed with monoclonal anti-GSH antibody (1:500) and/or polyclonal anti-actin antibody (1:1000). Identification of Glutathionylated Actin by Immunoprecipitation— Fibroblasts (resuspended in 150 lofH O and sonicated) were incu- bated with 50 l of anti-actin antibody (1 g)for1hat4 °C,and50 l of the resuspended volume of protein G PLUS-agarose were added to the solution and incubated at 4 °C for1hona rocker platform. The FIG.3. Fluorescence and light microscopy images of fibro- pellet was collected by centrifugation at 800  g for 5 min at 4 °C and blasts at the same magnification (40). Actin was visualized by washed four times with phosphate-buffered saline. After a final wash, rhodamine phalloidin staining. Clear fluorescence attenuation ap- the pellet was resuspended in 10 l of electrophoresis sample buffer, peared in fibroblasts of FRDA patients (B), together with enlargement boiled for 2 min, loaded onto a nonreducing 12% SDS-polyacrylamide of the cytoplasmic area and disarray of actin filaments, compared with gel for Western blot analysis, and revealed with anti-GSH antibody control cells (A). The same FRDA cells stained with hematoxylin and (1:500). eosin showed marked enlargement of cell size as well as abnormal Quantification of Glutathionylated Actin—The extent of glutathiony- shape (E) with respect to control cells (D). Rescue of fluorescence inten- lation was quantified by analyzing nitrocellulose filters with a Bio-Rad sity (C) and of cytoskeletal abnormalities (F) in FRDA cells was ob- Model GS-670 imaging densitometer. Data were analyzed using Bio- tained after incubation for 30 days at 37 °C with 10 mM GSH. TM Rad Molecular Analyst software (Version 1.3) and normalized to the quantity of protein loaded on the gels and to total glutathione content ical stages of the disease (35). Although the pathogenesis of in fibroblasts. FRDA is still unclear, one possibility is that the presence of Fluorescence Microscopy Analysis of Rhodamine Phalloidin-stained unbound (free) reactive iron, via the Fenton reaction, generates Fibroblasts—Cells were washed twice with phosphate-buffered saline, free radicals within the mitochondria, leading to oxidative fixed in 3.7% formaldehyde solution for 10 min at room temperature, damage. permeabilized with 0.1% Triton X-100, and stained with rhodamine phalloidin. Fluorescent images were monitored using a Zeiss micro- Thus, in light of accumulating evidence indicating a crucial scope (Axoskobe 50) equipped with epifluorescence and a 40 objective. role for glutathione in the regulation of cellular signaling in Cell Morphology—Cells grown on Falcon chamber slides (BD Bio- response to oxidative and nitrosative stress (36, 37), we ana- sciences) were washed twice with phosphate-buffered saline, fixed in lyzed the redox status of glutathione in fibroblasts of patients 3.7% formaldehyde solution for 10 min, and stained with Harris hema- with FRDA and identified a protein that undergoes glutathio- toxylin for 7 min. After treatment with 0.1% (v/v) HCl, cells were nylation in these patients. For this purpose, we cultured skin washed with H O for 10 min, stained with eosin for 20 s, dried with ethanol (70, 95, and 100%, v/v), and washed with xylol. Images were biopsies obtained from nine patients with FRDA and deter- monitored using the Axoskobe 50 microscope equipped with a mined total, free, reduced, oxidized, and protein-bound gluta- 40 objective. thione concentrations by high-pressure liquid chromatography Effect in Vivo of GSH, Ethyl Ester-reduced Glutathione (EE-GSH), (HPLC) analysis. In addition, we analyzed the glutathionylated and Acivicin on Actin Glutathionylation and Cell Morphology and Vi- protein pattern by Western blotting using a monoclonal anti- ability—Fibroblasts of three patients with FRDA were incubated for 30 GSH antibody. days at 37 °C with 10 mM (final concentration) GSH and 10 mM (final concentration) EE-GSH in both the absence and presence of 150 M EXPERIMENTAL PROCEDURES (final concentration) acivicin, a -glutamyltranspeptidase inhibitor. Materials—Monoclonal anti-GSH antibody was obtained from Viro- Cell lysates were analyzed by Western blotting as described above to gen. Polyclonal anti-actin antibody and 6-carboxyfluorescein (apoptosis determine the extent of actin glutathionylation. GSH-treated FRDA detection kit) were from Sigma. Rhodamine phalloidin was from Molec- cells were subjected to fluorescence and light microscopy by staining ular Probes, Inc. Protein G PLUS-agarose was from Santa Cruz Bio- with rhodamine phalloidin and hematoxylin/eosin, respectively (see Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42591 FIG.4. In vivo reversibility of actin glutathionylation by treatment with antioxidant compounds. Fibroblasts obtained from FRDA patients were treated in vivo with 10 mM GSH and 10 mM EE-GSH for 30 days at 37 °C. A, data obtained by HPLC analysis of the protein-bound (GS-Pro)/free GSH ratio (n  3). *, p  0.05. B, GSH/GSSG ratios as determined by HPLC analysis in controls (CTRL; n  4), in FRDA cells (n 9), and in GSH- and EE-GSH-treated FRDA fibroblasts (n  3; p  0.05 compared with untreated FRDA cells). C, Western blot analysis of untreated control fibroblasts (lane 1), untreated FRDA fibroblasts (lane 2), EE-GSH-treated FRDA cells (lane 3), and GSH-treated FRDA fibroblasts (lane 4). The blot is representative of one of three independent experiments for each condition tested. For details, see ‘‘Experimental Procedures.’’ above). Cell viability was measured using 6-carboxyfluorescein, a non- tios (Fig. 1B). The GSH/GSSG ratio was 129  18.8 in controls fluorescent compound that becomes fluorescent in live cells by the and decreased to 45  8.6 in FRDA fibroblasts (p  0.05). The action of esterases. Flow cytometric analyses were performed on a protein-bound/total GSH ratio was 0.05  0.01 in control cells FACSCalibur flow cytometer (BD Biosciences), and the results were and increased to 0.12  0.03 in FRDA cells (p  0.05). The analyzed using the CellQuest program (BD Biosciences). Effect in Vivo of FeSO Treatment on Actin Glutathionylation—The protein-bound/free GSH ratio was 0.056  0.013 in control effect of FeSO treatment was studied by incubating control fibroblasts fibroblasts and increased to 0.25  0.12 in FRDA patients (p in vivo with 100 M (final concentration) FeSO or H Oat37 °Cfor6, 4 2 0.05). These results indicate that FRDA fibroblasts undergo 24, 48, 96, and 192 h. Protein-bound/free GSH levels were determined oxidative stress, with the cellular redox equilibrium shifted by HPLC analysis as described above. Western blot analysis was per- formed on cells treated with 100 M FeSO at 37 °C for 24 and 192 h. toward more protein-bound glutathione with respect to fibro- Effect in Vivo of FeSO Treatment on Cell Morphology—Control 4 blasts of healthy subjects. fibroblasts were treated in vivo with 100 M (final concentration) FeSO Monoclonal anti-GSH antibody was used to investigate the for 1 month and subjected to fluorescence microscopy by staining with glutathionylation state of FRDA fibroblasts (Fig. 2). Western rhodamine phalloidin (see above). blot analysis of fibroblasts obtained from three patients re- RESULTS vealed a 42-kDa band (Fig. 2A), which was also detected by anti-actin antibody (Fig. 2B). To identify the 42-kDa protein, We analyzed the glutathione content in fibroblasts of nine we immunoprecipitated the fibroblast lysates with anti-actin FRDA patients and determined the GSH/GSSG ratios (Fig. 1A) and protein-bound/total GSH and protein-bound/free GSH ra- antibody and probed them with anti-GSH antibody. As shown 42592 Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts To investigate whether the unexpected increase in actin glu- tathionylation in FRDA fibroblasts is related to changes in the cytoskeletal organization, we stained fibroblasts with the fila- mentous actin indicator rhodamine phalloidin. The fluorescent images revealed a significant disarrangement of F-actin in FRDA patients (Fig. 3B), with clear attenuation of the fluores- cent signal compared with controls (Fig. 3A). Under light mi- croscopy, FRDA fibroblasts (Fig. 3E) appeared to be enlarged with respect to fibroblasts of healthy subjects (Fig. 3D). Taken together, these observations suggest that glutathionylation of actin leads to a disarray of actin filaments, inducing size and shape abnormalities in FRDA fibroblasts. To determine whether there is an in vivo reversibility of glutathionylation in FRDA fibroblasts, we treated FRDA cells with 10 mM (final concentration) GSH and 10 mM (final con- centration) EE-GSH for 30 days at 37 °C. Fig. 4A illustrates the results from HPLC analysis, showing a 17  3.7% decrease in the protein-bound/free GSH ratio in EE-GSH-treated FRDA fibroblasts and a 56  6.8% reduction in GSH-treated FRDA fibroblasts compared with untreated cells. HPLC analysis of GSH and GSSG levels (Fig. 4B) revealed increases in GSH/ GSSG ratios to 113  53.5 upon GSH treatment and to 115 57 upon EE-GSH treatment with respect to untreated FRDA cells (45  8.6; p  0.05), thus becoming comparable to those in controls (129  18.8). Furthermore, Western blot analysis showed a 20  5.5% decrease in glutathionylated proteins in EE-GSH-treated cells (Fig. 4C, lane 3)anda60  15% decrease in GSH-treated cells (lane 4) compared with untreated FRDA fibroblasts (lane 2). To elucidate how medium GSH is available to the cells, we performed in vivo experiments by treating FRDA fibroblasts with acivicin (150 M), an inhibitor of -glu- tamyltranspeptidase. Our findings show that acivicin blocked the protection obtained by GSH treatment, whereas EE-GSH, which is membrane-permeable, was not affected by the inhib- itor (data not shown). The fluorescent images of FRDA fibroblasts after 1 month of in vivo GSH treatment showed an increase in the rhodamine phalloidin signal of F-actin (Fig. 3C) and a rescue of cell size and shape (Fig. 3F). Flow cytometric analysis using the cell viability indicator 6-carboxyfluorescein showed a 15.5  5.6% (p  0.05) increase in the fluorescent signal in GSH-treated FRDA cells (Fig. 5C) with respect to untreated cells (Fig. 5B), comparable to that in controls (Fig. 5A). Finally, to determine whether there is an in vivo association between iron overload, which underlies the pathogenesis of FRDA, and actin glutathionylation, we treated control fibro- blasts with 100 M FeSO for 6, 24, 48, 96, and 192 h. HPLC analysis showed 1.12-, 1.34-, 1.44-, 1.85-, and 2.3-fold increases in the protein-bound/free GSH ratio, respectively, compared with H O treatments (Fig. 6A). Western blot analysis of fibro- blasts incubated with 100 M FeSO for 24 and 192 h at 37 °C showed 1.7-fold (Fig. 6B, lane 2) and 2-fold (lane 3) increases in glutathionylated actin, respectively, compared with H O- treated cells (lane 1). Moreover, to determine whether FeSO induces morphological changes characteristic of FRDA, we FIG.5. Cell morphology of fibroblast cell population as as- sessed by light scattering parameters with density plot of con- treated in vivo control cells with FeSO for 1 month without trol (A), untreated FRDA (B), and GSH-treated FRDA (C) cells obtaining any significant morphological changes resembling after staining with 6-carboxyfluorescein. Forward scatter (FSC) those in FRDA fibroblasts (Fig. 7). measures the relative size, whereas side scatter (SSC) is related to the relative granularity or internal complexity. DISCUSSION The prevailing hypothesis underlying the pathogenesis of in Fig. 2C, we obtained a 42-kDa band corresponding to gluta- FRDA supposes that frataxin is involved in the regulation of thionylated actin. The extent of glutathionylation was in- mitochondrial iron export and that impaired intramitochon- creased by 4.7-fold in FRDA patients with respect to controls drial iron metabolism results in iron overload and oxidative (7.47  3.5 versus 1.6  0.51; p  0.05) (Fig. 2D). Equal stress (1, 2). Iron deposits are observed consistently in some amounts of loaded protein were verified by Coomassie Blue heart myofibrils of FRDA patients upon autopsy, and recent staining of an equivalent SDS gel (data not shown). magnetic resonance imaging data indicate that iron also accu- Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42593 FIG.6. In vivo effect of FeSO treat- ments on protein glutathionylation. A, after incubating control cells with H O or 100 M FeSO for the indicated times, protein-bound (GS-Pro)/free GSH levels were determined by HPLC analysis. Re- sults represent the mean of three inde- pendent determinations. B, after treat- ment with FeSO , control fibroblasts were analyzed by Western blotting. Control cells were incubated at 37 °C for 192 h with H O(lane 1) or with 100 M FeSO 2 4 for 24 h (lane 2) and 192 h (lane 3). The blot is representative of one of at least three identical experiments for each con- dition tested. For details, see ‘‘Experimen- tal Procedures.’’ mulates in the dentate nucleus (9, 39, 40). In previous studies, redox equilibrium is shifted toward more protein-bound gluta- we reported a significant increase in glutathionyl-hemoglobin thione, with significant increases in protein-bound/total GSH in the blood of patients with FRDA, together with an imbalance and protein-bound/free GSH ratios. Furthermore, we used between the superoxide dismutase and glutathione peroxidase monoclonal anti-GSH antibody to investigate protein glutathio- antioxidant enzyme activities, thus supporting the presence of nylation in fibroblasts, and we found that proteins are gluta- a systemic oxidative stress in the disease (16, 17). Therefore, in thionylated in FRDA, with a 5-fold increase in FRDA patients. light of these recent advances in the pathogenesis of FRDA, our Protein glutathionylation occurs also in normal fibroblasts, study has focused on the analysis of glutathione metabolism in but to a lower extent. The fact that some proteins are consti- fibroblasts of FRDA patients. Furthermore, as the oxidized and tutively glutathionylated under basal conditions has already reduced glutathione redox ratios affect the state of glutathio- been observed by Fratelli et al. (37) in T lymphocytes and may nylated proteins, we also studied the glutathionylated protein indicate a regulatory role for glutathionylation in several pro- pattern in FRDA fibroblasts. tein functions. In normal liver, for instance, 20–30 nmol of Our findings show, for the first time, that fibroblasts of glutathione/g of liver are present as disulfides mixed with patients with FRDA undergo oxidative stress, with a signifi- proteins (41). cant decrease in the GSH/GSSG ratio. In addition, the cellular Many proteins can undergo glutathionylation under oxida- 42594 Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts Some of the proteins found to be glutathionylated belong to the class of cytoskeletal proteins, which are particularly abun- dant in cells. The supramolecular organization of these pro- teins depends on the presence of exposed sulfhydryl residues; the modification of these groups by glutathionylation could be relevant to their function, by either protecting them against irreversible oxidation or inhibiting polymerization (48–50). In this study, we have demonstrated that the glutathionyla- tion of actin caused an impairment of microfilaments dynamic in FRDA fibroblasts, as showed using the filamentous actin indicator rhodamine phalloidin. The fluorescent images re- vealed a marked and diffuse reduced signal of F-actin, probably due to disassembly of actin filaments. The same hematoxylin/ eosin-stained cells showed clear abnormalities in size and shape. Also, the viability of FRDA fibroblasts was 18.7  4.3% lower than that of control cells as measured by flow cytometric analysis using 6-carboxyfluorescein. Actin is one of the major cytoskeletal proteins, playing an important role in mediating the infrastructure and dynamics of the cytoplasmic matrix. Its polymerization is a dynamic process implicated in growth factor-mediated cytoskeletal changes. Wang et al. (30) directly linked the epidermal growth factor- mediated signaling pathway to in vivo de-glutathionylation of actin with an increase in F-actin, thus highlighting protein glutathionylation as a physiologically relevant regulatory mechanism in actin polymerization. Monitored by light scat- tering, the steady-state rate for non-glutathionylated actin po- lymerization was at least 5.6-fold faster than that for gluta- thionylated actin polymerization obtained in the presence of GSSG (30). Recently, Dalle-Donne et al. (50) demonstrated in vitro that glutathionylated actin has a decreased capacity to polymerize compared with native actin, with filament elonga- tion being inhibited. An impaired microfilament organization by glutathione binding has been reported in rabbit muscle, FIG.7. Fluorescent images of control fibroblasts after in vivo where the addition of glutathione at Cys of actin resulted in FeSO treatment. Control cells (A) were treated in vivo with 100 M FeSO for 30 days at 37 °C (B), and actin was visualized by rhodamine filaments with a diminished mechanical stability (51, 52). phalloidin staining. The glutathionylation of actin has been observed in several cells under varying conditions of oxidative stress, e.g. gastric mucosal cells treated with H O or diamide and phorbol my- 2 2 tive stress (36, 42, 43). Some proteins sustain different but ristate acetate-stimulated murine macrophages and human important functions, such as nucleophosmin (involved in the neutrophils (48, 53). H O treatment is also responsible for 2 2 assembly of ribosomal proteins), cyclophilin (a chaperonin in- actin glutathionylation in human epidermal carcinoma A431 volved in the proteasomal degradation of proteins), and the cells, where it regulates actin polymerization (30). heat shock proteins HSP60 and HSP70. Glyceraldehyde-3- A role of oxidized actin has been reported in some neuro- phosphate dehydrogenase is the major S-glutathionylated pro- degenerative diseases, such as in a mouse model of amyotro- tein in endothelial cells exposed to hydrogen peroxide and in phic lateral sclerosis, where axonal degeneration appears to be monocytes during the endogenous oxidative burst (23, 44). Cre- due to defective transport of components required for axonal atine kinase and glycogen phosphorylase b are also targets for maintenance (54). Actin oxidation was significantly higher S-glutathionylation in myocytes and cardiac tissue during cy- even in brain extracts of patients with Alzheimer’s disease, clic oxidative stress. Carbonic anhydrase III, glutathione S- suggesting that oxidative stress-induced injury may lead to the transferase, superoxide dismutase, hemoglobin, and bovine eye degeneration of neurons in the Alzheimer’s disease brain (55). lens crystalline become thiolated in cellular models of oxidative Glutathionylation is a redox-dependent reversible mecha- stress. Additional proteins, including fatty-acid synthase, 3-hy- nism (30, 50). Indeed, when we incubated fibroblasts with droxy-3-methylglutaryl-CoA reductase, aldose reductase, hu- pro-oxidants in vitro, we found a 4-fold increase in glutathio- man immunodeficiency virus-1 protease, and small HSP25, nylation in the presence of GSSG and a 2.5-fold increase with have been reported as potential targets in vitro for redox-de- H O . Glutathionylation was also reversed in vitro by excess pendent S-glutathionylation (36). Even c-Jun DNA binding 2 2 GSH or dithiothreitol (data not shown). To address the in vivo appears to be redox-regulated by glutathionylation (27, 28), reversibility of actin glutathionylation in FRDA, we treated and dopamine biosynthesis is also inhibited by S-glutathiony- fibroblasts with GSH for 1 month and evaluated the extent of lation during oxidative stress (45). In Parkinson’s disease, glutathionylation and cell morphology. Interestingly, we found monoamine oxidase-derived H O was shown to inhibit mito- 2 2 a 60% decrease in protein glutathionylation in treated fibro- chondrial respiration by glutathionylation of respiratory chain enzymes (46). Finally, direct glutathionylation of proteins by blasts upon analysis by Western blotting and a complete rescue of size and cell shape. Even cell viability resulted in a signifi- superoxide anion has been demonstrated in a study showing that S-glutathionylation of protein-tyrosine phosphatases mod- cant enhancement of the fluorescent signal in GSH-treated FRDA cells, becoming comparable to that in controls. ulates the phosphorylation state of cells and preserves protein function (47). GSH transport across cell membrane is not yet well defined, Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42595 Cooper, J. M. (2001) Ann. Neurol. 49, 590–596 although some studies have provided insight especially on GSH 14. Lerman-Sagie, T., Rustin, P., Lev, D., Yanoov, M., Leshinsky-Silver, E., Sagie, release, which seems to involve the family of multidrug resis- A., Ben-Gal, T., and Munnich, A. (2001) J. Inherit. Metab. Dis. 24, 28–34 15. Housse, A. O., Aggoun, Y., Bonnet, D., Sidi, D., Munnich, A., Rotig, A., and tance-associated proteins (32). Evidence for direct uptake of Rustin, P. (2002) Heart (Lond.) 87, 346–349 glutathione has been shown only in mitochondria, where the 16. Tozzi, G., Nuccetelli, M., Lo Bello, M., Bernardini, S., Bellincampi, L., Bal- dicarboxylate and 2-oxoglutarate carriers were identified in the lerini, S., Gaeta, L. M., Casali, C., Pastore, A., Federici, G., Bertini, E., and Piemonte, F. (2002) Arch. Dis. Child. 86, 376–380 inner membrane (56, 57). Therefore, to elucidate how medium 17. Piemonte, F., Pastore, A., Tozzi, G., Tagliacozzi, D., Santorelli, F. M., Carrozzo, GSH is available to the cells, we treated FRDA fibroblasts in R., Casali, C., Damiano, M., Federici, G., and Bertini, E. (2001) Eur. J. Clin. Investig. 31, 1007–1011 vivo with acivicin, an inhibitor of -glutamyltranspeptidase, 18. Gilbert, H. F. (1995) Methods Enzymol. 251, 8–28 and we found that acivicin blocked the protection obtained by 19. Gilbert, H. F. (1982) J. Biol. Chem. 257, 12086–12091 GSH treatment, whereas EE-GSH continued to act (data not 20. Ernest, M. J., and Kim, K. H. (1974) J. Biol. Chem. 249, 5011–5018 21. Nakashima, K., Horecker, B. L., and Pontremoli, S. (1970) Arch. Biochem. shown). Thus, GSH does not seem to be directly taken up by the Biophys. 141, 579–587 cells, whereas the action of EE-GSH, which is membrane- 22. Cappel, R. E., and Gilbert, H. F. (1989) J. Biol. Chem. 264, 9180–9187 23. Ravichandran, V., Seres, T., Moriguchi, T., Thomas, J. A., and Johnston, R. B. permeable, is not affected by the inhibitor. Therefore, the avail- (1994) J. Biol. Chem. 269, 25010–25015 ability of medium GSH to the cells seems to be mediated by 24. Ward, N. E., Pierce, D. S., Chung, S. E., Gravitt, K. R., and O’Brian, C. A. -glutamyltranspeptidase, although further studies will be (1998) J. Biol. Chem. 273, 12558–12566 25. Brandwein, H. J., Lewicki, J. A., and Murad, F. (1981) J. Biol. Chem. 256, necessary to better elucidate the mechanism underlying the in 2958–2962 vivo effect of glutathione treatments, also in light of the unex- 26. Silva, C. M., and Cidiowski, J. A. (1989) J. Biol. Chem. 264, 6638–6647 27. Klatt, P., Molina, E. S., Lacoba, M. C., Padilla, C. A., Mrtinez-Glaisteo, E., pected lesser effectiveness of EE-GSH with respect to GSH, on Barcena, J. A., and Lamas, S. (1999) FASEB J. 13, 1481–1490 the protein-bound GSH levels. 28. Klatt, P., Molina, E. P., and Lamas, S. (1999) J. Biol. Chem. 274, 15857–15864 Iron-mediated oxidative damage has been proposed to un- 29. Jahnegen-Hodge, J., Obin, M. S., Gong, X., Shang, F., Novell, T. R., Gong, J., Abasi, H., Blumberg, J., and Taylor, A. (1997) J. Biol. Chem. 272, derlie the pathogenesis of FRDA. Our findings directly link 28218–28226 iron overload and actin glutathionylation, as demonstrated by 30. Wang, J., Boja, E. S., Tan, W., Tekle, E., Fales, H. M., English, S., Mieyal, J. J., in vivo treatments of control fibroblasts with FeSO . HPLC and Cock, B. (2001) J. Biol. Chem. 276, 47763–47766 31. Reddy, S., Jones, A. D., Cross, C. E., Wong, P. S. 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Actin Glutathionylation Increases in Fibroblasts of Patients with Friedreich's Ataxia

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 43, Issue of October 24, pp. 42588–42595, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Actin Glutathionylation Increases in Fibroblasts of Patients with Friedreich’s Ataxia A POTENTIAL ROLE IN THE PATHOGENESIS OF THE DISEASE* Received for publication, February 21, 2003, and in revised form, August 8, 2003 Published, JBC Papers in Press, August 11, 2003, DOI 10.1074/jbc.M301872200 Anna Pastore‡, Giulia Tozzi§, Laura Maria Gaeta§, Enrico Bertini§, Valentina Serafini‡, Silvia Di Cesare¶, Valentina Bonetto**, Filippo Casoni, Rosalba Carrozzo§, Giorgio Federici‡, and Fiorella Piemonte§‡‡ From the ‡Laboratory of Biochemistry and the §Molecular Medicine and ¶Flow Cytometric Units, Children’s Hospital and Research Institute “Bambino Gesu`,” Piazza S. Onofrio, 4, 00165 Rome, Italy and the Dulbecco Telethon Institute, Pharmacological Research Institute “Mario Negri,” Via Eritrea, 62, 20157 Rome, Italy Increasing evidence suggests that iron-mediated oxi- and a point mutation (1–3). Data from yeast suggest that dative stress might underlie the development of neuro- frataxin deficiency results in iron accumulation within mito- degeneration in Friedreich’s ataxia (FRDA), an autoso- chondria and increased sensitivity to oxidative stress (4, 5). mal recessive ataxia caused by decreased expression of Mouse models for FRDA exhibit cardiomyopathy, sensory frataxin, a protein implicated in iron metabolism. In this nerve defects, and Fe-S enzyme deficiency followed by intrami- study, we demonstrate that, in fibroblasts of patients tochondrial iron deposits (6). Patients with FRDA have iron with FRDA, the cellular redox equilibrium is shifted deposits in the heart, increased mitochondrial iron in fibro- toward more protein-bound glutathione. Furthermore, blasts, and greater sensitivity to oxidative stress by pro-oxi- we found that actin is glutathionylated, probably as a dants such as FeCl and hydrogen peroxide (3, 7, 8). Further- result of the accumulation of reactive oxygen species, more, a defective mitochondrial respiratory chain has been generated by iron overload in the disease. Indeed, high- found in FRDA tissues, in association with iron accumulation pressure liquid chromatography analysis of control fi- and moderate decreases in mtDNA levels (9, 10). broblasts in vivo treated with FeSO showed a signifi- Iron is a crucial reagent in the Fenton reaction, as it can cant increase in the protein-bound/free GSH ratio, and react with mitochondrially generated superoxide anion (O )to Western blot analysis indicated a relevant rise in gluta- 2 produce the toxic hydroxyl radical (OH ), and iron-mediated thionylation. Actin glutathionylation contributes to im- oxidative stress has been hypothesized to underlie the patho- paired microfilament organization in FRDA fibroblasts. physiology of the disease. Increased levels of oxidative stress Rhodamine phalloidin staining revealed a disarray of actin filaments and a reduced signal of F-actin fluores- markers such as plasma malondialdehyde and urine 8-hy- cence. The same hematoxylin/eosin-stained cells showed droxy-2-deoxyguanosine have been found in patients with abnormalities in size and shape. When we treated FRDA FRDA, and improvement of cardiac and skeletal muscle bioen- fibroblasts with reduced glutathione, we obtained a ergetics has been observed after antioxidant treatment (11– complete rescue of cytoskeletal abnormalities and cell 15). Moreover, we found an impairment in vivo of the antioxi- viability. Thus, we conclude that oxidative stress may dant enzymes superoxide dismutase and glutathione induce actin glutathionylation and impairment of cy- peroxidase and decreased levels of free glutathione in the blood toskeletal functions in FRDA fibroblasts. of patients with FRDA (16, 17). In cells, exposure to acute oxidative stress triggers a se- quence of events characterized by depletion of antioxidant de- Oxidative stress has been proposed to underlie neurodegen- fenses and oxidative modification of proteins, lipids, and nu- eration in Friedreich’s ataxia (FRDA), the most common of the cleic acids. Protein thiols are particularly susceptible to hereditary ataxias, caused by severely reduced levels of oxidation and may represent important targets in redox signal- frataxin, a protein implicated in iron metabolism. FRDA is ing. Recently, protein glutathionylation has gained attention characterized by degeneration of the large sensory neurons and as a possible means of protein function redox regulation. One spinocerebellar tracts, cardiomyopathy, and increased inci- proposed mechanism leading to protein S-glutathionylation in dence of diabetes. Most patients (95%) are homozygous for the vivo is the thiol/disulfide exchange mechanism (18), which oc- hyperexpansion of a GAA repeat sequence in the first intron of curs when an oxidative insult changes the GSH/GSSG ratio the frataxin gene; a few are heterozygous for a GAA expansion and induces GSSG to bind to protein thiols. Several important enzymes, including phosphofructokinase (19), glycogen syn- thase (20), fructose-1,6-diphosphatase (21), 3-hydroxy-3-meth- * This work was supported in part by Grant R-03-30 from the ‘‘Fonda- zione Pierfranco e Luisa Mariani,’’ Italy. The costs of publication of this ylglutaryl-CoA reductase (22), glyceraldehyde-3-phosphate de- article were defrayed in part by the payment of page charges. This hydrogenase (23), protein kinase C (24), and guanylate cyclase article must therefore be hereby marked “advertisement” in accordance (25), and glucocorticoid receptors (26) are potentially influ- with 18 U.S.C. Section 1734 solely to indicate this fact. enced by the formation of protein adducts with glutathione. ** Assistant Telethon Scientist supported by Grant TCP.01010 from Telethon, Italy. Also transcription factors such as c-Jun appear to be redox- ‡‡ To whom correspondence should be addressed. Tel.: 390-6-6859- regulated by mechanisms that include protein S-thiolation (27, 2105; Fax: 390-6-6859-2024; E-mail: [email protected]. 28), and ubiquitin-activating enzymes become S-glutathiony- The abbreviations used are: FRDA, Friedreich’s ataxia; HPLC, lated, with a concomitant decrease in the ubiquitinylation high-pressure liquid chromatography; EE-GSH, ethyl ester-reduced glutathione. pathway, when cells are exposed to oxidants (29). A reversible 42588 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42589 FIG.2. Glutathionylated proteins in fibroblasts of FRDA pa- tients. A, glutathione bound to proteins was immunologically detected by Western blot analysis. Equal amounts of fibroblast lysates (40 g) were separated by SDS-PAGE and transferred to nitrocellulose. The glutathione conjugates were probed with monoclonal anti-GSH anti- body. Lane 1, control; lanes 2–4, FRDA fibroblasts obtained from three exemplifying patients. B, fibroblast lysates were analyzed by Western blotting using anti-actin antibody. Lane 1, control; lane 2, FRDA fibro- blasts (corresponding to the sample in lane 2 of A). C, anti-GSH anti- body was used for immunochemical detection of immunoprecipitated actin from FRDA fibroblasts. D, nitrocellulose filters were subjected to densitometric analysis and normalized by calculating the ratios be- tween mean optical density of FRDA patients (n  9) or controls (n  4) and total glutathione concentrations as obtained by HPLC analysis. glutathionylation was found to regulate actin polymerization in human epidermal carcinoma cells (30), and S-glutathionyla- tion, followed by inactivation, was reported for creatine kinase, a crucial source of ATP in myocytes, during oxidative stress (31). Furthermore, we recently found an increase in glutathio- nyl-hemoglobin in the blood of patients with FRDA, accompa- nied by a significant decrease in free glutathione (17). Free glutathione concentration, mainly represented by its reduced form, is a limiting factor in many detoxifying processes by protecting protein thiol groups from oxidation, directly as a free radical scavenger or as a cosubstrate for a number of important enzymes such as glutathione peroxidase and gluta- thione transferases (32). Under conditions of increased oxidant stress such as ischemia/reperfusion, chronic ethanol ingestion, tumor necrosis factor-induced cytotoxicity, and bile acid reten- tion in cholestasis, glutathione status is a critical factor in determining loss of mitochondrial function and cell viability as well as transcription factor activation and gene regulation (33). The relation existing among glutathione, oxidative stress, and FIG.1. HPLC analysis of glutathione status in FRDA fibro- neurodegeneration was recently reviewed by Schulz et al. (34), blasts. The GSH/GSSG ratios (A) and protein-bound (GS-Pro)/total and an important role for glutathione has been proposed for the (Tot) GSH and protein-bound/free GSH ratios (B) were determined in pathogenesis of Parkinson’s disease, where a decrease in GSH FRDA fibroblasts (n  9) and in controls (CTRL; n  4). For all concentrations in the substantia nigra was observed in preclin- experiments, p  0.05. For details, see ‘‘Experimental Procedures.’’ 42590 Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts technology. The BCA protein assay was obtained from Pierce. All ma- terials for cell cultures were from Invitrogen; all other chemicals were from Sigma. Cell Culture—Skin biopsies were taken from nine clinically affected (and genetically proven) FRDA patients (four males and five females) and four age-matched controls. Fibroblasts were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 g/ml streptomycin, 0.4% (v/v) amphotericin B (250 g/ml), and 1 mM sodium pyruvate at 37 °C in 5% CO . Fibroblasts were grown to 90% confluence. The assays were performed in triplicate, and each fibroblast strain was separately grown and processed twice. Cells were used at similar passage numbers. After washing with phos- phate-buffered saline, the cells were resuspended in 100 lofH O, sonicated three times for 2 s (Sonics Vibra Cell, Sonics & Material Inc., Newtown, CT), and subjected to biochemical analysis. HPLC Determination of Various Forms of Glutathione—The cells (treated differently) were sonicated three times for2sin0.1mlof0.1 M potassium phosphate buffer (pH 7.2). After sonication, 50 lof12% sulfosalicylic acid were added, and the GSH content in the acid-soluble fraction was determined (free GSH). The protein pellet was dissolved in 150 lof0.1 N NaOH, and protein-bound GSH was determined. To measure oxidized glutathione (GSSG), cells were sonicated three times for2sin0.1mlof0.1 M potassium phosphate buffer (pH 7.2) containing 10 mM N-ethylmaleimide. Total GSH was determined in fibroblast lysates before adding 12% sulfosalicylic acid. GSH levels were calcu- lated by subtracting GSSG concentrations from free GSH values. Pro- tein concentrations were quantified by the BCA protein assay. Derivat- ization and chromatography procedures were carried out as previously reported (38). Analysis of Glutathione Conjugates by Western Blotting—40 gof lysate sample were subjected to 12% SDS-polyacrylamide gel electro- phoresis, and the proteins were transferred to a nitrocellulose mem- brane overnight at 70 mA. The membrane was blocked with 5% nonfat dry milk in 100 mM NaCl and 10 mM Tris-HCl (pH 7.8) containing 0.1% Tween 20 for2hat room temperature and probed with monoclonal anti-GSH antibody (1:500) and/or polyclonal anti-actin antibody (1:1000). Identification of Glutathionylated Actin by Immunoprecipitation— Fibroblasts (resuspended in 150 lofH O and sonicated) were incu- bated with 50 l of anti-actin antibody (1 g)for1hat4 °C,and50 l of the resuspended volume of protein G PLUS-agarose were added to the solution and incubated at 4 °C for1hona rocker platform. The FIG.3. Fluorescence and light microscopy images of fibro- pellet was collected by centrifugation at 800  g for 5 min at 4 °C and blasts at the same magnification (40). Actin was visualized by washed four times with phosphate-buffered saline. After a final wash, rhodamine phalloidin staining. Clear fluorescence attenuation ap- the pellet was resuspended in 10 l of electrophoresis sample buffer, peared in fibroblasts of FRDA patients (B), together with enlargement boiled for 2 min, loaded onto a nonreducing 12% SDS-polyacrylamide of the cytoplasmic area and disarray of actin filaments, compared with gel for Western blot analysis, and revealed with anti-GSH antibody control cells (A). The same FRDA cells stained with hematoxylin and (1:500). eosin showed marked enlargement of cell size as well as abnormal Quantification of Glutathionylated Actin—The extent of glutathiony- shape (E) with respect to control cells (D). Rescue of fluorescence inten- lation was quantified by analyzing nitrocellulose filters with a Bio-Rad sity (C) and of cytoskeletal abnormalities (F) in FRDA cells was ob- Model GS-670 imaging densitometer. Data were analyzed using Bio- tained after incubation for 30 days at 37 °C with 10 mM GSH. TM Rad Molecular Analyst software (Version 1.3) and normalized to the quantity of protein loaded on the gels and to total glutathione content ical stages of the disease (35). Although the pathogenesis of in fibroblasts. FRDA is still unclear, one possibility is that the presence of Fluorescence Microscopy Analysis of Rhodamine Phalloidin-stained unbound (free) reactive iron, via the Fenton reaction, generates Fibroblasts—Cells were washed twice with phosphate-buffered saline, free radicals within the mitochondria, leading to oxidative fixed in 3.7% formaldehyde solution for 10 min at room temperature, damage. permeabilized with 0.1% Triton X-100, and stained with rhodamine phalloidin. Fluorescent images were monitored using a Zeiss micro- Thus, in light of accumulating evidence indicating a crucial scope (Axoskobe 50) equipped with epifluorescence and a 40 objective. role for glutathione in the regulation of cellular signaling in Cell Morphology—Cells grown on Falcon chamber slides (BD Bio- response to oxidative and nitrosative stress (36, 37), we ana- sciences) were washed twice with phosphate-buffered saline, fixed in lyzed the redox status of glutathione in fibroblasts of patients 3.7% formaldehyde solution for 10 min, and stained with Harris hema- with FRDA and identified a protein that undergoes glutathio- toxylin for 7 min. After treatment with 0.1% (v/v) HCl, cells were nylation in these patients. For this purpose, we cultured skin washed with H O for 10 min, stained with eosin for 20 s, dried with ethanol (70, 95, and 100%, v/v), and washed with xylol. Images were biopsies obtained from nine patients with FRDA and deter- monitored using the Axoskobe 50 microscope equipped with a mined total, free, reduced, oxidized, and protein-bound gluta- 40 objective. thione concentrations by high-pressure liquid chromatography Effect in Vivo of GSH, Ethyl Ester-reduced Glutathione (EE-GSH), (HPLC) analysis. In addition, we analyzed the glutathionylated and Acivicin on Actin Glutathionylation and Cell Morphology and Vi- protein pattern by Western blotting using a monoclonal anti- ability—Fibroblasts of three patients with FRDA were incubated for 30 GSH antibody. days at 37 °C with 10 mM (final concentration) GSH and 10 mM (final concentration) EE-GSH in both the absence and presence of 150 M EXPERIMENTAL PROCEDURES (final concentration) acivicin, a -glutamyltranspeptidase inhibitor. Materials—Monoclonal anti-GSH antibody was obtained from Viro- Cell lysates were analyzed by Western blotting as described above to gen. Polyclonal anti-actin antibody and 6-carboxyfluorescein (apoptosis determine the extent of actin glutathionylation. GSH-treated FRDA detection kit) were from Sigma. Rhodamine phalloidin was from Molec- cells were subjected to fluorescence and light microscopy by staining ular Probes, Inc. Protein G PLUS-agarose was from Santa Cruz Bio- with rhodamine phalloidin and hematoxylin/eosin, respectively (see Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42591 FIG.4. In vivo reversibility of actin glutathionylation by treatment with antioxidant compounds. Fibroblasts obtained from FRDA patients were treated in vivo with 10 mM GSH and 10 mM EE-GSH for 30 days at 37 °C. A, data obtained by HPLC analysis of the protein-bound (GS-Pro)/free GSH ratio (n  3). *, p  0.05. B, GSH/GSSG ratios as determined by HPLC analysis in controls (CTRL; n  4), in FRDA cells (n 9), and in GSH- and EE-GSH-treated FRDA fibroblasts (n  3; p  0.05 compared with untreated FRDA cells). C, Western blot analysis of untreated control fibroblasts (lane 1), untreated FRDA fibroblasts (lane 2), EE-GSH-treated FRDA cells (lane 3), and GSH-treated FRDA fibroblasts (lane 4). The blot is representative of one of three independent experiments for each condition tested. For details, see ‘‘Experimental Procedures.’’ above). Cell viability was measured using 6-carboxyfluorescein, a non- tios (Fig. 1B). The GSH/GSSG ratio was 129  18.8 in controls fluorescent compound that becomes fluorescent in live cells by the and decreased to 45  8.6 in FRDA fibroblasts (p  0.05). The action of esterases. Flow cytometric analyses were performed on a protein-bound/total GSH ratio was 0.05  0.01 in control cells FACSCalibur flow cytometer (BD Biosciences), and the results were and increased to 0.12  0.03 in FRDA cells (p  0.05). The analyzed using the CellQuest program (BD Biosciences). Effect in Vivo of FeSO Treatment on Actin Glutathionylation—The protein-bound/free GSH ratio was 0.056  0.013 in control effect of FeSO treatment was studied by incubating control fibroblasts fibroblasts and increased to 0.25  0.12 in FRDA patients (p in vivo with 100 M (final concentration) FeSO or H Oat37 °Cfor6, 4 2 0.05). These results indicate that FRDA fibroblasts undergo 24, 48, 96, and 192 h. Protein-bound/free GSH levels were determined oxidative stress, with the cellular redox equilibrium shifted by HPLC analysis as described above. Western blot analysis was per- formed on cells treated with 100 M FeSO at 37 °C for 24 and 192 h. toward more protein-bound glutathione with respect to fibro- Effect in Vivo of FeSO Treatment on Cell Morphology—Control 4 blasts of healthy subjects. fibroblasts were treated in vivo with 100 M (final concentration) FeSO Monoclonal anti-GSH antibody was used to investigate the for 1 month and subjected to fluorescence microscopy by staining with glutathionylation state of FRDA fibroblasts (Fig. 2). Western rhodamine phalloidin (see above). blot analysis of fibroblasts obtained from three patients re- RESULTS vealed a 42-kDa band (Fig. 2A), which was also detected by anti-actin antibody (Fig. 2B). To identify the 42-kDa protein, We analyzed the glutathione content in fibroblasts of nine we immunoprecipitated the fibroblast lysates with anti-actin FRDA patients and determined the GSH/GSSG ratios (Fig. 1A) and protein-bound/total GSH and protein-bound/free GSH ra- antibody and probed them with anti-GSH antibody. As shown 42592 Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts To investigate whether the unexpected increase in actin glu- tathionylation in FRDA fibroblasts is related to changes in the cytoskeletal organization, we stained fibroblasts with the fila- mentous actin indicator rhodamine phalloidin. The fluorescent images revealed a significant disarrangement of F-actin in FRDA patients (Fig. 3B), with clear attenuation of the fluores- cent signal compared with controls (Fig. 3A). Under light mi- croscopy, FRDA fibroblasts (Fig. 3E) appeared to be enlarged with respect to fibroblasts of healthy subjects (Fig. 3D). Taken together, these observations suggest that glutathionylation of actin leads to a disarray of actin filaments, inducing size and shape abnormalities in FRDA fibroblasts. To determine whether there is an in vivo reversibility of glutathionylation in FRDA fibroblasts, we treated FRDA cells with 10 mM (final concentration) GSH and 10 mM (final con- centration) EE-GSH for 30 days at 37 °C. Fig. 4A illustrates the results from HPLC analysis, showing a 17  3.7% decrease in the protein-bound/free GSH ratio in EE-GSH-treated FRDA fibroblasts and a 56  6.8% reduction in GSH-treated FRDA fibroblasts compared with untreated cells. HPLC analysis of GSH and GSSG levels (Fig. 4B) revealed increases in GSH/ GSSG ratios to 113  53.5 upon GSH treatment and to 115 57 upon EE-GSH treatment with respect to untreated FRDA cells (45  8.6; p  0.05), thus becoming comparable to those in controls (129  18.8). Furthermore, Western blot analysis showed a 20  5.5% decrease in glutathionylated proteins in EE-GSH-treated cells (Fig. 4C, lane 3)anda60  15% decrease in GSH-treated cells (lane 4) compared with untreated FRDA fibroblasts (lane 2). To elucidate how medium GSH is available to the cells, we performed in vivo experiments by treating FRDA fibroblasts with acivicin (150 M), an inhibitor of -glu- tamyltranspeptidase. Our findings show that acivicin blocked the protection obtained by GSH treatment, whereas EE-GSH, which is membrane-permeable, was not affected by the inhib- itor (data not shown). The fluorescent images of FRDA fibroblasts after 1 month of in vivo GSH treatment showed an increase in the rhodamine phalloidin signal of F-actin (Fig. 3C) and a rescue of cell size and shape (Fig. 3F). Flow cytometric analysis using the cell viability indicator 6-carboxyfluorescein showed a 15.5  5.6% (p  0.05) increase in the fluorescent signal in GSH-treated FRDA cells (Fig. 5C) with respect to untreated cells (Fig. 5B), comparable to that in controls (Fig. 5A). Finally, to determine whether there is an in vivo association between iron overload, which underlies the pathogenesis of FRDA, and actin glutathionylation, we treated control fibro- blasts with 100 M FeSO for 6, 24, 48, 96, and 192 h. HPLC analysis showed 1.12-, 1.34-, 1.44-, 1.85-, and 2.3-fold increases in the protein-bound/free GSH ratio, respectively, compared with H O treatments (Fig. 6A). Western blot analysis of fibro- blasts incubated with 100 M FeSO for 24 and 192 h at 37 °C showed 1.7-fold (Fig. 6B, lane 2) and 2-fold (lane 3) increases in glutathionylated actin, respectively, compared with H O- treated cells (lane 1). Moreover, to determine whether FeSO induces morphological changes characteristic of FRDA, we FIG.5. Cell morphology of fibroblast cell population as as- sessed by light scattering parameters with density plot of con- treated in vivo control cells with FeSO for 1 month without trol (A), untreated FRDA (B), and GSH-treated FRDA (C) cells obtaining any significant morphological changes resembling after staining with 6-carboxyfluorescein. Forward scatter (FSC) those in FRDA fibroblasts (Fig. 7). measures the relative size, whereas side scatter (SSC) is related to the relative granularity or internal complexity. DISCUSSION The prevailing hypothesis underlying the pathogenesis of in Fig. 2C, we obtained a 42-kDa band corresponding to gluta- FRDA supposes that frataxin is involved in the regulation of thionylated actin. The extent of glutathionylation was in- mitochondrial iron export and that impaired intramitochon- creased by 4.7-fold in FRDA patients with respect to controls drial iron metabolism results in iron overload and oxidative (7.47  3.5 versus 1.6  0.51; p  0.05) (Fig. 2D). Equal stress (1, 2). Iron deposits are observed consistently in some amounts of loaded protein were verified by Coomassie Blue heart myofibrils of FRDA patients upon autopsy, and recent staining of an equivalent SDS gel (data not shown). magnetic resonance imaging data indicate that iron also accu- Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42593 FIG.6. In vivo effect of FeSO treat- ments on protein glutathionylation. A, after incubating control cells with H O or 100 M FeSO for the indicated times, protein-bound (GS-Pro)/free GSH levels were determined by HPLC analysis. Re- sults represent the mean of three inde- pendent determinations. B, after treat- ment with FeSO , control fibroblasts were analyzed by Western blotting. Control cells were incubated at 37 °C for 192 h with H O(lane 1) or with 100 M FeSO 2 4 for 24 h (lane 2) and 192 h (lane 3). The blot is representative of one of at least three identical experiments for each con- dition tested. For details, see ‘‘Experimen- tal Procedures.’’ mulates in the dentate nucleus (9, 39, 40). In previous studies, redox equilibrium is shifted toward more protein-bound gluta- we reported a significant increase in glutathionyl-hemoglobin thione, with significant increases in protein-bound/total GSH in the blood of patients with FRDA, together with an imbalance and protein-bound/free GSH ratios. Furthermore, we used between the superoxide dismutase and glutathione peroxidase monoclonal anti-GSH antibody to investigate protein glutathio- antioxidant enzyme activities, thus supporting the presence of nylation in fibroblasts, and we found that proteins are gluta- a systemic oxidative stress in the disease (16, 17). Therefore, in thionylated in FRDA, with a 5-fold increase in FRDA patients. light of these recent advances in the pathogenesis of FRDA, our Protein glutathionylation occurs also in normal fibroblasts, study has focused on the analysis of glutathione metabolism in but to a lower extent. The fact that some proteins are consti- fibroblasts of FRDA patients. Furthermore, as the oxidized and tutively glutathionylated under basal conditions has already reduced glutathione redox ratios affect the state of glutathio- been observed by Fratelli et al. (37) in T lymphocytes and may nylated proteins, we also studied the glutathionylated protein indicate a regulatory role for glutathionylation in several pro- pattern in FRDA fibroblasts. tein functions. In normal liver, for instance, 20–30 nmol of Our findings show, for the first time, that fibroblasts of glutathione/g of liver are present as disulfides mixed with patients with FRDA undergo oxidative stress, with a signifi- proteins (41). cant decrease in the GSH/GSSG ratio. In addition, the cellular Many proteins can undergo glutathionylation under oxida- 42594 Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts Some of the proteins found to be glutathionylated belong to the class of cytoskeletal proteins, which are particularly abun- dant in cells. The supramolecular organization of these pro- teins depends on the presence of exposed sulfhydryl residues; the modification of these groups by glutathionylation could be relevant to their function, by either protecting them against irreversible oxidation or inhibiting polymerization (48–50). In this study, we have demonstrated that the glutathionyla- tion of actin caused an impairment of microfilaments dynamic in FRDA fibroblasts, as showed using the filamentous actin indicator rhodamine phalloidin. The fluorescent images re- vealed a marked and diffuse reduced signal of F-actin, probably due to disassembly of actin filaments. The same hematoxylin/ eosin-stained cells showed clear abnormalities in size and shape. Also, the viability of FRDA fibroblasts was 18.7  4.3% lower than that of control cells as measured by flow cytometric analysis using 6-carboxyfluorescein. Actin is one of the major cytoskeletal proteins, playing an important role in mediating the infrastructure and dynamics of the cytoplasmic matrix. Its polymerization is a dynamic process implicated in growth factor-mediated cytoskeletal changes. Wang et al. (30) directly linked the epidermal growth factor- mediated signaling pathway to in vivo de-glutathionylation of actin with an increase in F-actin, thus highlighting protein glutathionylation as a physiologically relevant regulatory mechanism in actin polymerization. Monitored by light scat- tering, the steady-state rate for non-glutathionylated actin po- lymerization was at least 5.6-fold faster than that for gluta- thionylated actin polymerization obtained in the presence of GSSG (30). Recently, Dalle-Donne et al. (50) demonstrated in vitro that glutathionylated actin has a decreased capacity to polymerize compared with native actin, with filament elonga- tion being inhibited. An impaired microfilament organization by glutathione binding has been reported in rabbit muscle, FIG.7. Fluorescent images of control fibroblasts after in vivo where the addition of glutathione at Cys of actin resulted in FeSO treatment. Control cells (A) were treated in vivo with 100 M FeSO for 30 days at 37 °C (B), and actin was visualized by rhodamine filaments with a diminished mechanical stability (51, 52). phalloidin staining. The glutathionylation of actin has been observed in several cells under varying conditions of oxidative stress, e.g. gastric mucosal cells treated with H O or diamide and phorbol my- 2 2 tive stress (36, 42, 43). Some proteins sustain different but ristate acetate-stimulated murine macrophages and human important functions, such as nucleophosmin (involved in the neutrophils (48, 53). H O treatment is also responsible for 2 2 assembly of ribosomal proteins), cyclophilin (a chaperonin in- actin glutathionylation in human epidermal carcinoma A431 volved in the proteasomal degradation of proteins), and the cells, where it regulates actin polymerization (30). heat shock proteins HSP60 and HSP70. Glyceraldehyde-3- A role of oxidized actin has been reported in some neuro- phosphate dehydrogenase is the major S-glutathionylated pro- degenerative diseases, such as in a mouse model of amyotro- tein in endothelial cells exposed to hydrogen peroxide and in phic lateral sclerosis, where axonal degeneration appears to be monocytes during the endogenous oxidative burst (23, 44). Cre- due to defective transport of components required for axonal atine kinase and glycogen phosphorylase b are also targets for maintenance (54). Actin oxidation was significantly higher S-glutathionylation in myocytes and cardiac tissue during cy- even in brain extracts of patients with Alzheimer’s disease, clic oxidative stress. Carbonic anhydrase III, glutathione S- suggesting that oxidative stress-induced injury may lead to the transferase, superoxide dismutase, hemoglobin, and bovine eye degeneration of neurons in the Alzheimer’s disease brain (55). lens crystalline become thiolated in cellular models of oxidative Glutathionylation is a redox-dependent reversible mecha- stress. Additional proteins, including fatty-acid synthase, 3-hy- nism (30, 50). Indeed, when we incubated fibroblasts with droxy-3-methylglutaryl-CoA reductase, aldose reductase, hu- pro-oxidants in vitro, we found a 4-fold increase in glutathio- man immunodeficiency virus-1 protease, and small HSP25, nylation in the presence of GSSG and a 2.5-fold increase with have been reported as potential targets in vitro for redox-de- H O . Glutathionylation was also reversed in vitro by excess pendent S-glutathionylation (36). Even c-Jun DNA binding 2 2 GSH or dithiothreitol (data not shown). To address the in vivo appears to be redox-regulated by glutathionylation (27, 28), reversibility of actin glutathionylation in FRDA, we treated and dopamine biosynthesis is also inhibited by S-glutathiony- fibroblasts with GSH for 1 month and evaluated the extent of lation during oxidative stress (45). In Parkinson’s disease, glutathionylation and cell morphology. Interestingly, we found monoamine oxidase-derived H O was shown to inhibit mito- 2 2 a 60% decrease in protein glutathionylation in treated fibro- chondrial respiration by glutathionylation of respiratory chain enzymes (46). Finally, direct glutathionylation of proteins by blasts upon analysis by Western blotting and a complete rescue of size and cell shape. Even cell viability resulted in a signifi- superoxide anion has been demonstrated in a study showing that S-glutathionylation of protein-tyrosine phosphatases mod- cant enhancement of the fluorescent signal in GSH-treated FRDA cells, becoming comparable to that in controls. ulates the phosphorylation state of cells and preserves protein function (47). GSH transport across cell membrane is not yet well defined, Glutathionylation of Actin in Friedreich’s Ataxia Fibroblasts 42595 Cooper, J. M. (2001) Ann. Neurol. 49, 590–596 although some studies have provided insight especially on GSH 14. Lerman-Sagie, T., Rustin, P., Lev, D., Yanoov, M., Leshinsky-Silver, E., Sagie, release, which seems to involve the family of multidrug resis- A., Ben-Gal, T., and Munnich, A. (2001) J. Inherit. Metab. Dis. 24, 28–34 15. Housse, A. O., Aggoun, Y., Bonnet, D., Sidi, D., Munnich, A., Rotig, A., and tance-associated proteins (32). Evidence for direct uptake of Rustin, P. 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