Báo cáo khoa học: Frataxin deficiency causes upregulation of mitochondrial Lon and ClpP proteases and severe loss of mitochondrial Fe–S proteins pot

Frataxin deficiency causes upregulation of mitochondrial Lon and ClpP proteases and severe loss of mitochondrial Fe–S proteins Blanche Guillon 1 , Anne-Laure Bulteau 2 , Marie Wattenhofer-Donze ´ 3,4 , Ste ´ phane Schmucker 3,5 , Bertrand Friguet 2 ,He ´ le ` ne Puccio 3,4,5,6,7 , Jean-Claude Drapier 1 and Ce ´ cile Bouton 1 1 Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France 2 Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite ´ Paris 7, France 3 IGBMC (Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire), Illkirch, France 4 Colle ` ge de France, Chaire de ge ´ ne ´ tique humaine, Illkirch, France 5 Universite ´ Louis Pasteur, Strasbourg, France 6 Inserm, U596, Illkirch, France 7 CNRS, UMR7104, Illkirch, France Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative and cardiodegenerative disease char- acterized by progressive ataxia and cardiomyopathy that is associated with deficit in Fe–S enzyme activities and abnormal cellular iron metabolism [1]. FRDA results from a greatly reduced level of the mitochon- drial protein frataxin, due to a large GAA repeat expansion in the gene, which inhibits the transcription of the frataxin gene through heterochromatin silencing of the locus [2]. Although the exact role of frataxin is still controversial, pathophysiological studies from patient autopsies demonstrated a specific loss of Fe–S protein activities, with accumulation of iron being thought to contribute to an increased level of oxidative stress. FRDA mouse models with a tissue-targeted frataxin deficiency have been developed to study the pathophysiology of the disease and the function of frataxin, and to test potential therapeutic agents [3]. Keywords ClpP; frataxin; Friedreich ataxia; iron-sulfur cluster; Lon protease Correspondence C. Bouton, ICSN-CNRS, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France Fax: +33 1 69 07 72 47 Tel: +33 1 69 82 30 10 E-mail: Cecile.bouton@icsn.cnrs-gif.fr (Received 21 October 2008, revised 4 December 2008, accepted 9 December 2008) doi:10.1111/j.1742-4658.2008.06847.x Friedreich ataxia (FRDA) is a rare hereditary neurodegenerative disease characterized by progressive ataxia and cardiomyopathy. The cause of the disease is a defect in mitochondrial frataxin, an iron chaperone involved in the maturation of Fe–S cluster proteins. Several human diseases, including cardiomyopathies, have been found to result from deficiencies in the activ- ity of specific proteases, which have important roles in protein turnover and in the removal of damaged or unneeded protein. In this study, using the muscle creatine kinase mouse heart model for FRDA, we show a clear progressive increase in protein levels of two important mitochondrial ATP- dependent proteases, Lon and ClpP, in the hearts of muscle creatine kinase mutants. These proteases have been shown to degrade unfolded and dam- aged proteins in the matrix of mitochondria. Their upregulation, which was triggered at a mid-stage of the disease through separate pathways, was accompanied by an increase in proteolytic activity. We also demonstrate a simultaneous and significant progressive loss of mitochondrial Fe–S pro- teins with no substantial change in their mRNA level. The correlative effect of Lon and ClpP upregulation on loss of mitochondrial Fe–S proteins dur- ing the progression of the disease may suggest that Fe–S proteins are potential targets of Lon and ClpP proteases in FRDA. Abbreviations DNP, 2,4-dinitrophenylhydrazone; ER, endoplasmic reticulum; FRDA, Friedreich ataxia; MCK, muscle creatine kinase; SDHA, succinate dehydrogenase complex subunit A; Yfh1p, yeast frataxin homolog. 1036 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS The cardiac conditional model, in which frataxin has been specifically deleted in striated muscles using a recombinase expressed under control of the muscle creatine kinase (MCK) promoter, and the animals in which are hereafter referred to as MCK mutants, reproduces important pathophysiological features and biochemical aspects of the human disease [4,5]. These animals show cardiodegeneration, deficiency of respira- tory chain complexes I–III and aconitases, and mito- chondrial iron accumulation, without the presence of major oxidative stress. This model was used to demon- strate that the deficit in mitochondrial Fe–S cluster enzyme activities is an early event in FRDA disease, followed by rapid cardiac dysfunction, whereas abnor- mal iron accumulation within the mitochondria occurs at a late stage, pointing to a role of frataxin in Fe–S cluster biogenesis [5]. Since then, many studies have reported the involvement of frataxin in the maturation of the Fe–S cluster proteins in yeast [6–10], mammals [11–14], Drosophila [15] and, recently, bacteria [16] and plants [17]. Frataxin, which has been the focus of extensive research in the yeast system, specifically interacts with Fe–S scaffold Isu1 ⁄ 2 proteins [10,18–20], and is thought to provide iron for the formation of the cluster. Evidence is accumulating that proteasome dysfunc- tion might be associated with cardiomyopathies in which accumulation of abnormal misfolded proteins may lead to the formation of potentially toxic aggre- gates [21]. In the mitochondrion, the main organelle affected in FRDA, various proteases also have important functions in protein quality control [22]. Among these, the ATP-stimulated Lon protease, which forms homo-oligomeric complexes, degrades misfolded and damaged proteins in the matrix space, similarly to the proteasome function in the cytoplasm [23]. By mediating complete proteolysis, Lon thereby prevents aggregation and deleterious effects on mito- chondrial functions. A second ATP-stimulated prote- ase, named ClpP, has also been identified in the matrix of mammalian cells [24] and associates with the ATPase ClpX subunits in vitro to effect its ATP- dependent proteolytic activity [25,26]. Although sub- strate specificity has not been defined yet, several studies have demonstrated that Fe–S cluster proteins can be preferential substrates for Lon and ⁄ or ClpXP proteases in different systems [27–30]. Indeed, it has recently been demonstrated in yeast that Fe–S cluster integrity in proteins is one of the major determinants of susceptibility to degradation by Pim1, the yeast homolog of human Lon protease [28]. By means of a proteomic approach using wild-type and Pim-1 mutant strains, the authors identified five Pim-1 substrate proteins, including two Fe–S proteins (the homoaconitase Lys4 and Yjl200c, a putative aconi- tase isozyme). Using an in organello degradation assay, they also demonstrated that improper assembly of Fe–S clusters on Yjl200c and aconitase (aco1) led to their increased susceptibility to degradation. Inter- estingly, in mammals, mitochondrial aconitase has been identified as a good proteolytic substrate for Lon under mild oxidative conditions [29]. Finally, one mutational study of ClpP performed in plants showed that the Rieske Fe–S protein can be a sub- strate for this protease [30]. In this study, we investigated whether mitochondrial Lon and ClpP proteases are regulated in the heart of conditional MCK mice during the progression of the FRDA cardiac disease. We found a progressive increase in mitochondrial Lon and ClpP protease expression and activity in cardiac tissues of the MCK mutant over the course of the disease. Moreover, the proteases are upregulated through two distinct mecha- nisms, as Lon upregulation is transcriptional, whereas that of ClpP is post-transcriptional, acting either by increasing its protein translation or by decreasing its rate of turnover. We also addressed the fate of several mitochondrial Fe–S proteins in cardiac tissue of MCK mutants, and showed an overall clearance in protein levels of key mitochondrial Fe–S cluster enzymes that followed the elevated mitochondrial ATP-stimulated proteolytic activity in this FRDA model. Results Modulation of Lon and ClpP expression in cardiac muscles of MCK mutants Using the MCK mutants, we investigated the effect of frataxin deficiency on the possible regulation of the two mitochondrial matrix ATP-dependent proteases. We first evaluated Lon and ClpP mRNA levels by quantitative real-time PCR from total RNA heart extracts of different age groups of control and MCK mutants. A significant and progressive increase in Lon mRNA levels was observed in mutant mice between 5 and 10 weeks of age, whereas ClpP mRNA levels were not affected (Fig. 1A). Indeed, the Lon protease mRNA level increased  2.5-fold at 5 weeks of age in the hearts of MCK mutants as compared with control mice, and continued to rise, increasing  4-fold at 10 weeks of age. We next investigated the possible regulation of both proteases in MCK mutants at the protein level. Immunoblot analysis using specific anti- bodies against peptides clearly showed a progressive B. Guillon et al. Upregulation of mitochondrial proteases in FRDA FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1037 increase in Lon protein content, starting at the interme- diate stage (5 weeks) of cardiomyopathy progression [5] in the MCK mutants (Fig. 1B). Quantitative immu- noblot analysis revealed  2.6-fold, 5.0-fold and 6.7- fold increases in Lon protein expression at 5, 7 and 10 weeks of age, respectively, in MCK mutants as compared with control mice. Interestingly, the ClpP protein level was also progressively enhanced, with  3-fold, 3.5-fold and 4.5-fold increases at 5, 7 and 10 weeks of age in MCK mutants, respectively, despite no change in mRNA levels (Fig. 1A,B). ATP-stimulated proteolytic activity of ClpP and Lon proteases in heart mitochondria of MCK mutants To investigate whether the increase in Lon and ClpP protein levels is accompanied by increased protein functionality, ATP-stimulated proteolytic activity, reflecting both ClpP and Lon activities [31], was measured in heart mitochondria from frataxin- deficient mice. The cytosolic contamination of mito- chondrial fractions was checked by performing A B Fig. 1. Regulation of mitochondrial Lon and ClpP protease expression in the hearts of control and MCK mutant mice. (A) Total RNA was isolated from the hearts of control and MCK mutant mice at 3, 5 and 10 weeks of age and used to measure Lon protease and ClpP mRNA levels by quantitative real-time PCR. The mRNA expression levels were expressed as fold change between MCK and control samples (value 1) and normalized to 18S ribosomal RNA. The experiment was repeated at least three times with independent RNA samples, and the average ± standard deviation of the three replicates is depicted in the bar graphs. Statistical analysis was performed using Student’s t-test: ***P < 0.0001. (B) Mitochondrial extracts from the hearts of control (C) and MCK (M) mice at 3, 5, 7 and 10 weeks of age were analyzed by immunoblotting with antibodies against Lon and ClpP. Immunolabeled protein bands of interest were then quantified using a ChemiDoc imaging system and QUANTITY ONE software (BioRad, Marne-La-Coquette, France), and were normalized using antibodies against prohibitin and ATP2 as mitochondrial loading controls. *P < 0.01. Upregulation of mitochondrial proteases in FRDA B. Guillon et al. 1038 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS immunoblotting using prohibitin as mitochondrial marker and vinculin and proteasome 20S subunit as cytosolic markers (Fig. 2A). As shown in Fig. 2A, very little cytosolic contamination was observed in the mitochondrial fraction. Values corresponding to ATP-dependent proteolytic activities in control and mutant mice (Table S1) were used to calculate the fold changes in ClpP and Lon protease activities found in MCK mutant versus control mice at 5, 7 and 8 weeks of age. We showed that ClpP ⁄ Lon protease activity, which was low in the heart mitochondria of 5-week-old MCK mutants, increased  2-fold to  2.5-fold between 7 and 8 weeks of age (Fig. 2B). Level of carbonylated proteins in heart mitochondria of MCK mutants at different ages Stimulation of Lon proteolytic activity that may depend on increases in carbonylated proteins has been reported in an in vivo cardiac ischemia–reperfusion model [32] and in yeast frataxin homolog (Yfh1p)-defi- cient yeast cells [33]. In MCK mutants, Seznec et al. [34] did not find any evidence of increased cellular oxi- dative stress. Rather, a reduction in oxidized proteins in the hearts of MCK mutants was detected from 7 to 10 weeks. In this previous report, carbonylated proteins were measured in total extracts of frataxin- deficient mice, leading to an underestimation of pos- sible oxidative stress in mitochondria. We therefore performed subcellular fractionation from cardiac tissue of wild-type and MCK mutant mice in order to detect carbonylated proteins in the mitochondrial protein fractions. In mitochondria of control mice, appreciable amounts of oxidized proteins were detected, probably due to the oxidative metabolism of mitochondria under normal conditions [35]. The total amount of oxi- dized proteins did not increase in frataxin-deficient mice as compared with control mice at any age tested (Fig. 2C). Carbonylated proteins of mitochondrial frac- tions were also quantified by ELISA using carbonylated standards, and the results indicated that their levels were similar in both control and MCK mutant mice at any age and represented < 0.1 nmolÆmg )1 of total proteins. Therefore, in contrast to the Yfh1p-deficient yeast model of FRDA, increase in Lon proteolytic activity in Fig. 2. Lon ⁄ ClpP protease activity and level of oxidized proteins in the heart mitochondria of MCK mutant mice. (A) Cytosolic contami- nation of the mitochondrial fraction was checked by immunoblot- ting with protein extracts (40 lg) from the first (500 g) and second (10 000 g) pellets using antibodies against vinculin, proteasome 20S and prohibitin. A representative result of cell fractionation is shown. C, control; M, MCK. (B) Mitochondrial fractions were pre- pared from the hearts of control and MCK mutant mice at 5, 7 and 8 weeks of age and assayed for ATP-dependent proteolytic activity. Two hearts of MCK mutant or control mice were pooled to mea- sure proteolytic activity per dot. Each triangle, diamond or square corresponds to the fold change in mitochondrial ATP-dependent proteolytic activity of MCK versus control mice at the different stages indicated. Detailed measures can be found in Table S1. (C) Carbonylated proteins of mitochondrial fractions (10 lg) from control (C) and MCK mutant (M) mice were detected after derivati- zation of their carbonyl groups using a solution of dinitrophenylhydr- azine, SDS ⁄ PAGE and immunoblotting using a primary antibody against DNP as described in Experimental procedures. A digitized image of Ponceau staining was used to check equal loading of each lane (not shown). Experiments were performed at least three times, and a representative result is shown. A B C B. Guillon et al. Upregulation of mitochondrial proteases in FRDA FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1039 the MCK ⁄ FRDA mouse model is not linked to accumu- lation of oxidized proteins. Regulation of mitochondria-encoded genes in MCK mutants at different ages Beside its protease function, it has been reported that, in vitro, Lon binds to specific regions of the light- strand and heavy-strand promoters of mitochondrial DNA [36,37], and this was recently confirmed in living cells, pointing to another function of this protease in the regulation of mitochondrial DNA replication and gene expression [38]. We therefore examined whether mitochondria-encoded gene expression was affected in MCK mutant hearts. The mRNA expression profile of 12 mitochondria-encoded genes involved in com- plexes I, III, IV and V of oxidative phosphorylation was determined in the hearts of wild-type and MCK mutant mice at different stages of the disease. Apart from ND3 at 5 weeks, there was no significant decrease or increase in gene expression of any of the mitochon- dria-encoded genes tested at 3 and 5 weeks of age in the hearts of MCK mutants as compared with control littermate mice (Table 1). In contrast, at a late stage, five genes of complex I, cytochrome b of complex III and two genes of complex IV were slightly but signi- ficantly downregulated in MCK mutants versus controls. Decrease in mitochondrial Fe–S protein levels in MCK mutants The effect of frataxin deficiency on the abundance of mitochondrial Fe–S cluster-containing proteins was also examined in the MCK mutants. Three Fe–S sub- units of complex I (NDUFS3), complex II (SDHB) and complex III (Rieske) of the respiratory chain were selected for immunoblot analysis, as well as ferrochela- tase, a [2Fe–2S] enzyme required for the last step in heme biosynthesis [39], and the [4Fe–4S] aconitase of the tricarboxylic acid cycle. A significant decrease in the protein level of every mitochondrial Fe–S protein tested was observed at 5 weeks in the heart of MCK mutants, where frataxin is completely deleted, as com- pared with control samples (Fig. 3A). Quantitative immunoblot analysis revealed a similar pattern in the time course of mitochondrial Fe–S cluster protein loss, whereas expression of the mitochondrial Atp2 b-sub- unit of the ATP synthase complex, which does not contain an Fe–S cluster, was not significantly affected in frataxin-deficient mice (Fig. 3B). At 3 weeks, the ini- tial stage of the disease, in which the only phenotype observed is the specific deficit in Fe–S enzyme activity, very little change in Fe–S protein levels was observed, suggesting a defect in Fe–S cluster assembly. The decrease in mitochondrial Fe–S cluster proteins was clearly apparent at 5 weeks ( 40–70% decrease) in the hearts of MCK mutants, a stage corresponding to the beginning of the cardiac dysfunction. Downregula- tion further decreased at 7 weeks, residual Fe–S protein levels reaching  30–20% in MCK mutants as compared with controls, and stabilizing at a plateau of  20% at 10 weeks. These results are in agreement with the strong enzymatic deficiency of aconitase and the respiratory chain previously observed. Protein expression levels of other subunits of the respiratory chain were also investigated (Fig. 4). The level of the hydrophilic succinate dehydrogenase complex sub- unit A (SDHA), the flavoprotein subunit of com- plex II, which is not an Fe–S cluster protein but which requires the Fe–S proteins to be properly folded into the complex [40], was also visibly reduced at 5 weeks by  40% as compared with control mice. The expres- sion of the ND6, which is a partner subunit of Fe–S complex I, was also significantly affected in frataxin- deficient mice (Fig. 4A,B). To determine whether the decrease in mitochondrial Fe–S proteins and partners was due to a transcrip- tional regulation, reverse transcription followed by real-time qPCR was performed in control and MCK mutants at 3, 5 and 10 weeks of age. As shown in Fig. 5, no significant change in the mRNA expression of mitochondrial aconitase and several subunits of the respiratory chain (Rieske, ND6 and SDHA) was observed in the hearts of either control or MCK mutant mice, despite a marked reduction in their pro- tein levels in the mutants. The ferrochelatase, Ndufs3 Table 1. mRNA expression of mitochondria-encoded genes in the heart of control versus MCK mutant mice at 3, 5 and 10 weeks of age. Cytb, cytochrome b; COX, cyclo-oxygenase. Mitochondrial subunits mRNA fold change difference (WT ⁄ MCK) 3 weeks 5 weeks 10 weeks ND1 0.83 ± 0.43 1.14 ± 0.09 1.22 ± 0.05 ND2 0.91 ± 0.47 1.08 ± 0.12 0.81 ± 0.03* ND3 0.77 ± 0.41 1.23 ± 0.12* 0.78 ± 0.09* ND4 0.79 ± 0.40 0.83 ± 0.06 0.74 ± 0.04* ND4L 0.94 ± 0.16 0.89 ± 0.07 0.63 ± 0.07* ND5 0.86 ± 0.27 0.81 ± 0.12 0.61 ± 0.06** ND6 0.84 ± 0.28 0.83 ± 0.09 0.87 ± 0.09 COX1 1.24 ± 0.19 0.88 ± 0.05 0.81 ± 0.07* COX2 0.93 ± 0.32 1.14 ± 0.22 1.08 ± 0.26 COX3 0.90 ± 0.31 0.99 ± 0.08 0.87 ± 0.08* ATPase 6 0.80 ± 0.43 1.10 ± 0.09 1.05 ± 0.16 Cytb 0.91 ± 0.31 0.87 ± 0.11 0.59 ± 0.04* *P < 0.05; **P < 0.001. Upregulation of mitochondrial proteases in FRDA B. Guillon et al. 1040 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS and Sdhb transcripts were slightly but significantly decreased at a late stage in frataxin-deficient mice (Fig. 5) [41], but this cannot explain the drastic change seen earlier at the protein level in the hearts of MCK mutants (Fig. 3). Discussion Disturbances of proteolytic systems have been associ- ated with various human diseases [42]. A defect in these enzymatic systems usually causes protein aggregation and subsequent cellular damage [43]. In the matrix of mitochondria, two ATP-stimulated serine proteases have been identified, namely ClpP and Lon proteases, which participate in the degradation of improperly folded and damaged proteins [23,44]. Whereas Lon is a homo-oligomeric complex, human ClpP forms a het- erocomplex in vitro with ClpX, an ATP-dependent AAA+ chaperone [45]. High expression levels of Lon and ClpP have been reported in energy-hungry tissues such as skeletal muscle and heart, suggesting an impor- tant mitochondrial function of these proteases in these tissues [46,47]. Little is known about Lon and ClpP in mammals, and to date, regulation of these proteases has never been studied in mitochondrial diseases. In the present article, we report the first demonstration that frataxin deficiency causes significant upregulation of both mitochondrial Lon and ClpP proteases in the car- diac mouse model for Friedreich ataxia. The increase in protease levels started at the mid-stage of the disease, and was rapidly followed by a boost of their proteolytic activity. We also show that Lon upregulation and ClpP upregulation in the hearts of MCK mutants operate A B Fig. 3. Levels of mitochondrial Fe–S cluster-containing proteins in cardiac muscle of control and MCK mutant mice. (A) Total protein extracts (20 lg) from control (C) and MCK mutant (M) mice at 3, 5, 7 and 10 weeks of age were analyzed by SDS ⁄ PAGE and immuno- blot with specific primary antibodies against frataxin, mitochondrial aconitase, ferrochelatase, three Fe–S subunits of complex I (NDUFS3), complex II (SDHB) and complex III (Rieske) and the ATP2 b-subunit of mitochondrial ATP synthase. (B) Immunolabeled protein bands of interest were quantified using a ChemiDoc imag- ing system and QUANTITY ONE software (BioRad), and were normal- ized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vinculin or b-tubulin as loading control. These experiments were performed at least three times independently, and representative data are shown. Statistical analysis was performed using Student’s t-test: **P < 0.001; ***P < 0.0001. A B Fig. 4. Levels of SDHA and ND6 respiratory chain subunits in the hearts of control and MCK mutant mice. (A) Total protein extracts (20 lg) from control (C) and MCK mutant (M) mice at 3, 5, 7 and 10 weeks of age were analyzed by immunoblot using specific pri- mary antibodies against the ND6 subunit of complex I and the flavoprotein (SDHA) of complex II. A representative result of three independent experiments is shown. (B) Immunolabeled protein bands of interest were quantified and normalized using vinculin as loading control. Statistical analysis was performed using Student’s t-test: **P < 0.001; ***P < 0.0001. B. Guillon et al. Upregulation of mitochondrial proteases in FRDA FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1041 through two distinct mechanisms. The increase in Lon protein level was due to transcriptional regulation, as the protein increase was mirrored at the mRNA level. The same does not hold true for ClpP, which did not exhibit a change in transcript level, suggesting transla- tional or post-translational regulation. Regarding Lon, although signals that trigger its upregulation in FRDA are unknown, some proposals can be put forward. The MCK mutants present signs of endoplasmic reticulum (ER) stress (H. Puccio, unpublished data) simulta- neously with the increase in Lon expression. As it has been reported that cells subjected to hypoxia or ER stress exhibit higher Lon mRNA levels [48], it is tempt- ing to suggest that ER stress leads to Lon protease upregulation in MCK mutants. Besides, depletion of ATP, which has been shown in tissues of FRDA patients [49], can lead to regulation of gene expression in some stressful situations [50,51]. As Lon proteolytic activity is stimulated up to nine-fold by ATP [44], it can be hypothesized that lack of ATP may compensate for low Lon activity by initiating upregulation of the Lon gene and protein expression. Regarding the ClpP protease, very little is known about its physiological function and regulation in mammals. One study reported ClpP gene upregulation after accumulation of unfolded proteins within the mitochondrial matrix, which appears to depend on CHOP and C ⁄ EBPb ele- ments identified in its promoter [52]. However, as the change in ClpP expression in MCK mutants occurred at a post-transcriptional level, protein accumulation in mitochondria, described by Zhao et al., is not the sig- nal that triggers ClpP protein upregulation in the MCK mutants. Lon protease has been described as a multifunctional enzyme, which behaves like an ATP-stimulated prote- ase, a chaperone or a regulator of mitochondrial DNA replication and gene expression [38,47,53]. We have shown that the major activity displayed by Lon in the heart of frataxin-deficient mice was its proteolytic activity, which contrasts with the small change in mito- chondria-encoded gene expression. In addition, the prominent accumulation of mitochondria observed in MCK mutants at 6–7 weeks, which becomes excessive at the final stage of the disease [34], may be related to high Lon expression. Indeed, two reports showed that the expression and activity of Lon were increased in cells with enhanced mitochondrial biogenesis [54] and that a population of Lon-deficient cells exhibited fewer mito- chondria [55]. Therefore, Lon could, at least in part, be responsible for the prominent accumulation of mito- chondria observed in the hearts of MCK mutants. In the bacterial and yeast systems, it has recently been shown that integrity of Fe–S clusters is a main determinant of susceptibility to Lon and ⁄ or ClpP deg- radation [27,28]. Interestingly, an important biochemi- cal feature associated with frataxin deficiency in Friedreich ataxia is the specific defect in Fe–S cluster enzyme activities, a very early step in the disease pro- cess [5,14,34]. This phenomenon was in part attributed to imperfect Fe–S protein maturation, as frataxin has been identified as an important component of the Fe–S cluster assembly machinery in mammals and other organisms [6,7,12,15–17]. In this study, we have shown that frataxin deficiency causes a severe protein loss for several mitochondrial Fe–S enzymes, contrib- uting to the overall Fe–S deficit in FRDA. The decrease in protein level at 5, 7 and 10 weeks was not due to transcriptional regulation, as mRNA levels showed no substantial difference between controls and mutants. According to structural studies, Fe–S sub- units and other proteins of complexes I and II actually fit into each other [40,56]. The complex I hydrophobic A B Fig. 5. Gene expression profile of mitochondrial Fe–S proteins and SDHA and ND6 subunits of the respiratory chain in cardiac muscle of wild-type and MCK mutant mice. Total RNA, which was extracted from the hearts of control and MCK mutant mice at 3, 5 and 10 weeks of age, was used to assess mRNA expression of genes encoding mitochondrial aconitase, ferrochelatase, NDUFS3, SDHB and Rieske proteins (A), and ND6 and SDHA proteins (B) by quantitative real-time PCR. The mRNA expression levels in MCK samples were expressed as fold change over controls (assigned the value of 1) and were normalized to 18S ribosomal RNA. Experi- ments were performed at least three times, and data are presented as mean ± standard deviation for three separate experiments. Sta- tistical analysis was performed using Student’s t-test. *P < 0.05; **P < 0.001. Upregulation of mitochondrial proteases in FRDA B. Guillon et al. 1042 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS subunits ND1–ND6 form a shell around the Fe–S protein fragments [57], and SDHA flavoprotein of complex II dimerizes with the Fe–S protein domain (Ip), forming a soluble catalytic heterodimer [40]. Interestingly, protein levels of ND6 and SDHA were also reduced. In contrast, protein expression of both ATP2, the b-subunit of mitochondrial ATP synthase, and of mitochondrial prohibitin, neither of which con- tains Fe–S clusters or is related to Fe–S proteins, was unaffected in mutant mice, indicating that frataxin deficiency specifically affects Fe–S proteins and their protein partners. These results are reminiscent of studies showing that Yfh1p-deficient yeast cells undergo degra- dation of mitochondrial aconitase [6] and that, in plants, depletion of chloroplastic NifS, another important com- ponent for Fe–S cluster biogenesis, decreases the abun- dance of several Fe–S proteins and partners [58]. By statistical analysis, we found that the time course of mitochondrial Fe–S protein loss in MCK mutants sig- nificantly correlates with the progressive increase in the levels of both mitochondrial Lon and ClpP proteases (Fig. S1). Taking all these data together, it is tempting to speculate that the frataxin-dependent defect in Fe–S cluster biogenesis leads to the formation of mitochon- drial apoenzymes, which are recognized as misfolded or low-stability proteins, and degraded by Lon and ⁄ or ClpP proteases. Although it is unlikely, we cannot exclude the possibility that aggregation of unfolded Fe–S protein also participates in the decrease of Fe–S protein assessed by immunoblot. Research on the devel- opment of specific Lon inhibitors has started very recently [59] and, when they are available, they will be very useful in discovering whether mitochondrial prote- ase activation is a deleterious or protective process in FRDA. To date, reports concerning the understanding of Lon cellular functions suggest a protective role of Lon against aggregation and intracellular accumulation of oxidized proteins in mitochondria [29,32]. Therefore, increased Lon and ClpP activity in MCK mutants, by preventing the accumulation of carbonylated proteins, may hide increased oxidative damage in MCK mutants. Specific inhibitors of Lon proteases will help to further characterize the Lon and ClpP cellular functions and identify whether Fe–S proteins are specific substrates of Lon protease in FRDA. Experimental procedures Animals MCK mutants were generated by crossing mice homozy- gous for a conditional allele of Frda (Frda L3 ⁄ L3 ) with mice heterozygous for the deletion of Frda exon 4 (Frda D ⁄ + ), which carries a tissue-specific Cre transgene under the con- trol of the MCK promoter [5]. In this study, mice carrying the transgene and conditional allele (L3 ⁄ L; MCK+) are called MCK mutants. Control mice were littermates having at least one normal frataxin-expressing allele. All methods employed in this work are in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Preparation of total protein extracts Using a glass homogenizer (Duall 21), a tissue homogenate was prepared from the heart of wild-type and MCK mutant mice in 100 mm Tris (pH 7.4) and 150 mm NaCl in the pres- ence of protease inhibitors (protease inhibitor cocktail set III; Calbiochem, Darmstadt, Germany). After centrifu- gation at 400 g for 10 min at 4 °C, red blood cells were lysed by resuspending the cell pellet in 800 lL of hypotonic solu- tion (Sigma, Saint-Quentin Fallavier, France). The resulting white pellet was then lysed in 100 mm Tris (pH 7.4) contain- ing 0.5% Triton X-100, with the protease inhibitor cocktail. After centrifugation at 10 000 g for 10 min at 4 °C, the supernatant was collected and protein content was deter- mined before storage at )80 °C for further measurements. Isolation of cardiac mitochondria Hearts of wild-type and MCK mutant mice were homo- genized in 210 mm mannitol, 5.0 mm Mops, 70 mm sucrose, and 1.0 mm EDTA (pH 7.4). The homogenate was centri- fuged at 500 g for 10 min at 4 °C to remove tissue frag- ments and cell nuclei, and the supernatant was recentrifuged at 10 000 g for 10 min at 4 °C to bring down the mitochondrial pellet. After two washings in homoge- nized buffer, the mitochondrial pellets were either stored at )80 °C for subsequent immunoblot analysis or immediately used for the measurement of ATP-dependent proteolytic activity as well as detection of carbonylated proteins. Immunoblot analysis Total protein extract (20–40 lg) or mitochondrial fraction (60 lg) was loaded on 10% or 12% SDS ⁄ PAGE gel (depending on the expected protein size), and proteins were transferred onto a Hybond nitrocellulose membrane. Spe- cific proteins were detected by immunoblotting with the indicated primary antibodies and peroxidase-conjugated secondary antibody (DAKO Cytomation, Glostrup, Denmark). Blots were developed with an enhanced chemilu- minescence detection system [Super Signal Pierce (Perbio Science, Berbie ` res, France) or Immobilon Western (Milli- pore, Saint-Quentin en Yvelines, France)]. B. Guillon et al. Upregulation of mitochondrial proteases in FRDA FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1043 Immunochemical reagents Rabbit polyclonal anti-peptide Lon protease serum was kindly provided by L. Sweda (Case Western Reserve Uni- versity, Cleveland, OH, USA). Mouse monoclonal IgG against complex I subunit NDUFS3 (NADH dehydroge- nase ubiquinone Fe–S protein), complex II subunit b (one Fe–S subunit of complex II), Rieske Fe–S protein subunit of complex III and the flavoprotein subunit of complex II were from MitoSciences (Eugene, OR, USA). Monoclonal anti-mitochondrial DNA-encoded ND6 subunit IgG (Mito- Sciences) was kindly provided by G. Dujardin (Centre de Ge ´ ne ´ tique Mole ´ culaire, Gif-sur-Yvette, France). Rabbit polyclonal sera were raised against synthetic peptides corre- sponding to amino acids 45–61 of mouse mitochondrial aconitase and to amino acids 410–422 of mouse ferrochela- tase. The serum against frataxin used was the 1250 poly- clonal serum [5]. Serum against ClpP raised against the peptide corresponding to amino acids 65–80 of human ClpP was kindly provided by L. Sweda (Oklahoma Medical Research Foundation, Oklahoma City, OK, USA). Sera against vinculin (Sigma) and prohibitin (Neomarkers, Fremont, CA, USA) were used as loading controls. Serum against the ATP2 b1-subunit of yeast ATP synthase was kindly provided by J. Velours (Institut de Biochimie et Ge ´ ne ´ tique Cellulaire, Bordeaux, France). ATP-stimulated protease activity ATP-stimulated Lon and ClpP protease activity was deter- mined using casein–fluorescein isothiocyanate (0.5 lgÆlL )1 ) as substrate [31]. Mitochondrial pellets (50–150 lg) were incubated in assay buffer containing 50 mm Tris (pH 7.9), 10 mm MgCl 2 ,1mm dithiothreitol and 0.1% Triton X-100 in the presence or absence of 8 mm ATP. Proteolysis of fluorescein isothiocyanate-labeled casein was then per- formed for 90 min at 37 °C. At incubation times of 0–90 min, an aliquot was collected and proteins were pre- cipitated by adding 8% trichloroacetic acid. After centrifu- gation at 15 000 g for 30 min, supernatant containing fluorescent peptides was recovered and neutralized by add- ing sodium borate (0.6 m final concentration, pH 10). Fluo- rescence was then measured with excitation ⁄ emission wavelengths of 495 ⁄ 515 nm. Activities were expressed as fluorescence units ⁄ min ⁄ mg protein, and a ratio between the ATP-dependent proteolytic activities found in MCK and wild-type mice was calculated. Carbonylated protein detection Carbonylated proteins were detected using the OxyBlot protein oxidation detection kit according to the manufac- turer’s protocol (Chemicon International, Temecula, CA, USA). Briefly, mitochondrial pellets were lysed in 100 mm Tris (pH 7.5), 0.5% Triton X-100 and 50 mm dithiothreitol. After centrifugation, clear lysate was denatured by adding 6% SDS, and the carbonyl groups in proteins were deriva- tized to 2,4-dinitrophenylhydrazone (DNP) using 1· dini- trophenylhydrazine solution for 15 min at 25 °C. After neutralization of the reaction, DNP-derivatized proteins were loaded on a 10% SDS ⁄ PAGE gel, transferred to a nitrocellulose membrane, and detected with a primary anti- body specific to the DNP moiety. Carbonylated proteins in the mitochondrial fractions from hearts of control and MCK mutant mice were measured using the protein car- bonyl ELISA kit according to the manufacturer’s instruc- tions (BioCell Corp., Auckland, New Zealand). Quantitative real-time PCR analysis Total RNA was extracted using the SV Total RNA Isolation System kit (Promega, Charbonnie ` res-les-bains, France), and 4 lg of total RNA was reverse-transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Courta- boeuf, France) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the Roche Light Cycler system and the FastStart DNA master plus SYBR green I kit (Roche Applied Sciences, Meylan, France). Lon protease (forward, 5¢-AGGATCTTGCCTTGTGT GGA-3¢; reverse, 5¢-TGGATGAGGAGCTGAGCAAG-3¢) ClpP (forward, 5¢-CACACCAAGCAGAGCCTACA-3¢; reverse, 5¢-CCCAGCAGAGGAAGTTTCAG-3¢), mitochon- drial aconitase (forward, 5¢-AGGAGTTTGGCCCTGTA CCT-3¢; reverse, 5¢-GCCTTGAATGGTCAGCTTGT-3¢), NDUFS3 (forward, 5¢-CTGTGGCAGCACGTAAGAAG-3¢; reverse, 5¢-ACTCATCAAGGCAGGACACC-3¢), SDHB (forward, 5¢-GGAGGGCAAGCAACAGTATC-3¢; reverse, 5¢-GCGTTCCTCTGTGAAGTCGT-3¢), Rieske (forward, 5¢-TGGTCTCCCAGTTTGTTTCC-3¢; reverse, 5¢-GCAGC TTCCTGGTCAATCTC-3¢) and SDHA (forward, 5¢-CAG AAGTCGATGCAGAACCA-3¢; reverse, 5 ¢-CGACCCGCA CTTTGTAATCT-3¢) sequence-specific primers were designed to span intron–exon boundaries to generate ampli- cons of approximately 100 bp. Values were normalized to the relative amounts of 18S ribosomal cDNA (forward, 5¢-CTGAGAAACGGCTACCACATC-3¢; reverse, 5¢-CGCT CCCAAGATCCAACTAC-3¢). Sequences of mitochondrial gene primers are listed in Table S2. Acknowledgements We wish to thank Alexandre Diet, Aurelien Bayot, Agnieszka Malinowska and Fabienne Pierre for techni- cal support, and Laurence Reutenauer for the genera- tion and genotyping of all the mice for this project. This work was supported by funds from the French National Agency for Research (ANR-05-MRAR-013- 01) and the French Medical Research Foundation Upregulation of mitochondrial proteases in FRDA B. Guillon et al. 1044 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS (H. Puccio). M. Wattenhofer-Donze ´ is an ATER at the Colle ` ge de France. References 1 Puccio H & Koenig M (2002) Friedreich ataxia: a para- digm for mitochondrial diseases. Curr Opin Genet Dev 12, 272–277. 2 Campuzano V, Montermini L, Molto MD, Pianese L, Cossee M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A et al. (1996) Friedreich’s ataxia: autoso- mal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271, 1423–1427. 3 Puccio H (2007) Conditional mouse models for Friedreich ataxia, a neurodegenerative disorder associ- ating cardiomyopathy. Handb Exp Pharmacol 178, 365–375. 4 Rotig A, de Lonlay P, Chretien D, Foury F, Koenig M, Sidi D, Munnich A & Rustin P (1997) Aconitase and mitochondrial iron–sulphur protein deficiency in Friedreich ataxia. Nat Genet 17, 215–217. 5 Puccio H, Simon D, Cossee M, Criqui-Filipe P, Tiziano F, Melki J, Hindelang C, Matyas R, Rustin P & Koenig M (2001) Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe–S enzyme deficiency followed by intramitochondrial iron deposits. Nat Genet 27, 181–186. 6 Chen OS, Hemenway S & Kaplan J (2002) Inhibition of Fe–S cluster biosynthesis decreases mitochondrial iron export: evidence that Yfh1p affects Fe–S cluster synthe- sis. Proc Natl Acad Sci USA 99, 12321–12326. 7 Muhlenhoff U, Richhardt N, Ristow M, Kispal G & Lill R (2002) The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe ⁄ S proteins. Hum Mol Genet 11, 2025–2036. 8 Zhang Y, Lyver ER, Knight SA, Pain D, Lesuisse E & Dancis A (2006) Mrs3p, Mrs4p, and frataxin provide iron for Fe–S cluster synthesis in mitochondria. J Biol Chem 281, 22493–22502. 9 Muhlenhoff U, Gerber J, Richhardt N & Lill R (2003) Components involved in assembly and dislocation of iron–sulfur clusters on the scaffold protein Isu1p. EMBO J 22, 4815–4825. 10 Ramazzotti A, Vanmansart V & Foury F (2004) Mitochondrial functional interactions between frataxin and Isu1p, the iron–sulfur cluster scaffold protein, in Saccharomyces cerevisiae. FEBS Lett 557, 215–220. 11 Yoon T & Cowan JA (2003) Iron–sulfur cluster biosyn- thesis. Characterization of frataxin as an iron donor for assembly of [2Fe–2S] clusters in ISU-type proteins. J Am Chem Soc 125, 6078–6084. 12 Stehling O, Elsasser HP, Bruckel B, Muhlenhoff U & Lill R (2004) Iron–sulfur protein maturation in human cells: evidence for a function of frataxin. Hum Mol Genet 13, 3007–3015. 13 Seznec H, Simon D, Bouton C, Reutenauer L, Hertzog A, Golik P, Procaccio V, Patel M, Drapier JC, Koenig M et al. (2005) Friedreich ataxia: the oxidative stress paradox. Hum Mol Genet 14, 463–474. 14 Martelli A, Wattenhofer-Donze M, Schmucker S, Bouvet S, Reutenauer L & Puccio H (2007) Frataxin is essential for extramitochondrial Fe–S cluster proteins in mammalian tissues. Hum Mol Genet 16, 2651–2658. 15 Anderson PR, Kirby K, Hilliker AJ & Phillips JP (2005) RNAi-mediated suppression of the mitochondrial iron chaperone, frataxin, in Drosophila. Hum Mol Genet 14, 3397–3405. 16 Layer G, Ollagnier-de-Choudens S, Sanakis Y & Fonte- cave M (2006) Iron–sulfur cluster biosynthesis: charac- terization of Escherichia coli CYaY as an iron donor for the assembly of [2Fe–2S] clusters in the scaffold IscU. J Biol Chem 281, 16256–16263. 17 Busi MV, Maliandi MV, Valdez H, Clemente M, Zabal- eta EJ, Araya A & Gomez-Casati DF (2006) Deficiency of Arabidopsis thaliana frataxin alters activity of mito- chondrial Fe–S proteins and induces oxidative stress. Plant J 48, 873–882. 18 Gerber J, Muhlenhoff U & Lill R (2003) An interaction between frataxin and Isu1 ⁄ Nfs1 that is crucial for Fe ⁄ S cluster synthesis on Isu1. EMBO Rep 4, 906–911. 19 Foury F, Pastore A & Trincal M (2007) Acidic residues of yeast frataxin have an essential role in Fe–S cluster assembly. EMBO Rep 8, 194–199. 20 Wang T & Craig EA (2008) Binding of yeast frataxin to the scaffold for Fe–S cluster biogenesis, Isu. J Biol Chem 283, 12674–12679. 21 Powell SR (2006) The ubiquitin–proteasome system in cardiac physiology and pathology. Am J Physiol Heart Circ Physiol 291, H1–H19. 22 Koppen M & Langer T (2007) Protein degradation within mitochondria: versatile activities of AAA prote- ases and other peptidases. Crit Rev Biochem Mol Biol 42, 221–242. 23 Ngo JK & Davies KJ (2007) Importance of the lon pro- tease in mitochondrial maintenance and the significance of declining lon in aging. Ann NY Acad Sci 1119, 78– 87. 24 de Sagarra MR, Mayo I, Marco S, Rodriguez-Vilarino S, Oliva J, Carrascosa JL & Castan JG (1999) Mito- chondrial localization and oligomeric structure of HClpP, the human homologue of E. coli ClpP. J Mol Biol 292, 819–825. 25 Santagata S, Bhattacharyya D, Wang FH, Singha N, Hodtsev A & Spanopoulou E (1999) Molecular cloning and characterization of a mouse homolog of bacterial ClpX, a novel mammalian class II member of the Hsp100 ⁄ Clp chaperone family. J Biol Chem 274, 16311–16319. B. Guillon et al. Upregulation of mitochondrial proteases in FRDA FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1045 [...]... Proteomic analysis of mitochondrial protein turnover: identification of novel substrate proteins of the matrix protease pim1 Mol Cell Biol 26, 762–776 29 Bota DA & Davies KJ (2002) Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism Nat Cell Biol 4, 674– 680 30 Majeran W, Wollman FA & Vallon O (2000) Evidence for a role of ClpP in the degradation of the chloroplast.. .Upregulation of mitochondrial proteases in FRDA B Guillon et al 26 Kang SG, Ortega J, Singh SK, Wang N, Huang NN, Steven AC & Maurizi MR (2002) Functional proteolytic complexes of the human mitochondrial ATPdependent protease, hClpXP J Biol Chem 277, 21095–21102 27 Mettert EL & Kiley PJ (2005) ClpXP-dependent proteolysis of FNR upon loss of its O2-sensing [4Fe–4S] cluster... oxidants and the role of proteolytic enzymes Biochem Soc Trans 21, 346–353 36 Fu GK & Markovitz DM (1998) The human LON protease binds to mitochondrial promoters in a singlestranded, site-specific, strand-specific manner Biochemistry 37, 1905–1909 37 Liu T, Lu B, Lee I, Ondrovicova G, Kutejova E & Suzuki CK (2004) DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein... the control of intracellular protein turnover: mitochondrial Lon protease inhibitors versus proteasome inhibitors Biochimie 90, 260–269 Supporting information The following supplementary material is available: Fig S1 The inverse correlation diagram of mitochondrial protein levels versus Lon and ClpP protease levels Table S1 ATP-dependent proteolytic activity in heart mitochondria of control and MCK mutant... expression of the mitochondrial ATP-dependent Lon protease FEBS Lett 444, 186–188 Bota DA, Ngo JK & Davies KJ (2005) Downregulation of the human Lon protease impairs mitochondrial structure and function and causes cell death Free Radic Biol Med 38, 665–677 Sazanov LA (2007) Respiratory complex I: mechanistic and structural insights provided by the crystal structure of the hydrophilic domain Biochemistry 46,... structure of mitochondrial respiratory membrane protein complex II Cell 121, 1043–1057 Schoenfeld RA, Napoli E, Wong A, Zhan S, Reutenauer L, Morin D, Buckpitt AR, Taroni F, Lonnerdal B, Ristow M et al (2005) Frataxin deficiency alters heme pathway transcripts and decreases mitochondrial heme metabolites in mammalian cells Hum Mol Genet 14, 3787–3799 Kato GJ (1999) Human genetic diseases of proteolysis... plaques, lipofuscin, ceroid, and ‘aggresomes’ during oxidative stress, aging, and disease Int J Biochem Cell Biol 36, 2519–2530 Bota DA & Davies KJ (2001) Protein degradation in mitochondria: implications for oxidative stress, aging and disease: a novel etiological classification of mitochondrial proteolytic disorders Mitochondrion 1, 33–49 Yu AY & Houry WA (2007) ClpP: a distinctive family of cylindrical... Riley M, Doolittle RF & Attardi G (1986) URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit Science 234, 614–618 Van Hoewyk D, Abdel-Ghany SE, Cohu CM, Herbert SK, Kugrens P, Pilon M & Pilon-Smits EA (2007) Chloroplast iron–sulfur cluster protein maturation Upregulation of mitochondrial proteases in FRDA requires the essential cysteine desulfurase CpNifS Proc Natl... energy-dependent serine proteases FEBS Lett 581, 3749–3757 Bross P, Andresen BS, Knudsen I, Kruse TA & Gregersen N (1995) Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene FEBS Lett 377, 249–252 Wang N, Gottesman S, Willingham MC, Gottesman MM & Maurizi MR (1993) A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease... control and MCK mutant mice at 5, 7 and 8 weeks of age Table S2 Oligonucleotide primers used to amplify mitochondria-encoded genes of the respiratory chain in cardiac muscle of control and MCK mutant mice This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied . Frataxin deficiency causes upregulation of mitochondrial Lon and ClpP proteases and severe loss of mitochondrial Fe–S proteins Blanche Guillon 1 ,. ClpP upregulation on loss of mitochondrial Fe–S proteins dur- ing the progression of the disease may suggest that Fe–S proteins are potential targets of Lon