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Human Molecular Genetics Advance Access originally published online on April 27, 2007
Human Molecular Genetics 2007 16(13):1534-1540; doi:10.1093/hmg/ddm102
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

In vivo maturation of human frataxin

Ivano Condò1, Natascia Ventura1, Florence Malisan1, Alessandra Rufini1, Barbara Tomassini1 and Roberto Testi1,2,*

1 Laboratory of Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Rome, Italy and 2 Fondazione S. Lucia, Rome, Italy

* To whom correspondence should be addressed at: Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133 Rome, Italy. Tel: +39 0672596503; Fax: +39 0672596505; Email: roberto.testi{at}uniroma2.it

Received December 21, 2006; Revised March 2, 2007; Accepted April 12, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The defective expression of frataxin causes the hereditary neurodegenerative disorder Friedreich's ataxia (FRDA). Human frataxin is synthesized as a 210 amino acid precursor protein, which needs proteolytic processing into mitochondria to be converted into the functional mature form. In vitro processing of human frataxin was previously described to yield a 155 amino acid mature form, corresponding to residues 56–210 (frataxin56–210). Here, we studied the maturation of frataxin by in vivo overexpression in human cells. Our data show that the main form of mature frataxin is generated by a proteolytic cleavage between Lys80 and Ser81, yielding a 130 amino acid protein (frataxin81–210). This maturation product corresponds to the endogenous frataxin detected in human heart, peripheral blood lymphocytes or dermal fibroblasts. Moreover, we demonstrate that frataxin81–210 is biologically functional, as it rescues aconitase defects in frataxin-deficient cells derived from FRDA patients. Importantly, our data indicate that frataxin56–210 can be produced in vivo when the primary 80–81 maturation site is unavailable, suggesting the existence of proteolytic mechanisms that can actively control the size of the mature product, with possible functional implications.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human frataxin is an ~17 kDa protein, whose deficiency causes Friedreich's ataxia (FRDA), a neurodegenerative disorder characterized by gait and limb ataxia, loss of proprioception, dysarthria, skeletal abnormalities, hypertrophic cardiomyopathy and increased incidence of diabetes (1). In the vast majority of patients (96–98%), the defective expression of frataxin is due to a homozygous GAA triplet repeat expansion within the first intron of the FXN gene, located on chromosome 9q13 (2). The hyperexpansion of GAA repeats determines the formation of a triple helix non-B DNA structure, resulting in an inhibition of frataxin mRNA transcription (3). Moreover, missense mutations are present in FRDA compound heterozygotes, representing 2–4% of patients, which carry an intronic GAA expansions on one FXN allele and a point mutation, mainly located at C-terminal region of frataxin, within exons of the other allele (4).

Frataxin is involved in several aspects of intracellular iron metabolism, such as biogenesis of heme (5) and iron–sulfur clusters (ISCs) (6), iron binding/storage (7) and iron chaperone activity (8). Consequently, frataxin-defective organisms, from unicellular yeast to humans, exhibit a plethora of metabolic disturbances caused by intramitochondrial iron accumulation (9,10), loss of ISC-dependent enzymes (11), reduced oxidative phosphorylation (12) and altered antioxidant defenses (13,14). Moreover, frataxin has a role in controlling cell survival, as underlined by reports documenting that frataxin-deficient cells are more sensitive to oxidative stress (15–17) and that evidence of both apoptotic and autophagic cell death are found in frataxin-deficient animal models (18,19).

The FXN gene encodes for a 210 amino acid protein, representing the precursor form of frataxin (20). The precursor protein contains an N-terminal transit sequence that directs its transport into the mitochondria. Here, a two-step proteolytic processing removes the transit sequence to produce the mature protein. Mitochondrial import and maturation of frataxin is highly conserved during evolution, as documented by several experimental approaches using the yeast, mouse and human frataxin homologs. The first clues on frataxin maturation emerged when physical interaction between mouse frataxin precursor and mouse mitochondrial processing peptidase (MPP) was observed (21). In this report, in vitro processing experiments show that recombinant rat MPP cleaves human frataxin to generate an intermediate form lacking the first 40 N-terminal residues. Subsequently, recombinant yeast MPP as well as recombinant rat MPP was shown to operate two sequential cleavages on human frataxin in vitro to produce the mature form (22). This two cleavages convert the frataxin precursor first into an ~19 kDa intermediate form and then into an ~17 kDa mature protein. The characterization of such processing products demonstrated that in vitro cleavages by MPP occur between Gly41 and Leu42 to yield the intermediate form and between residues Ala55 and Ser56 to generate the mature form of human frataxin (22). However, processing experiments aimed at the identification of the maturation sites in human living cells are still lacking. Here, we analyze the in vivo processing of frataxin in human cells. We found that the relevant cleavage responsible for the generation of the major form of mature frataxin in vivo occurs between Lys80 and Ser81. The resulting 130 amino acid protein is fully functional, as it can rescue aconitase defects in frataxin-deficient cells. Our data, moreover, indicate that the upstream 55–56 site, previously described in vitro, may not be normally used in vivo. However, it could be utilized in vivo when the primary 80–81 site is unavailable.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of mature frataxin in living cells
In a previous work (17), we generated a frataxin N-terminal deletion mutant lacking the first 55 amino acids (frataxin56–210) that remains extramitochondrial when expressed in human cell lines. Although corresponding to the mature frataxin, as defined by in vitro processing studies (22), when frataxin56–210 is transiently expressed in human cell lines, it migrates slower than the major processing product generated in vivo after similar expression of wild-type (WT) frataxin1–210. Similarly, when frataxin56–210 is stably expressed in frataxin-defective cells derived from FRDA patients, it generates a product that migrates slower than the major form produced in vivo after the expression of WT frataxin1–210 in the same cells (17). These observations raised the question of whether frataxin56–210 truly corresponds to the mature frataxin in vivo.

To identify the mature frataxin proteolytically generated in human living cells, SH-SY5Y neuroblastoma cells were transiently transfected with WT frataxin1–210 or with frataxin56–210 (Fig. 1A). The overexpression of frataxin1–210 allows us to detect precursor, intermediate and mature forms. The mature form co-migrates with the endogenous frataxin detectable in untransfected cells. The overexpression of frataxin56–210, on the contrary, produces a protein that migrates slower than mature frataxin (Fig. 1A). We also stably expressed WT frataxin1–210 or frataxin56–210 in HEK-293 cells. Unlike transient expression, which allows us to detect high levels of precursor and intermediate forms, the stable expression of WT frataxin1–210 results mainly in the detectable accumulation of the mature product. This clearly co-migrates with the endogenous frataxin detected in human heart, peripheral blood lymphocytes (PBLs) or dermal fibroblasts (Fig. 1B). Here again, the stable expression of frataxin56–210 in HEK-293 cells yields a product that migrates slower than the mature form generated from WT frataxin1–210. To further analyze this shorter maturation product, mitochondrial and cytosolic fractions were prepared from PBL and from HEK-293 cells stably expressing WT frataxin or frataxin56–210. As shown in Figure 1C, the mature frataxin recovered from both mitochondrial and cytosolic fractions of PBL and of HEK-293 cells expressing WT frataxin does not co-migrate with frataxin56–210. Together, these results suggest that both mitochondrial and extramitochondrial pools of the human mature frataxin generated in vivo are different from that previously identified by in vitro processing.


Figure 1
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  		Figure 1. Analysis of the human mature frataxin generated in living cells. Cell extracts were analyzed by 15% SDS–PAGE and immunoblot with anti-frataxin mAb. (A) Protein extracts from cultured SH-SY5Y cells (–) and SH-SY5Y cells transiently expressing WT frataxin1–210 (WT) or frataxin56–210 (56–210). (B) Protein extracts from human heart mitochondria (heart), human peripheral lymphocytes (PBL), human skin fibroblasts (fibr.) and HEK-293 cells stably expressing WT frataxin1–210 (WT) or frataxin56–210 (56–210). (C) Mitochondrial (M) and cytosolic (C) fractions from PBL and HEK-293 cells stably expressing WT frataxin1–210 (WT) or frataxin56–210 (56–210) were analyzed by 15% SDS–PAGE and immunoblot with anti-frataxin mAb.

 
N-terminal sequencing of the in vivo maturation product
HEK-293 cells stably transfected with WT frataxin1–210 were used to identify the in vivo maturation site of human frataxin. Frataxin was immunopurified from whole-cell extracts, resolved on SDS–PAGE and subjected to sequential Edman degradation (Fig. 2). N-terminal sequencing yielded the sequence SGTLGH, corresponding to residues 81–86 of human frataxin and indicating that the relevant cleavage occurs between Lys80 and Ser81. Interestingly, the predicted cleavage site matches the R-2 rule used by MPP to recognize most target sequences (23).


Figure 2
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  		Figure 2. N-terminal sequencing of mature frataxin. Western blot analysis of cell extracts from HEK-293 cells stably transfected with empty vector (–), WT frataxin1–210 (WT) or frataxin56–210 (56–210). Edman degradation analysis was performed on mature frataxin immunopurified from HEK-293/WT frataxin extracts as described in Materials and Methods. The figure shows the band that was sequenced and the position of the obtained N-terminal residues (underlined) within the frataxin sequence.

 
Analysis of in vivo processing of frataxin cleavage mutants
On the basis of the earlier-mentioned sequencing data, we generated a shorter N-terminal deletion mutant (frataxin81–210) and different site-specific mutants of frataxin1–210. To abolish the potential cleavage site between residues 80 and 81, a mutant (KO81) was constructed by the substitution of Arg79 and Lys80 with glycine residues. Likewise, to abolish the potential cleavage site Ala55–Ser56, a different mutant (KO56) was obtained by replacing the two arginine residues at positions 53 and 54 with glycines. Finally, an additional mutant (KO56 + 81) combining both mutations was also generated. These mutants were transiently transfected in SH-SY5Y cells, and their in vivo maturation was followed. As shown in Figure 3A, the overexpression of the WT frataxin1–210 in SH-SY5Y cells results in the accumulation of precursor, intermediate and mature forms, the latter co-migrating with the endogenous frataxin detectable in untransfected cells, whereas the overexpression of frataxin56–210 yields a slower migrating product. Importantly, the processing of KO56 mutant is indistinguishable from the processing of WT frataxin1–210, suggesting that the 55–56 cleavage site plays a minor role. As predicted, the frataxin81–210 deletion mutant gives a product perfectly corresponding to the major maturation product of the WT frataxin1–210 and to the endogenous frataxin (Fig. 3A). Accordingly, the KO81 mutant overaccumulates the intermediate form and it is unable to generate mature frataxin, indicating that the 80–81 cleavage site is relevant in vivo. Interestingly, the KO81 mutant also allows the accumulation of the 56–210 product, suggesting that the 55–56 site can be functional. Finally, the KO56 + 81 double mutant abrogates both the accumulation of the mature 81–210 product and the alternative 56–210 product and causes overaccumulation of the precursor (Fig. 3A). The severely perturbed maturation of KO81 and KO81 + 56 mutants also results in the appearance of faster migrating products. To address the subcellular distribution of frataxin mutants, mitochondrial and cytosolic fractions were obtained from SH-SY5Y cells transiently transfected as earlier (Fig. 3B). As expected, overexpression of the frataxin81–210 construct generates a product that remains extramitochondrial. Importantly, the frataxin56–210 product generated from the processing of the overexpressed KO81 mutant mainly resides in the mitochondria (Fig. 3B). This finding suggests that a genuine mitochondrial processing generates the alternative 56–210 product. Together, these data indicate that, in living cells, the primary cleavage site responsible for the generation of mature frataxin occurs between Lys80 and Ser81. However, when the 80–81 site is unavailable, the upstream 55–56 cleavage site can be activated.


Figure 3
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  		Figure 3. Proteolytic processing of frataxin cleavage mutants. (A) SH-SY5Y cells (–) were transiently transfected with WT frataxin1–210 (WT), frataxin56–210 (56–210), frataxin KO56 mutant (KO56), frataxin81–210 (81–210), frataxin KO81 mutant (KO81) or frataxin KO56 + 81 double mutant (KO56 + 81). Cell extracts were analyzed by 15% SDS–PAGE and immunoblot with anti-frataxin mAb. (B) Mitochondrial (M) and cytosolic (C) fractions from SH-SY5Y cells transiently transfected with the indicated constructs were analyzed by 15% SDS–PAGE and immunoblot with anti-frataxin mAb.

 
Frataxin 81–210 rescues aconitase defects in FRDA cells
To further verify that frataxin polypeptide corresponding to residues 81–210 represents an authentic mature protein, we analyzed its functional capability. To this purpose, frataxin-defective lymphoblasts derived from an FRDA patient were stably reconstituted with either the WT frataxin1–210 or the processing mutants. Aconitase activity was chosen as a functional readout of frataxin reconstitution, as it has been shown that frataxin-deficient cells have a deficit of numerous ISC-dependent proteins, including aconitase (11,24). Accordingly, cellular extracts from FRDA cells exhibit a significant decrease of aconitase activity with respect to control cells derived from a healthy brother of the patient (Fig. 4). Indeed, aconitase deficit is rescued in FRDA cells reconstituted with WT frataxin1–210. Aconitase activity is also restored in FRDA cells reconstituted with the KO56 mutant, which is capable of generating the 81–210 frataxin product, as well as in cells expressing the KO81 mutant, which generates the 56–210 product (Fig. 4). On the other hand, aconitase activity could not be restored in FRDA cells reconstituted with the KO56 + 81 mutant, unable to generate both maturation products. Measured as a control, the activity of malate dehydrogenase, another enzyme involved in the tricarboxylic acid cycle, but lacking ISC, did not change with the expression levels of mature frataxin (Fig. 4).


Figure 4
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  		Figure 4. Mature frataxin81–210 restores aconitase activity in FRDA cells. Aconitase and malate dehydrogenase assays were performed with cell lysates from lymphoblasts derived from an FRDA patient, from his healthy brother and from the patient lymphoblasts stably reconstituted with empty vector, WT (frataxin1–210), frataxin KO56 mutant, frataxin KO81 mutant or frataxin KO56 + 81 double mutant. Means ± 1 SD from three experiments are shown. Asterisk indicates P < 0.001.

 
Thus, our data strongly indicate that the 130 residue-long frataxin81–210 is biologically functional and it represents a genuine mature product in human cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Human frataxin is a highly conserved, nuclear-encoded protein, which needs proteolytic processing to be converted into the functional mature form. Indeed, in eukaryotes, frataxin is synthesized as a pre-protein containing an N-terminal signal peptide for its transport to the mitochondria. The yeast frataxin precursor Yfh1p is proteolytically maturated with two sequential cleavages by MPP (25). The first cleavage removes 20 N-terminal residues, containing the mitochondrial signal peptide, to generate an intermediate form. This product is then subjected to a second step that cleaves off a spacer sequence constituted by residues 21–51, yielding the 123 amino acid mature Yfh1p (25). The maturation of human frataxin precursor was described to occur through either a one- or a two-step reaction catalyzed by MPP. The one-step model was proposed on the basis of import and maturation of in vitro-translated precursor into purified yeast or rat mitochondria. These data, as also supported by in vitro-processing reactions with recombinant yeast or rat MPP, report a single cleavage product (26). The opposite two-step model derives from the analysis of frataxin cleavage by recombinant yeast or rat MPP and within isolated rat mitochondria (22). This latter study characterizes two MPP cleavage sites by N-terminal radiosequencing of the products of the in vitro-translated precursor processed by recombinant yeast enzyme. The first cleavage removes 41 N-terminal residues to generate the intermediate frataxin, whereas the second step removes a 14-residue linker peptide, yielding the 155 amino acid mature frataxin (frataxin56–210). However, both approaches make use of in vitro heterologous systems. Given the lack of information in living cells, we studied the maturation of human frataxin by in vivo overexpression in human cells. Although our data agree with the two-step processing model, they suggest a different identity for the human mature frataxin. We generated HEK-293 cells stably expressing WT frataxin1–210 or a truncated form corresponding to the previously described mature protein (frataxin56–210). Notably, the frataxin56–210 polypeptide does not co-migrate either with the mature form generated by frataxin1–210 expressing cells or with the endogenous frataxin detected in human primary cells or human tissues. Accordingly, Edman degradation analysis of immunopurified mature frataxin from HEK-293 cells demonstrates that the N-terminal sequence matches residues starting from Ser81 instead of Ser56. Interestingly, although not directly addressed in this work, the cleavage between Lys80 and Ser81 is a potential MPP target (27). In fact, the upstream sequence contains a proximal basic arginine at the P2 position (Arg79) and distal N-terminal basic residues, generally between 3 and 10 amino acids (Lys69 and Lys70) from the proximal Arg79. Moreover, this sequence shows a polar residue at position P3' (Thr83), but lacks a hydrophobic residue at position P1'. Accordingly, the glycine substitution of Arg79 and Lys80 completely blocks the last maturation step of the KO81 mutant. It is worthwhile mentioning that inhibition of processing at Ser81 site allows the accumulation of a novel cleavage product that co-migrates with the polypeptide 56–210. In fact, this form is absent in the processing of the KO56 + 81 mutant. It is therefore possible that the cryptic 55–56 site is used when the primary 80–81 site is somehow unavailable. This anyhow results in the generation of a functional frataxin, as frataxin56–210 from KO81 mutant can functionally reconstitute aconitase activity (Fig. 4) and resistance to oxidative stress in frataxin-defective cells derived from FRDA patients (17). The activation of a cryptic cleavage site, when the canonical site is abolished, has been described for the MPP-mediated maturation of ornithine transcarbamylase (28).

Our results show a clear discrepancy between in vivo and in vitro processing of human frataxin. A possible explanation could be represented by the preferential choice of a different processing site in heterologous systems. Rat and human MPPs show a high degree of sequence homology (23); nevertheless, a species-specific substrate recognition by this peptidase cannot be excluded. Moreover, the previously reported N-terminal sequencing of the human mature frataxin was performed on the product processed in vitro by yeast MPP (22).

A key information from our results is the ability of mature frataxin81–210 to rescue metabolic defects in frataxin-deficient cells. In fact, reconstitution of frataxin-defective cells derived from FRDA patients with the KO56 mutant, unable to produce frataxin56–210, but still competent to generate frataxin81–210, clearly restores the aconitase deficit. The defective activity of ISC-dependent proteins such as mitochondrial and cytosolic aconitase, as well as of the subunits of respiratory complexes, is clearly associated with frataxin deficiency in several model organisms and in FRDA patients (11,24,29,30). Moreover, many evidence support the direct function of frataxin in controlling aconitase activity by iron delivery and reactivation of its damaged ISC (8,31,32). Thus, frataxin81–210 is a functional processing product that is not generated from the degradation of frataxin56–210 in vivo, since no frataxin56–210 is made during the processing of the KO56 mutant. Accordingly, the transient or stable overexpression of frataxin56–210 in several cell types never resulted in the appearance of the 81–210 product (17 and this study).

It has been reported that recombinant human frataxin purified from Escherichia coli undergoes ‘spontaneous’ proteolysis yielding shorter forms devoid of the non-conserved N-terminal region (31,33–35). These more stable products have been used to define the structure of frataxin by NMR (33) and X-ray crystallography (34). It is important to note that an autoproteolytic form of frataxin was found to extend from residues 78 to 210 (7,35), intriguingly similar to the mature product identified in vivo by our study. This 78–210 form was shown to be fully functional as demonstrated by its ability to bind and to deliver ferrous iron for ISC (31) and heme biosynthesis (5). However, in vitro self-assembly experiments suggest a critical role played by residues spanning from 56 to 78 in frataxin polymers formation (36). The in vivo relevance of frataxin homopolymerization was recently analyzed in the yeast knockout model {Delta}yfh1 (37,38). Oligomerization-deficient Yfh1p mutants were shown to rescue the growth defect of {Delta}yfh1 cells, similar to the WT protein, and to interact with the Fe/S scaffold Isu (37), thus indicating that frataxin homopolymerization is dispensable in living yeast. However, in Yfh1p mutants lacking ferroxidase or mineralization activity, which are distinctive of the frataxin polymers, iron-induced oxidative stress is increased and life span is reduced, independently of the iron chaperone capability (38).

In humans, the in vivo role of frataxin polymers in iron storage and detoxification has not been clarified. Our data support the view that frataxin81–210 is the primary mature form generated in vivo, probably designed to act as a monomeric iron chaperone. However, we show that frataxin56–210 can be proteolytically produced from the precursor in vivo when the primary 80–81 site is blocked. This finding opens the possibility that, under conditions that would require enhanced frataxin-mediated iron storage or iron detoxification, the alternative 55–56 site could be activated to allow the production of a frataxin with oligomerization capability. Further analysis will be eventually required to elucidate the mechanisms that might control the molecular shift between the monomeric iron chaperone and the polymeric iron storage status of human frataxin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Frataxin expression constructs
The pIRES2/FXN1–210 and pIRES2/FXN56–210 constructs were previously described (17). The pIRES2/FXN81–210 construct was synthesized by PCR using the primers 5'-CTAGGAATTCATGTCTGGAACTTTGGGCCACCC-3' (EcoRI) and 5'-AGTTGGATCCGCATCAAGCATCTTTTCCGG-3' (BamHI), in order to remove the first 80 amino acid from frataxin precursor, and inserted in pIRES2-EGFP vector (BD Clontech). The FXN1–210 and FXN56–210 cDNAs were cloned into HindIII and BamHI sites of the vector pcDNA5/FRT (Invitrogen) to obtain the stable expression in Flp-In-293 cells (Invitrogen). The mutant constructs pIRES2/FXN_KO56, pIRES2/FXN_KO81 and pIRES2/FXN_KO56 + 81 were generated using the QuickChange site-directed mutagenesis kit (Stratagene) using pIRES2/FXN1–210 as template. All final constructs were verified by DNA sequencing.

Cell cultures and transfections
GM15850B lymphoblasts, from a clinically affected FRDA patient, homozygous for the GAA expansion in the FXN gene with alleles containing ~700 and 1050 repeats, and GM15851B lymphoblasts from a clinically unaffected brother of GM15850, with two FXN alleles in the normal range of GAA trinucleotide repeats, were obtained from NIGMS Human Genetic Cell Repository, Coriell Institute for Medical Research. Cells were maintained in RPMI 1640 medium supplemented with 15% FBS and transfected by electroporation. Briefly, 107 cells were incubated in 0.4 ml of RPMI 1640 for 10 min on ice with 30 µg of pIRES2/FNX mutant constructs. After electroporation at 260 V/950 µF, cells were left 30 min on ice and resuspended in 5 ml of RPMI 1640, 15% FBS. After 4 h, live cells were recovered by Lympholyte-H (Cedarlane Laboratories) density gradient centrifugation and replated. Stable transfectants were obtained from cultures in selection medium containing 500 µg/ml G418 (Invitrogen) for 15–20 days. Flp-In-293 cells (Invitrogen) are human embryonic kidney HEK-293 variants allowing the stable integration and expression of a transfected gene. Flp-In-293 cells were maintained in DMEM medium supplemented with 10% FBS and transfected with the calcium phosphate precipitation method. Briefly, cells were plated on 10 cm dishes and co-transfected with 10 µg total DNA. After 16 h, the medium was replaced with a fresh one. The 293 clones stably expressing FXN1–210 or FXN56–210 were obtained from cultures in selection medium containing 100 µg/ml hygromycin B (Invitrogen). Bone marrow neuroblastoma SH-SY5Y cells were maintained in RPMI 1640 medium containing 10% FBS and transiently transfected using Lipofectamine 2000 (Invitrogen), following the manufacturer's instructions. Human PBLs from different healthy donors were isolated by lymphoprep density gradient centrifugation at 800g. Cultured skin fibroblasts from healthy donors were provided by Dr G. Novelli (University of Rome ‘Tor Vergata’, Italy).

Purification and N-terminal sequencing of mature frataxin
Total cell lysates from Flp-In-293 cells stably expressing FXN WT were prepared in RIPA buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA) containing Complete protease inhibitor cocktail (Roche Diagnostics). Frataxin was immunoprecipitated from ~60 mg of the whole extract with mAb anti-frataxin 1G2 (Immunological Sciences) and protein G-sepharose beads (GE Healthcare Life Sciences). Immunocomplexes were then resolved by 15% SDS–PAGE, and coomassie-stained bands were excised from the gel. N-terminal amino acid sequence was determined by automated Edman degradation performed by Alta Bioscience (University of Birmingham, UK).

Western blotting
To prepare total protein lysates, cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA buffer supplemented with Complete protease inhibitor cocktail. Mitochondrial and cytosolic extracts were prepared as described (17). The human heart mitochondrial sample was prepared post-mortem from a donor (Immunological Sciences). Cell extracts were separated by 15% SDS–PAGE and analyzed by immunoblotting with mAb anti-frataxin (Immunological Sciences), mAb anti-{alpha}-Tubulin (Sigma) and anti-Mn SOD (StressGen) using ECL system detection (GE Healthcare Life Sciences).

Enzyme assays
Aconitase activity was measured spectrophotometrically at 340 nm by a coupled reaction of aconitase and isocitrate dehydrogenase. Total cell extracts were obtained by lysis in HDGC buffer (20 mM Hepes, pH 7.4, 1 mM DTT, 10% glycerol, 2 mM sodium citrate, 1% triton X-100) supplemented with Complete protease inhibitor cocktail. The assay reactions contained 100 µg of cell lysate in 50 mM Hepes, pH 7.4, 1 mM sodium citrate, 0.6 mM MnCl2, 0.2 mM NADP+ and 2 U/ml isocitrate dehydrogenase from porcine heart (Sigma-Aldrich). Malate dehydrogenase activity was assessed by following the oxidation of NADH at 340 nm. The reaction mixtures contained 50 µg of cell lysate in 50 mM Tris–HCl, pH 7.5, 0.1 mM NADH and 0.4 mM oxaloacetic acid.

For the calculation of the activities, 1 mU of enzyme was defined as the amount of protein that converted 1 nmol of NADP+ (aconitase) or NADH (malate dehydrogenase) in 1 min. Statistical analysis was performed using Student's t-test; all values are expressed as means ± SD.


   ACKNOWLEDGEMENTS
 
Thanks are due to Dario Serio for his technical assistance. This work has been supported by the Associazione Italiana Ricerca sul Cancro, the European Commission (Project TRANSDEATH), National Ataxia Foundation, Ataxia UK and Friedreich's Ataxia Research Alliance. The financial support of Telethon-Italy (Grant GGP06059) is gratefully acknowledged.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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