Normal and Friedreich ataxia cells express different isoforms of frataxin with complementary roles in iron-sulfur cluster assembly

Abstract

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN(42-210) and FXN(81-210). Previous reports suggested that FXN(42-210) is a transient processing intermediate, whereas FXN(81-210) represents the mature protein. However, we find that both FXN(42-210) and FXN(81-210) are present in control cell lines and tissues at steady-state, and that FXN(42-210) is consistently more depleted than FXN(81-210) in samples from FRDA patients. Moreover, FXN(42-210) and FXN(81-210) have strikingly different biochemical properties. A shorter N terminus correlates with monomeric configuration, labile iron binding, and dynamic contacts with components of the Fe-S cluster biosynthetic machinery, i.e. the sulfur donor complex NFS1·ISD11 and the scaffold ISCU. Conversely, a longer N terminus correlates with the ability to oligomerize, store iron, and form stable contacts with NFS1·ISD11 and ISCU. Monomeric FXN(81-210) donates Fe(2+) for Fe-S cluster assembly on ISCU, whereas oligomeric FXN(42-210) donates either Fe(2+) or Fe(3+). These functionally distinct FXN isoforms seem capable to ensure incremental rates of Fe-S cluster synthesis from different mitochondrial iron pools. We suggest that the levels of both isoforms are relevant to FRDA pathophysiology and that the FXN(81-210)/FXN(42-210) molar ratio should provide a useful parameter to optimize FXN augmentation and replacement therapies.

FIGURE 1.

Recombinant FXN isoforms used in this study and native FXN isoform profiles in human lymphoblastoid cells. A, DNA fragments, including an ATG codon in-frame with codons 42–210, 56–210, 78–210, or 81–210 of human frataxin (FXN), were expressed in E. coli and purified as described under “Experimental Procedures.” The accurate molecular mass (shown in parentheses) of each rec-FXN species were determined by electrospray ionization mass spectrometry. According to the length parameter rule (51), the initiator methionine was cleaved from rec-FXN^56–210 and rec-FXN^81–210 during expression in E. coli, whereas cleavage of the initiator methionine from rec-FXN^42–210 was achieved after substitution of alanine for leucine at position 42. This causes a slight increase in electrophoretic mobility relative to native FXN^42–210 (see Fig. 1B). Leucine 78 in rec-FXN^78–210 was not replaced and this protein retained the initiator methionine as expected without obvious effects on the electrophoretic mobility relative to native FXN^78–210 (see Fig. 1B). B, lymphoblastoid cell lines from the Coriell Cell Repository were as follows: GM07521 (female, 19 years old, normal GAA repeats), GM16200 (FRDA carrier, female, 34 years old, mother of GM16197, 830/normal GAA repeats), GM16197 (FRDA male, 14 years old, 760 and 830 GAA repeats), GM14907 (male, 28 years old, normal GAA repeats), GM16215 (FRDA carrier, female, 41 years old, mother of GM16214, 830/normal GAA repeats), and GM16214 (FRDA male, 15 years old, 600 and 700 GAA repeats). Cell extracts were prepared from exponentially growing cell cultures, and each sample (50 μg of total protein) was analyzed by Western blotting with PAC 2517 anti-FXN polyclonal antibody, following the procedure described under “Experimental Procedures.” Rec-FXN proteins (All rec, 50 pg each) were mixed together and used as standards. The double asterisk denotes a nonspecific cross-reacting band that unlike FXN isoforms is present in equivalent amounts in the controls, carriers, and patients; this band was recognized weakly by PAC 2517 and more strongly by PAC 2518, whereas it was not recognized by a monoclonal antibody (see supplemental Fig. S1). DLD, dihydrolipoamide dehydrogenase. Two different exposures of the same blot are shown.

FIGURE 2.

FXN isoform profiles in FRDA post-mortem tissues. A, autoptic samples from the indicated tissues were obtained for FRDA patients 1 (male, 39 years old, 934 and 249 GAA repeats) and 2 (male, 10 years old, 1016 and 1016 GAA repeats). Tissue extracts were prepared as described under “Experimental Procedures.” One hundred μg of total protein was loaded in each lane, and analyzed by Western blotting with PAC 2517 as described above. The triple asterisk denotes a band in cerebellum and spinal cord, which does not co-migrate with any of the known FXN isoforms; this band was also detected with PAC 2518 and the monoclonal antibody (supplemental Fig. S2c) and its significance is undetermined. The single asterisk denotes an abundant protein present in heart, probably myoglobin, which migrates close to FXN^56–210. In the hearts of both patients, the 14-kDa region of the blot was dominated by a strong band migrating between FXN^81–210 and FXN^78–210 (white asterisk). Further analyses using either PAC 2518 or the monoclonal antibody indicated that this band most likely consisted of low amounts of ∼14-kDa FXN that were detected by all antibodies, and larger amounts of a co-migrating protein that was only detected by PAC 2517 (supplemental Fig. S2c). DLD, dihydrolipoamide dehydrogenase. Two different exposures of the same blot are shown. B, comparison of autoptic samples of cerebellum from three non-FRDA individuals (males, 52 to 62 years old) and the two patients described above (100 μg of total protein in each case) as analyzed by Western blotting with PAC 2518.

FIGURE 3.

Quantification of FXN^42–210 and FXN^81–210 isoforms in lymphoblastoid cells and post-mortem tissues. A, densitometry data were obtained for 2 carriers and 2 patients (6 measurements per group from 4 different blots, of which two were probed with PAC 2517, one with PAC 2518, and one with the monoclonal antibody), and for 7 controls (13 measurements from 5 different blots, of which two were probed with PAC 2517, two with PAC 2518, and one with the monoclonal antibody). Shown are the mean ± S.D. p values were: *, p ≤ 2.3 × 10⁻⁶; **, p ≤ 0.0025; ***, p ≤ 2.7 × 10⁻⁵; ****, p ≤ 0.005. The molar ratio of FXN^81–210 to FXN^42–210 shown under each set of data were calculated from the mean values. B, densitometry data were obtained for patients 1 and 2 (3 measurements each from 3 different blots, of which two were probed with PAC 2517 and one with PAC 2518), and for 9 controls (18 measurements from 2 different blots, of which one was probed with PAC 2517 and one with PAC 2518). The 9 controls were males with ages ranging from 49 to 82 years. Shown are the mean ± S.D. p values were: *, p ≤ 2.9 × 10⁻⁵; **, p ≤ 4.5 × 10⁻⁵; ***, p ≤ 1.3 × 10⁻⁴; ****, p ≤ 1.5 × 10⁻⁴. The molar ratio of FXN^81–210 to FXN^42–210 shown under each set of data were calculated from the mean values. In both A and B, known amounts of rec-FXN^42–210 and rec-FXN^81–210 were analyzed side by side with unknown samples and served as internal standards, enabling conversion of densitometry values to pg of FXN protein per μg of total protein.

FIGURE 4.

Distributions of native and rec-FXN isoforms in cells. A and B, soluble extracts were prepared from two different lymphoblastoid cell lines from the Biospecimens Accessioning Processing Laboratory (Mayo Clinic) as described under “Experimental Procedures,” and ∼1 mg of total protein was analyzed by gel filtration chromatography on a Superdex 75 column. Fractions comprising the entire molecular mass fractionation range of the column were analyzed by Western blotting with PAC 2517. Rec-FXN proteins (All rec) were used as standards. The double asterisk denotes a nonspecific cross-reacting band as described in the legend to Fig. 1B. Native FXN^78–210 was eluted with apparent molecular mass of 44 kDa in both A and B; the significance of this result remains to be established. C, a culture of the yfh1Δ[FXN^1–210] strain was grown for ∼20 h at 30 °C in rich medium with galactose as the carbon source, after which mitochondria were isolated (32). The soluble mitochondrial fraction (∼5 mg of total protein) was analyzed by gel filtration chromatography and Western blotting as described above. D and E, the indicated recombinant FXN proteins were expressed in E. coli, and after sonication of bacterial cells and centrifugation, soluble cell extracts were analyzed by Superdex 75 gel filtration chromatography, SDS-PAGE, and staining with SYPRO Orange. MW, molecular weight markers.

FIGURE 5.

Oligomerization and Fe³⁺ chelation properties of rec-FXN^42–210 and rec-FXN^81–210. Purified low (A) and high (B) molecular weight (MW) pools of rec-FXN^42–210 (20 μm each) or (C) the rec-FXN^81–210 pool (40 μm), were incubated in the absence or presence of 10 molar eq of Fe²⁺, as described under “Experimental Procedures.” Each reaction was centrifuged for 5 min at 20,000 × g and the supernatant was directly analyzed by Superdex 200 (A and B) or Superdex 75 (C) gel filtration chromatography. In each chromatogram, −Iron and +Iron denote the iron-free and the iron-treated FXN protein. D, each purified rec-FXN protein or a commercial preparation of horse spleen ferritin (3 μm each) was incubated with 30 μm Fe²⁺ for 10 or 60 min under conditions that promote iron oxidation (10 mm HEPES-KOH, pH 7.3, at 30 °C); samples were then subjected to ultrafiltration in a Ultrafree-0.5 device (Millipore) at 10,000 × g for 10 min at 4 °C. Iron levels were measured in the concentrated (protein-bound iron), filterable (labile iron), and insoluble fractions with the chelator (α-α′-bipyridine) after addition of a strong reducing agent (dithionite) (29). Shown are the mean ± S.D. from 3 independent experiments.

FIGURE 6.

Interactions of rec-FXN isoforms with purified ISCU. A, in vitro pulldown assays were performed with a fixed concentration (4 μm or 400 pmol of FXN subunit) of iron-free (−) or iron-loaded (+) preparations of the indicated rec-FXN isoforms and increasing concentrations of ISCU_His in 20 mm HEPES-KOH, pH 7.4, 100 mm NaCl, 30 mm imidazole, 0.01% Triton X-100, 5 mm β-mercaptoethanol, as described in detail under supplemental Methods. After 60 min of incubation with Ni-NTA Superflow agarose beads (nickel beads), bound protein (100%) was separated on 14% BisTris SDS-PAGE (FXN^81–210) or 14% Tris glycine SDS-PAGE (FXN^42–210 or oligomeric FXN^42–210), and detected by staining with SYPRO Orange. S denotes 100 pmol of each rec-FXN protein used as a reference. 0 denotes control reactions containing nickel beads and the indicated FXN isoform but not ISCU_His. The reproducible results of two independent assays (a and b) are shown. B, schematic representation of the pulldown results shown in A.

FIGURE 7.

Interactions of rec-FXN isoforms with purified NFS1·ISD11. A, each FXN isoform (2 μm or 200 pmol of FXN subunit) was incubated with the indicated concentrations of _HisNFS1·ISD11. Pulldown assays were carried out and bound proteins analyzed using the same conditions described in the legend to Fig. 6A. 0 denotes control reactions containing nickel beads and the indicated FXN isoform but not _HisNFS1·ISD11. B, schematic representation of the pulldown results.

FIGURE 8.

Interactions of rec-FXN isoforms with purified ISCU and NFS1·ISD11. A, all indicated proteins were used at a final concentration of 2 μm. Pulldown assays were carried out and analyzed using the same conditions described in the legend to Fig. 6A. Both _HisNFS1·ISD11 complex and untagged ISCU were used in this experiment. The reproducible amounts of each rec-FXN isoform pulled down in three independent assays (a–c) are shown. In each set, the second lane (denoted by an asterisk under the lane) shows control reactions containing nickel beads and the indicated FXN isoform but not _HisNFS1·ISD11. B, schematic representation of the pulldown results shown in A. C, _HisNFS1·ISD11, untagged ISCU or all proteins together were incubated with nickel beads in binding buffer for 1 h at 4 °C, and the incubation was continued in the absence or presence of 20 μm Fe²⁺ at 37 °C for 30 min. The lanes denoted by an asterisk show control reactions containing nickel beads and ISCU but not _HisNFS1·ISD11. D, schematic representation of the pulldown results shown in C.

FIGURE 9.

Interactions of native FXN isoforms with native NFS1 and ISCU. Lymphoblastoid cell lysate was analyzed by Superdex 75 size exclusion chromatography. Fractions comprising the entire molecular mass fractionation range of the column were analyzed by Western blotting with (A) anti-NFS1 monoclonal antibody MyBioSource, (B) anti-FXN, or (C) anti-Isu1 (32) polyclonal antibodies. The high and low molecular weight fractions were pooled (HMW and LMW box, respectively), and immunoprecipitation was performed with PAC 2517 anti-FXN antibody immobilized on Protein A Magnetic beads, as described under “Experimental Procedures.” Aliquots of each pool (HMW or LMW) before immunoprecipitation (Input, ∼5% of total volume), the flow-through fraction (Not bound; ∼5% of total volume), and the affinity-purified fraction (Bound; 100% of total volume) were analyzed by Western blotting (WB) with anti-NFS1 monoclonal antibody (D) or anti-FXN monoclonal antibody (E) or anti-Isu1 polyclonal antibody (F). FXN and ISCU were detected in individual HMW fractions (B and C) but not in the HMW fraction pool (E and F, input and not Bound) because ∼4 times less total protein was loaded on the gel in the latter analysis. For the control (MOCK), the Protein A Magnetic beads without antibody were incubated overnight with non-fractionated lymphoblastoid cell lysate, and Input, Not bound, and Bound proteins were analyzed as described above.

FIGURE 10.

FXN isoforms differentially use Fe²⁺ or Fe³⁺ to support [2Fe-2S] assembly on ISCU. Rec-FXN proteins were incubated with 10 eq of Fe²⁺ for 20 min anaerobically (A) or for ∼12 h aerobically (B) in 20 mm HEPES-KOH, pH 7.3, 150 mm NaCl. The [2Fe-2S] cluster synthesis reaction was started with the addition of 50 μm iron, provided as FXN-bound or free iron, to anaerobic reactions containing 5 μm ISCU_His, 5 μm _HisNFS1·ISD11 complex, 5 mm dithiothreitol, and 2 mm l-cysteine in 20 mm HEPES-KOH, pH 7.3, 150 mm NaCl. Each plot represents the mean ± S.D. from 2 (oligomeric FXN^42–210) and 3 (FXN^81–210) independent experiments. Data fitting was performed using a sigmoidal equation in the SigmaPlot program. The inset shows the individual UV-visible absorption spectra for the completed anaerobic reactions containing ISCU + NFS1·ISD11 + l-Cys + Fe²⁺ (green), ISCU + NFS1·ISD11 + l-Cys + [oligomeric rec-FXN^42–210 + Fe²⁺] (red), and ISCU + NFS1·ISD11 + l-Cys + [rec-FXN^81–210 + Fe²⁺] (blue).

FIGURE 11.

Proposed functions of FXN isoforms in Fe-S cluster synthesis. The FXN precursor is processed by MPP to FXN^42–210, which can undergo further processing to FXN^81–210 or oligomerize. A, under basal conditions, most FXN^42–210 is cleaved to FXN^81–210, which controls the labile Fe²⁺ pool and supports the bulk of [2Fe-2S] cluster assembly through dynamic interactions with the NFS1·ISD11·ISCU complex. Stable complexes of oligomeric FXN and NFS1·ISD11 form in an iron-independent manner but may not significantly participate in Fe-S cluster assembly under basal conditions. B, under conditions of increased Fe-S cluster demand and increased mitochondrial iron uptake (e.g. increased mitochondrial biogenesis), a larger proportion of FXN^42–210 can be converted to oligomeric FXN^42–210, which can chelate any iron that exceeds the limited iron-binding capacity of FXN^81–210. Stable complexes between iron-loaded oligomeric FXN^42–210 and NFS1·ISD11 become an additional site of Fe-S cluster assembly on ISCU. I, II, and III, respiratory chain complexes I, II, and III; Aco, mitochondrial aconitase. Components involved in the transfer of [2Fe-2S] cluster from ISCU to Apo enzymes (52) are not shown.