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.
Highlights
The Friedreich ataxia (FRDA) locus encodes a mitochondrial protein designated frataxin (FXN), which is expressed at much lower levels in FRDA patients compared with normal individuals [2]
The controls contained similar levels of two major protein bands that co-migrated with rec-FXN42–210 and recFXN81–210, respectively
We focused on FXN42–210 and FXN81–210, the longest and shortest of the isoforms known to result from processing of FXN1–210 by mitochondrial processing peptidase (MPP) [19, 20, 22]
Summary
Expression and Purification of Recombinant Proteins Used in This Study—For expression of rec-FXN isoforms, E. coli strain BL21(DE3) (Novagen) was transformed with vector pETHF-42 (FXN cDNA coding for residues 42–210 in pET24aϩ vector), pETHF-56 (FXN cDNA coding for residues 56 –210 in pET24aϩ vector) [25, 29], pETHF-78 (FXN cDNA coding for residues 78 –210 in pET24aϩ vector), or pETHF-81 (FXN cDNA coding for residues 81–210 in pET24aϩ vector). Two DNA fragments corresponding to residues 56 – 458 of NFS1 and 6 –92 of ISD11 [32, 36] were cloned into the XhoI and BamHI sites of vector pET15b (Novagen), and the NcoI and EcoRI sites of vector pCDFDuet (Novagen), respectively This resulted in the addition of an N-terminal His tag followed by a thrombin cleavage site on NFS1. Frozen cell pellets were suspended in 400 l of ice-cold extraction buffer (1.5% lauryl maltoside, 100 mM NaCl, 25 mM HEPES-KOH, pH 7.3) with protease inhibitors as above, and incubated on ice for 30 min with periodical mixing. These samples were cleared of insoluble material by centrifugation at 20,000 ϫ g for 15 min at 4 °C. Electrophoresis was carried out at room temperature at 180 V for ϳ3.5 h, and continued at 260 V until the bromphenol blue reached the bottom of the separating gel, and further continued for an additional 1.5 h
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