Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes

Abstract

Severe combined deficiency of the 2-oxoacid dehydrogenases, associated with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, analysis of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs(∗)13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the production of [Fe-S] centers for the normal maturation of lipoate-containing 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.

Figure 1

Proposed Scheme for the Interaction of Human [Fe-S] Genes within the Mitochondria mNFS1 produces elemental sulfur from cysteine and makes it available to mISCU by interaction with this scaffold protein. LYRM4 is required for stabilization of mNFS1. Fe is provided by the chaperone frataxin (FXN) through an interaction with LYRM4 and mISCU, and [2Fe-2S] clusters are assembled upon each monomer of mISCU. These are then combined into [4Fe-4S] clusters. Ferredoxin provides the necessary reducing agents for these reactions from electrons initially provided by ferredoxin reductase (not shown). Cochaperones HSCB, HSPA9, and GLRX5 bind to ISCU and aid in the transfer of [2Fe-2S] clusters into recipient proteins. Only GLRX5 is required as a chaperone for [4Fe-4S] transfer. BOLA3 possibly functions by interacting with GLRX5. ISCA1,2 and IBA57 are required for the maturation of aconitase-like proteins and the catalytic activation of radical SAM enzymes (e.g., lipoic acid synthase). IND1 is a complex-I-specific assembly factor. mNFU1 is thought to act as an alternate scaffold, possibly for the assembly of a subset of [Fe-S] proteins. The E3BP and E2 subunits have covalently bound lipoic acid, which requires a complex synthesis process involving transfer of an octanoyl-ACP (derived from fatty acid biosynthesis) onto an apoprotein by LIPT2. LIAS catalyzes the lipoylation of the octanoylated apo-protein by using SAM and sulfur (potentially produced by the action of mNFS1). Ferredoxin (FDX1) is thought to provide the reducing power. ISCA1,2 and IBA57 are required for catalytic activation of LIAS. Lipoylated E2 is present as dihydrolipoamide S-acetyltransferase (DLAT) in PDHc, dihydrolipoamide S-succinyltransferase (DLST) in OGDH and dihydrolipoamide branched chain transacetylase (DBT) in BCKADHc. Lipoylated E3-binding protein is present in PDHc. Diseases associated with defective mammalian proteins are noted in italics.

Figure 2

Analysis of NFU1 in Family 1 (A) Sequence chromatograms are shown for gDNA regions of NFU1. The c.545G>A (p.Arg182Gln) mutation can be seen in affected individuals P1–P3 (II-1, II-2 and II-3); there is no mutation in unaffected sibling P4 (II-4). (B) The genomic structure of isoform 1 (cytosolic) and isoform 2 (mitochondrial) NFU1 is shown for individuals P1–P3 (II-1, II-2, and II-3). The c.545G>A mutation is identified. The normal alternate splicing that creates the two isoforms from the gDNA sequence is shown with solid lines, and the effect of the mutation on the splice site between exons 5 and 6 is demonstrated with dashed lines, yielding three possible transcripts. (C) PCR amplification from individual P1 (II-1, NFU1 mutation), P5 (II-1 in Figure 3, BOLA3 mutation), and control cDNA. In the control and individual P5 cDNA, two transcripts representing the cytosolic and mitochondrial isoforms can be seen. In individual P1, there is almost no full-length mitochondrial NFU1, and two smaller transcripts are present (a and b). There is less cytosolic NFU1 in individual P1, with one transcript variant visible (a). The mutant transcripts a and b are all identified in (A) and were all verified by sequencing. (D) Immunoblot of mitochondria showing the mitochondrial isoform of NFU1 with citrate synthase as a loading control.

Figure 3

Analysis of BOLA3 in Family 2 (A) Sequence chromatograms are shown for gDNA regions of BOLA3. The single base pair duplication (c.123dupA) can be seen in individual P5 (II-1). The parents (I-1, I-2) and unaffected sibling P6 (II-2) are all heterozygous. There is no mutation in NFU1 mutant individual P1 (II-1 in Figure 2). (B) The two isoforms of BOLA3 cDNA are shown with the mutation seen in P5 (II-1) highlighted.

Figure 4

Immunoblot Analysis of the Oxoacid Dehydrogenases and Respiratory Chain Subunits in NFU1, BOLA3, and ISCU Mutant Fibroblasts Immunoblots of 25 μg fibroblast mitochondria were immunoblotted with antibodies against NFU1, lipoate, complex I subunits (NDUFS1, NDUFV1, NDUFS2, NDUFA9, and NDUFA8), complex II subunit SDHA, complex III subunits (UQCRFS1, UQCRC1, and CYTB), and citrate synthase (CS).

Figure 5

Rescue of the Biochemical Deficiencies in NFU1 and BOLA3 Mutant Fibroblasts by Retroviral Expression of the Wild-Type cDNAs (A) Immunoblot of control and mutant NFU1 fibroblast mitochondria transduced with constructs expressing mitochondrial or cytosolic isoforms of NFU1were immunoblotted with antibodies against NFU1, lipoate, complex I subunit NDUFA9, complex II subunit SDHA, and actin. (B) BN-PAGE analysis of the same samples as in (A). (C) Immunoblot of control and mutant BOLA3 fibroblast mitochondria transduced with constructs expressing the BOLA3-1 and BOLA3-2 isoforms were immunoblotted with antibodies against NFU1, lipoate, complex I subunit NDUFA9, complex II subunit SDHA, and actin. (D) BN-PAGE analysis of the same samples as in (C).

Figure 6

Immunofluorescence analysis of Fibroblast Cells Transduced with Retroviral Constructs Expressing the Cytosolic and Mitochondrial Versions of NFU1 and the Two Isoforms of BOLA3 (A) SDHA was used as a mitochondrial marker and as a marker of the respiratory chain defect. The predicted mitochondrial isoform of NFU1 localizes to mitochondria, and expression of the cDNA rescues the SDHA defect in individual P1 (II-1 in Figure 1) cells. The cytosolic isoform shows diffuse staining in mutant fibroblasts and does not rescue the complex II defect. (B) BOLA3-1HA rescues the complex II defect of individual P5 and localizes to mitochondria, but BOLA3-2HA shows diffuse staining and does not rescue the defect.

Figure 7

Pyruvate Dehydrogenase Enzyme Activities for Control and Mutant NFU1 and BOLA3 Fibroblasts Native and DCA-activated PDHc enzyme activities were determined for control and mutant NFU1 fibroblast mitochondria transfected with mitochondrial and cytosolic transcripts of NFU1, and control and mutant BOLA3 fibroblast mitochondria transfected with BOLA3-1 and BOLA3-2 isoforms. Data are represented as mean ± SEM for four separate enzyme measurements.