Impact of the Mitochondrial Genetic Background in Complex III Deficiency

BACKGROUND: In recent years clinical evidence has emphasized the importance of the mtDNA genetic background that hosts a primary pathogenic mutation in the clinical expression of mitochondrial disorders, but little experimental confirmation has been provided. We have analyzed the pathogenic role of a novel homoplasmic mutation (m.15533 A>G) in the cytochrome b (MT-CYB) gene in a patient presenting with lactic acidosis, seizures, mild mental delay, and behaviour abnormalities. METHODOLOGY: Spectrophotometric analyses of the respiratory chain enzyme activities were performed in different tissues, the whole muscle mitochondrial DNA of the patient was sequenced, and the novel mutation was confirmed by PCR-RFLP. Transmitochondrial cybrids were constructed to confirm the pathogenicity of the mutation, and assembly/stability studies were carried out in fibroblasts and cybrids by means of mitochondrial translation inhibition in combination with blue native gel electrophoresis. PRINCIPAL FINDINGS: Biochemical analyses revealed a decrease in respiratory chain complex III activity in patient's skeletal muscle, and a combined enzyme defect of complexes III and IV in fibroblasts. Mutant transmitochondrial cybrids restored normal enzyme activities and steady-state protein levels, the mutation was mildly conserved along evolution, and the proband's mother and maternal aunt, both clinically unaffected, also harboured the homoplasmic mutation. These data suggested a nuclear genetic origin of the disease. However, by forcing the de novo functioning of the OXPHOS system, a severe delay in the biogenesis of the respiratory chain complexes was observed in the mutants, which demonstrated a direct functional effect of the mitochondrial genetic background. CONCLUSIONS: Our results point to possible pitfalls in the detection of pathogenic mitochondrial mutations, and highlight the role of the genetic mtDNA background in the development of mitochondrial disorders

Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance

Respiratory chains are crucial for cellular energy conversion and consist of multi-subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high-resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms

Residual enzyme activities of mitochondrial respiratory chain complexes in different tissues from the index patient.

<p>Enzyme activities are expressed as</p><p>*cU/U citrate synthase (CS) and</p><p>**nmol.min⁻¹.mg prot⁻¹. CS activity is expressed as mU/mg protein. Abnormal values are indicated in bold. nd, not determined. Complex I, CI; Complex II, CII; Complex III, CIII; Complex IV, CIV.</p

Genetic and structural analysis of the m.15533A>G mutation.

<p>(A) Electropherogram showing the nucleotide change in patient's muscle DNA, indicated by arrowheads. (B) PCR-RFLP analysis of the <i>MT-CYB</i> mutation. Uncut (wild-type) DNA consists of a 150 bp PCR product. The mutated sequence contains one <i>Dde</i>I restriction site that results in two products of 90 and 60 bp after digestion. The control sequence contains one <i>Mse</i>I restriction site that results in the same two products after digestion. C, wild-type control; F, proband's fibroblasts DNA; M, proband's muscle DNA; Cy, control cybrid; 1, 2 and 3 refer to three independent mutant cybrid clones. (C) Alignment of cytochrome b amino acid sequences from selected species. Asparagine at amino acid position 263 is indicated with an arrow. (D) Partial 3D-images of the cytochrome b protein. The left panel shows the interaction site between the asparagine 263 (indicated in red) of cytochrome b and the aspartate 2 of the cytochrome c1 subunit (coloured in green). This is the closest negatively charged side-chain of cytochrome c1, which is facing the same aqueous pocket at a distance of 11.25 Armstrongs. Asparagine at position 260 of cytochrome b is indicated in blue. The central panel shows the 3D-structure of the asparagine 263 (in blue) in controls. The right panel shows the predicted structural effect of the N263D substitution (in red) in the patient. The images were obtained using the Chimera software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012801#pone.0012801-Pettersen1" target="_blank">[20]</a>.</p

BN-PAGE analysis of mitochondrial respiratory chain complexes in control and mutant fibroblasts and cybrids.

<p>Mitochondrial particles were isolated as described in Methods and 40 µg of protein were analyzed on a 5–15% BN-polyacrylamide gel for the separation of multisubunit complexes. Western-blot analysis was performed using antibodies against the indicated OXPHOS subunits. CI, fully-assembled complex I. CIII₂, complex III dimer. CIV, complex IV. CIII₂+IV indicates the presence of the supercomplex containing complexes III and IV. C1 and C2, control fibroblasts. P, patient's fibroblasts. C, control cybrid. Two independent mutant cybrids are indicated as #1 and #2.</p

Assembly kinetics of respiratory chain complexes in control and mutant cybrids.

<p>Two different control cybrids belonging to haplogroup H, and two independent <i>MT-CYB</i> mutant clones were treated for 6 days with doxycycline (an inhibitor of mitochondrial translation), the medium was replaced by doxycycline-free medium and cells were collected at 0, 6, 15, 24, 48, 72, and 96 hours (indicated as t0–t16). SS indicates the steady-state expression levels of the respiratory chain complexes (A) Example of one control and one mutant clone. 40 µg of crude mitochondrial pellets were analyzed by BN-PAGE in combination with complex I and complex IV-IGA assays. (B) Duplicate gels were blotted and incubated with antibodies against the NDUFA9 complex I subunit, complex III core2 protein, complex IV COX5A subunit and complex II SDHA subunit. (C) The signals from the blots were quantified, expressed as percentage of the untreated cells (SS), normalized with the complex II SDHA subunit and plotted. The restoration curves constitute the mean values ± SD obtained from the two controls and the two independent mutant cybrids. Upper left panel, complex I assembly rates. Upper right panel, complex III assembly rates. Lower left panel, complex IV assembly rates. Lower right panel, supercomplex CIII₂+IV assembly kinetics.</p

Haplogroup affiliation and non-synonymous nucleotide changes of the mtDNA sequence from the index patient.

a<p>rCRS refers to the revised Cambridge reference sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012801#pone.0012801-Andrews1" target="_blank">[16]</a>, which belongs to haplogroup H2a.</p