COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2.

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

Primary coenzyme Q10 deficiency presenting as fatal neonatal multiorgan failure.

Coenzyme Q10 deficiency is a clinically and genetically heterogeneous disorder, with manifestations that may range from fatal neonatal multisystem failure, to adult-onset encephalopathy. We report a patient who presented at birth with severe lactic acidosis, proteinuria, dicarboxylic aciduria, and hepatic insufficiency. She also had dilation of left ventricle on echocardiography. Her neurological condition rapidly worsened and despite aggressive care she died at 23 h of life. Muscle histology displayed lipid accumulation. Electron microscopy showed markedly swollen mitochondria with fragmented cristae. Respiratory-chain enzymatic assays showed a reduction of combined activities of complex I+III and II+III with normal activities of isolated complexes. The defect was confirmed in fibroblasts, where it could be rescued by supplementing the culture medium with 10 µM coenzyme Q10. Coenzyme Q10 levels were reduced (28% of controls) in these cells. We performed exome sequencing and focused the analysis on genes involved in coenzyme Q10 biosynthesis. The patient harbored a homozygous c.545T>G, p.(Met182Arg) alteration in COQ2, which was validated by functional complementation in yeast. In this case the biochemical and morphological features were essential to direct the genetic diagnosis. The parents had another pregnancy after the biochemical diagnosis was established, but before the identification of the genetic defect. Because of the potentially high recurrence risk, and given the importance of early CoQ10 supplementation, we decided to treat with CoQ10 the newborn child pending the results of the biochemical assays. Clinicians should consider a similar management in siblings of patients with CoQ10 deficiency without a genetic diagnosis.

Genetic bases and clinical manifestations of coenzyme Q10 (CoQ 10) deficiency.

Coenzyme Q(10) is a remarkable lipid involved in many cellular processes such as energy production through the mitochondrial respiratory chain (RC), beta-oxidation of fatty acids, and pyrimidine biosynthesis, but it is also one of the main cellular antioxidants. Its biosynthesis is still incompletely characterized and requires at least 15 genes. Mutations in eight of them (PDSS1, PDSS2, COQ2, COQ4, COQ6, ADCK3, ADCK4, and COQ9) cause primary CoQ(10) deficiency, a heterogeneous group of disorders with variable age of onset (from birth to the seventh decade) and associated clinical phenotypes, ranging from a fatal multisystem disease to isolated steroid resistant nephrotic syndrome (SRNS) or isolated central nervous system disease. The pathogenesis is complex and related to the different functions of CoQ(10). It involves defective ATP production and oxidative stress, but also an impairment of pyrimidine biosynthesis and increased apoptosis. CoQ(10) deficiency can also be observed in patients with defects unrelated to CoQ(10) biosynthesis, such as RC defects, multiple acyl-CoA dehydrogenase deficiency, and ataxia and oculomotor apraxia.Patients with both primary and secondary deficiencies benefit from high-dose oral supplementation with CoQ(10). In primary forms treatment can stop the progression of both SRNS and encephalopathy, hence the critical importance of a prompt diagnosis. Treatment may be beneficial also for secondary forms, although with less striking results.In this review we will focus on CoQ(10) biosynthesis in humans, on the genetic defects and the specific clinical phenotypes associated with CoQ(10) deficiency, and on the diagnostic strategies for these conditions.

Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q10 deficiency.

Human COQ6 encodes a monooxygenase which is responsible for the C5-hydroxylation of the quinone ring of coenzyme Q (CoQ). Mutations in COQ6 cause primary CoQ deficiency, a condition responsive to oral CoQ10 supplementation. Treatment is however still problematic given the poor bioavailability of CoQ10. We employed S. cerevisiae lacking the orthologous gene to characterize the two different human COQ6 isoforms and the mutations found in patients. COQ6 isoform a can partially complement the defective yeast, while isoform b, which lacks part of the FAD-binding domain, is inactive but partially stable, and could have a regulatory/inhibitory function in CoQ10 biosynthesis. Most mutations identified in patients, including the frameshift Q461fs478X mutation, retain residual enzymatic activity, and all patients carry at least one hypomorphic allele, confirming that the complete block of CoQ biosynthesis is lethal. These mutants are also partially stable and allow the assembly of the CoQ biosynthetic complex. In fact treatment with two hydroxylated analogues of 4-hydroxybenzoic acid, namely, vanillic acid or 3-4-hydroxybenzoic acid, restored the respiratory growth of yeast Δcoq6 cells expressing the mutant huCOQ6-isoa proteins. These compounds, and particularly vanillic acid, could therefore represent an interesting therapeutic option for COQ6 patients.

Copper and bezafibrate cooperate to rescue cytochrome c oxidase deficiency in cells of patients with SCO2 mutations.

BACKGROUND: Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients' cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene. METHODS: We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production.. RESULTS: Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 µM BZF, and peaked at 400 µM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 µM CuCl2 and 200 µM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells. CONCLUSIONS: These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.

Haploinsufficiency of COQ4 causes coenzyme Q10 deficiency.

BACKGROUND: COQ4 encodes a protein that organises the multienzyme complex for the synthesis of coenzyme Q(10) (CoQ(10)). A 3.9 Mb deletion of chromosome 9q34.13 was identified in a 3-year-old boy with mental retardation, encephalomyopathy and dysmorphic features. Because the deletion encompassed COQ4, the patient was screened for CoQ(10) deficiency. METHODS: A complete molecular and biochemical characterisation of the patient's fibroblasts and of a yeast model were performed. RESULTS: The study found reduced COQ4 expression (48% of controls), CoQ(10) content and biosynthetic rate (44% and 43% of controls), and activities of respiratory chain complex II+III. Cells displayed a growth defect that was corrected by the addition of CoQ(10) to the culture medium. Knockdown of COQ4 in HeLa cells also resulted in a reduction of CoQ(10.) Diploid yeast haploinsufficient for COQ4 displayed similar CoQ deficiency. Haploinsufficency of other genes involved in CoQ(10) biosynthesis does not cause CoQ deficiency, underscoring the critical role of COQ4. Oral CoQ(10) supplementation resulted in a significant improvement of neuromuscular symptoms, which reappeared after supplementation was temporarily discontinued. CONCLUSION: Mutations of COQ4 should be searched for in patients with CoQ(10) deficiency and encephalomyopathy; patients with genomic rearrangements involving COQ4 should be screened for CoQ(10) deficiency, as they could benefit from supplementation.