Carlos Santos-Ocaña

COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q10 biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q10 treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q10-related forms of SRNS and hearing loss can be molecularly identified and potentially treated.

Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants.

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery inSaccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q6 by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-lesscoq7 mutants derived from four distinct laboratory yeast strains. Q6 supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the fourcoq7 mutant strains. Following culture in medium containing dextrose, the added Q6 was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q6 in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q6. These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q6 into the plasma membrane. The two coq7 mutant strains rescued by Q6 supplementation for growth on ethanol contained mitochondrial Q6 levels similar to wild type. However, the content of Q6 in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q6, succinate-cytochrome creductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q6 content, succinate-cytochromec reductase activity, and steady state levels of the cytochrome c 1 polypeptide. These studies show that transport of extracellular Q6 to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc 1complex is unstable. These results indicate that delivery of exogenous Q6 to mitochondria is required fore activity and stability of the bc 1 complex in yeast coqmutants.

Haploinsufficiency of COQ4 causes coenzyme Q10 deficiency

Background COQ4 encodes a protein that organises the multienzyme complex for the synthesis of coenzyme Q10 (CoQ10). 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 CoQ10 deficiency. Methods A complete molecular and biochemical characterisation of the patients fibroblasts and of a yeast model were performed. Results The study found reduced COQ4 expression (48% of controls), CoQ10 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 CoQ10 to the culture medium. Knockdown of COQ4 in HeLa cells also resulted in a reduction of CoQ10. Diploid yeast haploinsufficient for COQ4 displayed similar CoQ deficiency. Haploinsufficency of other genes involved in CoQ10 biosynthesis does not cause CoQ deficiency, underscoring the critical role of COQ4. Oral CoQ10 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 CoQ10 deficiency and encephalomyopathy; patients with genomic rearrangements involving COQ4 should be screened for CoQ10 deficiency, as they could benefit from supplementation.

Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration in Saccharomyces cerevisiae and lacks antioxidant activity

Caenorhabditis elegans clk-1 mutants cannot produce coenzyme Q9 and instead accumulate demethoxy-Q9 (DMQ9). DMQ9 has been proposed to be responsible for the extended lifespan of clk-1 mutants, theoretically through its enhanced antioxidant properties and its decreased function in respiratory chain electron transport. In the present study, we assess the functional roles of DMQ6 in the yeast Saccharomyces cerevisiae. Three mutations designed to mirror the clk-1 mutations of C. elegans were introduced into COQ7, the yeast homologue of clk-1: E233K, predicted to disrupt the di-iron carboxylate site considered essential for hydroxylase activity; L237Stop, a deletion of 36 amino acid residues from the carboxyl terminus; and P175Stop, a deletion of the carboxyl-terminal half of Coq7p. Growth on glycerol, quinone content, respiratory function, and response to oxidative stress were analyzed in each of the coq7 mutant strains. Yeast strains lacking Q6 and producing solely DMQ were respiratory deficient and unable to support 6either NADH-cytochrome c reductase or succinate-cytochrome c reductase activities. DMQ6 failed to protect cells against oxidative stress generated by H2O2 or linolenic acid. Thus, in the yeast model system, DMQ does not support respiratory activity and fails to act as an effective antioxidant. These results suggest that the life span extension observed in the C. elegans clk-1 mutants cannot be attributed to the presence of DMQ per se.

Is coenzyme Q a key factor in aging

Coenzyme Q (Q) is a key component for bioenergetics and antioxidant protection in the cell. During the last years, research on diseases linked to Q-deficiency has highlighted the essential role of this lipid in cell physiology. Q levels are also affected during aging and neurodegenerative diseases. Therefore, therapies based on dietary supplementation with Q must be considered in cases of Q deficiency such as in aging. However, the low bioavailability of dietary Q for muscle and brain obligates to design new mechanisms to increase the uptake of this compound in these tissues. In the present review we show a complete picture of the different functions of Q in cell physiology and their relationship to age and age-related diseases. Furthermore, we describe the problems associated with dietary Q uptake and the mechanisms currently used to increase its uptake or even its biosynthesis in cells. Strategies to increase Q levels in tissues are indicated.

Genetic Evidence for Coenzyme Q Requirement in Plasma Membrane Electron Transport

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3Δ, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q6 and contained greatly diminished levels of NADH–ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q6 in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase. Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q6 and NADH–ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3Δ strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3Δ strain grown in the presence of coenzyme Q6. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3–14. The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3Δ strain. Ascorbate free radical reductase was not altered significantly in mutants atp2Δ and cor1Δ which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3Δ mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH–ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.

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.

Hydroxylation of demethoxy-Q6 constitutes a control point in yeast coenzyme Q6 biosynthesis

Abstract.Coenzyme Q is a lipid molecule required for respiration and antioxidant protection. Q biosynthesis in Saccharomyces cerevisiae requires nine proteins (Coq1p–Coq9p). We demonstrate in this study that Q levels are modulated during growth by its conversion from demethoxy-Q (DMQ), a late intermediate. Similar conversion was produced when cells were subjected to oxidative stress conditions. Changes in Q6/DMQ6 ratio were accompanied by changes in COQ7 gene mRNA levels encoding the protein responsible for the DMQ hydroxylation, the penultimate step in Q biosynthesis pathway. Yeast coq null mutant failed to accumulate any Q late biosynthetic intermediate. However, in coq7 mutants the addition of exogenous Q produces the DMQ synthesis. Similar effect was produced by over-expressing ABC1/COQ8. These results support the existence of a biosynthetic complex that allows the DMQ6 accumulation and suggest that Coq7p is a control point for the Q biosynthesis regulation in yeast.

NQR1 controls lifespan by regulating the promotion of respiratory metabolism in yeast.

The activity and expression of plasma membrane NADH coenzyme Q reductase is increased by calorie restriction (CR) in rodents. Although this effect is well‐established and is necessary for CRs ability to delay aging, the mechanism is unknown. Here we show that the Saccharomyces cerevisiae homolog, NADH‐Coenzyme Q reductase 1 (NQR1), resides at the plasma membrane and when overexpressed extends both replicative and chronological lifespan. We show that NQR1 extends replicative lifespan in a SIR2‐dependent manner by shifting cells towards respiratory metabolism. Chronological lifespan extension, in contrast, occurs via an SIR2‐independent decrease in ethanol production. We conclude that NQR1 is a key mediator of lifespan extension by CR through its effects on yeast metabolism and discuss how these findings could suggest a function for this protein in lifespan extension in mammals.

Mitochondrial responsibility in ageing process: innocent, suspect or guilty

Ageing is accompanied by the accumulation of damaged molecules in cells due to the injury produced by external and internal stressors. Among them, reactive oxygen species produced by cell metabolism, inflammation or other enzymatic processes are considered key factors. However, later research has demonstrated that a general mitochondrial dysfunction affecting electron transport chain activity, mitochondrial biogenesis and turnover, apoptosis, etc., seems to be in a central position to explain ageing. This key role is based on several effects from mitochondrial-derived ROS production to the essential maintenance of balanced metabolic activities in old organisms. Several studies have demonstrated caloric restriction, exercise or bioactive compounds mainly found in plants, are able to affect the activity and turnover of mitochondria by increasing biogenesis and mitophagy, especially in postmitotic tissues. Then, it seems that mitochondria are in the centre of metabolic procedures to be modified to lengthen life- or health-span. In this review we show the importance of mitochondria to explain the ageing process in different models or organisms (e.g. yeast, worm, fruitfly and mice). We discuss if the cause of aging is dependent on mitochondrial dysfunction of if the mitochondrial changes observed with age are a consequence of events taking place outside the mitochondrial compartment.