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Coenzyme Q10/ COQ10 , an essential cofactor in the electron-transport chain is involved in ATP production. Primary COQ10 deficiency is clinically and genetically a heterogeneous group of mitochondrial disorders caused by defects in the COQ10 synthesis pathway. Its mode of inheritance is autosomal recessive and it is characterized by metabolic abnormalities and multisystem involvement including neurological features. Mutations in 10 genes have been identified concerning this group of diseases, so far. Among those, variants of the COQ7 gene are very rare and confined to three patients with Asian ancestry. Here, we present the clinical features and results of whole-exome sequencing (WES) of three Iranian unrelated families affected by primary COQ10 deficiency. Three homozygous variants in COQ2, COQ4, and COQ7 genes were identified. Candidate variants of the COQ2 and COQ4 genes were novel and associated with the cerebellar signs and multisystem involvement, whereas, the known variant in COQ7 was associated with a mild phenotype that was initially diagnosed as hereditary spastic paraplegia (HSP). This variant has already been reported in a Canadian girl with similar presentations that also originated from Iran suggesting both patients may share a common ancestor. Due to extensive heterogeneity in this group of disorders, and overlap with other mitochondrial/neurological disorders, WES may be helpful to distinguish primary coenzyme Q10 deficiency from other similar conditions. Given that some features of primary coenzyme Q10 deficiency may improve with exogenous COQ10 , early diagnosis is very important.

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined ‘complex Q’ metabolon. Here we present a structure and functional analyses of a substrate- and NADH-bound oligomeric complex comprised of two complex Q subunits: the hydroxylase COQ7, which performs the penultimate step in CoQ biosynthesis, and the prenyl lipid-binding protein COQ9. We reveal that COQ7 adopts a modified ferritin-like fold with an extended hydrophobic access channel whose substrate binding capacity is enhanced by COQ9. Using molecular dynamics simulations, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for CoQ intermediates to translocate from within the bilayer to the proteins’ lipid-binding sites. Two such tetramers assemble into a soluble octamer, closed like a capsid, with lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors and coordinate subsequent synthesis steps toward producing mature CoQ.

Coenzyme Q (CoQ) is a lipid-like mobile electron transporter of the mitochondrial respiratory chain. Patients with partial loss-of-function mutations in the CoQ biosynthesis pathway suffer from partial primary CoQ deficiency (MIM 607426). This leads to mitochondrial dysfunction, which presents like mitochondrial disease syndrome (MDS). In addition, many other conditions, including MDS itself, lead to secondary CoQ deficiency. We sought to identify drugs that can alleviate the consequences of the mitochondrial dysfunction that is associated with CoQ deficiency. Loss of the CoQ-biosynthetic enzyme COQ7 prevents CoQ synthesis but leads to the accumulation of the biosynthetic intermediate demethoxyubiquinone (DMQ). Coq7-knockout mouse embryonic fibroblasts (MEFs) die when rapid ATP generation from glycolysis is prevented. We screened for drugs that could rescue cell death under these conditions. All compounds that were identified inhibit mTOR signaling. In the CoQ-deficient cells, the beneficial action mTOR inhibition appears to be mediated by inhibition of protein translation rather than by stimulation of autophagy. We further studied the Coq7-knockout cells to better determine under which conditions mTOR inhibition could be beneficial. We established that Coq7-knockout cells remain capable of a low level of mitochondrial respiration mediated by DMQ. To obtain more profound mitochondrial dysfunction, we created double-knockout mutant MEFs lacking both Coq7, as well as Pdss2, which is required for sidechain synthesis. These cells make neither CoQ nor DMQ, and their extremely small residual respiration depends on uptake of CoQ from the culture medium. Although these cells are healthy in the presence of sufficient glucose for glycolysis and do not require uridine or pyruvate supplementation, mTOR inhibitors were unable to prevent their death in the absence of sufficient glycolysis. We conclude that, for reasons that remain to be elucidated, the energy-sparing benefits of the inhibition of mTOR signaling require a minimally functional respiratory chain.

Coenzyme Q10 (CoQ10) or ubiquinone is one of the two electron carriers in the mitochondrial respiratory chain which has an essential role in the process of oxidative phosphorylation. Defects in CoQ10 synthesis are usually associated with the impaired function of CoQ10–dependent complexes I, II and III. The recessively transmitted CoQ10 deficiency has been associated with a number of phenotypically and genetically heterogeneous groups of disorders manifesting at variable age of onset. The infantile, multisystemic presentation is usually caused by mutations in genes directly involved in CoQ10 biosynthesis. To date, mutations in COQ1 (PDSS1 and PDSS2), COQ2, COQ4, COQ6, COQ7, COQ8A/ADCK3, COQ8B/ADCK4, and COQ9 genes have been identified in patients with primary form of CoQ10 deficiency. Here we report novel mutations in the COQ4 gene, which were identified in an infant with profound mitochondrial disease presenting with perinatal seizures, hypertrophic cardiomyopathy and severe muscle CoQ10 deficiency.

Coenzyme Q10 (CoQ10) is an essential cofactor of the mitochondrial oxidative phosphorylation (OXPHOS) system and its deficiency has important implications for several inherited metabolic disorders of childhood. The biosynthesis of CoQ10 is a complicated process, which involves at least 12 different enzymes. One of the metabolic intermediates that are formed during CoQ10 biosynthesis is the molecule 6-demethoxyubiquinone (6-DMQ). This CoQ precursor is processed at the level of COQ7 and COQ9. We selected this metabolite as a marker substance for metabolic analysis of cell lines with inherited genetic defects (COQ2, COQ4, COQ7 and COQ9) or siRNA knockdown in CoQ biosynthesis enzymes using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). In COQ4, COQ7 and COQ9 deficient cell lines, we detected significantly elevated levels of 6-DMQ. This suggests a functional interplay of these proteins. However, additional siRNA studies demonstrated that elevated 6-DMQ levels are not an exclusive marker of the COQ7/COQ9 enzymatic step of CoQ10 biosynthesis but constitute a more general phenomenon that occurs in disorders impairing the function or stability of the CoQ-synthome. To further investigate the interdependence of CoQ10 biosynthesis enzyme expression, we performed immunoblotting in various cell lines with CoQ10 deficiency, indicating that COQ4, COQ7 and COQ9 protein expression levels are highly regulated depending on the underlying defect. Supplementation of cell lines with synthetic CoQ precursor compounds demonstrated beneficial effects of 2,4-dihydroxybenzoic acid in COQ7 and COQ9 deficiency. Moreover, vanillic acid selectively stimulated CoQ10 biosynthesis and improved cell viability in COQ9 deficiency. However, compounds tested in this study failed to rescue COQ4 deficiency.

Primary ubiquinone (co‐enzyme Q) deficiency results in a wide range of clinical features due to mitochondrial dysfunction. Here, we analyse and characterize two mutations in the ubiquinone biosynthetic gene COQ7. One mutation from the only previously identified patient (V141E), and one (L111P) from a 6‐year‐old girl who presents with spasticity and bilateral sensorineural hearing loss. We used patient fibroblast cell lines and a heterologous expression system to show that both mutations lead to loss of protein stability and decreased levels of ubiquinone that correlate with the severity of mitochondrial dysfunction. The severity of L111P is enhanced by the particular COQ7 polymorphism (T103M) that the patient carries, but not by a mitochondrial DNA mutation (A1555G) that is also present in the patient and that has been linked to aminoglycoside‐dependent hearing loss. We analysed treatment with the unnatural biosynthesis precursor 2,4‐dihydroxybenzoate (DHB), which can restore ubiquinone synthesis in cells completely lacking the enzymatic activity of COQ7. We find that the treatment is not beneficial for every COQ7 mutation and its outcome depends on the extent of enzyme activity loss.

Coenzyme Q is an essential lipid with redox capacity that is present in all organisms. In yeast its biosynthesis depends on a multiprotein complex in which Coq7 protein has both catalytic and regulatory functions. Coq7 modulates CoQ6 levels through a phosphorylation cycle, where dephosphorylation of three amino acids (Ser/Thr) by the mitochondrial phosphatase Ptc7 increases the levels of CoQ6. Here we analyzed the role of Ptc7 and the phosphorylation state of Coq7 in yeast mitochondrial function. The conversion of the three Ser/Thr to alanine led to a permanently active form of Coq7 that caused a 2.5-fold increase of CoQ6 levels, albeit decreased mitochondrial respiratory chain activity and oxidative stress resistance capacity. This resulted in an increase in endogenous ROS production and shortened the chronological life span (CLS) compared to wild type. The null PTC7 mutant (ptc7∆) strain showed a lower biosynthesis rate of CoQ6 and a significant shortening of the CLS. The reduced CLS observed in ptc7Δ was restored by the overexpression of PTC7 but not by the addition of exogenous CoQ6. Overexpression of PTC7 increased mitophagy in a wild type strain. This finding suggests an additional Ptc7 function beyond the regulation of CoQ biosynthesis. Genetic disruption of PTC7 prevented mitophagy activation in conditions of nitrogen deprivation. In brief, we show that, in yeast, Ptc7 modulates the adaptation to respiratory metabolism by dephosphorylating Coq7 to supply newly synthesized CoQ6, and by activating mitophagy to remove defective mitochondria at stationary phase, guaranteeing a proper CLS in yeast.

Given that the associated clinical manifestations of ubiquinone (UQ, or coenzyme Q) deficiency diseases are highly heterogeneous and complicated, effective new research tools for UQ homeostasis studies are awaited. We set out to develop human COQ7 inhibitors that interfere with UQ synthesis. Systematic structure-activity relationship development starting from a screening hit compound led to the identification of highly potent COQ7 inhibitors that did not disturb physiological cell growth of human normal culture cells. These new COQ7 inhibitors may serve as useful tools for studying the balance between UQ supplementation pathways: de novo UQ synthesis and extracellular UQ uptake.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi). It was originally a Latin American endemic health problem, but now is expanding worldwide as a result of increasing migration. The currently available drugs for Chagas disease, benznidazole and nifurtimox, provoke severe adverse effects, and thus the development of new drugs is urgently required. Ubiquinone (UQ) is essential for respiratory chain and redox balance in trypanosomatid protozoans, therefore we aimed to provide evidence that inhibitors of the UQ biosynthesis have trypanocidal activities. In this study, inhibitors of the human COQ7, a key enzyme of the UQ synthesis, were tested for their trypanocidal activities because they were expected to cross-react and inhibit trypanosomal COQ7 due to their genetic homology. We show the trypanocidal activity of a newly found human COQ7 inhibitor, an oxazinoquinoline derivative. The structurally similar compounds were selected from the commercially available compounds by 2D and 3D ligand-based similarity searches. Among 38 compounds selected, 12 compounds with the oxazinoquinoline structure inhibited significantly the growth of epimastigotes of T. cruzi. The most effective 3 compounds also showed the significant antitrypanosomal activity against the mammalian stage of T. cruzi at lower concentrations than benznidazole, a commonly used drug today. We found that epimastigotes treated with the inhibitor contained reduced levels of UQ9. Further, the growth of epimastigotes treated with the inhibitors was partially rescued by UQ10 supplementation to the culture medium. These results suggest that the antitrypanosomal mechanism of the oxazinoquinoline derivatives results from inhibition of the trypanosomal UQ synthesis leading to a shortage of the UQ pool. Our data indicate that the UQ synthesis pathway of T. cruzi is a promising drug target for Chagas disease.

Mutations of many PDSS and COQ genes are associated with primary coenzyme Q10 (CoQ10) deficiency, whereas mitochondrial DNA (mtDNA) mutations might cause secondary CoQ10 deficiency. Previously, we found that COQ5 and COQ9 proteins are present in different protein complexes in the mitochondria in human 143B cells and demonstrated that COQ5 and COQ9 knockdown suppresses CoQ10 levels. In the present study, we characterized other PDSS and COQ proteins and examined possible crosstalk among various PDSS and COQ proteins. Specific antibodies and mitochondrial localization of mature proteins for these proteins, except PDSS1 and COQ2, were identified. Multiple isoforms of PDSS2 and COQ3 were observed. Moreover, PDSS1, PDSS2, and COQ3 played more important roles in maintaining the stability of the other proteins. Protein complexes containing PDSS2, COQ3, COQ4, COQ6, or COQ7 protein in the mitochondria were detected. Two distinct PDSS2-containing protein complexes could be identified. Transient knockdown of these genes, except COQ6 and COQ8, decreased CoQ10 levels, but only COQ7 knockdown hampered mitochondrial respiration and caused increased ubiquinol:ubiquinone ratios and accumulation of a putative biosynthetic intermediate with reversible redox property as CoQ10. Furthermore, suppressed levels of PDSS2 and various COQ proteins (except COQ3 and COQ8A) were found in cybrids containing the pathogenic mtDNA A8344G mutation or in FCCP-treated 143B cells, which was similar to our previous findings for COQ5. These novel findings may prompt the elucidation of the putative CoQ synthome in human cells and the understanding of these PDSS and COQ protein under physiological and pathological conditions.

Background Primary coenzyme Q10 (CoQ10) deficiencies are clinically and genetically heterogeneous group of disorders associated with defects of genes involved in the CoQ10 biosynthesis pathway. COQ7‐associated CoQ10 deficiency is very rare and only two cases have been reported. Methods and Results We report a patient with encephalo‐myo‐nephro‐cardiopathy, persistent lactic acidosis, and basal ganglia lesions resulting in early infantile death. Using whole exome sequencing, we identified compound heterozygous variants in the COQ7 gene consisting of a deletion insertion resulting in frameshift [c.599_600delinsTAATGCATC, p.(Lys200Ilefs*56)] and a missense substitution [c.319C>T, p.(Arg107Trp), NM_016138.4]. Skin fibroblast studies showed decreased combined complex II + III activity and reduction in CoQ10 level. Conclusion This third patient presenting with lethal encephalo‐myo‐nephro‐cardiopathy represents the severe end of this ultra‐rare mitochondrial disease caused by biallelic COQ7 mutations. The response to CoQ10 supplement is poor and alternative treatment strategies should be developed for a more effective management of this disorder.

Coenzyme Q (CoQ) is a well-studied molecule, present in every cell membrane in the body, best known for its roles as a mitochondrial electron transporter and a potent membrane anti-oxidant. Much of the previous work was done in vitro in yeast and more recent work has suggested that CoQ may have additional roles prompting calls for a re-assessment of its role using in vivo systems in mammals. Here we investigated the putative role of Coenzyme Q in ethanol-induced effects in vivo using BXD RI mice. We examined hippocampal expression of Coq7 in saline controls and after an acute ethanol treatment, noting enriched biologic processes and pathways following ethanol administration. We also identified 45 ethanol-related phenotypes that were significantly correlated with Coq7 expression, including six phenotypes related to conditioned taste aversion and ethanol preference. This analysis highlights the need for further investigation of Coq7 and related genes in vivo as well as previously unrecognized roles that it may play in the hippocampus.

Mitohormesis is an adaptive response induced by a mild mitochondrial stress that promotes longevity and metabolic health in different organisms. This mechanism has been proposed as the cause of the increase in the survival in Coq7+/− (Mclk1+/−) mice, which show hepatic reduction of COQ7, early mitochondrial dysfunction and increased oxidative stress. Our study shows that the lack of COQ9 in Coq9Q95X mice triggers the reduction of COQ7, COQ6 and COQ5, which results in an increase in life expectancy. However, our results reveal that the hepatic CoQ levels are not decreased and, therefore, neither mitochondrial dysfunction or increased oxidative stress are observed in liver of Coq9Q95X mice. These data point out the tissue specific differences in CoQ biosynthesis. Moreover, our results suggest that the effect of reduced levels of COQ7 on the increased survival in Coq9Q95X mice may be due to mitochondrial mechanisms in non-liver tissues or to other unknown mechanisms., This work was supported by grants from Ministerio de Economía Competitividad, Spain, and the ERDF (Grant Number SAF2015-65786-R), from the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (grant number P10-CTS-6133) and from the University of Granada (grant reference “UNETE”, UCE-PP2017-06). AHG is a “FPU fellow” from the Ministerio de Educación Cultura y Deporte, Spain. MLS was a predoctoral fellow from the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía. LCL was supported by the “Ramón y Cajal” National Programme, Ministerio de Economía y Competitividad, Spain (RYC-2011-07643).

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors—among nature’s most hydrophobic molecules—must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q6 (IDMQ6) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of 13C6-pABA are provided, the coq5-5 mutant accumulates both 13C6-imino-demethyl-demethoxy-Q6 (13C6-IDDMQ6) and 13C6-demethyl-demethoxy-Q6 (13C6-DDMQ6). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and 13C6- pABA labeling leads to the absence of 13C6-DDMQ6, and the nitrogen-containing intermediates 13C6-4-AP and 13C6-IDDMQ6 persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q6 content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.

BACKGROUND: Coenzyme Q is an essential mitochondrial electron carrier, redox cofactor and a potent antioxidant in the majority of cellular membranes. Coenzyme Q deficiency has been associated with a range of metabolic diseases, as well as with some drug treatments and ageing. METHODS: We used whole exome sequencing (WES) to investigate patients with inherited metabolic diseases and applied a novel ultra-pressure liquid chromatography-mass spectrometry approach to measure coenzyme Q in patient samples. RESULTS: We identified a homozygous missense mutation in the COQ7 gene in a patient with complex mitochondrial deficiency, resulting in severely reduced coenzyme Q levels We demonstrate that the coenzyme Q analogue 2,4-dihydroxybensoic acid (2,4DHB) was able to specifically bypass the COQ7 deficiency, increase cellular coenzyme Q levels and rescue the biochemical defect in patient fibroblasts. CONCLUSION: We report the first patient with primary coenzyme Q deficiency due to a homozygous COQ7 mutation and a potentially beneficial treatment using 2,4DHB.

Coenzyme Q10 (CoQ10) is a redox molecule critical for the proper function of energy metabolism and antioxidant defenses. Despite its essential role in cellular metabolism, the regulation of CoQ10 biosynthesis in humans remains mostly unknown. Herein, we determined that PPTC7 is a regulatory protein of CoQ10 biosynthesis required for human cell survival. We demonstrated by in vitro approaches that PPTC7 is a bona fide protein phosphatase that dephosphorylates the human COQ7. Expression modulation experiments determined that human PPTC7 dictates cellular CoQ10 content. Using two different approaches (PPTC7 over-expression and caloric restriction), we demonstrated that PPTC7 facilitates and improves the human cell adaptation to respiratory conditions. Moreover, we determined that the physiological role of PPTC7 takes place in the adaptation to starvation and pro-oxidant conditions, facilitating the induction of mitochondrial metabolism while preventing the accumulation of ROS. Here we unveil the first post-translational mechanism regulating CoQ10 biosynthesis in humans and propose targeting the induction of PPTC7 activity/expression for the treatment of CoQ10-related mitochondrial diseases., The research group is funded by the Andalusian Government as the BIO177 group through FEDER funds (European Commission), by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (FIS PI14/01962 and FIS PI17/01286).

Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA) in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB). To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model.

The longevity gene clk-1/coq7 encodes an enzyme that is essential for the biosynthesis of coenzyme Q (CoQ) in mitochondria and regulates the lifespan and behavioral timing in Caenorhabditis elegans and the chronological lifespan in fission yeast. However, whether the mammalian clk-1/coq7 ortholog (clk-1) regulates these phenotypes in mammals remains to be fully evaluated due to the embryonic lethality of clk-1-deficient (clk-1(-/-)) mice. To investigate whether clk-1 regulates biological functions, such as growth and heartbeat, through CoQ in mouse embryos, we cultivated the cells and hearts of clk-1(-/-) mouse embryos at embryonic day 10.5 (E10.5) for at least 10 days in the presence of fetal bovine serum. In embryonic cells, cardiomyocytes, and hearts, the growth and heart rates were significantly slowed in clk-1(-/-) compared with wild-type or heterozygous mouse tissues. Moreover, frequent apoptosis and a significant reduction in mitochondrial functions, including membrane potential and ATP production, were observed in the clk-1(-/-) cells and hearts. The slowed growth and heart rates and the reduced mitochondrial function of clk-1(-/-) embryonic cells and hearts in culture were almost completely rescued by the administration of exogenous CoQ(10). The results indicate that clk-1 regulates growth and heart rates through CoQ-mediated mitochondrial functions in mouse embryos.

MCLK1 and COQ3 are mitochondrial enzymes necessary for ubiquinone biosynthesis, but only MCLK1 also regulates the partitioning of ubiquinone between mitochondrial membranes and affects longevity in mice., Mclk1 (also known as Coq7) and Coq3 code for mitochondrial enzymes implicated in the biosynthetic pathway of ubiquinone (coenzyme Q or UQ). Mclk1+/− mice are long-lived but have dysfunctional mitochondria. This phenotype remains unexplained, as no changes in UQ content were observed in these mutants. By producing highly purified submitochondrial fractions, we report here that Mclk1+/− mice present a unique mitochondrial UQ profile that was characterized by decreased UQ levels in the inner membrane coupled with increased UQ in the outer membrane. Dietary-supplemented UQ10 was actively incorporated in both mitochondrial membranes, and this was sufficient to reverse mutant mitochondrial phenotypes. Further, although homozygous Coq3 mutants die as embryos like Mclk1 homozygous null mice, Coq3+/− mice had a normal lifespan and were free of detectable defects in mitochondrial function or ubiquinone distribution. These findings indicate that MCLK1 regulates both UQ synthesis and distribution within mitochondrial membranes.

Coenzyme Q (CoQ) is a lipid-soluble antioxidant, with the oxidized (ubiquinone) and the reduced (ubiquinol) forms, and also an electron carrier for the electron transport chain. CoQ6 is synthesized by at least nine proteins (Coq1--9), and many of them form the Coq protein complex in the mitochondria in yeast. CoQ is present in the form of CoQ10 in humans. Yeast Coq1 corresponds to PDSS1 and PDSS2 proteins in humans. Mutations in PDSS or COQ genes are associated with primary CoQ10 deficiency. Secondary CoQ10 deficiency is associated with mitochondrial DNA mutation. We have demonstrated that the human COQ5, present as a mature protein without mitochondrial targeting sequence in the mitochondria, is required for maintaining CoQ10 levels. The maturation of COQ5 protein and the stability of the COQ5-containing protein complex in the mitochondria were suppressed by the conditions of mitochondrial energy deficiency. Here we further investigated the roles of COQ3 and COQ7 proteins in maintaining CoQ10 levels and in the mitochondria. Our results showed that both COQ3 and COQ7 were required for maintaining CoQ10 levels in human 143B cells, but the degree of the decrease in CoQ10 levels was greater after COQ7 knockdown than that after COQ3 knockdown. After COQ7 knockdown, but not after COQ3 knockdown, the ubiquinol-10:ubiquinone-10 ratio was increased and the peaks corresponding to the putative substrate of the COQ7 enzyme, 5-demethoxyubiquinone-10 (DMQ10/DMQ10H2), appeared on the HPLC chromatograms. The CoQ10 levels in the mitochondria were also decreased after COQ3 or COQ7 knockdown, and the putative DMQ10 was present in the mitochondria after COQ7 knockdown. Moreover, COQ3 or COQ7 knockdown caused decreased mitochondrial respiratory functions. Only COQ3 knockdown could decrease the stability of PDSS2 and several COQ proteins. Furthermore, mature forms of COQ3 and COQ7 were found in the mitochondria. Levels of the mature COQ7 protein, but not COQ3, were suppressed in 143B cells treated with the chemical uncoupler FCCP and in the cybrids harboring a pathogenic mitochondrial DNA mutation, which both had diminished CoQ10 levels. All these novel findings may further elucidate the functions of COQ proteins and their roles in human diseases.

Coenzyme Q (ubiquinone or Q) is a lipid electron and proton carrier in the electron transport chain. In yeast Saccharomyces cerevisiae eleven genes, designated COQ1 through COQ9 , YAH1 and ARH1 , have been identified as being required for Q biosynthesis. One of these genes, COQ8 ( ABC1 ), encodes an atypical protein kinase, containing six (I, II, III, VIB, VII, and VIII) of the twelve motifs characteristically present in canonical protein kinases. Here we characterize seven distinct Q-less coq8 yeast mutants and show that unlike the coq8 null mutant, each maintained normal steady-state levels of the Coq8 polypeptide. The phosphorylation states of Coq polypeptides were determined with two-dimensional gel analyses. Coq3p, Coq5p, and Coq7p were phosphorylated in a Coq8p-dependent manner. Expression of a human homolog of Coq8p, ADCK3(CABC1) bearing an amino-terminal yeast mitochondrial leader sequence, rescued growth of yeast coq8 mutants on medium containing a nonfermentable carbon source and partially restored biosynthesis of Q 6 . The phosphorylation state of several of the yeast Coq polypeptides was also rescued, indicating a profound conservation of yeast Coq8p and human ADCK3 protein kinase function in Q biosynthesis.

The development of neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington disease is strongly age-dependent. Discovering drugs that act on the high rate of aging in older individuals could be a means of combating these diseases. Reduction of the activity of the mitochondrial enzyme CLK-1 (also known as COQ7) slows down aging in Caenorhabditis elegans and in mice. Clioquinol is a metal chelator that has beneficial effects in several cellular and animal models of neurodegenerative diseases as well as on Alzheimer disease patients. Here we show that clioquinol inhibits the activity of mammalian CLK-1 in cultured cells, an inhibition that can be blocked by iron or cobalt cations, suggesting that chelation is involved in the mechanism of action of clioquinol on CLK-1. We also show that treatment of nematodes and mice with clioquinol mimics a variety of phenotypes produced by mutational reduction of CLK-1 activity in these organisms. These results suggest that the surprising action of clioquinol on several age-dependent neurodegenerative diseases with distinct etiologies might result from a slowing down of the aging process through action of the drug on CLK-1. Our findings support the hypothesis that pharmacologically targeting aging-associated proteins could help relieve age-dependent diseases.

23 páginas, 8 figuras, 2 tablas., 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., The work was supported by the Spanish Ministerio de Ciencia y Tecnología, Grant BFU2005–03017/BMC, and by NIH GM45952 to CFC.

Coenzyme Q10 (CoQ10) is essential for ATP production in the mitochondria, and is an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, but none have yet been isolated and characterized. Here we purified a CoQ10-binding protein from human urine and identified it to be saposin B, a housekeeping protein necessary for sphingolipid hydrolysis in lysosomes. We confirmed that cellular saposin B binds CoQ10 in human sperm and the hepatoma cell line HepG2 by using saposin B monoclonal antibody. The molar ratios of CoQ10 to saposin B were estimated to be 0.22 in urine, 0.003 in HepG2, and 0.12 in sperm. We then confirmed that aqueous saposin B extracts CoQ10 from hexane to form a saposin B-CoQ10 complex. Lipid binding affinity to saposin B decreased in the following order: CoQ10>CoQ9>CoQ7>>α-tocopherol>>cholesterol (no binding). The CoQ10-binding affinity to saposin B increased with pH, with maximal binding seen at pH 7.4. On the other hand, the CoQ10-donating activity of the saposin B-CoQ10 complex to erythrocyte ghost membranes increased with decreasing pH. These results suggest that saposin B binds and transports CoQ10 in human cells.

Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ(10) deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ(10) deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ(10) function are implicated. CoQ(10) deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ(10) supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

et al., Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3′-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ biosynthesis., This research was supported by grants from the Spanish Ministry of Health, Instituto de Salud Carlos III (ISCIII), FIS PI14–01962 to PN, the Spanish Ministry of Economy and Competitively, SAF2013–41945-R to JMC, and the Intramural Research Program of the NIA, NIH (KA, MG).

Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.

Coenzyme Q (Q) functions in the mitochondrial respiratory chain and serves as a lipophilic antioxidant. There is increasing interest in the use of Q as a nutritional supplement. Although, the physiological significance of Q is extensively investigated in eukaryotes, ranging from yeast to human, the eukaryotic Q biosynthesis pathway is best characterized in the budding yeast Saccharomyces cerevisiae. At least ten genes (COQ1-COQ10) have been shown to be required for Q biosynthesis and function in respiration. This review highlights recent knowledge about the endogenous synthesis of Q in eukaryotes, with emphasis on S. cerevisiae as a model system.

By regulating metabolism, mitochondria supply cells with energy, modulate cellular redox balance, and affect organismal longevity. CLK-1 in worms and COQ7 in humans are targeted to mitochondria, are cleaved to become trapped in these organelles, and then function in the biosynthesis of ubiquinone, a cofactor in the electron transport chain. Worms and mice lacking the respective genes exhibit altered mitochondrial metabolism and increased life span. Monaghan et al . detected the uncleaved form of these proteins in the nucleus. The proportion of the uncleaved, nuclear form was increased by exposing worms or HeLa cells to hydrogen peroxide and was reduced by exposure to an antioxidant. Replacing COQ7 with a R28A mutant that could not localize to the nucleus reduced proliferation without affecting ubiquinone production. Adding a single copy of a nuclear-targeted, truncation mutant of CLK-1 (CLK-1 nuc ) in clk-1 -null worms did not rescue ubiquinone production but did reduce the amount of reactive oxygen species (ROS). Paraquat causes redox stress by inhibiting mitochondrial respiration, and more CLK-1 nuc worms survived paraquat exposure than clk-1 -null worms.CLK-1 nuc partially rescued and wild-type CLK-1 completely rescued the increased life span of clk-1 -null worms. Nuclear CLK-1 or COQ7 stimulated the expression of some genes (for example, that encoding mitochondrial glutaminase) and repressed that of others (for example, those encoding the oxidoreductase WWOX, superoxide dismutase, and the proapoptotic mitochondrial protease HTRA2). Expression of the gene encoding the proapoptotic mitochondrial protease HTRA2 was increased in cells expressing only R28A COQ7 (mitochondrial COQ7), and redox-stress-induced death of these cells with only was reduced by inhibition of this protease. Nuclear CLK-1 or COQ7 inhibited the UPR mt (mitochondrial unfolded protein response). Collectively, the data suggested that the balance of nuclear and mitochondrial CLK-1 and COQ7 influences the redox status of the cell and mitochondrial stress responses to control life span. R. M. Monaghan, R. G. Barnes, K. Fisher, T. Andreou, N. Rooney, G. B. Poulin, A. J. Whitmarsh, A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity. Nat. Cell Biol. 17 , 782–792 (2015). [PubMed]

Untreated or poorly controlled arterial hypertension induced development of pathologic left ventricular hypertrophy (LVH), a common finding in hypertensive patients and a strong predictor of cardiovascular morbidity and mortality. The proteomic approach is a powerful technique to analyze a complex mixture of proteins in various settings. An experimental model of hypertension-induced early LVH was performed in spontaneously hypertensive rats, and the cardiac protein pattern compared with the normotensive Wistar Kyoto counterpart was analyzed. Fifteen altered protein spots were shown in the early stage of LVH. Compared with a previous animal model of established and regressed LVH, three protein spots were common in both models. These three altered protein spots corresponded to two unique proteins that were identified as Calsarcin-1 (CS-1) and ubiquinone biosynthesis protein COQ7 homolog. CS-1 is a negative regulator of the calcineurin/NF-AT pathway. Because upregulation in the expression levels of this protein was observed, the activation level of NF-kappaB by oxidative stress as an alternative pathway was investigated. It was found that antihypertensive therapies partially decreased oxidative stress and normalized the activation of NF-kappaB in the kidneys and aorta NF-kappaB activation but just moderately in the heart. This could be due to the interaction of any specific cardiac protein with any component of the NF-kappaB pathway. In this sense, CS-1 could be a good candidate because it is expressed preferentially in heart, to a lesser extent in smooth muscle cells, but not in kidney. Further investigations are necessary to elucidate the exact role of CS-1 and ubiquinone biosynthesis protein COQ7 in the setting of hypertension-induced LVH.

Coenzyme Q (ubiquinone or Q) functions in the respiratory electron transport chain and serves as a lipophilic antioxidant. In the budding yeast Saccharomyces cerevisiae, Q biosynthesis requires nine Coq proteins (Coq1–Coq9). Previous work suggests both an enzymatic activity and a structural role for the yeast Coq7 protein. To define the functional roles of yeast Coq7p we test whether Escherichia coli ubiF can functionally substitute for yeast COQ7. The ubiF gene encodes a flavin-dependent monooxygenase that shares no homology to the Coq7 protein and is required for the final monooxygenase step of Q biosynthesis in E. coli. The ubiF gene expressed at low copy restores growth of a coq7 point mutant (E194K) on medium containing a non-fermentable carbon source, but fails to rescue a coq7 null mutant. However, expression of ubiF from a multicopy vector restores growth and Q synthesis for both mutants, although with a higher efficiency in the point mutant. We attribute the more efficient rescue of the coq7 point mutant to higher steady state levels of the Coq3, Coq4, and Coq6 proteins and to the presence of demethoxyubiquinone, the substrate of UbiF. Coq7p co-migrates with the Coq3 and Coq4 polypeptides as a high molecular mass complex. Here we show that addition of Q to the growth media also stabilizes the Coq3 and Coq4 polypeptides in the coq7 null mutant. The data suggest that Coq7p, and the lipid quinones (demethoxyubiquinone and Q) function to stabilize other Coq polypeptides.

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q2. Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q6/mol of protein. We propose that Coq10p is a Q6-binding protein and that in the coq10 mutant Q6 it is not able to act as an electron carrier, possibly because of improper localization.

Inactivation of the Caenorhabditis elegans gene clk-1, which is required for ubiquinone biosynthesis, increases lifespan by an insulin signaling-independent mechanism. We find that homozygous inactivation of mclk1, the mouse ortholog of clk-1, yields ES cells that are protected from oxidative stress and damage to DNA. Moreover, in the livers of old mclk1+/- mice, hepatocytes that have lost mclk1 expression by loss of heterozygosity undergo clonal expansion, suggesting that their resistance to stress allows them to outcompete cells that still express the gene. mclk1+/- mice, whose growth and fertility are normal, also display a substantial increase in lifespan in each of three different genetic backgrounds. These observations indicate that the distinct mechanism by which clk-1/mclk1 affects lifespan is evolutionarily conserved from nematodes to mammals and is not tied to a particular anatomy or physiology.

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ 7 , CoQ 8 , CoQ 9 , and CoQ 10 , as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p -hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell.