Brigitte Meunier

A Nonsense Mutation in COQ9 Causes Autosomal-Recessive Neonatal-Onset Primary Coenzyme Q10 Deficiency: A Potentially Treatable Form of Mitochondrial Disease

Coenzyme Q(10) is a mobile lipophilic electron carrier located in the inner mitochondrial membrane. Defects of coenzyme Q(10) biosynthesis represent one of the few treatable mitochondrial diseases. We genotyped a patient with primary coenzyme Q(10) deficiency who presented with neonatal lactic acidosis and later developed multisytem disease including intractable seizures, global developmental delay, hypertrophic cardiomyopathy, and renal tubular dysfunction. Cultured skin fibroblasts from the patient had a coenzyme Q(10) biosynthetic rate of 11% of normal controls and accumulated an abnormal metabolite that we believe to be a biosynthetic intermediate. In view of the rarity of coenzyme Q(10) deficiency, we hypothesized that the disease-causing gene might lie in a region of ancestral homozygosity by descent. Data from an Illumina HumanHap550 array were analyzed with BeadStudio software. Sixteen regions of homozygosity >1.5 Mb were identified in the affected infant. Two of these regions included the loci of two of 16 candidate genes implicated in human coenzyme Q(10) biosynthesis. Sequence analysis demonstrated a homozygous stop mutation affecting a highly conserved residue of COQ9, leading to the truncation of 75 amino acids. Site-directed mutagenesis targeting the equivalent residue in the yeast Saccharomyces cerevisiae abolished respiratory growth.

A Missense Mutation of Cytochrome Oxidase Subunit II Causes Defective Assembly and Myopathy

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast

Whereas the majority of disease‐related mitochondrial DNA mutations exhibit significant biochemical and clinical heterogeneity, mutations within the mitochondrially encoded human cytochrome b gene (MTCYB) are almost exclusively associated with isolated complex III deficiency in muscle and a clinical presentation involving exercise intolerance. Recent studies have shown that a small number of MTCYB mutations are associated with a combined enzyme complex defect involving both complexes I and III, on account of the fact that an absence of assembled complex III results in a dramatic loss of complex I, confirming a structural dependence between these two complexes. We present the biochemical and molecular genetic studies of a patient with both muscle and brain involvement and a severe reduction in the activities of both complexes I and III in skeletal muscle due to a novel mutation in the MTCYB gene that predicts the substitution (Arg318Pro) of a highly conserved amino acid. Consistent with the dramatic biochemical defect, Western blotting and BN‐PAGE experiments demonstrated loss of assembled complex I and III subunits. Biochemical studies of the equivalent amino‐acid substitution (Lys319Pro) in the yeast enzyme showed a loss of enzyme activity and decrease in the steady‐state level of bc1 complex in the mutant confirming pathogenicity.

Predicted structure and possible ionmotive mechanism of the sodium-linked NADH-ubiquinone oxidoreductase of Vibrio alginolyticus.

Two groups have now published sequences of the six genes contained in the operon coding for the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus. Sequence analyses indicate that this enzyme is unrelated to other known respiratory NADH dehydrogenases. A search for cofactor motifs suggests that the enzyme contains only one FAD, a ferredoxin‐type iron sulphur centre, and the NADH‐binding site. These are all located on NqrF, a subunit that can be recognized as a new member of a large diverse family of NAD(P)H‐oxidizing flavoenzymes. A possible model of ion‐coupling is presented, based upon this new information.

Acridinediones: Selective and Potent Inhibitors of the Malaria Parasite Mitochondrial bc1 Complex

The development of drug resistance to affordable drugs has contributed to a global increase in the number of deaths from malaria. This unacceptable situation has stimulated research for new drugs active against multidrug-resistant Plasmodium falciparum parasites. In this regard, we show here that deshydroxy-1-imino derivatives of acridine (i.e., dihydroacridinediones) are selective antimalarial drugs acting as potent (nanomolar Ki) inhibitors of parasite mitochondrial bc1 complex. Inhibition of the bc1 complex led to a collapse of the mitochondrial membrane potential, resulting in cell death (IC50 ∼15 nM). The selectivity of one of the dihydroacridinediones against the parasite enzyme was some 5000-fold higher than for the human bc1 complex, significantly higher (∼200 fold) than that observed with atovaquone, a licensed bc1-specific antimalarial drug. Experiments performed with yeast manifesting mutations in the bc1 complex reveal that binding is directed to the quinol oxidation site (Qo) of the bc1 complex. This is supported by favorable binding energies for in silico docking of dihydroacridinediones to P. falciparum bc1 Qo. Dihydroacridinediones represent an entirely new class of bc1 inhibitors and the potential of these compounds as novel antimalarial drugs is discussed.

Formation of the redox cofactor centers during Cox1 maturation in yeast cytochrome oxidase.

ABSTRACT The biogenesis of cytochrome c oxidase initiates with synthesis and maturation of the mitochondrion-encoded Cox1 subunit prior to the addition of other subunits. Cox1 contains redox cofactors, including the low-spin heme a center and the heterobimetallic heme a3:CuB center. We sought to identify the step in the maturation of Cox1 in which the redox cofactor centers are assembled. Newly synthesized Cox1 is incorporated within one early assembly intermediate containing Mss51 in Saccharomyces cerevisiae. Subsequent Cox1 maturation involves the progression to downstream assembly intermediates involving Coa1 and Shy1. We show that the two heme a cofactor sites in Cox1 form downstream of Mss51- and Coa1-containing Cox1 intermediates. These Cox1 intermediates form normally in cells defective in heme a biosynthesis or in cox1 mutant strains with heme a axial His mutations. In contrast, the Shy1-containing Cox1 assembly intermediate is perturbed in the absence of heme a. Heme a3 center formation in Cox1 appears to be chaperoned by Shy1. CuB site formation occurs near or at the Shy1-containing Cox1 assembly intermediate also. The CuB metallochaperone Cox11 transiently interacts with Shy1 by coimmunoprecipitation. The Shy1-containing Cox1 complex is markedly attenuated in cells lacking Cox11 but is partially restored with a nonfunctional Cox11 mutant. Thus, formation of the heterobimetallic CuB:heme a3 site likely occurs in the Shy1-containing Cox1 complex.

Endochin-like quinolones are highly efficacious against acute and latent experimental toxoplasmosis

Toxoplasma gondii is a widely distributed protozoan pathogen that causes devastating ocular and central nervous system disease. We show that the endochin-like quinolone (ELQ) class of compounds contains extremely potent inhibitors of T. gondii growth in vitro and is effective against acute and latent toxoplasmosis in mice. We screened 50 ELQs against T. gondii and selected two lead compounds, ELQ-271 and ELQ-316, for evaluation. ELQ-271 and ELQ-316, have in vitro IC50 values of 0.1 nM and 0.007 nM, respectively. ELQ-271 and ELQ-316 have ED50 values of 0.14 mg/kg and 0.08 mg/kg when administered orally to mice with acute toxoplasmosis. Moreover, ELQ-271 and ELQ-316 are highly active against the cyst form of T. gondii in mice at low doses, reducing cyst burden by 76–88% after 16 d of treatment. To investigate the ELQ mechanism of action against T. gondii, we demonstrate that endochin and ELQ-271 inhibit cytochrome c reduction by the T. gondii cytochrome bc1 complex at 8 nM and 31 nM, respectively. We also show that ELQ-271 inhibits the Saccharomyces cerevisiae cytochrome bc1 complex, and an M221Q amino acid substitution in the Qi site of the protein leads to >100-fold resistance. We conclude that ELQ-271 and ELQ-316 are orally bioavailable drugs that are effective against acute and latent toxoplasmosis, likely acting as inhibitors of the Qi site of the T. gondii cytochrome bc1 complex.

Mutational Analysis of Cytochrome b at the Ubiquinol Oxidation Site of Yeast Complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.

Recapitulation in Saccharomyces cerevisiae of Cytochrome b Mutations Conferring Resistance to Atovaquone in Pneumocystis jiroveci

ABSTRACT Pneumocystis jiroveci (human-derived P. carinii) is an opportunistic pathogenic fungus which causes pneumonia and is life-threatening in immunocompromised individuals. Spontaneously acquired resistance to atovaquone, a hydroxynaphthoquinone that is used to treat P. jiroveci infections, was linked to mutations in the mitochondrially encoded cytochrome b gene. Because P. jiroveci cannot be easily cultivated, we have developed Saccharomyces cerevisiae as an alternative system to study atovaquone resistance mutations. In this work, we introduced seven mutations linked with atovaquone resistance in P. jiroveci into the S. cerevisiae cytochrome b gene. The effects of the mutations on the respiratory function and on the sensitivity to the inhibitor were then characterized. Six of the reported mutations lowered the sensitivity of the S. cerevisiae bc1 complex to atovaquone, while one mutation had no effect on the drug resistance. These results were confirmed by monitoring the in vivo resistance of S. cerevisiae mutants which carried both the cytochrome b mutations and a deletion of the ABC transporter genes, allowing the drug to bypass the weakened efflux pump system. S. cerevisiae thus provides an easy-to-use system to characterize in vivo and in vitro cytochrome b mutations reported in pathogens and to assess their role in drug resistance.

Human Disease-related Mutations in Cytochrome b Studied in Yeast

Several mutations in the mitochondrially encoded cytochrome b have been reported in patients. To characterize their effect, we introduced six “human” mutations, namely G33S, S152P, G252D, Y279C, G291D, and Δ252-259 in the highly similar yeast cytochrome b. G252D showed wild type behavior in standard conditions. However, Asp-252 may interfere with structural lipid and, in consequence, destabilize the enzyme assembly, which could explain the pathogenicity of the mutation. The mutations G33S, S152P, G291D, and Δ252-259 were clearly pathogenic. They caused a severe decrease of the respiratory function and altered the assembly of the iron-sulfur protein in the bc1 complex, as observed by immunodetection. Suppressor mutations that partially restored the respiratory function impaired by S152P or G291D were found in or close to the hinge region of the iron-sulfur protein, suggesting that this region may play a role in the stable binding of the subunit to the bc1 complex. Y279C caused a significant decrease of the bc1 function and perturbed the quinol binding. The EPR spectra showed an altered signal, indicative of a lower occupancy of the Qo site. The effect of human mutation of residue 279 was confirmed by another change, Y279A, which had a more severe effect on Qo site properties. Thus by using yeast as a model system, we identified the molecular basis of the respiratory defect caused by the disease mutations in cytochrome b.