David Pacheu-Grau

MITRAC links mitochondrial protein translocation to respiratory-chain assembly and translational regulation.

Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state.

Mitochondrial antibiograms in personalized medicine

Some ribosomal antibiotics used in clinical practice to fight pathogenic bacteria can provoke serious adverse drug reactions in patients. Sensitivity to the antibiotics is a multifactorial trait but the genetic variation of sensitive individuals to off-target effects of the drugs might be one of the factors contributing to this condition. Thus, the protein synthesis apparatus of mitochondria is similar to that of bacteria because of its endosymbiotic origin and, therefore, mitochondrial ribosomes are frequently unintended off-targets of these antibiotics. Because of the limitations of epidemiologic studies of pharmacogenomics, we constructed 25 transmitochondrial cell lines using platelets from individuals belonging to high-frequency European mitochondrial DNA (mtDNA) haplogroups and grew them in the absence or presence of commonly used ribosomal antibiotics. Next, we analyzed the mitochondrial synthesis of proteins and the mitochondrial oxygen consumption to ascertain whether some side effects of ribosomal drugs are due to their interaction with particular mtDNA haplogroup-defining polymorphisms. The amount of mitochondrial translation products, the p.MT-CO1/succinate dehydrogenase subunit A ratio and the ratio of respiratory complex IV quantity to citrate synthase (CS)-specific activity were significantly lower, after the treatment with linezolid, in cybrids harboring the highly frequent m.3010A allele. These results suggest that mitochondrial antibiograms should be implemented for at least the most frequent mitochondrial ribosomal RNA (rRNA) polymorphisms and combinations of polymorphisms and the most frequently used ribosomal antibiotics. In this way, we would obtain individualized barcodes for antibiotic therapy, avoid the side effects of the antibiotics and enable appropriate personalized medicine.

Cooperation between COA6 and SCO2 in COX2 Maturation during Cytochrome c Oxidase Assembly Links Two Mitochondrial Cardiomyopathies

Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency.

Influence of mtDNA genetic variation on antibiotic therapy

The human mitochondrial genome (mtDNA) encodes 13 polypeptides of the oxidative phosphorylation system, the main pathway for energy production in the cell, and 24 RNAs (2 rRNAs and 22 tRNAs) that are required for their translation in mitochondrial ribosomes. Detailed phylogenetic ana lysis of small subunit rRNA sequences consistently indicates that mitochondria derive from a-proteobacteria [1,2]. These microorganisms include a wide variety of human pathogens that can be fought with antibiotics. The bacterial ribosome is the target of more than half of all antibiotic classes used in the clinic [3]. Unfortunately, many ribosomal antibiotics can affect the mitochondrial protein synthesis [4]. Recently, it has been shown that fibroblasts from patients with mitochondrial translation defects, which are caused by mutations in the nuclear-encoded components of the mitochondrial translation system, are more vulnerable to the toxic side effects associated with certain translationtargeted antibiotics [5]. Patients with nuclear mutations in genes encoding components of the mitochondrial translation system are rare, but patients with mtDNA pathologic mutations in these types of genes are more frequent. For instance, the prevalence of the m.3243A>G/MT-TL1 (mt-tRNA) mutation in the white Australian population is 236 in 100,000 individuals (one in 424 individuals harbors this mutation) [6]. Furthermore, more than 250 different pathogenic mtDNA mutations have been identified [7]. Therefore, the number of people at risk for developing toxic side effects after therapy with translation-targeted antibiotics may be substantial. For example, erythromycin may have hastened a bioenergetic crisis in the optic nerve of a patient with the m.11778G>A mutation in a key component (p.MT-ND4) of the oxidative phosphorylation system, which resulted in bilateral vision loss and optic nerve atrophy. This case underscores the importance of being cautious with the use of drugs that might interfere with the mitochondrial protein synthesis in individuals with an underlying mitochondrial dysfunction [8].

FK506 affects mitochondrial protein synthesis and oxygen consumption in human cells

FK506 is an important immunosuppressive medication. However, it can provoke neurotoxicity, nephrotoxicity, and diabetes as adverse side effects. The decrease in oxygen consumption of rat cells treated with pharmacologically relevant concentrations of FK506, along with other evidences, has insinuated that some of the toxic effects are probably caused by drug-induced mitochondrial dysfunction at the level of gene expression. To confirm this suggestion, we have analyzed cell respiration and mitochondrial protein synthesis in human cell lines treated with FK506. This drug provokes an important decrease in oxygen consumption, accompanied by a slight reduction in the synthesis of mitochondria DNA-encoded proteins. These results are similar to those triggered by rapamycin, another macrolide with immunosuppressive properties, therefore insinuating a common toxic pathway.

Mitochondrial pharma-Q-genomics: Targeting the OXPHOS cytochrome b

Genetic variation in human cytochrome b generates structurally different coenzyme Q binding pockets, affects the coupling efficiency of the oxidative phosphorylation system and susceptibility to different medical conditions. As modification of coupling efficiency has already been shown to have therapeutic interest, these structural differences might be used to develop new drugs and allow for personalized medicine, giving rise to a new field: mitochondrial pharmacogenomics.

COX16 promotes COX2 metallation and assembly during respiratory complex IV biogenesis

Cytochrome c oxidase of the mitochondrial oxidative phosphorylation system reduces molecular oxygen with redox equivalent-derived electrons. The conserved mitochondrial-encoded COX1- and COX2-subunits are the heme- and copper-center containing core subunits that catalyze water formation. COX1 and COX2 initially follow independent biogenesis pathways creating assembly modules with subunit-specific, chaperone-like assembly factors that assist in redox centers formation. Here, we find that COX16, a protein required for cytochrome c oxidase assembly, interacts specifically with newly synthesized COX2 and its copper center-forming metallochaperones SCO1, SCO2, and COA6. The recruitment of SCO1 to the COX2-module is COX16- dependent and patient-mimicking mutations in SCO1 affect interaction with COX16. These findings implicate COX16 in CuA-site formation. Surprisingly, COX16 is also found in COX1-containing assembly intermediates and COX2 recruitment to COX1. We conclude that COX16 participates in merging the COX1 and COX2 assembly lines.

Mutations of the mitochondrial carrier translocase channel subunit TIM22 cause early-onset mitochondrial myopathy

&NA; Protein import into mitochondria is facilitated by translocases within the outer and the inner mitochondrial membranes that are dedicated to a highly specific subset of client proteins. The mitochondrial carrier translocase (TIM22 complex) inserts multispanning proteins, such as mitochondrial metabolite carriers and translocase subunits (TIM23, TIM17A/B and TIM22), into the inner mitochondrial membrane. Both types of substrates are essential for mitochondrial metabolic function and biogenesis. Here, we report on a subject, diagnosed at 1.5 years, with a neuromuscular presentation, comprising hypotonia, gastroesophageal reflux disease and persistently elevated serum and Cerebrospinal fluid lactate (CSF). Patient fibroblasts displayed reduced oxidative capacity and altered mitochondrial morphology. Using trans‐mitochondrial cybrid cell lines, we excluded a candidate variant in mitochondrial DNA as causative of these effects. Whole‐exome sequencing identified compound heterozygous variants in the TIM22 gene (NM_013337), resulting in premature truncation in one allele (p.Tyr25Ter) and a point mutation in a conserved residue (p.Val33Leu), within the intermembrane space region, of the TIM22 protein in the second allele. Although mRNA transcripts of TIM22 were elevated, biochemical analyses revealed lower levels of TIM22 protein and an even greater deficiency of TIM22 complex formation. In agreement with a defect in carrier translocase function, carrier protein amounts in the inner membrane were found to be reduced. This is the first report of pathogenic variants in the TIM22 pore‐forming subunit of the carrier translocase affecting the biogenesis of inner mitochondrial membrane proteins critical for metabolite exchange.

Pharmacologic concentrations of linezolid modify oxidative phosphorylation function and adipocyte secretome

The oxidative phosphorylation system is important for adipocyte differentiation. Therefore, xenobiotics inhibitors of the oxidative phosphorylation system could affect adipocyte differentiation and adipokine secretion. As adipokines impact the overall health status, these xenobiotics may have wide effects on human health. Some of these xenobiotics are widely used therapeutic drugs, such as ribosomal antibiotics. Because of its similarity to the bacterial one, mitochondrial translation system is an off-target for these compounds. To study the influence of the ribosomal antibiotic linezolid on adipokine production, we analyzed its effects on adipocyte secretome. Linezolid, at therapeutic concentrations, modifies the levels of apolipoprotein E and several adipokines and proteins related with the extracellular matrix. This antibiotic also alters the global methylation status of human adipose tissue-derived stem cells and, therefore, its effects are not limited to the exposure period. Besides their consequences on other tissues, xenobiotics acting on the adipocyte oxidative phosphorylation system alter apolipoprotein E and adipokine production, secondarily contributing to their systemic effects.