Editorial: Mitochondrial OXPHOS System: Emerging Concepts and Technologies and Role in Disease.

Mitochondria are eukaryotic organelles responsible for generating the main bulk of ATP, the cellular energy currency, via the process of oxidative phosphorylation (OXPHOS). The OXPHOS system is unique because it comprises subunits of dual genetic origin, encoded in the mitochondrial and the nuclear genomes. Therefore, to form the multimeric membrane-bound complexes responsible for this energy production process, proteins translated inside the organelle must be assembled in coordination with those expressed in the cytosol and imported into mitochondria by using a sophisticated import and translocation machiner

Role of COQ4 on mitochondrial DNA maintenance

Resumen del póster presentado en Mitochondrial Medicine, celebrado en Hinxton (Inglaterra) del 09 al 11 de mayo de 2018.Coenzyme Q (CoQ) is a lipidic molecule composed by a hydroquinone head and an isoprenoid chain. Since its discovery, several functions have been assigned to CoQ, being the transfer of electrons from complexes I and II to complex III in the mitochondrial respiratory chain the best known. CoQ also receives electrons from other dehydrogenases involved in different cellular processes and it is a potent membrane antioxidant. CoQ is endogenously synthesized by a set of enzymes forming a biosynthetic complex in the mitochondrial inner membrane, which has been mostly studied in yeast models. Defects in any of the genes coding for these proteins result in reduced levels of CoQ and, consequently, defects in energy production. COQ4 is one of the proteins involved in CoQ biosynthesis, but its exact enzymatic activity is still unknown. COQ4 KO HEK 293T-Rex/Flp-In cells generated by CRISPR/Cas9, as well as patient fibroblasts carrying mutations in COQ4 show the accumulation of a yet uncharacterised biosynthetic intermediate that lacks redox activity. Two candidate molecules have emerged from mass spectrometry analysis performed to identify this intermediate. On the other hand, the KO cells show a surprising phenotype related to mtDNA metabolism which may be due either to the lack of de novo synthesis of CoQ, to the biosynthetic complex instability itself, to the presence of the intermediate, or to a different and yet not characterized role of COQ4. Altogether, these results indicate a possible double function of the COQ4 protein

Loss of COX4I1 leads to combined respiratory chain deficiency and impaired mitochondrial protein synthesis

The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI

Loss of COX4I1 Leads to Combined Respiratory Chain Deficiency and Impaired Mitochondrial Protein Synthesis.

Funder: Akademie Věd České Republiky; Grant(s): RVO:67985823The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI

Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in Mpv17 knockout mice

In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly affecting the liver and the nervous system. To gain insight into physiopathology of MPV17-related disease, we investigated an available Mpv17 knockout animal model. We found severe mtDNA depletion in liver and, albeit to a lesser extent, in skeletal muscle, whereas hardly any depletion was detected in brain and kidney, up to 1 year after birth. Mouse embryonic fibroblasts did show mtDNA depletion, but only after several culturing passages, or in a serumless culturing medium. In spite of severe mtDNA depletion, only moderate decrease in respiratory chain enzymatic activities, and mild cytoarchitectural alterations, were observed in the Mpv17−/− livers, but neither cirrhosis nor failure ever occurred in this organ at any age. The mtDNA transcription rate was markedly increased in liver, which could contribute to compensate the severe mtDNA depletion. This phenomenon was associated with specific downregulation of Mterf1, a negative modulator of mtDNA transcription. The most relevant clinical features involved skin, inner ear and kidney. The coat of the Mpv17−/− mice turned gray early in adulthood, and 18-month or older mice developed focal segmental glomerulosclerosis (FSGS) with massive proteinuria. Concomitant degeneration of cochlear sensory epithelia was reported as well. These symptoms were associated with significantly shorter lifespan. Coincidental with the onset of FSGS, there was hardly any mtDNA left in the glomerular tufts. These results demonstrate that Mpv17 controls mtDNA copy number by a highly tissue- and possibly cytotype-specific mechanism

Cooperative assembly of the mitochondrial respiratory chain

Deep understanding of the pathophysiological role of the mitochondrial respiratory chain (MRC) relies on a well-grounded model explaining how its biogenesis is regulated. The lack of a consistent framework to clarify the modes and mechanisms governing the assembly of the MRC complexes and supercomplexes (SCs) works against progress in the field. The plasticity model was postulated as an attempt to explain the coexistence of mammalian MRC complexes as individual entities and associated in SC species. However, mounting data accumulated throughout the years question the universal validity of the plasticity model as originally proposed. Instead, as we argue here, a cooperative assembly model provides a much better explanation to the phenomena observed when studying MRC biogenesis in physiological and pathological settings

Mitochondrial gene expression is regulated at multiple levels and differentially in the heart and liver by thyroid hormones

Biogenesis of the oxidative phosphorylation system (OXPHOS) requires the coordinated expression of the nuclear and the mitochondrial genomes. Thyroid hormones play an important role in cell growth and differentiation and are one of the main effectors in mitochondrial biogenesis. To determine how mtDNA expression is regulated, we have investigated the response of two different tissues, the heart and liver, to the thyroid hormone status in vivo and in vitro. We show here that mtDNA expression is a tightly regulated process and that several levels of control can take place simultaneously. In addition, we show that the mechanisms operating in the control of mtDNA expression and their relevance differ between the two tissues, being gene dosage important only in heart while transcription rate and translation efficiency have more weight in liver cells. Another interesting difference is the lack of a direct effect of thyroid hormones on heart mitochondrial transcription

Tissue-specific differences in mitochondrial activity and biogenesis

Each cell type develops and maintains a specific oxidative phosphorylation (OXPHOS) capacity to satisfy its metabolic and energetic demands. This implies that there are differences between tissues in mitochondrial number, function, protein composition and morphology. The OXPHOS system biogenesis requires the coordinated expression of both mitochondrial and nuclear genomes. Mitochondrial DNA (mtDNA) expression can be regulated at different levels (replication, transcription, translation and post-translational levels) to contribute to the final observed OXPHOS activities. By analyzing five mammalian tissues, we evaluated the differences in the cellular amount of mtDNA and its correlation with the final observed mitochondrial activity. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved

Isolation of mitochondria for biogenetical studies: An update

The use of good quality preparations of isolated mitochondria is necessary when studying the mitochondrial biogenetical activities. This article explains a fast and simple method for the purification of mammalian mitochondria from different tissues and cultured cells, that is suitable for the analysis of many aspects of the organelle's biogenesis. The mitochondria isolated following the protocol described here, are highly active and capable of DNA. RNA and protein synthesis. Mitochondrial tRNA aminoacylation, mtDNA-protein interactions and specific import of added proteins into the organelles, can also be studied using this kind of preparations. (C) 2009 Elsevier B.V. and Mitochondria Research Society. All rights reserved