Mitochondrial complex III Rieske Fe-S protein processing and assembly.

Regulation of the mitochondrial respiratory chain biogenesis is a matter of great interest because of its implications for mitochondrial disease. One of the mitochondrial disease genes recently discovered associated to encephalopathy and mitochondrial complex III (cIII) deficiency is TTC19. Our study of TTC19-deficient human and mouse models, has led us to propose a post-assembly quality control role or 'husbandry' function for this factor that is linked to Rieske Fe-S protein (UQCRFS1). UQCRFS1 is the last incorporated cIII subunit, and its presence is essential for enzymatic activity. During UQCRFS1 assembly, the precursor is cleaved and its N-terminal part remains bound to the complex, between the two core subunits (UQCRC1 and UQCRC2). In the absence of TTC19 there is a prominent accumulation of these UQCRFS1-derived N-terminal fragments that proved to be detrimental for cIII function. In this article we will discuss some ideas around the UQCRFS1 processing and assembly and its importance for the regulation of cIII activity and biogenesis

Organización modular de la cadena respitatoria de mamíferos y optogenética mitocondrial

Proponemos que la cadena respiratoria (CR) de mamíferos se organiza en módulos formados por elementos individuales o asociados entre sí, lo que determina la existencia de diferentes rutas para el flujo de electrones. Esta composición de la cadena respiratoria de mamíferos es acorde al recientemente modelo de plasticidad propuesto para la misma, según el cual, la organización del sistema se podría modular atendiendo a las necesidades fisiológicas o la demanda bioenergética de las células. Además hemos identificado por primera vez una proteína (Cox7a2l) que actúa como factor de ensamblaje de supercomplejos, esencial en la interacción entre CIII y CIV. En un último apartado, pretendemos abordar el estudio de la CR desde una aproximación de optogenética, a través de la expresion de la Bacteriorrodopsina en la membrana interna mitocondrial

Cavitating Leukoencephalopathy With Posterior Predominance Caused by a Deletion in the APOPT1 Gene in an Indian Boy.

A 5-year-old Indian boy presented with subacute onset regression of milestones associated with seizures and spasticity. The symptoms started after an attack of measles. The magnetic resonance imaging (MRI) of the brain showed cavitating leukodystrophy with posterior predominance. Molecular analysis of the APOPT1 gene, a recently described gene associated with mitochondrial leukodystrophy, showed the patient to be homozygous for a 12.82-kilobase deletion, including coding exon 3. Deletion of exon 3 produces a frameshift, predicting the translation of a truncated protein (p.Glu121Valfs*4). The patient was started on mitochondrial cocktail regimen of thiamine, riboflavin, coenzyme Q and carnitine. Although he initially showed some improvement, he died 6 months after the onset of his illness.Genetic testing for the patient was done as part of the APOPT1 research project funded by MRC (MRC-QQR grant 2015-2020 and ERC advanced grant ERC FP7-322424

SURF1 knockout cloned pigs: early onset of a severe lethal phenotype

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1−/− mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1−/− and SURF1−/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1−/− pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1−/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1−/− tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1−/− skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1−/− piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics

COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency.

BACKGROUND: Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. METHODS: Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. RESULTS: We found compound heterozygous mutations in COA7: a paternal c.410A>G, p.Y137C, and a maternal c.287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. CONCLUSIONS: We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested.Supported by Telethon-Italy grant GGP15041 (to DG); Telethon-Italy Network of Genetic Biobank grant GTB12001J; ERC advanced grant ERC FP7-322424 (to MZ). MRC QQR grant MC_UP_1002/1

Knockdown of APOPT1/COA8 Causes Cytochrome c Oxidase Deficiency, Neuromuscular Impairment, and Reduced Resistance to Oxidative Stress in Drosophila melanogaster.

Cytochrome c oxidase (COX) deficiency is the biochemical hallmark of several mitochondrial disorders, including subjects affected by mutations in apoptogenic-1 (APOPT1), recently renamed as COA8 (HGNC:20492). Loss-of-function mutations are responsible for a specific infantile or childhood-onset mitochondrial leukoencephalopathy with a chronic clinical course. Patients deficient in COA8 show specific COX deficiency with distinctive neuroimaging features, i.e., cavitating leukodystrophy. In human cells, COA8 is rapidly degraded by the ubiquitin-proteasome system, but oxidative stress stabilizes the protein, which is then involved in COX assembly, possibly by protecting the complex from oxidative damage. However, its precise function remains unknown. The CG14806 gene (dCOA8) is the Drosophila melanogaster ortholog of human COA8 encoding a highly conserved COA8 protein. We report that dCOA8 knockdown (KD) flies show locomotor defects, and other signs of neurological impairment, reduced COX enzymatic activity, and reduced lifespan under oxidative stress conditions. Our data indicate that KD of dCOA8 in Drosophila phenocopies several features of the human disease, thus being a suitable model to characterize the molecular function/s of this protein in vivo and the pathogenic mechanisms associated with its defects

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

Defective PITRM1 mitochondrial peptidase is associated with Aβ amyloidotic neurodegeneration.

Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (Aβ). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested in vitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1(+/-) heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of Aβ-positive amyloid deposits. Our results show that PITRM1 is responsible for significant Aβ degradation and that impairment of its activity results in Aβ accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.Cariplo2011‐0526 ERCFP7‐322424 Swedish Research Council Helse Vest911810 Forening for muskelsyke Italian Ministry of HealthGR‐2010‐2306‐75

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila

Abstract: Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research

CEDAR, an online resource for the reporting and exploration of complexome profiling data

Complexome profiling is an emerging ‘omics’ approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the “minimum information required for a complexome profiling experiment” (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems