Purpose: Retinal ischemia (RI) and progressive neuronal death are sight-threatening conditions. Mitochondrial (mt) dysfunction and fusion/fission processes have been suggested to play a role in the pathophysiology of RI. This study focuses on changes in the mt parameters of the neuroretina, retinal pigment epithelium (RPE) and choroid in a porcine high intraocular pressure (IOP)-induced RI minipig model. Methods: In one eye, an acute IOP elevation was induced in minipigs and compared to the other control eye. Activity and amount of respiratory chain complexes (RCC) were analyzed by spectrophotometry and Western blot, respectively. The coenzyme Q10 (CoQ10) content was measured using HPLC, and the ultrastructure of the mt was studied via transmission electron microscopy. The expression of selected mt-pathway genes was determined by RT-PCR. Results: At a functional level, increased RCC I activity and decreased total CoQ10 content were found in RPE cells. At a protein level, CORE2, a subunit of RCC III, and DRP1, was significantly decreased in the neuroretina. Drp1 and Opa1, protein-encoding genes responsible for mt quality control, were decreased in most of the samples from the RPE and neuroretina. Conclusions: The eyes of the minipig can be considered a potential RI model to study mt dysfunction in this disease. Strategies targeting mt protection may provide a promising way to delay the acute damage and onset of RI.

• Klíčová slova
• coenzyme Q10, minipig, mitochondrial dysfunction, retinal ischemia,
• MeSH
• glaukom * metabolismus   MeSH
• ischemie metabolismus   MeSH
• karcinom z renálních buněk * metabolismus   MeSH
• miniaturní prasata MeSH
• mitochondrie metabolismus   MeSH
• nádory ledvin * metabolismus   MeSH
• nitrooční tlak MeSH
• prasata MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• COX, COX4, OXPHOS, biogenesis interdependency, cI, cIV, cIV assembly, complex I, complexome profiling, knock-out, mitochondria, mitochondrial protein synthesis,
• MeSH
• glykolýza MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mitochondriální nemoci metabolismus   MeSH
• mitochondriální proteiny biosyntéza   MeSH
• oxidativní fosforylace MeSH
• podjednotky proteinů metabolismus   MeSH
• proteosyntéza * MeSH
• respirační komplex IV metabolismus   MeSH
• spotřeba kyslíku MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• COX4I1 protein, human MeSH Prohlížeč
• mitochondriální proteiny MeSH
• podjednotky proteinů MeSH
• respirační komplex IV MeSH

Cytochrome c oxidase (COX), the terminal enzyme of mitochondrial electron transport chain, couples electron transport to oxygen with generation of proton gradient indispensable for the production of vast majority of ATP molecules in mammalian cells. The review summarizes current knowledge of COX structure and function of nuclear-encoded COX subunits, which may modulate enzyme activity according to various conditions. Moreover, some nuclear-encoded subunits posess tissue-specific and development-specific isoforms, possibly enabling fine-tuning of COX function in individual tissues. The importance of nuclear-encoded subunits is emphasized by recently discovered pathogenic mutations in patients with severe mitopathies. In addition, proteins substoichiometrically associated with COX were found to contribute to COX activity regulation and stabilization of the respiratory supercomplexes. Based on the summarized data, a model of three levels of quaternary COX structure is postulated. Individual structural levels correspond to subunits of the i) catalytic center, ii) nuclear-encoded stoichiometric subunits and iii) associated proteins, which may constitute several forms of COX with varying composition and differentially regulated function.

• MeSH
• buněčné jádro enzymologie   genetika   MeSH
• genom MeSH
• lidé MeSH
• mitochondriální nemoci enzymologie   patologie   MeSH
• mitochondrie enzymologie   genetika   MeSH
• orgánová specificita MeSH
• podjednotky proteinů MeSH
• respirační komplex IV genetika   metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• podjednotky proteinů MeSH
• respirační komplex IV MeSH

Skeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model.This article has an associated First Person interview with the first author of the paper.

• Klíčová slova
• Biomarkers, Disease development, HD large animal model, Huntington's disease, Mitochondrial function, Skeletal muscle, Ultrastructure,
• MeSH
• DNA metabolismus   MeSH
• energetický metabolismus * MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc metabolismus   patologie   MeSH
• kosterní svaly metabolismus   ultrastruktura   MeSH
• lidé MeSH
• miniaturní prasata MeSH
• mitochondriální proteiny metabolismus   MeSH
• modely nemocí na zvířatech * MeSH
• mutace MeSH
• oxidativní fosforylace MeSH
• prasata MeSH
• progrese nemoci MeSH
• protein huntingtin genetika   MeSH
• svalové mitochondrie metabolismus   ultrastruktura   MeSH
• tělesná hmotnost MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• HTT protein, human MeSH Prohlížeč
• mitochondriální proteiny MeSH
• protein huntingtin MeSH

Huntington's disease (HD) is a progressive neurodegenerative disorder primarily affecting the basal ganglia and is caused by expanded CAG repeats in the huntingtin gene. Except for CAG sizing, mitochondrial and nuclear DNA (mtDNA and nDNA) parameters have not yet proven to be representative biomarkers for disease and future therapy. Here, we identified a general suppression of genes associated with aerobic metabolism in peripheral blood mononuclear cells (PBMCs) from HD patients compared to controls. In HD, the complex II subunit SDHB was lowered although not sufficiently to affect complex II activity. Nevertheless, we found decreased level of factors associated with mitochondrial biogenesis and an associated dampening of the mitochondrial DNA damage frequency in HD, implying an early defect in mitochondrial activity. In contrast to mtDNA, nDNA from HD patients was four-fold more modified than controls and demonstrated that nDNA integrity is severely reduced in HD. Interestingly, the level of nDNA damage correlated inversely with the total functional capacity (TFC) score; an established functional score of HD. Our data show that PBMCs are a promising source to monitor HD progression and highlights nDNA damage and diverging mitochondrial and nuclear genome responses representing early cellular impairments in HD.

• MeSH
• dospělí MeSH
• Huntingtonova nemoc genetika   patologie   MeSH
• leukocyty mononukleární metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mitochondriální DNA analýza   genetika   MeSH
• mitochondrie metabolismus   patologie   MeSH
• mladý dospělý MeSH
• nestabilita genomu * MeSH
• poškození DNA * MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.

• Klíčová slova
• Cytochrome c oxidase, Doxycycline, Leigh syndrome, Pulse-chase, Respiratory supercomplexes, SURF1(−/−) mouse knockout,
• MeSH
• druhová specificita MeSH
• fibroblasty metabolismus   patologie   MeSH
• Leighova nemoc genetika   metabolismus   patologie   MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• orgánová specificita MeSH
• respirační komplex IV genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• respirační komplex IV MeSH
• Surf-1 protein MeSH Prohlížeč

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

• MeSH
• apoptóza MeSH
• ATPázy spojené s různými buněčnými aktivitami MeSH
• genový knockdown MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• lidé MeSH
• metaloendopeptidasy metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální proteiny MeSH
• mitochondrie metabolismus   MeSH
• NADH, NADPH oxidoreduktasy metabolismus   MeSH
• proliferace buněk * MeSH
• proteasy závislé na ATP metabolismus   MeSH
• proteasy metabolismus   MeSH
• protein - isoformy metabolismus   MeSH
• respirační komplex I MeSH
• respirační komplex IV metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   metabolismus   MeSH
• transport elektronů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATPázy spojené s různými buněčnými aktivitami MeSH
• GTP-fosfohydrolasy MeSH
• metaloendopeptidasy MeSH
• mitochondriální proteiny MeSH
• NADH, NADPH oxidoreduktasy MeSH
• NDUFB6 protein, human MeSH Prohlížeč
• OPA1 protein, human MeSH Prohlížeč
• proteasy závislé na ATP MeSH
• proteasy MeSH
• protein - isoformy MeSH
• respirační komplex I MeSH
• respirační komplex IV MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• YME1 protein, S cerevisiae MeSH Prohlížeč
• YME1L1 protein, human MeSH Prohlížeč