Using cultivation at different oxygen tensions, the molar ratio of cardiolipin to haem a in cells of Paracoccus denitrificans was varied systematically from 30 to 300. The molecular activity of cytochrome aa3 (with N, N, N', N'-tetramethyl-p-phenylenediamine as substrate) remained unchanged in this interval, this ruling out any regulatory effect of physiological cardiolipin levels on the terminal oxidase. Titration of anaerobically grown cells with cyanide indicated the presence of cytochrome aa3 which accounted for about 1/4 of the total electron flow from TMPD to oxygen.

• MeSH
• aerobióza MeSH
• anaerobióza MeSH
• hem metabolismus   MeSH
• kardiolipiny metabolismus   MeSH
• kyslík metabolismus   MeSH
• Paracoccus denitrificans růst a vývoj   metabolismus   MeSH
• respirační komplex IV metabolismus   MeSH
• substrátová specifita MeSH
• tetramethylfenylendiamin MeSH
• transport elektronů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hem MeSH
• kardiolipiny MeSH
• kyslík MeSH
• respirační komplex IV MeSH
• tetramethylfenylendiamin 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

• Klíčová slova
• CYTOCHROME OXIDASE *, HOUSEFLIES *, INSECTICIDES *, SUCCINATE DEHYDROGENASE *,
• MeSH
• insekticidy * MeSH
• moucha domácí * MeSH
• oxidace-redukce * MeSH
• respirační komplex II * MeSH
• respirační komplex IV * MeSH
• sukcinátdehydrogenasa * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• insekticidy * MeSH
• respirační komplex II * MeSH
• respirační komplex IV * MeSH
• sukcinátdehydrogenasa * MeSH

The Trypanosoma brucei cytochrome c oxidase (respiratory complex IV) is a very divergent complex containing a surprisingly high number of trypanosomatid-specific subunits with unknown function. To gain insight into the functional organization of this large protein complex, the expression of three novel subunits (TbCOX VII, TbCOX X and TbCOX 6080) were down-regulated by RNA interference. We demonstrate that all three subunits are important for the proper function of complex IV and the growth of the procyclic stage of T. brucei. These phenotypes were manifested by the structural instability of the complex when these indispensible subunits were repressed. Furthermore, the impairment of cytochrome c oxidase resulted in other severe mitochondrial phenotypes, such as a decreased mitochondrial membrane potential, reduced ATP production via oxidative phoshorylation and redirection of oxygen consumption to the trypanosome-specific alternative oxidase, TAO. Interestingly, the inspected subunits revealed some disparate phenotypes, particularly regarding the activity of cytochrome c reductase (respiratory complex III). While the activity of complex III was down-regulated in RNAi induced cells for TbCOX X and TbCOX 6080, the TbCOX VII silenced cell line actually exhibited higher levels of complex III activity and elevated levels of ROS formation. This result suggests that the examined subunits may have different functional roles within complex IV of T. brucei, perhaps involving the ability to communicate between sequential enzymes in the respiratory chain. In summary, by characterizing the function of three hypothetical components of complex IV, we are able to assign these proteins as genuine and indispensable subunits of the procyclic T. brucei cytochrome c oxidase, an essential component of the respiratory chain in these evolutionary ancestral and medically important parasites.

• MeSH
• fenotyp * MeSH
• genový knockdown MeSH
• kvarterní struktura proteinů MeSH
• mitochondriální protonové ATPasy metabolismus   MeSH
• mitochondrie enzymologie   MeSH
• oxidace-redukce MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• respirační komplex III genetika   metabolismus   MeSH
• respirační komplex IV genetika   metabolismus   MeSH
• RNA interference MeSH
• spotřeba kyslíku MeSH
• stabilita enzymů MeSH
• Trypanosoma brucei brucei enzymologie   genetika   růst a vývoj   MeSH
• zdroje energie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální protonové ATPasy MeSH
• podjednotky proteinů MeSH
• protozoální proteiny MeSH
• respirační komplex III MeSH
• respirační komplex IV MeSH

The loss of Surf1 protein leads to a severe COX deficiency manifested as a fatal neurodegenerative disorder, the Leigh syndrome (LS(COX)). Surf1 appears to be involved in the early step of COX assembly but its function remains unknown. The aim of the study was to find out how SURF1 gene mutations influence expression of OXPHOS and other pro-mitochondrial genes and to further characterize the altered COX assembly. Analysis of fibroblast cell lines from 9 patients with SURF1 mutations revealed a 70% decrease of the COX complex content to be associated with 32-54% upregulation of respiratory chain complexes I, III and V and accumulation of Cox5a subunit. Whole genome expression profiling showed a general decrease of transcriptional activity in LS(COX) cells and indicated that the adaptive changes in OXPHOS complexes are due to a posttranscriptional compensatory mechanism. Electrophoretic and WB analysis showed that in mitochondria of LS(COX) cells compared to controls, the assembled COX is present entirely in a supercomplex form, as I-III₂-IV supercomplex but not as larger supercomplexes. The lack of COX also caused an accumulation of I-III₂ supercomplex. The accumulated Cox5a was mainly present as a free subunit. We have found out that the major COX assembly subcomplexes accumulated due to SURF1 mutations range in size between approximately 85-140kDa. In addition to the originally proposed S2 intermediate they might also represent Cox1-containing complexes lacking other COX subunits. Unlike the assembled COX, subcomplexes are unable to associate with complexes I and III.

• MeSH
• buněčné extrakty MeSH
• buněčné linie MeSH
• cyklooxygenasa 1 genetika   metabolismus   MeSH
• cyklooxygenasa 2 genetika   metabolismus   MeSH
• deficit cytochrom-c-oxidázy genetika   metabolismus   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• Leighova nemoc genetika   metabolismus   MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• mutace genetika   MeSH
• oxidativní fosforylace MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• respirační komplex I genetika   metabolismus   MeSH
• respirační komplex III genetika   metabolismus   MeSH
• respirační komplex IV genetika   metabolismus   MeSH
• transport elektronů fyziologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• buněčné extrakty MeSH
• COX5B protein, human MeSH Prohlížeč
• cyklooxygenasa 1 MeSH
• cyklooxygenasa 2 MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• podjednotky proteinů MeSH
• PTGS1 protein, human MeSH Prohlížeč
• PTGS2 protein, human MeSH Prohlížeč
• respirační komplex I MeSH
• respirační komplex III MeSH
• respirační komplex IV MeSH
• Surf-1 protein MeSH Prohlížeč

Cytochrome c oxidase (CIV) deficiency is among the most common childhood mitochondrial disorders. The diagnosis of this deficiency is complex, and muscle biopsy is used as the gold standard of diagnosis. Our aim was to minimize the patient burden and to test the use of a dipstick immunocapture assay (DIA) to determine the amount of CIV in non-invasively obtained buccal epithelial cells. Buccal smears were obtained from five children with Leigh syndrome including three children exhibiting a previously confirmed CIV deficiency in muscle and fibroblasts and two children who were clinical suspects for CIV deficiency; the smear samples were analysed using CI and CIV human protein quantity dipstick assay kits. Samples from five children of similar age and five adults were used as controls. Analysis of the controls demonstrated that only samples of buccal cells that were frozen for a maximum of 4 h after collection provide accurate results. All three patients with confirmed CIV deficiency due to mutations in the SURF1 gene exhibited significantly lower amounts of CIV than the similarly aged controls; significantly lower amounts were also observed in two new patients, for whom later molecular analysis also confirmed pathologic mutations in the SURF1 gene. We conclude that DIA is a simple, fast and sensitive method for the determination of CIV in buccal cells and is suitable for the screening of CIV deficiency in non-invasively obtained material from children who are suspected of having mitochondrial disease.

• MeSH
• deficit cytochrom-c-oxidázy diagnóza   enzymologie   genetika   MeSH
• dospělí MeSH
• elektromyografie MeSH
• epitelové buňky enzymologie   MeSH
• fibroblasty enzymologie   MeSH
• imunosorpční techniky * MeSH
• kojenec MeSH
• kultivované buňky MeSH
• Leighova nemoc diagnóza   enzymologie   genetika   MeSH
• lidé MeSH
• membránové proteiny nedostatek   genetika   MeSH
• mitochondriální proteiny nedostatek   genetika   MeSH
• mutační analýza DNA MeSH
• neprospívání etiologie   MeSH
• předškolní dítě MeSH
• reagenční papírky * MeSH
• respirační komplex I analýza   MeSH
• respirační komplex IV analýza   MeSH
• sekvenční delece MeSH
• studie případů a kontrol MeSH
• svalová hypotonie etiologie   MeSH
• svalové mitochondrie enzymologie   MeSH
• tremor etiologie   MeSH
• ústní sliznice patologie   MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH
• Názvy látek
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• reagenční papírky * MeSH
• respirační komplex I MeSH
• respirační komplex IV MeSH
• Surf-1 protein MeSH Prohlížeč

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

This paper describes data related to a research article entitled "Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects" [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1 (-/-) ) and control (SURF1 (+/+) ) mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX), to reversible inhibition of mitochondrial translation in SURF1 (-/-) mouse and SURF1 patient fibroblast cell lines.

• Klíčová slova
• COX, Cytochrome c oxidase, Cytochrome c oxidase, DOX, doxycycline, Doxycycline, Knockout, Respiratory chain, SURF1,
• Publikační typ
• časopisecké články MeSH

Dysfunction of mitochondria induced by ischemia is considered to be a key event triggering neuronal cell death after brain ischemia. Here we report the effect of ischemia-reperfusion on mitochondrial protein synthesis and activity of cytochrome c oxidase (EC 1.9.3.1, COX). By performing 4-vessel occlusion model of global brain ischemia, we have observed that 15 min of global ischemia led to the inhibition of COX subunit I (COXI) synthesis to 56 % of control. After 1, 3 and 24 h of reperfusion, COXI synthesis was inhibited to 46, 50 and 72 % of control, respectively. Depressed synthesis of COXI was not a result of either diminished transcription of COXI gene or increased proteolytic degradation of COXI, since both Northern hybridization and Western blotting did not show significant changes in COXI mRNA and protein level. Thus, ischemia-reperfusion affects directly mitochondrial translation machinery. In addition, ischemia in duration of 15 min and consequent 1, 3 and 24 h of reperfusion led to the inhibition of COX activity to 90.3, 80.3, 81.9 and 83.5 % of control, respectively. Based on our data, we suggest that inhibition of COX activity is rather caused by ischemia-induced modification of COX polypeptides than by inhibition of mitochondrial translation.

• MeSH
• časové faktory MeSH
• down regulace MeSH
• genetická transkripce MeSH
• ischemie mozku komplikace   enzymologie   MeSH
• krysa rodu Rattus MeSH
• messenger RNA metabolismus   MeSH
• mitochondriální proteiny biosyntéza   genetika   MeSH
• mitochondrie enzymologie   MeSH
• modely nemocí na zvířatech MeSH
• mozková kůra enzymologie   MeSH
• posttranslační úpravy proteinů MeSH
• potkani Wistar MeSH
• regulace genové exprese enzymů MeSH
• reperfuzní poškození enzymologie   etiologie   MeSH
• respirační komplex IV biosyntéza   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• mitochondriální proteiny MeSH
• respirační komplex IV MeSH

Mammalian CcO (cytochrome c oxidase) is a hetero-oligomeric protein complex composed of 13 structural subunits encoded by both the mitochondrial and nuclear genomes. To study the role of nuclear-encoded CcO subunits in the assembly and function of the human complex, we used stable RNA interference of COX4, COX5A and COX6A1, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)-293 cells. Knockdown of Cox4, Cox5a and Cox6a resulted in reduced CcO activity, diminished affinity of the residual enzyme for oxygen, decreased holoCcO and CcO dimer levels, increased accumulation of CcO subcomplexes and gave rise to an altered pattern of respiratory supercomplexes. An analysis of the patterns of CcO subcomplexes found in both knockdown and overexpressing cells identified a novel CcO assembly intermediate, identified the entry points of three late-assembled subunits and demonstrated directly the essential character as well as the interdependence of the assembly of Cox4 and Cox5a. The ectopic expression of the heart/muscle-specific isoform of the Cox6 subunit (COX6A2) resulted in restoration of both CcO holoenzyme and activity in COX6A1-knockdown cells. This was in sharp contrast with the unaltered levels of COX6A2 mRNA in these cells, suggesting the existence of a fixed expression programme. The normal amount and function of respiratory complex I in all of our CcO-deficient knockdown cell lines suggest that, unlike non-human CcO-deficient models, even relatively small amounts of CcO can maintain the normal biogenesis of this respiratory complex in cultured human cells.

• MeSH
• buněčné jádro metabolismus   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• respirační komplex IV genetika   metabolismus   MeSH
• RNA interference MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• COX7B protein, human MeSH Prohlížeč
• messenger RNA MeSH
• podjednotky proteinů MeSH
• respirační komplex IV MeSH