Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

• Klíčová slova
• NDUFA6, complex I, complexome profiling, mitochondrial disease,
• MeSH
• alely MeSH
• fenotyp MeSH
• fibroblasty patologie   MeSH
• genetická heterogenita MeSH
• kojenec MeSH
• lidé MeSH
• mitochondriální nemoci genetika   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondrie genetika   MeSH
• mutace genetika   MeSH
• respirační komplex I nedostatek   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• respirační komplex I MeSH

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b6f (Cytb6f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb6f at the antenna side of PSI. The rectangular-shaped Cytb6f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1-3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb6f, may improve regulation of electron transport by the control of binding partners and distances in small domains.

• Klíčová slova
• Cytochrome b(6)f complex, Electron microscopy, NDH, Photosystem I, Supercomplex,
• MeSH
• Arabidopsis metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• chloroplasty metabolismus   MeSH
• fotosyntéza fyziologie   MeSH
• fotosystém I - proteinový komplex metabolismus   MeSH
• komplex cytochromů b6f metabolismus   MeSH
• NADH-dehydrogenasa metabolismus   MeSH
• oxidace-redukce MeSH
• světlo MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• transport elektronů fyziologie   MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl MeSH
• fotosystém I - proteinový komplex MeSH
• komplex cytochromů b6f MeSH
• NADH-dehydrogenasa MeSH
• světlosběrné proteinové komplexy MeSH

Complex I, i.e. proton-pumping NADH:quinone oxidoreductase, is an essential component of the mitochondrial respiratory chain but produces superoxide as a side-reaction. However, conditions for maximum superoxide production or its attenuation are not well understood. Unlike for Complex III, it has not been clear whether a Complex I-derived superoxide generation at forward electron transport is sensitive to membrane potential or protonmotive force. In order to investigate this, we used Amplex Red for H(2)O(2) monitoring, assessing the total mitochondrial superoxide production in isolated rat liver mitochondria respiring at state 4 as well as at state 3, namely with exclusive Complex I substrates or with Complex I substrates plus succinate. We have shown for the first time, that uncoupling diminishes rotenone-induced H(2)O(2) production also in state 3, while similar attenuation was observed in state 4. Moreover, we have found that 5-(N-ethyl-N-isopropyl) amiloride is a real inhibitor of Complex I H(+) pumping (IC(50) of 27 microM) without affecting respiration. It also partially prevented suppression by FCCP of rotenone-induced H(2)O(2) production with Complex I substrates alone (glutamate and malate), but nearly completely with Complexes I and II substrates. Sole 5-(N-ethyl-N-isopropyl) amiloride alone suppressed 20% and 30% of total H(2)O(2) production, respectively, under these conditions. Our data suggest that Complex I mitochondrial superoxide production can be attenuated by uncoupling, which means by acceleration of Complex I H(+) pumping due to the respiratory control. However, when this acceleration is prevented by 5-(N-ethyl-N-isopropyl) amiloride inhibition, no attenuation of superoxide production takes place.

• MeSH
• amilorid analogy a deriváty   farmakologie   MeSH
• biologické modely MeSH
• buněčné dýchání účinky léků   MeSH
• jaterní mitochondrie účinky léků   enzymologie   MeSH
• krysa rodu Rattus MeSH
• kyselina glutamová farmakologie   MeSH
• kyselina jantarová farmakologie   MeSH
• maláty farmakologie   MeSH
• peroxid vodíku metabolismus   MeSH
• potkani Wistar MeSH
• protonové pumpy metabolismus   MeSH
• respirační komplex I metabolismus   MeSH
• rozpřahující látky farmakologie   MeSH
• superoxidy metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amilorid MeSH
• ethylisopropylamiloride MeSH Prohlížeč
• kyselina glutamová MeSH
• kyselina jantarová MeSH
• maláty MeSH
• malic acid MeSH Prohlížeč
• peroxid vodíku MeSH
• protonové pumpy MeSH
• respirační komplex I MeSH
• rozpřahující látky MeSH
• superoxidy MeSH

Recent findings suggest that apoptotic protein apoptosis-inducing factor (AIF) may also play an important non-apoptotic function inside mitochondria. AIF was proposed to be an important component of respiratory chain complex I that is the major producer of superoxide radical. The possible role of AIF is still controversial. Superoxide production could be used as a valuable measure of complex I function, because the majority of superoxide is produced there. Therefore, we employed superoxide-specific mitochondrial fluorescence dye for detection of superoxide production. We studied an impact of AIF knockdown on function of mitochondrial complex I by analyzing superoxide production in selected cell lines. Our results show that tumoral telomerase-positive (TP) AIF knockdown cell lines display significant increase in superoxide production in comparison to control cells, while a non-tumoral cell line and tumoral telomerase-negative cell lines with alternative lengthening of telomeres (ALT) show a decrease in superoxide production. According to these results, we can conclude that AIF knockdown disrupts function of complex I and therefore increases the superoxide production in mitochondria. The distinct effect of AIF depletion in various cell lines could result from recently discovered activity of telomerase in mitochondria of TP cancer cells, but this hypothesis needs further investigation.

• MeSH
• buněčné linie MeSH
• faktor vyvolávající apoptózu genetika   fyziologie   MeSH
• fenantridiny farmakologie   MeSH
• fluorescenční barviva farmakologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• nádorové buněčné linie MeSH
• počítačové zpracování obrazu MeSH
• respirační komplex I metabolismus   MeSH
• superoxidy metabolismus   MeSH
• telomerasa metabolismus   MeSH
• telomery ultrastruktura   MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine MeSH Prohlížeč
• faktor vyvolávající apoptózu MeSH
• fenantridiny MeSH
• fluorescenční barviva MeSH
• respirační komplex I MeSH
• superoxidy MeSH
• telomerasa MeSH

BACKGROUND: Maternally inherited complex I deficiencies due to mutations in MT-ND genes represent a heterogeneous group of multisystem mitochondrial disorders (MD) with a unfavourable prognosis. The aim of the study was to characterize the impact of the mutations in MT-ND genes, including the novel m.13091 T > C variant, on the course of the disease, and to analyse the activities of respiratory chain complexes, the amount of protein subunits, and the mitochondrial energy-generating system (MEGS) in available muscle biopsies and cultivated fibroblasts. METHODS: The respiratory chain complex activities were measured by spectrophotometry, MEGS were analysed using radiolabelled substrates, and protein amount by SDS-PAGE or BN-PAGE in muscle or fibroblasts. RESULTS: In our cohort of 106 unrelated families carrying different mtDNA mutations, we found heteroplasmic mutations in the genes MT-ND1, MT-ND3, and MT-ND5, including the novel variant m.13091 T > C, in 13 patients with MD from 12 families. First symptoms developed between early childhood and adolescence and progressed to multisystem disease with a phenotype of Leigh or MELAS syndromes. MRI revealed bilateral symmetrical involvement of deep grey matter typical of Leigh syndrome in 6 children, cortical/white matter stroke-like lesions suggesting MELAS syndrome in 3 patients, and a combination of cortico-subcortical lesions and grey matter involvement in 4 patients. MEGS indicated mitochondrial disturbances in all available muscle samples, as well as a significantly decreased oxidation of [1-14C] pyruvate in fibroblasts. Spectrophotometric analyses revealed a low activity of complex I and/or complex I + III in all muscle samples except one, but the activities in fibroblasts were mostly normal. No correlation was found between complex I activities and mtDNA mutation load, but higher levels of heteroplasmy were generally found in more severely affected patients. CONCLUSIONS: Maternally inherited complex I deficiencies were found in 11% of families with mitochondrial diseases in our region. Six patients manifested with Leigh, three with MELAS. The remaining four patients presented with an overlap between these two syndromes. MEGS, especially the oxidation of [1-14C] pyruvate in fibroblasts might serve as a sensitive indicator of functional impairment due to MT-ND mutations. Early onset of the disease and higher level of mtDNA heteroplasmy were associated with a worse prognosis.

• Klíčová slova
• Complex I, Leigh syndrome, MEGS, MELAS syndrome, MT-ND genes, Mitochondria, mtDNA,
• MeSH
• biopsie MeSH
• dítě MeSH
• dospělí MeSH
• fibroblasty metabolismus   MeSH
• kojenec MeSH
• kosterní svaly metabolismus   MeSH
• kultivované buňky MeSH
• Leighova nemoc genetika   MeSH
• lidé MeSH
• magnetická rezonanční tomografie MeSH
• mitochondriální DNA * MeSH
• mitochondriální nemoci genetika   MeSH
• mladiství MeSH
• mozek diagnostické zobrazování   patologie   MeSH
• mutace * MeSH
• novorozenec MeSH
• respirační komplex I nedostatek   genetika   metabolismus   MeSH
• syndrom MELAS genetika   MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mitochondriální DNA * MeSH
• respirační komplex I MeSH

The requirement of complex I (NADH:ubiquionone oxidoreductase) for respiration in Trypanosoma brucei is controversial. Recent identification of homologues of its subunits in mitochondrial proteome resolved a question of its presence or absence. However, with one exception, no data have been available concerning the function(s) of complex I or its subunits. Here we present a functional RNAi study of three (NUBM, NUKM, NUEM) putative subunits of this complex. Although no changes were detected in growth, mitochondrial membrane potential or reactive oxygen species production in cell lines depleted for target transcript, the NUBM and NUKM RNAi knock-downs showed decreased specific NADH:ubiquinone oxidoreductase activity. Moreover, glycerol gradients of all cell lines revealed the presence of two distinct peaks of NADH dehydrogenase activity, with shifted sensitivity to inhibitors of complex I upon RNAi induction. Thus complex I is not only present in the procyclic stage of T. brucei 29-13 strain, but it does participate in electron transport chain.

• MeSH
• buněčné dýchání MeSH
• genový knockdown MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• mikrobiální viabilita * MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační komplex I genetika   metabolismus   MeSH
• Trypanosoma brucei brucei enzymologie   růst a vývoj   metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá interferující RNA MeSH
• podjednotky proteinů MeSH
• reaktivní formy kyslíku MeSH
• respirační komplex I MeSH

Hydrogenosomes are double-membraned ATP-producing and hydrogen-producing organelles of diverse anaerobic eukaryotes. In some versions of endosymbiotic theory they are suggested to be homologues of mitochondria, but alternative views suggest they arose from an anaerobic bacterium that was distinct from the mitochondrial endosymbiont. Here we show that the 51-kDa and 24-kDa subunits of the NADH dehydrogenase module in complex I, the first step in the mitochondrial respiratory chain, are active in hydrogenosomes of Trichomonas vaginalis. Like mitochondrial NADH dehydrogenase, the purified Trichomonas enzyme can reduce a variety of electron carriers including ubiquinone, but unlike the mitochondrial enzyme it can also reduce ferredoxin, the electron carrier used for hydrogen production. The presence of NADH dehydrogenase solves the long-standing conundrum of how hydrogenosomes regenerate NAD+ after malate oxidation. Phylogenetic analyses show that the Trichomonas 51-kDa homologue shares common ancestry with the mitochondrial enzyme. Recruitment of complex I subunits into a H2-producing pathway provides evidence that mitochondria and hydrogenosomes are aerobic and anaerobic homologues of the same endosymbiotically derived organelle.

• MeSH
• aerobióza MeSH
• anaerobióza MeSH
• biologické modely MeSH
• fylogeneze MeSH
• maláty metabolismus   MeSH
• mitochondrie enzymologie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• NAD metabolismus   MeSH
• NADH-dehydrogenasa chemie   metabolismus   MeSH
• organely enzymologie   metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• respirační komplex I chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• symbióza MeSH
• Trichomonas vaginalis cytologie   enzymologie   metabolismus   MeSH
• vodík metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• maláty MeSH
• malic acid MeSH Prohlížeč
• NAD MeSH
• NADH-dehydrogenasa MeSH
• podjednotky proteinů MeSH
• protozoální proteiny MeSH
• respirační komplex I MeSH
• vodík MeSH

The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.

• MeSH
• alely MeSH
• dospělí MeSH
• exom genetika   MeSH
• Fanconiho syndrom genetika   patologie   MeSH
• genetická predispozice k nemoci MeSH
• heterozygot MeSH
• homozygot MeSH
• ledviny metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mapování chromozomů MeSH
• mitochondriální nemoci genetika   metabolismus   patologie   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondrie metabolismus   patologie   MeSH
• mutace MeSH
• plíce metabolismus   patologie   MeSH
• respirační komplex I genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Kanada MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• NDUFAF6 protein, human MeSH Prohlížeč
• respirační komplex I MeSH

OBJECTIVES: Inhibition of the enzyme acetylcholinesterase (AChE) is the main mechanism both of therapeutic action of drugs for the treatment of Alzheimer's disease and toxic action of organophosphorus compounds. Various types of oximes reactivate AChE and are commonly used as antidotes against organophosphates (pesticides, nerve agents). METHODS: Effects both of AChE inhibitors (tacrine, 7-methoxytacrine) and oximes (pralidoxime, trimedoxime, obidoxime, methoxime, HI-6) on Complex I of electron transport chain (ETC) were examined. The enzyme activity was measured spectrophotometrically in crude mitochondrial fraction isolated from pig brain. RESULTS: Our results showed statistically significant Complex I inhibition by tacrine, other drugs did not affect the enzyme activity significantly. CONCLUSIONS: These observations suggest the possibility of tacrine-induced side effects related to disturbance in ETC. On the contrary, it seems that oximes do not affect cellular energetic metabolism.

• MeSH
• cholinesterasové inhibitory farmakologie   MeSH
• elektronový transportní řetězec antagonisté a inhibitory   účinky léků   MeSH
• energetický metabolismus účinky léků   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• mozek - chemie účinky léků   MeSH
• oximy farmakologie   MeSH
• prasata MeSH
• reaktivátory cholinesterázy farmakologie   MeSH
• respirační komplex I metabolismus   MeSH
• takrin analogy a deriváty   farmakologie   MeSH
• techniky in vitro MeSH
• transport elektronů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 7-methoxytacrine MeSH Prohlížeč
• cholinesterasové inhibitory MeSH
• elektronový transportní řetězec MeSH
• oximy MeSH
• reaktivátory cholinesterázy MeSH
• respirační komplex I MeSH
• takrin MeSH

The biological conversion of light energy into chemical energy is performed by a flexible photosynthetic machinery located in the thylakoid membranes. Photosystems I and II (PSI and PSII) are the two complexes able to harvest light. PSI is the last complex of the electron transport chain and is composed of multiple subunits: the proteins building the catalytic core complex that are well conserved between oxygenic photosynthetic organisms, and, in green organisms, the membrane light-harvesting complexes (Lhc) necessary to increase light absorption. In plants, four Lhca proteins (Lhca1-4) make up the antenna system of PSI, which can be further extended to optimize photosynthesis by reversible binding of LHCII, the main antenna complex of photosystem II. Here, we used biochemistry and electron microscopy in Arabidopsis to reveal a previously unknown supercomplex of PSI with LHCII that contains an additional Lhca1-a4 dimer bound on the PsaB-PsaI-PsaH side of the complex. This finding contradicts recent structural studies suggesting that the presence of an Lhca dimer at this position is an exclusive feature of algal PSI. We discuss the features of the additional Lhca dimer in the large plant PSI-LHCII supercomplex and the differences with the algal PSI. Our work provides further insights into the intricate structural plasticity of photosystems.

• Klíčová slova
• Arabidopsis thaliana, LHCII, Lhca, light-harvesting complex, photosynthesis, photosystem I,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• elektronová mikroskopie MeSH
• elektronový transportní řetězec metabolismus   MeSH
• fosforylace MeSH
• fotosyntéza MeSH
• fotosystém I - proteinový komplex metabolismus   MeSH
• fotosystém II - proteinový komplex metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny vázající chlorofyl metabolismus   MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elektronový transportní řetězec MeSH
• fotosystém I - proteinový komplex MeSH
• fotosystém II - proteinový komplex MeSH
• LHCA1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteiny vázající chlorofyl MeSH