Sulfhydryl oxidase Erv1 is a ubiquitous and conserved protein of the mitochondrial intermembrane space that plays a role in the transport of small sulfur-containing proteins. In higher eukaryotes, Erv1 interacts with the mitochondrial import protein Mia40. However, Trypanosoma brucei lacks an obvious Mia40 homologue in its genome. Here we show by tandem affinity purification and mass spectrometry that in this excavate protist, Erv1 functions without a Mia40 homologue and most likely any other interaction partner. Down-regulation of TbErv1 caused a reduction of the mitochondrial membrane potential already within 24h to less than 50% when compared with control cells. The depletion of TbErv1 was accompanied by accumulation of trCOIV precursor, with a concomitant reduction of aconitase activity both in the cytosol and mitochondrion. Overall, TbErv1 seems to have a role in the mitochondrial translocation and Fe-S cluster assembly in the organelle.

• MeSH
• hmotnostní spektrometrie MeSH
• mapování interakce mezi proteiny MeSH
• mitochondrie enzymologie   metabolismus   MeSH
• oxidoreduktasy metabolismus   MeSH
• proteiny obsahující železo a síru metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• Trypanosoma brucei brucei enzymologie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

The respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH : ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH : ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH : ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD+ regeneration in the mitochondrion of procyclic T. brucei.

• MeSH
• cytosol enzymologie   MeSH
• intracelulární membrány metabolismus   MeSH
• membránové potenciály MeSH
• mitochondrie enzymologie   MeSH
• NAD metabolismus   MeSH
• NADH-dehydrogenasa genetika   metabolismus   MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• respirační komplex I MeSH
• spotřeba kyslíku MeSH
• transport elektronů MeSH
• Trypanosoma brucei brucei enzymologie   genetika   růst a vývoj   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins.

• MeSH
• membránové proteiny metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální proteiny metabolismus   fyziologie   MeSH
• mitochondrie metabolismus   MeSH
• transport proteinů MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Trypanosoma brucei brucei metabolismus   fyziologie   MeSH
• Trypanosoma metabolismus   fyziologie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• anionty metabolismus   MeSH
• lipidové dvojvrstvy * MeSH
• mastné kyseliny metabolismus   MeSH
• mitochondriální ADP/ATP-translokasy metabolismus   MeSH
• protony * MeSH
• Publikační typ
• časopisecké články MeSH

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
• genový knockdown MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• lidé MeSH
• metaloendopeptidasy metabolismus   MeSH
• mitochondriální membrány metabolismus   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 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

Mitochondria primarily serve as source of cellular energy through the Krebs cycle and beta-oxidation to generate substrates for oxidative phosphorylation. Redox reactions are used to transfer electrons through a gradient to their final acceptor, oxygen, and to pump hydrogen protons into the intermembrane space. Then, ATP synthase uses the electrochemical gradient to generate adenosine triphosphate (ATP). During these processes, reactive oxygen species (ROS) are generated. ROS are highly reactive molecules with important physiological functions in cellular signaling. Mitochondria play a crucial role in intracellular calcium homeostasis and serve as transient calcium stores. High levels of both, ROS and free cytosolic calcium, can damage mitochondrial and cellular structures and trigger apoptosis. Impaired mitochondrial function has been described in many psychiatric diseases, including mood disorders, in terms of lowered mitochondrial membrane potential, suppressed ATP formation, imbalanced Ca(2+) levels and increased ROS levels. In vitro models have indicated that mood stabilizers affect mitochondrial respiratory chain complexes, ROS production, ATP formation, Ca(2+) buffering and the antioxidant system. Most studies support the hypothesis that mitochondrial dysfunction is a primary feature of mood disorders. The precise mechanism of action of mood stabilizers remains unknown, but new mitochondrial targets have been proposed for use as mood stabilizers and mitochondrial biomarkers in the evaluation of therapy effectiveness.

• MeSH
• duševní poruchy farmakoterapie   patofyziologie   MeSH
• energetický metabolismus MeSH
• kyselina valproová MeSH
• lidé MeSH
• lithium MeSH
• mitochondrie účinky léků   fyziologie   MeSH
• psychotropní léky farmakologie   terapeutické užití   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• signální transdukce MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O(2), unexpectedly find O(2) and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O(2) by TbERV1 is not dependent upon a simple O(2) channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes.

• MeSH
• cytochromy c chemie   MeSH
• fylogeneze MeSH
• genový knockdown MeSH
• kinetika MeSH
• kyslík chemie   MeSH
• mitochondriální proteiny chemie   genetika   MeSH
• mitochondrie enzymologie   ultrastruktura   MeSH
• molekulární evoluce MeSH
• mutageneze cílená MeSH
• oxidace-redukce MeSH
• oxidancia MeSH
• oxidoreduktasy chemie   genetika   MeSH
• protozoální proteiny chemie   genetika   MeSH
• RNA interference MeSH
• sbalování proteinů MeSH
• substituce aminokyselin MeSH
• transport proteinů MeSH
• Trypanosoma brucei brucei cytologie   enzymologie   MeSH
• zduření mitochondrií MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mitochondrial contact site and cristae organization system (MICOS) is a multiprotein complex responsible for cristae formation. Even though cristae are found in all mitochondria capable of oxidative phosphorylation, only Mic10 and Mic60 appear to be conserved throughout eukaryotes. The remaining 4 or 5 known MICOS subunits are specific to the supergroup Opisthokonta, which includes yeast and mammals that are the only organisms in which this complex has been analyzed experimentally. We have isolated the MICOS from Trypanosoma brucei, a member of the supergroup Excavata that is profoundly diverged from opisthokonts. We show that it is required for the maintenance of the unique discoidal cristae that typify excavates, such as euglenids and kinetoplastids, the latter of which include trypanosomes. The trypanosome MICOS consists of 9 subunits, most of which are essential for normal growth. Unlike in opisthokonts, it contains two distinct Mic10 orthologs and an unconventional putative Mic60 that lacks a mitofilin domain. Interestingly, one of the essential trypanosomatid-specific MICOS subunits called TbMic20 is a thioredoxin-like protein that appears to be involved in import of intermembrane space proteins, including respiratory chain complex assembly factors. This result points to trypanosome MICOS coordinating cristae shaping and population of its membrane with proteins involved in respiration, the latter via the catalytic activity of TbMic20. Thus, trypanosome MICOS allows us to define which of its features are conserved in all eukaryotes and decipher those that represent lineage-specific adaptations.

• MeSH
• mitochondriální membrány fyziologie   MeSH
• multiproteinové komplexy metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• Trypanosoma brucei brucei fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

• MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• mutace MeSH
• proteiny chemie   genetika   farmakologie   MeSH
• protonové ATPasy antagonisté a inhibitory   MeSH
• regulace genové exprese * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• skot MeSH
• Trypanosoma brucei brucei enzymologie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA.

• MeSH
• apoptóza účinky léků   MeSH
• chemorezistence MeSH
• cílená molekulární terapie * MeSH
• cisplatina farmakologie   MeSH
• kyselina jantarová metabolismus   MeSH
• lidé MeSH
• mitochondrie účinky léků   enzymologie   MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny antagonisté a inhibitory   MeSH
• neuroblastom patologie   MeSH
• oxidace-redukce MeSH
• protinádorové látky farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační komplex II antagonisté a inhibitory   MeSH
• screeningové testy protinádorových léčiv MeSH
• spotřeba kyslíku účinky léků   MeSH
• superoxidy metabolismus   MeSH
• synergismus léků MeSH
• thenoyltrifluoraceton farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH