The identification and quantification of mitochondrial effects of novel antipsychotics (brexpiprazole, cariprazine, loxapine, and lurasidone) were studied in vitro in pig brain mitochondria. Selected parameters of mitochondrial metabolism, electron transport chain (ETC) complexes, citrate synthase (CS), malate dehydrogenase (MDH), monoamine oxidase (MAO), mitochondrial respiration, and total ATP and reactive oxygen species (ROS) production were evaluated and associated with possible adverse effects of drugs. All tested antipsychotics decreased the ETC activities (except for complex IV, which increased in activity after brexpiprazole and loxapine addition). Both complex I- and complex II-linked respiration were dose-dependently inhibited, and significant correlations were found between complex I-linked respiration and both complex I activity (positive correlation) and complex IV activity (negative correlation). All drugs significantly decreased mitochondrial ATP production at higher concentrations. Hydrogen peroxide production was significantly increased at 10 μM brexpiprazole and lurasidone and at 100 μM cariprazine and loxapine. All antipsychotics acted as partial inhibitors of MAO-A, brexpiprazole and loxapine partially inhibited MAO-B. Based on our results, novel antipsychotics probably lacked oxygen uncoupling properties. The mitochondrial effects of novel antipsychotics might contribute on their adverse effects, which are mostly related to decreased ATP production and increased ROS production, while MAO-A inhibition might contribute to their antidepressant effect, and brexpiprazole- and loxapine-induced MAO-B inhibition might likely promote neuroplasticity and neuroprotection. The assessment of drug-induced mitochondrial dysfunctions is important in development of new drugs as well as in the understanding of molecular mechanism of adverse or side drug effects.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- antipsychotika klasifikace farmakologie MeSH
- chinolony farmakologie MeSH
- elektronový transportní řetězec účinky léků MeSH
- energetický metabolismus účinky léků MeSH
- inhibitory MAO farmakologie MeSH
- loxapin farmakologie MeSH
- lurasidon hydrochlorid farmakologie MeSH
- mitochondrie účinky léků metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- piperaziny farmakologie MeSH
- prasata MeSH
- reaktivní formy kyslíku metabolismus MeSH
- receptory neurotransmiterů účinky léků MeSH
- spotřeba kyslíku účinky léků MeSH
- thiofeny farmakologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné dýchání účinky léků MeSH
- buněčné linie MeSH
- elektrony MeSH
- glukosa farmakologie MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- metabolické sítě a dráhy účinky léků MeSH
- metabolomika * MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie účinky léků metabolismus MeSH
- oxidace-redukce MeSH
- oxidoreduktasy metabolismus MeSH
- prolin metabolismus MeSH
- proteom metabolismus MeSH
- protozoální proteiny metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rostlinné proteiny metabolismus MeSH
- signální transdukce MeSH
- transkriptom genetika MeSH
- transport elektronů účinky léků MeSH
- Trypanosoma brucei brucei účinky léků genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.
- MeSH
- adenosin metabolismus MeSH
- adenosintrifosfát biosyntéza MeSH
- bourec metabolismus virologie MeSH
- deoxyglukosa farmakologie MeSH
- energetický metabolismus MeSH
- glykolýza účinky léků genetika MeSH
- hmyzí proteiny metabolismus MeSH
- infekce DNA virem metabolismus virologie MeSH
- interakce hostitele a patogenu imunologie MeSH
- mezibuněčné signální peptidy a proteiny metabolismus MeSH
- nukleopolyhedroviry fyziologie MeSH
- purinergní receptory P1 genetika metabolismus MeSH
- replikace viru účinky léků MeSH
- Sf9 buňky MeSH
- Spodoptera MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Swim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. However, the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms that the upper fraction of bull sperm has not only improved motility compared to the cells in the lower fraction but also shows higher metabolic rates and longer flagella. This pattern was consistent across media of two different levels of viscosity. We conclude that the motility-based separation of the swim-up technique is also reflected in underlying metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- bazální metabolismus * MeSH
- bičík spermie metabolismus MeSH
- chov MeSH
- flagella metabolismus MeSH
- kinetika MeSH
- motilita spermií fyziologie MeSH
- oxidativní fosforylace MeSH
- skot MeSH
- spermie metabolismus MeSH
- spotřeba kyslíku MeSH
- viskozita MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ritonavir (RIT) is a widely used antiviral drug that acts as an HIV protease inhibitor with emerging potential in anticancer therapies. RIT causes inhibition of P-glycoprotein, which plays an important role in multidrug resistance (MDR) in cancer cells when overexpressed. Moreover, RIT causes mitochondrial dysfunction, leading to decreased ATP production and reduction of caveolin I expression, which can affect cell migration and tumor progression. To increase its direct antitumor activity, decrease severe side effects induced by the use of free RIT and improve its pharmacokinetics, ritonavir 5-methyl-4-oxohexanoate (RTV) was synthesized and conjugated to a tumor-targeted polymer carrier based on a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. Here we demonstrated that polymer-bound RTV enhanced the internalization of polymer-RTV conjugates, differing in RTV content from 4 to 15 wt%, in HeLa cancer cells compared with polymer without RTV. The most efficient influx and internalization properties were determined for the polymer conjugate bearing 11 wt% of RTV. This conjugate was internalized by cells using both caveolin- and clathrin-dependent endocytic pathways in contrast to the RTV-free polymer, which was preferentially internalized only by clathrin-mediated endocytosis. Moreover, we found the co-localization of the RTV-conjugate with mitochondria and a significant decrease of ATP production in treated cells. Thus, the impact on mitochondrial mechanism can influence the function of ATP-dependent P-glycoprotein and also the cell viability of MDR cancer cells. Overall, this study demonstrated that the polymer-RTV conjugate is a promising polymer-based nanotherapeutic, suitable for antitumor combination therapy with other anticancer drugs and a potential mitochondrial drug delivery system.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- antitumorózní látky aplikace a dávkování chemie MeSH
- chemorezistence účinky léků MeSH
- endocytóza účinky léků MeSH
- HeLa buňky MeSH
- kaveolin 1 biosyntéza genetika MeSH
- klathrin farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- methakryláty chemie MeSH
- nanostruktury chemie MeSH
- P-glykoprotein účinky léků metabolismus MeSH
- polymery MeSH
- ritonavir aplikace a dávkování analogy a deriváty chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
We have investigated the effect of lipid composition on interactions between cytochrome bo 3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo 3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ~80 nm.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- aktivace enzymů MeSH
- biochemické jevy MeSH
- biologické modely MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- protonové ATPasy chemie metabolismus MeSH
- protonové pumpy chemie metabolismus MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The mitochondrial network provides the central cell's energetic and regulatory unit, which besides ATP and metabolite production participates in cellular signaling through regulated reactive oxygen species (ROS) production and various protein/ion fluxes. The inner membrane forms extensive folds, called cristae, i.e. cavities enfolded from and situated perpendicularly to its inner boundary membrane portion, which encompasses an inner cylinder within the outer membrane tubule. Mitochondrial cristae ultramorphology reflects various metabolic, physiological or pathological states. Since the mitochondrion is typically a predominant superoxide source and generated ROS also serve for the creation of information redox signals, we review known relationships between ROS generation within the respiratory chain complexes of cristae and cristae morphology. Notably, it is emphasized that cristae shape is governed by ATP-synthase dimers, MICOS complexes, OPA1 isoforms and the umbrella of their regulation, and also dependent on local protonmotive force (electrical potential component) in cristae. Cristae are also affected by redox-sensitive kinases/phosphatases or p66SHC. ATP-synthase dimers decrease in the inflated intracristal space, diminishing pH and hypothetically having minimal superoxide formation. Matrix-released signaling superoxide/H2O2 is predominantly integrated along mitochondrial tubules, whereas the diffusion of intracristal signaling ROS species is controlled by crista junctions, the widening of which enables specific retrograde redox signaling such as during hypoxic cell adaptation. Other physiological cases of H2O2 release from the mitochondrion include the modulation of insulin release in pancreatic β-cells, enhancement of insulin signaling in peripheral tissues, signaling by T-cell receptors, retrograde signaling during the cell cycle and cell differentiation, specifically that of adipocytes.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- lidé MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondrie metabolismus MeSH
- signální transdukce * MeSH
- superoxidy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- ATP-synthetasa (komplexy) metabolismus MeSH
- buňky Hep G2 MeSH
- down regulace MeSH
- fyziologická adaptace fyziologie MeSH
- interakční proteinové domény a motivy MeSH
- kyslík * MeSH
- lidé MeSH
- mitochondriální dynamika fyziologie MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- mitochondrie fyziologie MeSH
- multiproteinové komplexy fyziologie MeSH
- podjednotky proteinů MeSH
- regulace genové exprese fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- fibroblasty metabolismus MeSH
- fosfáty chemie metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- makromolekulární látky chemie metabolismus MeSH
- mitochondriální ADP/ATP-translokasy chemie genetika metabolismus MeSH
- mitochondriální protonové ATPasy chemie genetika metabolismus MeSH
- mitochondrie metabolismus MeSH
- novorozená zvířata MeSH
- potkani Wistar MeSH
- srdeční mitochondrie metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
