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1.
Nucleic Acids Res ; 48(2): 605-632, 2020 01 24.
Article in English | MEDLINE | ID: mdl-31799603

ABSTRACT

Mitochondria participate in metabolism and signaling. They adapt to the requirements of various cell types. Publicly available expression data permit to study expression dynamics of genes with mitochondrial function (mito-genes) in various cell types, conditions and organisms. Yet, we lack an easy way of extracting these data for mito-genes. Here, we introduce the visual data mining platform mitoXplorer, which integrates expression and mutation data of mito-genes with a manually curated mitochondrial interactome containing ∼1200 genes grouped in 38 mitochondrial processes. User-friendly analysis and visualization tools allow to mine mitochondrial expression dynamics and mutations across various datasets from four model species including human. To test the predictive power of mitoXplorer, we quantify mito-gene expression dynamics in trisomy 21 cells, as mitochondrial defects are frequent in trisomy 21. We uncover remarkable differences in the regulation of the mitochondrial transcriptome and proteome in one of the trisomy 21 cell lines, caused by dysregulation of the mitochondrial ribosome and resulting in severe defects in oxidative phosphorylation. With the newly developed Fiji plugin mitoMorph, we identify mild changes in mitochondrial morphology in trisomy 21. Taken together, mitoXplorer (http://mitoxplorer.ibdm.univ-mrs.fr) is a user-friendly, web-based and freely accessible software, aiding experimental scientists to quantify mitochondrial expression dynamics.


Subject(s)
Computational Biology , Data Mining , Mitochondria/genetics , Software , Gene Expression Regulation/genetics , Humans , Mutation/genetics , Oxidative Phosphorylation , Proteome/genetics , Transcriptome/genetics
2.
Mol Cell ; 67(4): 711-723.e7, 2017 Aug 17.
Article in English | MEDLINE | ID: mdl-28820965

ABSTRACT

The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium/metabolism , Drug Discovery/methods , High-Throughput Screening Assays , Mitochondria/drug effects , Mitoxantrone/pharmacology , Saccharomyces cerevisiae/drug effects , Aequorin/metabolism , Animals , Calcium Channel Blockers/chemistry , Calcium Channels/genetics , Calcium Channels/metabolism , Dose-Response Relationship, Drug , Energy Metabolism/drug effects , HEK293 Cells , HeLa Cells , Humans , Kinetics , Lactic Acid/metabolism , Mannitol/metabolism , Membrane Potentials , Mice, Transgenic , Mitochondria/metabolism , Mitoxantrone/chemistry , Models, Molecular , Molecular Structure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Structure-Activity Relationship , Sucrose/metabolism , Xenopus laevis
3.
Proc Natl Acad Sci U S A ; 109(12): 4497-502, 2012 Mar 20.
Article in English | MEDLINE | ID: mdl-22393005

ABSTRACT

Bid-induced mitochondrial membrane permeabilization and cytochrome c release are central to apoptosis. It remains a mystery how tiny amounts of Bid synchronize the function of a large number of discrete organelles, particularly in mitochondria-rich cells. Looking at cell populations, the rate and lag time of the Bid-induced permeabilization are dose-dependent, but even very low doses lead eventually to complete cytochrome c release. By contrast, individual mitochondria display relatively rapid and uniform kinetics, indicating that the dose dependence seen in populations is due to a spreading of individual events in time. We report that Bid-induced permeabilization and cytochrome c release regularly demonstrate a wave-like pattern, propagating through a cell at a constant velocity without dissipation. Such waves do not depend on caspase activation or permeability transition pore opening. However, reactive oxygen species (ROS) scavengers suppressed the coordination of cytochrome c release and also inhibited Bid-induced cell death, whereas both superoxide and hydrogen peroxide sensitized mitochondria to Bid-induced permeabilization. Thus, Bid engages a ROS-dependent, local intermitochondrial potentiation mechanism that amplifies the apoptotic signal as a wave.


Subject(s)
BH3 Interacting Domain Death Agonist Protein/metabolism , Mitochondrial Membranes/metabolism , Adenylate Kinase/metabolism , Calcium/metabolism , Cell Death , Cytochromes c/metabolism , Flow Cytometry/methods , Green Fluorescent Proteins/metabolism , Humans , Membrane Potential, Mitochondrial , Mitochondria/metabolism , Permeability , Reactive Oxygen Species/metabolism , Signal Transduction , Superoxides/chemistry , Time Factors
4.
Am J Physiol Heart Circ Physiol ; 301(5): H1907-15, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21856920

ABSTRACT

Propagation of ryanodine receptor (RyR2)-derived Ca(2+) signals to the mitochondrial matrix supports oxidative ATP production or facilitates mitochondrial apoptosis in cardiac muscle. Ca(2+) transfer likely occurs locally at focal associations of the sarcoplasmic reticulum (SR) and mitochondria, which are secured by tethers. The outer mitochondrial membrane and inner mitochondrial membrane (OMM and IMM, respectively) also form tight focal contacts (contact points) that are enriched in voltage-dependent anion channels, the gates of OMM for Ca(2+). Contact points could offer the shortest Ca(2+) transfer route to the matrix; however, their alignment with the SR-OMM associations remains unclear. Here, in rat heart we have studied the distribution of mitochondria-associated SR in submitochondrial membrane fractions and evaluated the colocalization of SR-OMM associations with contact points using transmission electron microscopy. In a sucrose gradient designed for OMM purification, biochemical assays revealed lighter fractions enriched in OMM only and heavier fractions containing OMM, IMM, and SR markers. Pure OMM fractions were enriched in mitofusin 2, an ~80 kDa mitochondrial fusion protein and SR-mitochondrial tether candidate, whereas in fractions of OMM + IMM + SR, a lighter (~50 kDa) band detected by antibodies raised against the NH(2) terminus of mitofusin 2 was dominating. Transmission electron microscopy revealed mandatory presence of contact points at the junctional SR-mitochondrial interface versus a random presence along matching SR-free OMM segments. For each SR-mitochondrial junction at least one tether was attached to contact points. These data establish the contact points as anchorage sites for the SR-mitochondrial physical coupling. Close coupling of the SR, OMM, and IMM is likely to provide a favorable spatial arrangement for local ryanodine receptor-mitochondrial Ca(2+) signaling.


Subject(s)
Calcium Signaling , Cell Communication , Mitochondria, Heart/metabolism , Mitochondrial Membranes/metabolism , Myocardium/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Cell Fractionation , Cells, Cultured , GTP Phosphohydrolases/deficiency , GTP Phosphohydrolases/genetics , Male , Membrane Proteins/metabolism , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Mitochondria, Heart/ultrastructure , Mitochondrial Membranes/ultrastructure , Mitochondrial Proteins/metabolism , Myocardium/ultrastructure , NADP/metabolism , Oxidoreductases/metabolism , Rats , Rats, Sprague-Dawley , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/ultrastructure , Time Factors
5.
J Biol Chem ; 283(47): 32771-80, 2008 Nov 21.
Article in English | MEDLINE | ID: mdl-18790739

ABSTRACT

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.


Subject(s)
Calcium/metabolism , Mitochondria/metabolism , Myocardium/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Kinetics , Male , Membrane Potentials , Microscopy, Confocal , Microscopy, Electron, Transmission , Models, Biological , Protein Structure, Tertiary , Rats , Rats, Sprague-Dawley , Ryanodine Receptor Calcium Release Channel/metabolism , Thapsigargin/chemistry
6.
Cell Calcium ; 40(5-6): 553-60, 2006.
Article in English | MEDLINE | ID: mdl-17074387

ABSTRACT

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.


Subject(s)
Apoptosis/physiology , Calcium Signaling/physiology , Calcium/metabolism , Mitochondria/physiology , Animals , Calcium Channels/drug effects , Calcium Channels/metabolism , Endoplasmic Reticulum/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Sarcoplasmic Reticulum/metabolism
7.
J Biol Chem ; 281(25): 17347-17358, 2006 Jun 23.
Article in English | MEDLINE | ID: mdl-16597621

ABSTRACT

Cell function depends on the distribution of cytosolic and mitochondrial factors across the outer mitochondrial membrane (OMM). Passage of metabolites through the OMM has been attributed to the voltage-dependent anion-selective channel (VDAC), which can form a large conductance and permanently open a channel in lipid bilayers. However, recent data indicate that the transport of metabolites through the OMM is controlled in the cells. Recognizing that the bilayer studies had been commonly conducted at supraphysiological [Ca2+] and [K+], we determined the effect of Ca2+ on VDAC activity. In liposomes, the purified VDAC displays Ca2+-dependent control of the molecular cut-off size and shows Ca2+-regulated Ca2+ permeability in the physiological [Ca2+] range. In bilayer experiments, at submicromolar [Ca2+], the purified VDAC or isolated OMM does not show sustained large conductance but rather exhibits gating between a nonconducting state and various subconductance states. Ca2+ addition causes a reversible increase in the conductance and may evoke channel opening to full conductance. Furthermore, single cell imaging data indicate that Ca2+ may facilitate the cation and ATP transport across the OMM. Thus, the VDAC gating is dependent on the physiological concentrations of cations, allowing the OMM to control the passage of ions and some small molecules. The OMM barrier is likely to decrease during the calcium signal.


Subject(s)
Calcium/metabolism , Cell Membrane/metabolism , Mitochondrial Membranes/metabolism , Voltage-Dependent Anion Channels/chemistry , Voltage-Dependent Anion Channels/metabolism , Animals , Calcium/chemistry , Lipid Bilayers/chemistry , Liposomes/chemistry , Liposomes/metabolism , Liver/metabolism , Mitochondria, Liver/metabolism , Models, Biological , Potassium/chemistry , Protein Binding , Rats
8.
Arch Biochem Biophys ; 405(1): 104-111, 2002 Sep 01.
Article in English | MEDLINE | ID: mdl-12176063

ABSTRACT

We investigated the effects of calcium on the oxidative metabolism and steroidogenic activity of human term placental mitochondria. Submicromolar Ca(2+) concentrations stimulated state 3 oxygen consumption with 2-oxoglutarate and isocitrate and activated the 2-oxoglutarate and the NAD-isocitrate dehydrogenases by diminishing their Michaelis-Menten constants. Ca(2+) inhibited NADP-isocitrate dehydrogenase (NADP-ICDH) and the synthesis of progesterone. The NADP-ICDH maximal velocity was threefold higher than that of NAD-ICDH and had a threefold lower K(m) for isocitrate than NAD-ICDH. Isocitrate but not malate or 2-oxoglutarate supported progesterone synthesis. Calcium inhibition of progesterone synthesis was observed with isocitrate but not with malate or 2-oxoglutarate. Tight regulation of NADP-isocitrate dehydrogenase by calcium ions suggests that this enzyme plays an important role in placental mitochondrial metabolism.


Subject(s)
Calcium/metabolism , Mitochondria/metabolism , Placenta/metabolism , Dose-Response Relationship, Drug , Humans , Isocitrate Dehydrogenase/chemistry , Kinetics , Models, Biological , NADP/chemistry , Oxygen Consumption , Progesterone/biosynthesis , Progesterone/pharmacology , Steroids/metabolism , Time Factors
9.
Int J Biochem Cell Biol ; 34(8): 992-1003, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12007637

ABSTRACT

This study evaluated the effect of Ca2+ on the extramitochondrial hydrolysis of ATP and ADP by the extramitochondrial ATPase in isolated mitochondria and submitochondrial particles (SMPs) from human term placenta. The effect of different oxidizable substrates on the hydrolysis of ATP and ADP in the presence of sucrose or K+ was evaluated. Ca2+ increased phosphate release from ATP and ADP, but this stimulation showed different behavior depending on the oxidizable substrate present in the incubation media. Ca2+ stimulated the hydrolysis of ATP and ADP in the presence of sucrose. However, Ca2+ did not stimulate the hydrolysis of ADP in the medium containing K+. Ca2+ showed inhibition depending on the respiratory substrate. This study suggests that the energetic state of mitochondria controls the extramitochondrial ATPase activity, which is modulated by Ca2+ and respiratory substrates.


Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Calcium/metabolism , Mitochondria/metabolism , Placenta/metabolism , Female , Humans , Hydrolysis , Ketoglutaric Acids/pharmacology , Malates/pharmacology , Mitochondria/drug effects , Potassium Chloride/pharmacology , Pregnancy
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