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The Journal of Biological Chemistry Jul 2019Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial...
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-C]glucose to [C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.
Topics: Acetyl Coenzyme A; Acyltransferases; Animals; Carbon; Cardiolipins; Cell Line; Citric Acid Cycle; Energy Metabolism; Enzyme Activation; Lipids; Mice; Mice, Knockout; Mitochondria; Pyruvate Carboxylase; Pyruvate Dehydrogenase Complex; Transcription Factors
PubMed: 31186346
DOI: 10.1074/jbc.RA119.009037 -
Biochimica Et Biophysica Acta.... Nov 2020The physical and functional organisation of the OXPHOS system in mitochondria in vivo remains elusive. At present, different models of OXPHOS arrangement, representing...
The physical and functional organisation of the OXPHOS system in mitochondria in vivo remains elusive. At present, different models of OXPHOS arrangement, representing either highly ordered respiratory strings or, vice versa, a set of randomly dispersed supercomplexes and respiratory complexes, have been suggested. In the present study, we examined a supramolecular arrangement of the OXPHOS system in pea shoot mitochondria using digitonin solubilisation of its constituents, which were further analysed by classical BN-related techniques and a multidimensional gel electrophoresis system when required. As a result, in addition to supercomplexes IIII, IIIIIV and IIIIV, dimer V, and individual complexes I-V previously detected in plant mitochondria, new OXPHOS structures were also revealed. Of them, (1) a megacomplex (IIIIIIV)n including complex II, (2) respirasomes IIIIIV with two copies of complex I and dimeric complex III, (3) a minor new supercomplex IVVa comigrating with IIII, and (4) a second minor form of ATP synthase, Va, were found. The activity of singular complexes I, IV, and V was higher than the activity of the associated forms. The detection of new supercomplex IVVa, along with assemblies IIII and IIIIIV, prompted us to suggest the occurrence of in vivo oxphosomes comprising complexes I, III, IV, and V. The putative oxphosome's stoichiometry, historical background, assumed functional significance, and subcompartmental location are discussed herein.
Topics: Mitochondria; Mitochondrial Proteins; Mitochondrial Proton-Translocating ATPases; Multienzyme Complexes; Oxidative Phosphorylation; Pisum sativum; Plant Proteins; Plant Shoots
PubMed: 32663476
DOI: 10.1016/j.bbabio.2020.148264 -
Biochimie Apr 2023Accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of D-2-hydroxyglutaric aciduria type I and, particularly, of D-2-hydroxyglutaric aciduria...
Accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of D-2-hydroxyglutaric aciduria type I and, particularly, of D-2-hydroxyglutaric aciduria type II (D2HGA2). D2HGA2 is a metabolic inherited disease caused by gain-of-function mutations in the gene isocitrate dehydrogenase 2. It is clinically characterized by neurological abnormalities and a severe cardiomyopathy whose pathogenesis is still poorly established. The present work investigated the potential cardiotoxicity D-2-HG, by studying its in vitro effects on a large spectrum of bioenergetics parameters in heart of young rats and in cultivated H9c2 cardiac myoblasts. D-2-HG impaired cellular respiration in purified mitochondrial preparations and crude homogenates from heart of young rats, as well as in digitonin-permeabilized H9c2 cells. ATP production and the activities of cytochrome c oxidase (complex IV), alpha-ketoglutarate dehydrogenase, citrate synthase and creatine kinase were also inhibited by D-2-HG, whereas the activities of complexes I, II and II-III of the respiratory chain, glutamate, succinate and malate dehydrogenases were not altered. We also found that this organic acid compromised mitochondrial Ca retention capacity in heart mitochondrial preparations and H9c2 myoblasts. Finally, D-2-HG reduced the viability of H9c2 cardiac myoblasts, as determined by the MTT test and by propidium iodide incorporation. Noteworthy, L-2-hydroxyglutaric acid did not change some of these measurements (complex IV and creatine kinase activities) in heart preparations, indicating a selective inhibitory effect of the enantiomer D. In conclusion, it is presumed that D-2-HG-disrupts mitochondrial bioenergetics and Ca retention capacity, which may be involved in the cardiomyopathy commonly observed in D2HGA2.
Topics: Rats; Animals; Calcium; Cell Survival; Energy Metabolism; Creatine Kinase; Cardiomyopathies
PubMed: 36372308
DOI: 10.1016/j.biochi.2022.11.004 -
Chemical Research in Toxicology Nov 2022Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the...
Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the medium () can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log -0.07-6.84), in the culture medium () and cells () after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished mass balance model that combined relevant equations from similar models published by others. The total for all chemicals except tamoxifen (TAM) were similar to the . This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) for the low/no protein binding chemicals were similar to the , whereas values of all moderately to highly protein-bound chemicals were less than 30% of the . Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the was the same as the . The for all other chemicals tended to increase over time and were all 2- to 274-fold higher than . Measurements of , using a digitonin method to release cytosol, compared well with (using a freeze-thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total within 30% of the measured values for 11 chemicals. The free of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of values, with a median overprediction of 3- to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between and total and free and , which were also relatively well predicted by the mass balance model.
Topics: Mice; Animals; Acetaminophen; Hydrophobic and Hydrophilic Interactions; Protein Binding; Cell Culture Techniques
PubMed: 36264934
DOI: 10.1021/acs.chemrestox.2c00128 -
The Journal of General and Applied... Sep 2019After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these...
After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.
Topics: Biological Transport; Endoplasmic Reticulum; Golgi Apparatus; Mannosyltransferases; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 30700649
DOI: 10.2323/jgam.2018.09.001 -
Computational and Structural... 2022Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and...
Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and -mutant high-throughput screening (HTS) data. We matched essential genes from text-mining and mutant genes from the COSMIC and CCLE HTS datasets to build a prediction model of SL gene pairs. CCLE gene expression data were used to enrich the essential-mutant SL gene pairs using Spearman's correlation coefficient and literature mining. In total, 223 essential trigger terms were extracted and ranked. The threshold of the essential gene score ( ) was set to 10. We identified 586 genes essential for the SL prediction model of colon cancer. Seven essential -mutant SL gene pairs were identified in our model, including -/----/ and - gene pairs. Using -mutant HTS data validation, we identified two potential SL gene pairs, including the (essential gene)- (mutant gene) pair and - pair in the DLD-1 colon cancer cell line (Spearman's correlation values = 0.004786 and 0.00249, respectively). Based on further annotations by PubChem, we observed that digitonin targeted the complex comprising , especially in -mutated HCT116 cancer cells. Moreover, we experimentally demonstrated that exhibited selective vulnerability in -mutant colorectal cancer. We used literature mining and HTS data to identify candidates for SL targets for mutant colon cancer.
PubMed: 36212540
DOI: 10.1016/j.csbj.2022.09.025 -
Methods in Molecular Biology (Clifton,... 2020In eukaryotes, most mRNAs that encode secretory or membrane-bound proteins are translated by ribosomes associated with the surface of the endoplasmic reticulum (ER)....
In eukaryotes, most mRNAs that encode secretory or membrane-bound proteins are translated by ribosomes associated with the surface of the endoplasmic reticulum (ER). Other such mRNAs are tethered to the ER by mRNA receptors. However, there has been much debate as to whether all mRNAs, regardless of their encoded polypeptide, are anchored to the ER at some low level. Here we describe a protocol to visualize ER-associated mRNAs in tissue culture cells by single-molecule fluorescence in situ hybridization (smFISH). Using this protocol, we have established that a subset of all mRNAs, regardless of whether they encode secretory or cytosolic proteins, are ER associated in a ribosome-dependent manner.
Topics: Animals; Cell Line; Cytosol; Digitonin; Endoplasmic Reticulum; Humans; In Situ Hybridization, Fluorescence; Membrane Proteins; Optical Imaging; RNA, Messenger; Ribosomes; Single Molecule Imaging
PubMed: 32710402
DOI: 10.1007/978-1-0716-0712-1_3 -
Pesticide Biochemistry and Physiology Mar 2020Imidacloprid (IMD) is a neonicotinoid insecticide widely used in crops, pets, and on farm animals for pest control, which can cause hepatotoxicity in animals and humans....
Imidacloprid (IMD) is a neonicotinoid insecticide widely used in crops, pets, and on farm animals for pest control, which can cause hepatotoxicity in animals and humans. In a previous study using isolated rat liver mitochondria, we observed that IMD inhibited the activity of FF-ATP synthase. The aim of this study was to evaluate the effects of IMD on rat isolated hepatocytes and perfused rat liver, besides the influence of its biotransformation on the toxicological potential. For the latter goal, rats were pretreated with dexamethasone or phenobarbital, two classical cytochrome P-450 stimulators, before hepatocytes isolation or liver perfusion. IMD (150 and 200 μM) reduced state 3 mitochondrial respiration in digitonin-permeabilized cells that were energized with glutamate plus malate but did not dissipate the mitochondrial membrane potential. In intact (non-permeabilized) hepatocytes, the intracellular ATP concentration and cell viability were reduced when high IMD concentrations were used (1.5-3.0 mM), and only in cells isolated from dexamethasone-pretreated rats, revealing that IMD biotransformation increases its toxicity and that IMD itself affects isolated mitochondria or mitochondria in permeabilized hepatocytes in concentrations that do not affect mitochondrial function in intact hepatocytes. Coherently, in the prefused liver, IMD (150 and 250 μM) inhibited gluconeogenesis from alanine, but without affecting oxygen consumption and urea production, indicating that such effect was not of mitochondrial origin. The gluconeogenesis inhibition was incomplete and occurred only when the rats were pretreated with phenobarbital, signs that IMD biotransformation was involved in the observed effect. Our findings reveal that changes in hepatic energy metabolism may be acutely implicated in the hepatotoxicity of IMD only when animals and humans are exposed to high levels of this compound, and that IMD metabolites seem to be the main cause for its toxicity.
Topics: Animals; Biotransformation; Hepatocytes; Liver; Neonicotinoids; Nitro Compounds; Rats
PubMed: 32284125
DOI: 10.1016/j.pestbp.2020.01.011 -
International Journal of Molecular... Mar 2021Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5'end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been...
Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5'end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP-host interactions further, we expressed TP in (). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.
Topics: Active Transport, Cell Nucleus; Adenoviridae; Cell Nucleus; Cytosol; DNA; Escherichia coli; Genome, Viral; Green Fluorescent Proteins; HEK293 Cells; HeLa Cells; Humans; Microscopy, Confocal; Nuclear Localization Signals; Protein Binding; Viral Proteins; alpha Karyopherins; beta Karyopherins
PubMed: 33804953
DOI: 10.3390/ijms22073310 -
SLAS Discovery : Advancing Life... May 2024We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of...
We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC values between 100 nM and 1 μM in cells, which align with the EC values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.
PubMed: 38797286
DOI: 10.1016/j.slasd.2024.100165