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MBio Apr 2021Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the...
Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of , the etiologic agent of Chagas disease. Endogenous tagging of () and with 3× showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of and genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized -KO and -KO epimastigotes showed reduced O consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. -KO and -KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in -KOs. Overall, MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes. is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual () and knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.
Topics: Anion Transport Proteins; Biological Transport; CRISPR-Cas Systems; DNA Replication; Gene Knockout Techniques; Mitochondrial Membrane Transport Proteins; Protozoan Proteins; Pyruvic Acid; Trypanosoma cruzi
PubMed: 33824204
DOI: 10.1128/mBio.00540-21 -
Current Protocols in Cytometry Apr 2019Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the...
Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the oxidized form of nicotinamide adenine dinucleotide (NAD ). The availability of free NAD can affect the activities of NAD -consuming enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family members. Many methods for measuring the NAD available to these enzymes are limited because they cannot determine free NAD as it exists in various subcellular compartments distinctly from bound NAD or NADH. Here, an approach to express the sensor in mammalian cells, monitor NAD -dependent fluorescence intensity changes using flow cytometry approaches, and analyze data obtained is described. The benefit of flow cytometry approaches with the NAD sensor is the ability to monitor compartmentalized free NAD fluctuations simultaneously within many cells, which greatly facilitates analyses and calibration. © 2018 by John Wiley & Sons, Inc.
Topics: Acrylamides; Biosensing Techniques; Calibration; Digitonin; Enzyme Inhibitors; Flow Cytometry; Fluorescence; HeLa Cells; Humans; Hydrogen-Ion Concentration; Intracellular Space; Mitochondria; NAD; Nicotinamide Phosphoribosyltransferase; Piperidines; Small Molecule Libraries; Statistics as Topic
PubMed: 30556645
DOI: 10.1002/cpcy.54 -
Quarterly Reviews of Biophysics Mar 2021Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron...
Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.
Topics: Cryoelectron Microscopy; Electrons; Membrane Proteins; Surface-Active Agents
PubMed: 33785082
DOI: 10.1017/S0033583521000044 -
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 -
PLoS Neglected Tropical Diseases Feb 2021In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal,...
In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.
Topics: Life Cycle Stages; Microbodies; Nucleotides; Sugars; Trypanosoma brucei brucei
PubMed: 33592041
DOI: 10.1371/journal.pntd.0009132 -
Science Bulletin Mar 2024Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce...
Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionic for the incomplete understanding of silkworm spinning in vivo. Here, we found that amphipol and digitonin stabilized the structure of natural silk fibroin (NSF) by a large-scale screening in vitro, and then studied the close-to-native ultrastructure and hierarchical assembly of NSF in the silk gland lumen. Our study showed that NSF formed reversible flexible nanofibrils mainly composed of random coils with a sedimentation coefficient of 5.8 S and a diameter of about 4 nm, rather than a micellar or rod-like structure assembled by the aggregation of globular NSF molecules. Metal ions were required for NSF nanofibril formation. The successive pH decrease from posterior silk gland (PSG) to anterior silk gland (ASG) resulted in a gradual increase in NSF hydrophobicity, thus inducing the sol-gelation transition of NSF nanofibrils. NSF nanofibrils were randomly dispersed from PSG to ASG-1, and self-assembled into anisotropic herringbone patterns at ASG-2 near the spinneret ready for silkworm spinning. Our findings reveal the controlled self-assembly mechanism of the multi-scale hierarchical architecture of NSF from nanofibrils to herringbone patterns programmed by metal ions and pH gradient, which provides novel insights into the spinning mechanism of silk-secreting animals and bioinspired design of high-performance fibers.
Topics: Animals; Bombyx; Silk; Fibroins; Solvents; Metals; Hydrogen-Ion Concentration
PubMed: 38245448
DOI: 10.1016/j.scib.2023.12.050 -
Biochimica Et Biophysica Acta.... Jun 2018The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most...
The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the FF-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V and V) and different respiratory supercomplexes (I/IV, I/III, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption.
Topics: Algal Proteins; Detergents; Digitonin; Electron Transport; Electron Transport Complex I; Electron Transport Complex III; Electron Transport Complex IV; Gene Expression; Glucosides; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Protein Binding; Volvocida
PubMed: 29540299
DOI: 10.1016/j.bbabio.2018.03.004 -
Cell Jul 2017Voltage-gated sodium (Na) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNa1.4, the Na channel from electric eel, in...
Voltage-gated sodium (Na) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNa1.4, the Na channel from electric eel, in complex with the β1 subunit at 4.0 Å resolution. The immunoglobulin domain of β1 docks onto the extracellular L5 and L6 loops of EeNa1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSD). The VSDs exhibit "up" conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule. Structural comparison with closed NaPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.
Topics: Amino Acid Sequence; Animals; Cryoelectron Microscopy; Electrophorus; Fish Proteins; Models, Molecular; Protein Domains; Sequence Alignment; Voltage-Gated Sodium Channels
PubMed: 28735751
DOI: 10.1016/j.cell.2017.06.039 -
Cells Apr 2024The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their...
The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.
Topics: Animals; Mice; NIH 3T3 Cells; Nanoparticles; RAW 264.7 Cells; Cell Survival; Holography; Quantitative Phase Imaging
PubMed: 38667312
DOI: 10.3390/cells13080697 -
Biochimica Et Biophysica Acta Jan 2016Membranes prepared from rat brain were treated with increasing concentrations of cationic, neutral, anionic and zwitterionic surfactants. Potent inactivation of...
Membranes prepared from rat brain were treated with increasing concentrations of cationic, neutral, anionic and zwitterionic surfactants. Potent inactivation of [(3)H]MK-801 binding to NMDA receptors (NRs) was provided by the cation cetyl pyridinium (IC50 25 μM) and the neutral digitonin (IC50 37 μM). A 2 h incubation of rat brain membranes at 24°C with 100 μM of the neutral Triton X-100 resulted in about 50% reversible inhibition (without inactivation). Reversible inhibition was also effected by the anion deoxycholate (IC50 700 μM), and by the zwitterions N-lauryl sulfobetaine (12-SB(±), 400 μM) and CHAPS (1.5 mM), with inactivation at higher concentrations. Keeping the NR cation channel in the closed state significantly protected against inactivation by cations and by 12-SB(±), but not by the other detergents. Inactivation depended differentially on the amount of the membranes, on the duration of the treatment, and on the temperature. Varying the amount of membranes by a factor 8 yielded for cetyl trimethylammonium (16-NMe3(+)) IC50s of inactivation from 10 to 80 μM, while for deoxycholate the IC50 of inactivation was 1.2 mM for all tissue quantities. Some compounds inactivated within a few min (16-NMe3(+), digitonin, CHAPS), while inactivation by others took at least half an hour (Triton X-100, deoxycholate, 12-SB(±)). These last 3 ones also exhibited the steepest temperature dependence. Knowledge about the influence of various parameters is helpful in selecting appropriate conditions allowing the treatment of brain membranes with amphiphiles without risking irreversible inactivation.
Topics: Animals; Cell Membrane; Cerebral Cortex; Cetrimonium; Cetrimonium Compounds; Cholic Acids; Deoxycholic Acid; Detergents; Digitonin; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Male; Octoxynol; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate
PubMed: 26518518
DOI: 10.1016/j.bbamem.2015.10.021