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Frontiers in Molecular Biosciences 2023TMEM16 family proteins are involved in a variety of functions, including ion transport, phospholipid scrambling, and the regulation of membrane proteins. Among them,...
TMEM16 family proteins are involved in a variety of functions, including ion transport, phospholipid scrambling, and the regulation of membrane proteins. Among them, TMEM16F has dual functions as a phospholipid scramblase and a nonselective ion channel. TMEM16F is widely expressed and functions in platelet activation during blood clotting, bone formation, and T cell activation. Despite the functional importance of TMEM16F, the modulators of TMEM16F function have not been sufficiently studied. In this study, we generated TMEM16F-specific affibodies by performing phage display with brain-specific TMEM16F (hTMEM16F) variant 1 purified from GnTi cells expressing this variant in the presence of digitonin as a detergent. Purified human TMEM16F protein, which was proficient in transporting phospholipids in a Ca-dependent manner in proteoliposomes, was coated onto plates and then the phage library was added to fish out TMEM16F-binding affibodies. For the validation of interaction between affibodies and TMEM16F proteins, ELISA, bio-layer interferometry, and size exclusion chromatography were conducted. As a result, the full sequences of 38 candidates were acquired from 98 binding candidates. Then, we selected 10 candidates and purified seven of them from expressing these candidates. Using various assays, we confirmed that two affibodies bound to human TMEM16F with high affinity. These affibodies can be useful for therapeutical and diagnostic applications of TMEM16F-related cancer and neurodegenerative diseases. Future studies will be required to investigate the effects of these affibodies on TMEM16F function.
PubMed: 38274091
DOI: 10.3389/fmolb.2023.1319251 -
Virologica Sinica Jun 2021Zika virus (ZIKV) is associated with severe birth defects and Guillain-Barré syndrome and no approved vaccines or specific therapies to combat ZIKV infection are...
Zika virus (ZIKV) is associated with severe birth defects and Guillain-Barré syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine (HHT), bruceine D (BD), dihydroartemisinin (DHA) and digitonin (DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549. The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.
Topics: Animals; Antiviral Agents; Chlorocebus aethiops; High-Throughput Screening Assays; Humans; Vero Cells; Virus Replication; Zika Virus; Zika Virus Infection
PubMed: 33231855
DOI: 10.1007/s12250-020-00316-0 -
Immunology Jul 2018Interleukin-1β (IL-1β) is a potent mediator of innate immunity commonly up-regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface...
Interleukin-1β (IL-1β) is a potent mediator of innate immunity commonly up-regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL-1β initiates a signalling cascade that cooperatively induces the expression of canonical IL-1 target genes such as IL-8 and IL-6. Here, we present galectin-3 as a novel regulator of IL-1β responses in corneal keratinocytes. Using the SNAP-tag system and digitonin semi-permeabilization, we show that recombinant exogenous galectin-3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin-3, but not a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, exacerbates the response to IL-1β by stimulating the secretion of inflammatory cytokines. The activity of galectin-3 could be reduced by a novel d-galactopyranoside derivative targeting the conserved galactoside-binding site of galectins and did not involve interaction with IL-1 receptor 1 or the induction of endogenous IL-1β. Consistent with these observations, we demonstrate that small interfering RNA-mediated suppression of endogenous galectin-3 expression is sufficient to impair the IL-1β-induced secretion of IL-8 and IL-6 in a p38 mitogen-activated protein kinase-independent manner. Collectively, our findings provide a novel role for galectin-3 as an amplifier of IL-1β responses during epithelial inflammation through an as yet unidentified mechanism.
Topics: Cells, Cultured; Endocytosis; Galectin 3; Humans; Interleukin-1beta; Keratinocytes; Keratitis; Protein Binding
PubMed: 29359328
DOI: 10.1111/imm.12899 -
STAR Protocols Mar 2024Cleavage under targets & release using nuclease (CUT&RUN) is a technique for identifying genomic sites where proteins or histone modifications are present in chromatin...
Cleavage under targets & release using nuclease (CUT&RUN) is a technique for identifying genomic sites where proteins or histone modifications are present in chromatin in permeabilized cells. Here, we present a fluorescence-based protocol to quantitatively titrate CUT&RUN buffer components, for efficient cell permeabilization and retention of target epitopes on chromatin. We describe steps for capturing cells on concanavalin A beads and using a fluorescently labeled secondary antibody to titrate concentrations of digitonin and NaCl in CUT&RUN buffers. We then detail procedures for fluorescence imaging to identify optimal conditions. For complete details on the use and execution of this protocol, please refer to Lerner et al..
Topics: Chromatin; Antibodies; Endonucleases; Epitopes; Genomics
PubMed: 38329880
DOI: 10.1016/j.xpro.2024.102866 -
Bio-protocol Jul 2018The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild...
The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein complexes are further solubilized with stronger detergent, β-DM, and subsequently separated on an orthogonal 2D-BN-PAGE to release smaller protein subcomplexes from the higher-order supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of thylakoid protein complexes from .
PubMed: 34395737
DOI: 10.21769/BioProtoc.2899 -
The Journal of Biological Chemistry Jun 2023Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic...
Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a K for its substrate CDP-diacylglycerol of 72.20 μM with a V of 0.079 nmol/(μg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.
Topics: Candida albicans; CDPdiacylglycerol-Serine O-Phosphatidyltransferase; Detergents; Digitonin
PubMed: 37116705
DOI: 10.1016/j.jbc.2023.104756 -
Journal of Virology Feb 2022After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid...
After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly in both interphase and mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export competent but deficient in viral capsid disassembly, in both interphase and mitotic cells.
Topics: Active Transport, Cell Nucleus; Adenoviridae; Adenoviridae Infections; Capsid; Cell Line; Genome, Viral; Host-Pathogen Interactions; Humans; Karyopherins; Microtubules; Models, Molecular; Mutation; Nuclear Envelope; Protein Conformation; Protein Transport; Receptors, Cytoplasmic and Nuclear; Structure-Activity Relationship; Virus Replication; Exportin 1 Protein
PubMed: 34757845
DOI: 10.1128/JVI.01273-21 -
Plant Physiology Jul 2019The Antarctic psychrophile sp. UWO241 evolved in a permanently ice-covered lake whose aquatic environment is characterized not only by constant low temperature and high...
The Antarctic psychrophile sp. UWO241 evolved in a permanently ice-covered lake whose aquatic environment is characterized not only by constant low temperature and high salt but also by low light during the austral summer coupled with 6 months of complete darkness during the austral winter. Since the UWO241 genome indicated the presence of and protein kinases, we examined protein phosphorylation and the state transition phenomenon in this psychrophile. Light-dependent [γ-P]ATP labeling of thylakoid membranes from sp. UWO241 exhibited a distinct low temperature-dependent phosphorylation pattern compared to despite comparable levels of the Stt7 protein kinase. The sequence and structure of the UWO241 Stt7 kinase domain exhibits substantial alterations, which we suggest predisposes it to be more active at low temperature. Comparative purification of PSII and PSI combined with digitonin fractionation of thylakoid membranes indicated that UWO241 altered its thylakoid membrane architecture and reorganized the distribution of PSI and PSII units between granal and stromal lamellae. Although UWO241 grown at low salt and low temperature exhibited comparable thylakoid membrane appression to that of at its optimal growth condition, UWO241 grown under its natural condition of high salt resulted in swelling of the thylakoid lumen. This was associated with an upregulation of PSI cyclic electron flow by 50% compared to growth at low salt. Due to the unique 77K fluorescence emission spectra of intact UWO241 cells, deconvolution was necessary to detect enhancement in energy distribution between PSII and PSI, which was sensitive to the redox state of the plastoquinone pool and to the NaCl concentrations of the growth medium. We conclude that a reorganization of PSII and PSI in UWO241 results in a unique state transition phenomenon that is associated with altered protein phosphorylation and enhanced PSI cyclic electron flow. These data are discussed with respect to a possible PSII-PSI energy spillover mechanism that regulates photosystem energy partitioning and quenching.
Topics: Algal Proteins; Amino Acid Sequence; Antarctic Regions; Chlamydomonas; Chlamydomonas reinhardtii; Chlorophyll; Cold Temperature; Light; Microscopy, Electron, Transmission; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Protein Domains; Protein Kinases; Sequence Homology, Amino Acid; Species Specificity; Spectrometry, Fluorescence; Thylakoids
PubMed: 31019005
DOI: 10.1104/pp.19.00411 -
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