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International Journal of Molecular... Oct 2020Conformational conversion of the cellular prion protein, PrP, into the abnormally folded isoform, PrP, is a key pathogenic event in prion diseases. However, the exact...
Conformational conversion of the cellular prion protein, PrP, into the abnormally folded isoform, PrP, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrP, designated Tg(PrP∆91-106)/ mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/ mice were resistant to RML, 22L and FK-1 prions, neither producing PrP∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrP∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrP∆91-104 after incubation with BSE-PrP-prions but not with RML- and 22L-PrP-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrP∆91-104 even after incubation with RML- and 22L-PrP-prions. These results suggest that residues 91-106 or 91-104 of PrP are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrP into PrP.
Topics: Animals; Baculoviridae; Base Sequence; Brain; Cattle; Cloning, Molecular; Disease Susceptibility; Encephalopathy, Bovine Spongiform; Gene Expression; Injections, Intraventricular; Mice; Mice, Transgenic; PrPC Proteins; PrPSc Proteins; Proteostasis Deficiencies; Recombinant Proteins; Scrapie; Sequence Deletion; Species Specificity
PubMed: 33019549
DOI: 10.3390/ijms21197260 -
International Journal of Molecular... Mar 2021This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and...
This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.
Topics: Cell Membrane; Cell Membrane Permeability; Dose-Response Relationship, Drug; Ion Channel Gating; Ion Channels; Lipid Bilayers; Membrane Lipids; Membrane Potentials; Molecular Structure; Phase Transition; Saponins
PubMed: 33804648
DOI: 10.3390/ijms22063167 -
Frontiers in Physiology 2021In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca, mitochondria undergo permeability transition (PT) leading to the...
In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.
PubMed: 34950052
DOI: 10.3389/fphys.2021.773839 -
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 -
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 -
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