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The Analyst Sep 2021Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have...
Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.
Topics: Biological Phenomena; Digitonin; Humans; Proto-Oncogene Proteins c-akt; Signal Transduction; Single-Cell Analysis
PubMed: 34351328
DOI: 10.1039/d1an00751c -
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 -
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 -
Journal of Virology Mar 2022To gain more information about the nature of virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the...
To gain more information about the nature of virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. Members of the infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.
Topics: Animals; Birnaviridae; Birnaviridae Infections; Cell Line; Chickens; Infectious bursal disease virus; Microscopy, Electron; Poultry Diseases; RNA, Viral; Viral Replication Compartments; Viral Structural Proteins; Virion; Virus Replication
PubMed: 35138130
DOI: 10.1128/jvi.02024-21 -
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 -
Journal of Immunology (Baltimore, Md. :... May 2023Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial...
Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.
Topics: Male; Female; Animals; Mice; NF-kappa B; C-Reactive Protein; Endotoxemia; Mice, Inbred C57BL; Liver; NF-kappa B p50 Subunit; Immunity, Innate
PubMed: 36946778
DOI: 10.4049/jimmunol.2200900 -
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 -
Journal of Applied Crystallography Apr 2020Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is...
Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is inhomogeneous and requires lengthy pre-treatment for reliable downstream use. Glyco-diosgenin (GDN) is a recently introduced synthetic surfactant with features that mimic digitonin. It is available in homogeneously pure form. GDN is proving to be a useful detergent, particularly in the area of single-particle cryo-electron microscopic studies of membrane integral proteins. With a view to using it as a detergent for crystallization trials by the or lipid cubic phase method, it was important to establish the carrying capacity of the cubic mesophase for GDN. This was quantified in the current study using small-angle X-ray scattering for mesophase identification and phase microstructure characterization as a function of temperature and GDN concentration. The data show that the lipid cubic phase formed by hydrated monoolein tolerates GDN to concentrations orders of magnitude in excess of those used for membrane protein studies. Thus, having GDN in a typical membrane protein preparation should not deter use of the method for crystallogenesis.
PubMed: 32280324
DOI: 10.1107/S1600576720002289 -
MethodsX 2019Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the...
Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the assessment of such mitochondrial activities in several working models. Pollen tube mitochondrial studies represent one example of this, where tests have been often restricted due the physical impossibility of performing experiments with isolated mitochondria in enough quantities. Here we detail a method to measure mitochondrial respiratory chain activity and calcium transport in tobacco pollen tubes. ••
PubMed: 31406686
DOI: 10.1016/j.mex.2019.07.023 -
Molecules (Basel, Switzerland) Oct 2021Saponins, a diverse group of natural compounds, offer an interesting pool of derivatives with biomedical application. In this study, three structurally related...
Spirostanol Saponins from Flowers of and Related Compounds Indicating Cytotoxic Activity and Affecting Nitric Oxide Production Inhibitory Effect in Peritoneal Macrophages.
Saponins, a diverse group of natural compounds, offer an interesting pool of derivatives with biomedical application. In this study, three structurally related spirostanol saponins were isolated and identified from the leek flowers of L. (garden leek). Two of them were identical with the already known leek plant constituents: aginoside (1) and 6-deoxyaginoside (2). The third one was identified as new component of ; however, it was found identical with yayoisaponin A (3) obtained earlier from a mutant of elephant garlic L. It is a derivative of the aginoside (1) with additional glucose in its glycosidic chain, identified by MS and NMR analysis as (2α, 3β, 6β, 25)-2,6-dihydroxyspirostan-3-yl β-D-glucopyranosyl-(1 → 3)-β-D-glucopranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosyl]-(1 → 4)-β-D-galactopyranoside, previously reported also under the name alliporin. The leek native saponins were tested together with other known and structurally related saponins (tomatonin and digitonin) and with their related aglycones (agigenin and diosgenin) for in vitro cytotoxicity and for effects on NO production in mouse peritoneal cells. The highest inhibitory effects were exhibited by 6-deoxyaginoside. The obtained toxicity data, however, closely correlated with the suppression of NO production. Therefore, an unambiguous linking of obtained bioactivities of saponins with their expected immunobiological properties remained uncertain.
Topics: Allium; Animals; Cell Line; Cell Survival; Flowers; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Molecular Conformation; Nitric Oxide; Saponins; Spirostans
PubMed: 34770942
DOI: 10.3390/molecules26216533