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Archives of Biochemistry and Biophysics Sep 2020To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the...
To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the biological activity and/or perturb oligomerization of individual MPs. Nanodisc scaffold peptide (NSP), an amphipathic peptide analog of ApoA1, was recently shown to reconstitute detergent solubilized MPs into peptidiscs in vitro. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also known as sterol O-acyltransferase 1 (SOAT1), plays a key role in cellular cholesterol storage in various cell types and is a drug target to treat multiple human diseases. ACAT1 contains nine transmembrane domains (TMDs) and primarily forms a homotetramer in vitro and in intact cells; deletion of the N-terminal dimerization domain produces a homodimer with full retention in catalytic activity. ACAT1 is prone to inactivation by numerous detergents. Here we pursued the use of NSP to overcome the detergent-induced inactivation of ACAT1 by generating near detergent-free ACAT1 peptidiscs. Based on native-PAGE analysis, we showed that NSP reconstitutes ACAT1 into soluble peptidiscs, in which ACAT1 exists predominantly in oligomeric states greater than a homotetramer. The formation of these higher-order oligomeric states was independent of the N-terminal dimerization domain, suggesting that the oligomerization is mediated through hydrophobic interactions of multiple ACAT1 subunits. ACAT1 peptidiscs were still susceptible to heat-mediated inactivation, presumably due to the residual detergent (CHAPS) bound to ACAT1. We then conditioned ACAT1 with phosphatidylcholine (PC) to replace CHAPS prior to the formation of ACAT1 peptidiscs. The results showed, when PC was included, ACAT1 was present mainly in higher-order oligomeric states with greater enzymatic activity. With PC present, the enzymatic activity of ACAT1 peptidiscs was protected from heat-mediated inactivation. These results support the use of NSP to create a near detergent-free solution of ACAT1 in peptidiscs for various in vitro studies. Our current results also raise the possibility that, under certain conditions, ACAT1 may form higher-order oligomeric states in vivo.
Topics: Amino Acid Sequence; Animals; CHO Cells; Cholic Acids; Cricetulus; Detergents; Digitonin; Humans; Peptides; Protein Domains; Protein Multimerization; Sterol O-Acyltransferase; Surface-Active Agents
PubMed: 32735863
DOI: 10.1016/j.abb.2020.108518 -
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 -
Biochimica Et Biophysica Acta.... Oct 2020Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed... (Comparative Study)
Comparative Study
Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.
Topics: Animals; Calorimetry; Cell Membrane Permeability; Electron Transport Complex IV; HEK293 Cells; Humans; Metabolism; Mice; Saponins
PubMed: 32598881
DOI: 10.1016/j.bbabio.2020.148251 -
International Journal of Biological... Nov 2020The mosquito Aedes aegypti L. is a vector transmitting diseases such as dengue, chikungunya and Zika virus fever. The water-soluble lectin from Moringa oleifera Lam....
The mosquito Aedes aegypti L. is a vector transmitting diseases such as dengue, chikungunya and Zika virus fever. The water-soluble lectin from Moringa oleifera Lam. seeds (WSMoL) is larvicidal, ovicidal and can stimulate oviposition in A. aegypti. This study aimed to investigate whether WSMoL could bind to membrane proteins from A. aegypti legs. Initially, proteins from the legs were extracted using sodium deoxycholate, digitonin, dodecyl sodium sulfate (SDS) or Triton X-100. The protein concentration was found to be higher in the extract obtained using Triton X-100, which was applied to a WSMoL-Sepharose column. The adsorbed proteins were evaluated using gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE) in presence of SDS. The similarity in the sequences of adsorbed proteins with those available in databases was determined. The proteins adsorbed on the matrix were eluted forming a single peak. Gel filtration chromatography and SDS-PAGE revealed the presence of proteins with molecular masses of approximately 20 kDa and polypeptide bands of 17.0 and 23.7 kDa, respectively. MS/MS analysis indicated similarity between these proteins and ABC carriers, which are expressed in the legs of mosquitos. WSMoL could bind to membrane proteins in the legs of A. aegypti females and induce oviposition through these interactions.
Topics: Aedes; Animals; Insect Proteins; Membrane Proteins; Moringa oleifera; Oviposition; Plant Lectins
PubMed: 32585265
DOI: 10.1016/j.ijbiomac.2020.06.189 -
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 -
Journal of Neurochemistry Apr 2020Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function...
Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na and Ca sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na and Ca signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na and Ca signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na and Ca waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na and Ca signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.
Topics: Acid Sensing Ion Channels; Animals; Cerebral Cortex; Energy Metabolism; Homeostasis; Hydrogen-Ion Concentration; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Neurons; Signal Transduction
PubMed: 31976561
DOI: 10.1111/jnc.14971 -
Journal of Colloid and Interface Science Mar 2020The present paper discusses the use of monolayers of lipid mixtures mimicking the composition of biological membranes of bacteria, erythrocyte and yeast in the context...
The present paper discusses the use of monolayers of lipid mixtures mimicking the composition of biological membranes of bacteria, erythrocyte and yeast in the context of the anti-bacterial, hemolytic and anti-fungal activity of saponins. Saponins are plant-produced glycosidic biosurfactants with either steroidal or triterpenoidal aglycone. In the present study we used digitonin as a representative steroidal saponin (extracted from Digitalis purpurea) and a mixture of triterpenoid saponins from Quillaja saponaria Molina. The effect of saponins was studied first on monolayers consisting of single lipids characteristic for the given type of biological membrane, and then - on model mixed lipid monolayers. Finally, the monolayers were formed from total lipid extracts of natural cell membranes (E. coli and S. cerevisiae) to verify the results obtained in the simplified models. The effect of saponins on monolayers was studied by a combination of surface pressure relaxation, infrared reflection - absorption spectroscopy (IRRAS) and fluorescence microscopy. In line with expectations, sterols (cholesterol and ergosterol) play a major role in the saponin-lipid interactions in monolayers, which may explain especially the hemolytic and antifungal properties of saponins. In contrast, bacterial membranes are devoid of sterols, although the presence of similar compounds may be responsible for their affinity to saponins. Nevertheless, the effect of saponins on bacterial models is less pronounced than for the erythrocyte or fungal ones.
Topics: Anti-Bacterial Agents; Antifungal Agents; Cell Membrane; Erythrocytes; Escherichia coli; Humans; Microbial Sensitivity Tests; Microscopy, Fluorescence; Molecular Structure; Particle Size; Saccharomyces cerevisiae; Saponins; Steroids; Surface Properties; Triterpenes
PubMed: 31874308
DOI: 10.1016/j.jcis.2019.12.014 -
Cell Structure and Function Jan 2020The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding...
The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.
Topics: Cell Membrane; Glycosylation; Membrane Proteins; Receptors, Cell Surface; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31787665
DOI: 10.1247/csf.19021 -
Identification of Novel Natural Products as Effective and Broad-Spectrum Anti-Zika Virus Inhibitors.Viruses Nov 2019Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic...
Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic agents have, so far, been approved for the treatment of ZIKV infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat ZIKV-caused diseases. After screening a natural product library, we have herein identified four natural products with anti-ZIKV activity in Vero E6 cells, including gossypol, curcumin, digitonin, and conessine. Except for curcumin, the other three natural products have not been reported before to have anti-ZIKV activity. Among them, gossypol exhibited the strongest inhibitory activity against almost all 10 ZIKV strains tested, including six recent epidemic human strains. The mechanistic study indicated that gossypol could neutralize ZIKV infection by targeting the envelope protein domain III (EDIII) of ZIKV. In contrast, the other natural products inhibited ZIKV infection by targeting the host cell or cell-associated entry and replication stages of ZIKV. A combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-ZIKV compound, exhibited significant combinatorial inhibitory effects against three ZIKV human strains tested. Importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (DENV) human strains in vitro. Taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against ZIKV and other flaviviruses, such as DENV.
Topics: Alkaloids; Animals; Antiviral Agents; Biological Products; Cell Survival; Chlorocebus aethiops; Curcumin; Dengue Virus; Digitonin; Drug Synergism; Gossypol; Humans; Molecular Structure; Vero Cells; Zika Virus; Zika Virus Infection
PubMed: 31684080
DOI: 10.3390/v11111019 -
Proceedings of the National Academy of... Oct 2019Cytochrome oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a...
Cytochrome oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.
Topics: Animals; Cardiolipins; Cattle; Crystallography, X-Ray; Digitonin; Electron Transport; Electron Transport Complex I; Electron Transport Complex IV; Hydrogen Bonding; Hydrogen-Ion Concentration; Mitochondria, Heart; Mitochondrial Membranes; Molecular Conformation; Oxidation-Reduction; Oxygen; Phospholipids; Phosphorus; Protein Binding; Protein Conformation; Protein Multimerization
PubMed: 31533957
DOI: 10.1073/pnas.1907183116