Pub Date : 2025-09-22DOI: 10.1016/j.bbamem.2025.184459
Brandon S. Veron , Kyle Lethcoe , Robert O. Ryan
The SpyCatcher/SpyTag system represents a unique technology that allows facile conjugation of proteins via formation of a covalent isopeptide bond between the 113 residue SpyCatcher protein and a 16 residue SpyTag peptide. Herein this technology was adapted to incorporate miniature bilayer membranes, termed nanodisks (ND). Fusion proteins comprised of apolipoprotein (apo) A-I/SpyTag peptide and SpyCatcher/maltose binding protein (MBP), respectively, were expressed and purified. Upon incubation of apoA-I:SpyTag fusion protein with SpyCatcher:MBP fusion protein, a covalent adduct was formed. ApoA-I:SpyTag formulated into ND particles with cardiolipin (CL) or phosphatidylcholine retained the ability to form an adduct with SpyCatcher:MBP. This adduct was then immobilized on amylose agarose resin beads through a binding interaction with the MBP component. Upon incubation of cytochrome c with immobilized CL ND, but not with phosphatidylcholine ND, cytochrome c binding occurred. When immobilized cytochrome c CL ND were incubated with buffer containing CaCl2, cytochrome c dissociated and was recovered in the supernatant fraction obtained after pelleting the amylose agarose beads. Subsequent incubation of the amylose agarose beads with 10 mM maltose revealed that nearly all of the cytochrome c had been released from the beads. The data are consistent with the known ability of calcium to form an ionic interaction with the two negatively charged phosphates in the polar head group of CL. Given the number of ligand-membrane interactions that occur in nature, immobilized ND provide a novel means to probe them.
{"title":"Immobilized nanodisks for study of ligand binding interactions","authors":"Brandon S. Veron , Kyle Lethcoe , Robert O. Ryan","doi":"10.1016/j.bbamem.2025.184459","DOIUrl":"10.1016/j.bbamem.2025.184459","url":null,"abstract":"<div><div>The SpyCatcher/SpyTag system represents a unique technology that allows facile conjugation of proteins via formation of a covalent isopeptide bond between the 113 residue SpyCatcher protein and a 16 residue SpyTag peptide. Herein this technology was adapted to incorporate miniature bilayer membranes, termed nanodisks (ND). Fusion proteins comprised of apolipoprotein (apo) A-I/SpyTag peptide and SpyCatcher/maltose binding protein (MBP), respectively, were expressed and purified. Upon incubation of apoA-I:SpyTag fusion protein with SpyCatcher:MBP fusion protein, a covalent adduct was formed. ApoA-I:SpyTag formulated into ND particles with cardiolipin (CL) or phosphatidylcholine retained the ability to form an adduct with SpyCatcher:MBP. This adduct was then immobilized on amylose agarose resin beads through a binding interaction with the MBP component. Upon incubation of cytochrome <em>c</em> with immobilized CL ND, but not with phosphatidylcholine ND, cytochrome <em>c</em> binding occurred. When immobilized cytochrome <em>c</em> CL ND were incubated with buffer containing CaCl<sub>2</sub>, cytochrome <em>c</em> dissociated and was recovered in the supernatant fraction obtained after pelleting the amylose agarose beads. Subsequent incubation of the amylose agarose beads with 10 mM maltose revealed that nearly all of the cytochrome <em>c</em> had been released from the beads. The data are consistent with the known ability of calcium to form an ionic interaction with the two negatively charged phosphates in the polar head group of CL. Given the number of ligand-membrane interactions that occur in nature, immobilized ND provide a novel means to probe them.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184459"},"PeriodicalIF":2.5,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145118696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-11DOI: 10.1016/j.bbamem.2025.184458
Valentina A. Iunusova , Nikita A. Orlov , Oksana V. Nekrasova , Alexey V. Feofanov , Alexander A. Vassilevski , Alexey I. Kuzmenkov
KcsA, a potassium channel from Streptomyces lividans, is one of the most extensively studied transmembrane proteins. Despite significant research in structural biology, relatively few ligands of KcsA have been identified. One such ligand is Hui1, an artificial peptide derived from a phage display screening using a combinatorial library constructed relying on several sea anemone toxins. In this study, we engineered a fluorescent probe by fusing Hui1 with enhanced green fluorescent protein (eGFP), creating the first fluorescence-based tool to visualize prokaryotic ion channels. The eGFP–Hui1 chimera was successfully produced in Escherichia coli and purified using chromatographic techniques. Our study revealed a direct interaction between KcsA, also recombinantly expressed in E. coli, and the fluorescent chimera. Furthermore, we demonstrated that both Hui1 and tetraethylammonium can effectively displace the chimera from its complex with KcsA, confirming the specificity of the binding interaction. This approach opens new avenues for pharmacological and structural investigations, including the development of novel antimicrobial agents and high-throughput ligand screening.
{"title":"Targeting prokaryotic ion channel by a chimera of fluorescent protein and artificial peptide toxin","authors":"Valentina A. Iunusova , Nikita A. Orlov , Oksana V. Nekrasova , Alexey V. Feofanov , Alexander A. Vassilevski , Alexey I. Kuzmenkov","doi":"10.1016/j.bbamem.2025.184458","DOIUrl":"10.1016/j.bbamem.2025.184458","url":null,"abstract":"<div><div>KcsA, a potassium channel from <em>Streptomyces lividans</em>, is one of the most extensively studied transmembrane proteins. Despite significant research in structural biology, relatively few ligands of KcsA have been identified. One such ligand is Hui1, an artificial peptide derived from a phage display screening using a combinatorial library constructed relying on several sea anemone toxins. In this study, we engineered a fluorescent probe by fusing Hui1 with enhanced green fluorescent protein (eGFP), creating the first fluorescence-based tool to visualize prokaryotic ion channels. The eGFP–Hui1 chimera was successfully produced in <em>Escherichia coli</em> and purified using chromatographic techniques. Our study revealed a direct interaction between KcsA, also recombinantly expressed in <em>E. coli</em>, and the fluorescent chimera. Furthermore, we demonstrated that both Hui1 and tetraethylammonium can effectively displace the chimera from its complex with KcsA, confirming the specificity of the binding interaction. This approach opens new avenues for pharmacological and structural investigations, including the development of novel antimicrobial agents and high-throughput ligand screening.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184458"},"PeriodicalIF":2.5,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-11DOI: 10.1016/j.bbamem.2025.184451
Mark J. Arcario , Vikram Dalal , David Fan , Fong-Fu Hsu , Wayland W.L. Cheng
Nanodiscs, soluble membrane mimetics composed of an amphipathic membrane scaffold protein encircling a lipid bilayer, are widely used in biophysical and structural studies of membrane proteins. Because many membrane proteins are responsive to their membrane environment, through specific protein–lipid interactions and bulk membrane shape and structure, it is important to understand the properties of lipid bilayers contained within nanodiscs in order to interpret studies using this technology. Nanodiscs are known to alter lipid properties, such as membrane thickness and melting temperature, and interactions with the nanodisc rim have been hypothesized to produce local perturbations in lipid structure and dynamics. Larger nanodiscs should compensate for this effect with a larger unperturbed area. To test this hypothesis, we examined the lipid bilayer properties of several lipids (DMPC, DPPC, POPC, DSPC) and soy polar lipid in circularized nanodiscs of 11 nm to 50 nm diameter using the environmentally-sensitive fluorophore, Laurdan. In nanodiscs containing a single lipid type, as nanodisc size increased, lipid packing, melting temperature, and cooperativity better approximated the properties of that lipid in large unilamellar vesicles (LUVs). In spNW50 (50 nm nanodisc), the lipid packing and melting temperature were indistinguishable from LUVs. However, nanodiscs containing soy polar lipids did not follow this trend suggesting that complex lipid mixtures may produce preferential incorporation of lipids into the nanodisc or nonhomogeneous distribution of lipids within the nanodisc.
{"title":"Examining the thermotropic properties of large circularized nanodiscs","authors":"Mark J. Arcario , Vikram Dalal , David Fan , Fong-Fu Hsu , Wayland W.L. Cheng","doi":"10.1016/j.bbamem.2025.184451","DOIUrl":"10.1016/j.bbamem.2025.184451","url":null,"abstract":"<div><div>Nanodiscs, soluble membrane mimetics composed of an amphipathic membrane scaffold protein encircling a lipid bilayer, are widely used in biophysical and structural studies of membrane proteins. Because many membrane proteins are responsive to their membrane environment, through specific protein–lipid interactions and bulk membrane shape and structure, it is important to understand the properties of lipid bilayers contained within nanodiscs in order to interpret studies using this technology. Nanodiscs are known to alter lipid properties, such as membrane thickness and melting temperature, and interactions with the nanodisc rim have been hypothesized to produce local perturbations in lipid structure and dynamics. Larger nanodiscs should compensate for this effect with a larger unperturbed area. To test this hypothesis, we examined the lipid bilayer properties of several lipids (DMPC, DPPC, POPC, DSPC) and soy polar lipid in circularized nanodiscs of 11 nm to 50 nm diameter using the environmentally-sensitive fluorophore, Laurdan. In nanodiscs containing a single lipid type, as nanodisc size increased, lipid packing, melting temperature, and cooperativity better approximated the properties of that lipid in large unilamellar vesicles (LUVs). In spNW50 (50 nm nanodisc), the lipid packing and melting temperature were indistinguishable from LUVs. However, nanodiscs containing soy polar lipids did not follow this trend suggesting that complex lipid mixtures may produce preferential incorporation of lipids into the nanodisc or nonhomogeneous distribution of lipids within the nanodisc.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184451"},"PeriodicalIF":2.5,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145046028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-11DOI: 10.1016/j.bbamem.2025.184454
Olivier Soubias , Jonathan D. Nickels , Kirk G. Hines , Walter E. Teague , John K. Northup , John Katsaras , Klaus Gawrisch
Although the oligomeric states of G-protein-coupled receptors (GPCRs) and interactions with cognate G proteins are central to their signal transduction capabilities, they remain poorly defined. In this study, we used small-angle neutron scattering (SANS) and a neutron contrast matching approach to elucidate the oligomeric states of the archetypal GPCR, rhodopsin, and its interaction with the G protein transducin (Gt). At a rhodopsin/lipid molar ratio of 1/360, we found that dark-adapted rhodopsin exists as a monomer, a finding consistent with its high functional activity measured upon photoactivation by spectrophotometry and the rate of catalyzed [35S]-GTP-γ-S exchange. Following light activation, we observed that rhodopsin forms a stable 1:1 stoichiometric complex with Gt, the structure of which is consistent with recent cryo-EM data. In contrast, activated rhodopsin in the absence of Gt showed a propensity to form higher order oligomers. This research underscores the concentration-dependent nature of rhodopsin oligomerization and establishes SANS and the ability to produce appropriately contrast-matched samples, as a robust strategy for characterizing integral membrane protein interactions under biologically relevant conditions.
{"title":"Small angle neutron scattering study of rhodopsin oligomerization and G-protein coupling in a physiologically relevant lipid membrane","authors":"Olivier Soubias , Jonathan D. Nickels , Kirk G. Hines , Walter E. Teague , John K. Northup , John Katsaras , Klaus Gawrisch","doi":"10.1016/j.bbamem.2025.184454","DOIUrl":"10.1016/j.bbamem.2025.184454","url":null,"abstract":"<div><div>Although the oligomeric states of G-protein-coupled receptors (GPCRs) and interactions with cognate G proteins are central to their signal transduction capabilities, they remain poorly defined. In this study, we used small-angle neutron scattering (SANS) and a neutron contrast matching approach to elucidate the oligomeric states of the archetypal GPCR, rhodopsin, and its interaction with the G protein transducin (G<sub>t</sub>). At a rhodopsin/lipid molar ratio of 1/360, we found that dark-adapted rhodopsin exists as a monomer, a finding consistent with its high functional activity measured upon photoactivation by spectrophotometry and the rate of catalyzed [<sup>35</sup>S]-GTP-γ-S exchange. Following light activation, we observed that rhodopsin forms a stable 1:1 stoichiometric complex with G<sub>t</sub>, the structure of which is consistent with recent cryo-EM data. In contrast, activated rhodopsin in the absence of G<sub>t</sub> showed a propensity to form higher order oligomers. This research underscores the concentration-dependent nature of rhodopsin oligomerization and establishes SANS and the ability to produce appropriately contrast-matched samples, as a robust strategy for characterizing integral membrane protein interactions under biologically relevant conditions.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184454"},"PeriodicalIF":2.5,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-10DOI: 10.1016/j.bbamem.2025.184457
Aleksandra Walewska , Paulina Kosikowska-Adamus , Anna Wardowska , Grzegorz Bulaj , Emilia Sikorska
Human β-defensins are disulfide-rich peptides that exhibit broad-spectrum antimicrobial activity against bacteria, fungi, and certain viruses, while also exerting immunomodulatory effects. As naturally occurring host defense peptides in the human body, they are less likely to induce adverse immune reactions or toxicity compared to synthetic drugs. Efficient chemical synthesis of cysteine-rich peptides is critical for drug lead optimization studies, yet in many cases is limiting by the low yields of correctly formed disulfide bonds. Herein, we present novel β-defensin 3 analogues with engineered disulfide bonds, designed to simplify and improve oxidative folding while preserving antimicrobial and antifungal activities. Our results suggest that the judicious replacement of cysteine residues with alanines or selenocysteines may facilitate the development of defensin-based drug leads with enhanced pharmacological properties, addressing their therapeutic potential to combat multidrug resistance.
{"title":"Improving chemical synthesis and the antimicrobial activity of human defensins through disulfide bond engineering of HBD-3","authors":"Aleksandra Walewska , Paulina Kosikowska-Adamus , Anna Wardowska , Grzegorz Bulaj , Emilia Sikorska","doi":"10.1016/j.bbamem.2025.184457","DOIUrl":"10.1016/j.bbamem.2025.184457","url":null,"abstract":"<div><div>Human β-defensins are disulfide-rich peptides that exhibit broad-spectrum antimicrobial activity against bacteria, fungi, and certain viruses, while also exerting immunomodulatory effects. As naturally occurring host defense peptides in the human body, they are less likely to induce adverse immune reactions or toxicity compared to synthetic drugs. Efficient chemical synthesis of cysteine-rich peptides is critical for drug lead optimization studies, yet in many cases is limiting by the low yields of correctly formed disulfide bonds. Herein, we present novel β-defensin 3 analogues with engineered disulfide bonds, designed to simplify and improve oxidative folding while preserving antimicrobial and antifungal activities. Our results suggest that the judicious replacement of cysteine residues with alanines or selenocysteines may facilitate the development of defensin-based drug leads with enhanced pharmacological properties, addressing their therapeutic potential to combat multidrug resistance.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184457"},"PeriodicalIF":2.5,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145046027","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The integral matrix protein M2 of the influenza A virus (H3N2) has been proposed to form a proton-selective channel, and is a target of the antiviral drug amantadine hydrochloride (Am). A significant increase in the number of drug-resistant strains containing the predominant M2-S31N mutant have emerged. We have previously developed a stoichiometric analysis of oligomeric states of membrane proteins by utilizing a coiled-coil method and fluorescence resonance energy transfer phenomenon and demonstrated that full-length M2-WT proteins in living cells formed a dimer at neutral pH, which was converted to a tetramer at acidic pH. In the present study, we revealed that the M2-S31N mutant stably formed dimers independent of pH, which was stabilized by multiple interactions between the protomers. We also found that neither the channel activity nor the oligomeric state of S31N were affected by Am. Molecular dynamics (MD) simulations revealed that Asn31 and Ile35 are involved in proton conduction via steady interactions with cholesterol. These results indicate that Am resistance could be attributed to a change in the arrangement of helices interfering with drug binding.
{"title":"Steady dimer formation by the S31N mutant of influenza A virus M2 protein in living cell membranes","authors":"Kenichi Kawano , Tyuji Hoshino , Yoshiaki Yano , Katsumi Matsuzaki","doi":"10.1016/j.bbamem.2025.184456","DOIUrl":"10.1016/j.bbamem.2025.184456","url":null,"abstract":"<div><div>The integral matrix protein M2 of the influenza A virus (H3N2) has been proposed to form a proton-selective channel, and is a target of the antiviral drug amantadine hydrochloride (Am). A significant increase in the number of drug-resistant strains containing the predominant M2-S31N mutant have emerged. We have previously developed a stoichiometric analysis of oligomeric states of membrane proteins by utilizing a coiled-coil method and fluorescence resonance energy transfer phenomenon and demonstrated that full-length M2-WT proteins in living cells formed a dimer at neutral pH, which was converted to a tetramer at acidic pH. In the present study, we revealed that the M2-S31N mutant stably formed dimers independent of pH, which was stabilized by multiple interactions between the protomers. We also found that neither the channel activity nor the oligomeric state of S31N were affected by Am. Molecular dynamics (MD) simulations revealed that Asn31 and Ile35 are involved in proton conduction <em>via</em> steady interactions with cholesterol. These results indicate that Am resistance could be attributed to a change in the arrangement of helices interfering with drug binding.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184456"},"PeriodicalIF":2.5,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145038976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-08DOI: 10.1016/j.bbamem.2025.184450
Éder Jéferson Souza Cardoso , Ellyêssa Nascimento Borges , Kleber Santiago Freitas e Silva , Rodrigo Saar Gomes , Fabiana Taniguchi Boni , Jhon Kennedy Alves Pereira , Paula Maria Alexandre Mangoba , Luciano Morais Lião , Ricardo Menegatti , Antonio Alonso
Three antileishmanial compounds incorporating a butylated hydroxytoluene (BHT) moiety and an acrylate-based Michael acceptor scaffold were rationally designed from the lead structures LQFM064 and LQFM332, which feature a chalcone-derived core. Their activities against Leishmania (L.) amazonensis were evaluated. Acrylate derivatives (5), (6), and (7) displayed IC50 values comparable to miltefosine, while showing markedly lower cytotoxicity toward J774.A1 and differentiated THP-1 macrophages, along with reduced hemolytic potential. Spin-label electron paramagnetic resonance (EPR) spectroscopy revealed that treatment with these compounds induces membrane rigidity after 24 h in a concentration-dependent manner. This effect is unlikely due to direct membrane interaction, as it does not occur after short incubations or at low concentrations, suggesting a correlation with oxidative stress, such as lipid peroxidation and/or protein oxidation, likely triggered by elevated reactive oxygen species (ROS) production. In contrast, no oxidative stress-induced membrane rigidity was detected in uninfected macrophages, suggesting that nitric oxide production may mitigate oxidative damage in these cells. However, significant membrane rigidity was observed in Leishmania-infected macrophages at concentrations slightly above the IC50 for amastigotes, indicating that the compounds may selectively target infected macrophages. Additionally, compound (5) exhibited moderate cytotoxicity in the rapidly proliferating J774.A1 macrophage line but displayed very low cytotoxicity in differentiated, non-proliferative THP-1 macrophages. Overall, this study suggests that the primary mechanisms underlying the antileishmanial activity of these compounds are associated with their effects on the parasite plasma membrane, potentially leading to ionic leakage, subsequent disruption of mitochondrial membrane potential, and enhanced ROS generation.
{"title":"Antileishmanial, cytotoxic activities, and membrane rigidity effects of three synthetic compounds","authors":"Éder Jéferson Souza Cardoso , Ellyêssa Nascimento Borges , Kleber Santiago Freitas e Silva , Rodrigo Saar Gomes , Fabiana Taniguchi Boni , Jhon Kennedy Alves Pereira , Paula Maria Alexandre Mangoba , Luciano Morais Lião , Ricardo Menegatti , Antonio Alonso","doi":"10.1016/j.bbamem.2025.184450","DOIUrl":"10.1016/j.bbamem.2025.184450","url":null,"abstract":"<div><div>Three antileishmanial compounds incorporating a butylated hydroxytoluene (BHT) moiety and an acrylate-based Michael acceptor scaffold were rationally designed from the lead structures LQFM064 and LQFM332, which feature a chalcone-derived core. Their activities against <em>Leishmania</em> (<em>L</em>.) <em>amazonensis</em> were evaluated. Acrylate derivatives (<strong>5</strong>), (<strong>6</strong>), and (<strong>7</strong>) displayed IC<sub>50</sub> values comparable to miltefosine, while showing markedly lower cytotoxicity toward J774.A1 and differentiated THP-1 macrophages, along with reduced hemolytic potential. Spin-label electron paramagnetic resonance (EPR) spectroscopy revealed that treatment with these compounds induces membrane rigidity after 24 h in a concentration-dependent manner. This effect is unlikely due to direct membrane interaction, as it does not occur after short incubations or at low concentrations, suggesting a correlation with oxidative stress, such as lipid peroxidation and/or protein oxidation, likely triggered by elevated reactive oxygen species (ROS) production. In contrast, no oxidative stress-induced membrane rigidity was detected in uninfected macrophages, suggesting that nitric oxide production may mitigate oxidative damage in these cells. However, significant membrane rigidity was observed in <em>Leishmania</em>-infected macrophages at concentrations slightly above the IC<sub>50</sub> for amastigotes, indicating that the compounds may selectively target infected macrophages. Additionally, compound (<strong>5</strong>) exhibited moderate cytotoxicity in the rapidly proliferating J774.A1 macrophage line but displayed very low cytotoxicity in differentiated, non-proliferative THP-1 macrophages. Overall, this study suggests that the primary mechanisms underlying the antileishmanial activity of these compounds are associated with their effects on the parasite plasma membrane, potentially leading to ionic leakage, subsequent disruption of mitochondrial membrane potential, and enhanced ROS generation.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184450"},"PeriodicalIF":2.5,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145010033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-08DOI: 10.1016/j.bbamem.2025.184455
Leilismara Sousa Nogueira , Bárbara Martins Cordeiro , Gilvânia Aparecida Rabelo Cordeiro , Ana Carolina Pacheco Ramos , Douglas Donizetti Raimundo , Grazielle Aparecida Silva Maia , Cristiane de Oliveira Renó , Leonardo Nunes Medeiros , Luciana Estéfani Drumont Carvalho , Vanessa Faria Cortes , Hérica de Lima Santos , Carlos Frederico Leite Fontes , Leandro Augusto Barbosa
Lactoferrin (Lf) is an iron-binding glycoprotein involved in various biological functions, including iron metabolism and immune response. Bovine lactoferrin (bLf) has gained attention due to its potential therapeutic applications. This study investigates the effects of bLf on human erythrocyte membranes, focusing on Na,K-ATPase (NKA) modulation. Whole blood and erythrocyte ghost membranes were treated with different concentrations of bLf, and multiple assays were performed to assess enzyme activity, lipid peroxidation, and lipid membrane composition. First, we demonstrate that Fe+2 is not able to increase the activity of the NKA. Bovine lactoferrin, on the other hand, significantly increases NKA activity, particularly affecting the α3 isoform. Additionally, bLf treatment led to an increase in total phospholipids and cholesterol content, suggesting modifications in the erythrocyte membrane. Molecular docking analysis revealed a strong interaction between bLf and α3-NKA, but not α1 isoform, supporting a potential regulatory role. Furthermore, bLf reduced Fe3+ and Thiobarbituric Acid Reactive Substances (TBARS) levels, indicating a protective effect against oxidative stress. These findings suggest that bLf modulates erythrocyte membrane properties, and could be specific to α3-NKA activation, reinforcing the role of Fe+3 in modulating the NKA activity.
乳铁蛋白（Lf）是一种铁结合糖蛋白，参与多种生物功能，包括铁代谢和免疫反应。牛乳铁蛋白（bLf）因其潜在的治疗应用而备受关注。本研究探讨了bLf对人红细胞膜的影响，重点是Na， k - atp酶（NKA）的调节。用不同浓度的bLf处理全血和红细胞鬼膜，并进行多项测定，以评估酶活性、脂质过氧化和脂质膜组成。首先，我们证明了Fe+2不能增加NKA的活性。另一方面，牛乳铁蛋白显著增加NKA活性，特别是影响α3亚型。此外，bLf治疗导致总磷脂和胆固醇含量增加，表明红细胞膜发生了改变。分子对接分析显示，bLf与α3-NKA之间存在较强的相互作用，但与α1亚型不存在相互作用，支持其潜在的调控作用。此外，bLf降低了Fe3+和硫代巴比妥酸反应物质（TBARS）水平，表明对氧化应激具有保护作用。这些结果表明，bLf可以调节红细胞膜性质，并且可能特异性地激活α3-NKA，从而增强了Fe+3在调节NKA活性中的作用。
{"title":"Evaluation of the effects of bovine lactoferrin on the membrane of human erythrocytes","authors":"Leilismara Sousa Nogueira , Bárbara Martins Cordeiro , Gilvânia Aparecida Rabelo Cordeiro , Ana Carolina Pacheco Ramos , Douglas Donizetti Raimundo , Grazielle Aparecida Silva Maia , Cristiane de Oliveira Renó , Leonardo Nunes Medeiros , Luciana Estéfani Drumont Carvalho , Vanessa Faria Cortes , Hérica de Lima Santos , Carlos Frederico Leite Fontes , Leandro Augusto Barbosa","doi":"10.1016/j.bbamem.2025.184455","DOIUrl":"10.1016/j.bbamem.2025.184455","url":null,"abstract":"<div><div>Lactoferrin (Lf) is an iron-binding glycoprotein involved in various biological functions, including iron metabolism and immune response. Bovine lactoferrin (bLf) has gained attention due to its potential therapeutic applications. This study investigates the effects of bLf on human erythrocyte membranes, focusing on Na,K-ATPase (NKA) modulation. Whole blood and erythrocyte ghost membranes were treated with different concentrations of bLf, and multiple assays were performed to assess enzyme activity, lipid peroxidation, and lipid membrane composition. First, we demonstrate that Fe<sup>+2</sup> is not able to increase the activity of the NKA. Bovine lactoferrin, on the other hand, significantly increases NKA activity, particularly affecting the α3 isoform. Additionally, bLf treatment led to an increase in total phospholipids and cholesterol content, suggesting modifications in the erythrocyte membrane. Molecular docking analysis revealed a strong interaction between bLf and α3-NKA, but not α1 isoform, supporting a potential regulatory role. Furthermore, bLf reduced Fe<sup>3+</sup> and Thiobarbituric Acid Reactive Substances (TBARS) levels, indicating a protective effect against oxidative stress. These findings suggest that bLf modulates erythrocyte membrane properties, and could be specific to α3-NKA activation, reinforcing the role of Fe<sup>+3</sup> in modulating the NKA activity.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184455"},"PeriodicalIF":2.5,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145032579","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-08DOI: 10.1016/j.bbamem.2025.184452
Maria Hoernke , Shuai Shi , Alasdair T.M. Hubbard , Nina Geringer , Fabio Strati , Chen Shen , Christian Wölk , Richard D. Harvey
Using the stable synthetic analogue 3-aza-dehydroxylysyl-phosphatidylglycerol (3adLPG), the putative role of native staphylococcal LPG in inhibiting the antibiotic daptomycin from binding to its target phosphatidylglycerol (PG), was investigated with respect to interfacial interactions between these lipids, daptomycin, and calcium ions. The influence of lipid monolayer/bilayer composition and interfacial ion concentrations upon the structure and integrity of model membranes were probed after daptomycin challenge using a combination of surface x-ray scattering techniques and fluorescence assays. In models representing the membrane composition of the daptomycin susceptible phenotype consisting of PG/3adLPG in a 7:3 M ratio, calcium ions drive the formation of two separate phases; Ca2+ cross-linked PG/PG pairs and PG/3adLPG ion pairs. Daptomycin is able to bind directly to the lipids in the PG/PG phase and increases the amount of interfacial Ca2+ ions to a level sufficient to displace 3adLPG from ion pairs with PG, and thus binds to its target PG. In bilayers with mixed chain lipids, daptomycin leads to pronounced membrane perturbations and enhanced permeability. Sequestering all of the available PG into PG/3adLPG ion pairs, therefore, would represent a putative daptomycin non-susceptible membrane. Daptomycin binding and the extent of subsequent lipid structural changes are reduced in these membranes. This implies that in bacteria, native LPG biosynthesis would need to ensure either an equivalence or an excess in relation to membrane PG content, in order for this mechanism alone to significantly contribute to daptomycin resistance.
{"title":"Daptomycin membrane activity is modulated by the localized interplay between calcium ions and phospholipids in monolayers and bilayers containing a lysyl-phosphatidylglycerol analogue","authors":"Maria Hoernke , Shuai Shi , Alasdair T.M. Hubbard , Nina Geringer , Fabio Strati , Chen Shen , Christian Wölk , Richard D. Harvey","doi":"10.1016/j.bbamem.2025.184452","DOIUrl":"10.1016/j.bbamem.2025.184452","url":null,"abstract":"<div><div>Using the stable synthetic analogue 3-aza-dehydroxylysyl-phosphatidylglycerol (3adLPG), the putative role of native staphylococcal LPG in inhibiting the antibiotic daptomycin from binding to its target phosphatidylglycerol (PG), was investigated with respect to interfacial interactions between these lipids, daptomycin, and calcium ions. The influence of lipid monolayer/bilayer composition and interfacial ion concentrations upon the structure and integrity of model membranes were probed after daptomycin challenge using a combination of surface x-ray scattering techniques and fluorescence assays. In models representing the membrane composition of the daptomycin susceptible phenotype consisting of PG/3adLPG in a 7:3 M ratio, calcium ions drive the formation of two separate phases; Ca<sup>2+</sup> cross-linked PG/PG pairs and PG/3adLPG ion pairs. Daptomycin is able to bind directly to the lipids in the PG/PG phase and increases the amount of interfacial Ca<sup>2+</sup> ions to a level sufficient to displace 3adLPG from ion pairs with PG, and thus binds to its target PG. In bilayers with mixed chain lipids, daptomycin leads to pronounced membrane perturbations and enhanced permeability. Sequestering all of the available PG into PG/3adLPG ion pairs, therefore, would represent a putative daptomycin non-susceptible membrane. Daptomycin binding and the extent of subsequent lipid structural changes are reduced in these membranes. This implies that in bacteria, native LPG biosynthesis would need to ensure either an equivalence or an excess in relation to membrane PG content, in order for this mechanism alone to significantly contribute to daptomycin resistance.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184452"},"PeriodicalIF":2.5,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145032551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-08DOI: 10.1016/j.bbamem.2025.184447
Jesse W. Sandberg , Grace Brannigan
The Envelope protein (E protein) of SARS-CoVs 1 and 2 has been implicated in the viral budding process and maintaining the spherical shape of the virus, but direct evidence linking the protein to long-range membrane deformation is still lacking. Computational predictions from molecular simulation have offered conflicting results, some showing long-range E-induced membrane curvature and others showing only local deformations. In the present study, we determine the mechanism driving these deformations by modulating the degree of hydrophobic mismatch between protein and membrane. We observe that certain barostat and restraint settings, common in coarse-grained MD simulations, can prevent equilibration of the membrane area. Our results indicate that the E protein does not induce long-range curvature, but does exhibit severe local deformations that are exacerbated by hydrophobic mismatch. These deformations occur in conjunction with local leaflet thickness asymmetry, suggesting asymmetry and curvature couple to reduce the free energy cost of a deformed membrane.
{"title":"SARS-CoV E protein couples asymmetric leaflet thickness and curvature deformations","authors":"Jesse W. Sandberg , Grace Brannigan","doi":"10.1016/j.bbamem.2025.184447","DOIUrl":"10.1016/j.bbamem.2025.184447","url":null,"abstract":"<div><div>The Envelope protein (E protein) of SARS-CoVs 1 and 2 has been implicated in the viral budding process and maintaining the spherical shape of the virus, but direct evidence linking the protein to long-range membrane deformation is still lacking. Computational predictions from molecular simulation have offered conflicting results, some showing long-range E-induced membrane curvature and others showing only local deformations. In the present study, we determine the mechanism driving these deformations by modulating the degree of hydrophobic mismatch between protein and membrane. We observe that certain barostat and restraint settings, common in coarse-grained MD simulations, can prevent equilibration of the membrane area. Our results indicate that the E protein does not induce long-range curvature, but does exhibit severe local deformations that are exacerbated by hydrophobic mismatch. These deformations occur in conjunction with local leaflet thickness asymmetry, suggesting asymmetry and curvature couple to reduce the free energy cost of a deformed membrane.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 8","pages":"Article 184447"},"PeriodicalIF":2.5,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145026664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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