Journal of Membrane Biology最新文献

{"title":"Integrating GlycoSHIELD Modeling and DNA-PAINT SMLM to Map the Glycosylation-Dependent Distribution of the Na,K-ATPase.","authors":"Bruno Stojcic, Piotr Draczkowski, Joan Patrick, Mezida Saeed, Hjalmar Brismar","doi":"10.1007/s00232-026-00396-1","DOIUrl":"10.1007/s00232-026-00396-1","url":null,"abstract":"<p><p>The cell surface localization of the Na,K-ATPase (sodium pump) is required for maintaining transmembrane electrochemical gradients. While glycosylation of the β1 subunit facilitates trafficking from the endoplasmic reticulum to the plasma membrane, its role in nanoscale surface organization is not characterized. This study employed GlycoSHIELD computational modeling and DNA-PAINT single-molecule localization microscopy (SMLM) to evaluate how N-glycans influence pump distribution. In-silico simulations indicated that N-glycans sequester the protein core, providing a steric shield that increases with structural complexity. To investigate this experimentally, glycosylation-deficient mutants (3NQ) were generated and confirmed via immunoblotting. Quantitative SMLM analysis of A498 cells demonstrated that wild-type pumps exhibit higher localization density and form larger (144 nm) and more frequent clusters than 3NQ mutants (109 nm). These results indicate that N-glycosylation promotes stable enzyme clustering, supporting a galectin-lattice mechanism of organization rather than steric repulsion.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13468990/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148727200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Real-time Structural Tracking of Slow P-type ATPase Dynamics.","authors":"K Magkakis, F Sabzian-Molaei, F Orädd, T Plivelic, M Andersson","doi":"10.1007/s00232-026-00389-0","DOIUrl":"10.1007/s00232-026-00389-0","url":null,"abstract":"<p><p>Time-resolved X-ray solution scattering (TR-XSS) provides direct access to protein structural dynamics but has largely been restricted from the microsecond range up to approximately 100 milliseconds. As a result, slower enzymatic systems, including many P-type ATPases, remain difficult to probe. Here, we extend the temporal reach of TR-XSS by sequentially positioning radiation damage-free acquisition windows to enable capturing structural evolution across sub-second to second timescales. Implemented at the CoSAXS beamline at MAX IV Laboratory, this strategy enables continuous tracking of slow protein dynamics while preserving structural sensitivity. Using adenylate kinase (AdK) as a benchmark, we observed a single conformational transition accompanied by signal amplitude decay. In contrast, application to the prokaryotic P-type ATPase LMCA1 revealed clear evolution in scattering profiles, consistent with sequential conformational transitions. Kinetic analysis identified two transitions on the 140 ms and 660 ms timescales, which correspond monitoring rise and decay of a rate-limiting step which can symbolize intermediate dynamics in a slow transport cycle. The results demonstrate that extended-time TR-XSS can resolve multi-step reaction pathways in slow membrane proteins. The approach broadens the accessible timescale of TR-XSS and establishes a general framework for studying slow conformational dynamics in P-type ATPases and related systems.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461796/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Conserved Fusion Peptide Sequence of SARS-CoVs is Insensitive to Ca²⁺ in Inducing Lipid Mixing in Model Membranes.","authors":"Priyanka Mahapatra, Hirak Chakraborty","doi":"10.1007/s00232-026-00395-2","DOIUrl":"10.1007/s00232-026-00395-2","url":null,"abstract":"<p><p>The viral envelope fuses with the host cell membrane to initiate viral infection. In the case of SARS-CoVs, the fusion process is catalyzed by the spike (S) protein. The S1 subunit interacts with the host cell receptor and assists in docking the viral particle at the cell surface, whereas the fusion process is induced by the insertion of a conserved hydrophobic motif at the N-terminal of the S2 subunit, known as a fusion peptide, into the host cell. Current experimental findings suggest that the fusion peptide of SARS-CoVs displays a strong Ca²⁺-dependent membrane binding and fusogenic activity, indicating that Ca²⁺ functions as a critical cofactor during viral entry. Nevertheless, many earlier studies fail to clearly differentiate between fusion driven directly by Ca²⁺ and that mediated by the fusion peptide, often due to the absence of appropriate controls for different Ca²⁺ concentrations. In this work, we have assessed the impact of the consensus fusion peptide of SARS-CoVs in polyethylene glycol (PEG)-mediated fusion of small unilamellar vesicles with varying Ca²⁺ concentration, where appropriate control experiments have been carried out in the absence of the peptide. Our results show that the peptide has no significant effect on Ca²⁺ in inducing lipid mixing of model membranes.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148686441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanistic Insights into Autoinhibition of the Human Flippase ATP8B1.","authors":"Michelle Juknaviciute Laursen, Mathilde Roth, Poul Nissen, Charlott Stock, Thibaud Dieudonné","doi":"10.1007/s00232-026-00391-6","DOIUrl":"10.1007/s00232-026-00391-6","url":null,"abstract":"<p><p>P4-ATPases are lipid flippases that maintain membrane phospholipid asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet, an essential process for membrane integrity, trafficking and signaling. Several P4-ATPases are tightly regulated by autoinhibitory N- and C-terminal extensions, yet the molecular basis of this regulation remains incompletely understood. Here, we investigated the autoinhibition mechanism of the human flippase ATP8B1 using trans-inhibition assays with synthetic peptides derived from its C-terminal tail. Using purified C-terminally truncated ATP8B1-CDC50A, we systematically assessed the inhibitory properties of peptides corresponding to distinct segments of the C-terminus. We show that the distal disordered region of the C-terminal tail significantly contributes to autoinhibition, likely through transient interactions with the cytosolic domains. We further identify a critical interaction between R1228 in the C-terminal tail and E219 in the A-domain, whose disruption markedly reduces inhibitory potency. In addition, we demonstrate that a minimal peptide spanning residues 1216-1228, which bridges the A- and N-domains in the autoinhibited conformation, is sufficient to inhibit ATPase activity. Together, these results refine the molecular description of ATP8B1 autoinhibition, open the way for structure-based activation strategies and provide insight into conserved regulatory mechanisms among P4-ATPases.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13415658/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148609704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of Time, Temperature, and Conformational Changes on the Characterization of Cardiotonic Steroid-Na⁺/K⁺-ATPase Interactions.","authors":"Pedro Azalim-Neto, Leandro Barbosa, Cristoforo Scavone, Luis Eduardo M Quintas","doi":"10.1007/s00232-026-00385-4","DOIUrl":"10.1007/s00232-026-00385-4","url":null,"abstract":"<p><p>Cardiotonic steroids (CTS) have gained prominence due to various mechanisms of action proposed or demonstrated to explain their effects, including their known classical and non-classical signaling pathways mediated by Na⁺/K⁺-ATPase (NKA) binding. For the characterization, interaction kinetics have been used to better translate in vivo efficacy and selectivity. In addition, temperature effects assessed by thermodynamic analysis provide deeper insight into intrinsic efficacy. However, the impact of conformational changes on both kinetic and thermodynamic parameters of CTS binding has been largely overlooked. This review discusses how these kinetic effects relate to efficacy and selectivity in the experimental characterization of CTS-NKA interactions. We focus on the most straightforward and high-throughput methods for describing CTS-NKA interaction, such as enzymatic activity inhibition, looking at dephosphorylation activity of NKA cycle, and compare them with gold-standard methods and its limitations. Finally, we include some statistical considerations for data analysis and future perspectives. This will provide better insight into the efficacy of this class of inhibitors/agonists targeting the NKA in drug discovery.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13407939/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148594271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro Reconstitution of Syncytin-2 and MFSD2A Reveals their Functional Tethering in Membrane Fusion Initiation.","authors":"Zhan Xiang, Lu Xu, Ying Li, Sha Sun","doi":"10.1007/s00232-026-00387-2","DOIUrl":"10.1007/s00232-026-00387-2","url":null,"abstract":"<p><p>Syncytin-2, a human endogenous retrovirus-derived fusogen, is essential for placental syncytiotrophoblast formation by mediating trophoblast cell-cell fusion. While Syncytin-2 shares structural similarities with viral envelope proteins, the molecular mechanisms underlying its receptor recognition and membrane fusion remain poorly characterized, largely due to the lack of in vitro reconstitution systems. Here, we established an in vitro reconstitution system to investigate the initial steps of Syncytin-2-mediated membrane fusion. We purified Syncytin-2 and its receptor MFSD2A and confirmed their interaction through co-immunoprecipitation and pull-down assays. Both proteins were successfully reconstituted into liposomes and exhibited specific tethering dependent on the receptor-fusogen interaction, which is the first committed step of fusion. This study provides an in vitro reconstitution of a placental fusogen-receptor pair, establishing a powerful platform for dissecting the molecular mechanism of syncytin-mediated cell-cell fusion and shedding light on understanding placenta development at the molecular level.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148388304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Basis of SERCA Inhibition by Derivatives of di-tert-butylhydroquinone Revealed by X-ray Crystallography.","authors":"Ryuta Kanai, Ayami Hirata, Chikashi Toyoshima, Stefan Paula","doi":"10.1007/s00232-026-00388-1","DOIUrl":"10.1007/s00232-026-00388-1","url":null,"abstract":"<p><p>The small molecule di-tert-butylhydroquinone (BHQ) is a potent inhibitor of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). It inhibits the enzyme by stabilizing it in its E2 conformation and thereby preventing the transition to the E1 state. To define the structural determinants underlying BHQ inhibition, we examined ten BHQ derivatives with systematic modifications of polar and hydrophobic substituents of different shapes and sizes. Inhibitory potencies were determined in ATPase activity assays, and crystal structures of SERCA1a in complex with eight of the ten derivatives were obtained at resolutions of 2.6 Å or better, aided by co-crystallization of thapsigargin to stabilize the E2 state. The structures revealed that the compounds bind in a pocket formed by transmembrane helices M1-M4, in which the ligand is stabilized by CH-π interactions between the central benzene ring and Leu61 and Pro312, by hydrogen bonds - preferentially involving Asp59 - and by extensive van der Waals contacts mediated by two butyl substituents. Analysis of the SERCA/inhibitor poses showed that while at least one hydrogen bond is required for binding, inhibitory potency correlates strongly with the number and quality of van der Waals contacts mediated by appropriately positioned alkyl groups. Compounds lacking alkyl substituents or containing oversized or mispositioned groups displayed markedly reduced potencies. Although no derivative exceeded BHQ in inhibitory activity, structural analysis suggests that repositioning hydroxy groups on the benzene ring or modifying alkyl substituents may further optimize binding affinity and thus inhibitory potency. Together, these results define the key molecular features of SERCA inhibition by BHQ analogs and suggest strategies for further optimization of this inhibitor class.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148347246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fungal Extracellular Vesicles are Recoverable Across Variable Ultracentrifugation Speeds but Display Species-specific Profiles of Sedimentation.","authors":"Luisa J Jozefowicz, Bárbara T Bezerra, Eva Veres, Zóra Szilovics, Cássia M Souza, Amanda C Camillo-Andrade, Hellen G G Santos, Paulo C Carvalho, Marlon D M Santos, Flavia C G Reis, Attila Gacser, Marcio L Rodrigues","doi":"10.1007/s00232-026-00386-3","DOIUrl":"10.1007/s00232-026-00386-3","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are central components of fungal biology, yet their isolation commonly relies on ultracentrifugation protocols originally developed for mammalian systems. In Cryptococcus, most EVs carry the capsular polysaccharide glucuronoxylomannan (GXM), raising the possibility that vesicle density, and therefore sedimentation, could be influenced by polysaccharide content. Here, we systematically evaluated EV recovery across sequential ultracentrifugation speeds (20,000 × g to 100,000 × g) in Cryptococcus deuterogattii, Candida auris, and Candida parapsilosis. In C. deuterogattii, GXM was detected across all ultracentrifugation fractions, and each fraction efficiently transferred polysaccharide to acapsular cells, demonstrating that its presence is independent of fraction density and does not correlate with sedimentation behavior. In the three fungal species, transmission electron microscopy and nanoparticle tracking analysis confirmed that bona fide EVs are recoverable across all centrifugation speeds. Qualitative and quantitative proteomic analyses revealed largely overlapping protein compositions among fractions within each species. However, proteomic profiles differed between species. In C. deuterogattii and C. auris, fractions displayed similar proteomic and predicted protein-protein interaction signatures across centrifugation speeds. In contrast, C. parapsilosis exhibited a clear partitioning pattern, with low-speed fractions (20,000 × g and 40,000 × g) clustering together and differing from higher-speed fractions (60,000-100,000 × g). These findings demonstrate that fungal EVs are recoverable across a broad range of ultracentrifugation speeds, but their sedimentation behavior is species-specific. Our study highlights the need for tailored EV isolation strategies and cautions against assuming methodological equivalence across fungal pathogens.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13275532/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148266844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Polyproline Modulates Membrane Translocation of Arginine-Rich Cell-Penetrating Peptides: Insights from Molecular Dynamics Simulations.","authors":"Seungho Choe, Afira Mariam, Maheen Ejaz","doi":"10.1007/s00232-026-00381-8","DOIUrl":"10.1007/s00232-026-00381-8","url":null,"abstract":"<p><p>Recent experiments have shown that incorporating polyproline segments into arginine (R)-rich cell-penetrating peptides (CPPs) enhances membrane penetration. Here, we employ molecular dynamics (MD) simulations combined with the weighted ensemble approach to investigate how a polyproline segment influences the free-energy barrier for membrane translocation in the designed peptide P9R9. Our results indicate that the extended, conformationally constrained nature of the P9 segment facilitates early membrane engagement and promotes the formation of a hydrated translocation pathway. This behavior is associated with a reduced desolvation penalty during insertion of the arginine-rich (R9) segment. Consistent with this interpretation, the solvent-accessible surface area (SASA) of R9 exhibits a non-monotonic trend, suggesting partial rehydration within the membrane interior. Together, these findings support a sequential translocation mechanism in which P9 interacts with the membrane prior to R9, thereby facilitating subsequent insertion and lowering the free-energy barrier relative to peptides lacking polyproline segments. This work provides a molecular-level perspective on how polyproline segments modulate membrane translocation and offers useful insights for designing more effective CPPs.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148177141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Peculiarities of Phosphatidylserine Externalization by Nano- and Microsecond Electric Pulses.","authors":"Mantas Silkunas, Alayna Enos, Iurii Semenov, Giedre Silkuniene, Eleni Zivla, Olga N Pakhomova, Andrei G Pakhomov","doi":"10.1007/s00232-026-00384-5","DOIUrl":"10.1007/s00232-026-00384-5","url":null,"abstract":"<p><p>Phosphatidylserine (PS) externalization is a hallmark of apoptosis but can also be triggered within seconds by pulsed electric fields (PEFs). Whether PS reaches the outer leaflet by lateral migration through electropores or indirectly through Ca²⁺-dependent signaling and scramblase activation remains debated. We used total internal reflection fluorescence (TIRF) microscopy with Annexin V-Alexa Fluor 568 and/or bovine lactadherin-FITC to resolve PS externalization in HEK293 cells placed on an indium tin oxide (ITO) transparent electrode. PEF exposures produced discrete PS-positive puncta (\"freckles\") within ~ 10 s after a 20-µs pulse (1.2-2.5 kV/cm) or with a ~ 1-min delay after a 400-ns pulse (9.3-13.7 kV/cm). Their number gradually increased, reaching up to 80-120 freckles per 100 µm² of cell membrane area within 5 min. Freckles were round, brightest in the center, and well described by a Gaussian intensity profile, with a consistent full width at half maximum (FWHM) of ~ 0.35 µm despite up to ~ 50-fold differences in peak intensity. Freckle formation was inhibited in Ca²⁺-free medium but could be partially restored by applying 20-µs PEFs at higher strengths. Many freckles moved laterally and axially in an apparently stochastic manner, whereas others remained spatially confined. The uniform size and shape of freckles and their mobility patterns suggested that they are PS-positive vesicles rather than PS membrane patches. Further analyses identified freckles as extracellular vesicles of ~ 200-nm estimated diameter whose formation is triggered by applying PEFs at intensities above the electroporation threshold.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":"259 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2026-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13236760/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148158908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}