搜索结果：The Journal of membrane biology

Membrane fusion is the first step in the infection process of the enveloped viruses. Enveloped viruses fuse either at the cell surface or enter the cell through endocytosis and transfer their internal genetic materials by fusing with the endosomal membrane at acidic pH. In this work, we have evaluated the effect of the Dengue virus fusion peptide (DENV FP) on the polyethylene glycol (PEG)-mediated lipid mixing of vesicles (hemifusion formation) at pH 5 and pH 7.4 with varying cholesterol concentrations. We have demonstrated that the DENV FP promotes hemifusion formation during the fusion of small unilamellar vesicles (SUVs) mainly at pH 5.0. Moreover, the fusion process demonstrates a strong correlation between fusogenicity and the amount of membrane cholesterol. We have further evaluated the partitioning ability of the peptide in three different membranes at pH 5.0 and pH 7.4. The fusogenic ability of the peptide at pH 5.0 is associated with the composition-dependent binding affinity of the peptide to the membrane. The depth-dependent fluorescence probes are used to evaluate membrane organization and dynamics utilizing steady-state and time-resolved fluorescence spectroscopic techniques. Our results show that the DENV FP promotes hemifusion formation by fluidizing the interfacial region of the membrane.

Cell-penetrating peptides (CPPs) are increasingly used for delivering cargo into cells, but the mechanisms of their membrane crossing remain poorly understood, even for shorter cationic hydrophobic peptides. This study explores how cationic hydrophobic peptides with various combinations of cationic (K and R) and hydrophobic (W and I) amino acids interact with lipid bilayers made of 90% neutral lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC) and 10% anionic lipids (either 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (PG) or 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (PS)). Specifically, it was found that changing the hydrophobic segment from W to I significantly influences turbidity during the pretransition for the PC/PG mixture. Conversely, in the PC/PS mixture, the interaction with I-peptides causes changes in turbidity during the main phase transition, regardless of whether the cationic segment is R or K. Molecular features analyzed through FTIR spectroscopy revealed significant differences in the vibrations of methylene groups within hydrocarbon chains. In the presence of I-peptides, regardless of whether they are R or K derivatives, a notably higher number of kink conformers were observed compared to W-peptides. Although W-peptides tend to form aggregates due to the insertion of their hydrophobic segments into the lipid bilayer, they appear to maintain the membrane's integrity and the organization of lipids. In contrast, the branched side chains of the I-segment induce out-of-plane movements in the hydrocarbon chains. As the initial interaction between the peptide and the lipid membrane is crucial for its translocation, these findings provide insights into the molecular events occurring before translocation and emphasize the specifics that make it unique to CPPs.

Dysfunction of the main inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is the underlying reason behind many neurological disorders including Alzheimer's and Huntington's diseases, autism spectrum disorders, anxiety, depression, hypertension, and cardiovascular diseases, among others. Here, we address neurotransmitter-induced alterations of synaptosomal and model membrane electrical properties for elucidating membrane-related biophysical mechanisms of neurological disorders. We focus on membrane surface characteristics of the pinched off nerve endings synaptosomes, which for decades have been a powerful tool in neurobiology. Microelectrophoretic measurements of GABA-treated negatively charged synaptosomes from rat cerebral cortex reveal lower negative zeta potential as a result of reduced electrical charge on the membrane surface at (1-4 h) after isolation. Conversely, enhancement of the surface parameters of synaptosomes (17-22 h) post isolation is obtained due to additional negatively exposed groups on the surface of the vesicles. The electrical properties of bilayer lipid membranes are probed by electrochemical impedance spectroscopy, reporting as light increase of the membrane electrical capacitance in the presence of GABA, likely related to membrane thinning and dielectric permittivity alterations. The neurotransmitter inhibits sodium-potassium as well as the total ATPase activity and slightly enhances magnesium-ATPase of native synaptic membranes. At low (pM) GABA concentrations the activity of acetylcholinesterase (AChE) in synaptic membranes increases. AChE inhibition is reported at higher GABA concentrations. The relation between the surface electrical properties of cells and the enzymatic activity of brain ATPases and AChE, as examined here, are expected to be helpful in the elucidation of membrane-mediated molecular mechanisms relevant to neurological disorders and conditions.

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.

We have recently reported the observation of closed-loop fluid-fluid immiscibility in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid membranes containing either 25-hydroxycholesterol (25HCH) or 27-hydroxycholesterol (27HCH) (Kamal et al. Proc Natl Acad Sci 120(25):2216002120, https://doi.org/10.1073/pnas.2216002120 ,  2023). Here we extend these studies to membranes of symmetric, saturated phospholipids having chain length varying from 12 to 16 carbon atoms. The phase behavior of the PC-25HCH and PC-27HCH membranes were probed by using fluorescence microscopy, atomic force microscopy and small angle X-ray scattering. 25HCH is found to induce fluid-fluid coexistence in these membranes for lipid chains lengths varying from 12 to 15 C atoms, whereas 27HCH induces similar phase behavior for chain lengths varying from 13 to 16. Thus the occurrence of fluid-fluid coexistence in these membranes depends both on the position of the -OH group in the oxysterol side chain as well as the membrane thickness. Our earlier studies have indicated that the phase behavior of these binary membranes can be understood in terms of the temperature-dependent orientation of the oxysterol in the membrane. Our present results suggest that the relative energies of these orientations depend on the membrane thickness and the oxysterol structure. Observation of fluid-fluid immiscibility in different saturated lipid model membranes containing 25HCH or 27HCH shows the generic nature of this phase behavior.

The group of inhibitors known as Potassium Competitive Acid Blockers (PCABs) has become one of the main topics of current research into reducing gastric H,K-ATPase activity. The design of novel PCABs relies on structure-based drug design strategies that integrate structural data with molecular dynamics simulations. A key aspect in conventional molecular dynamics simulations is the assignment of residue charge states, since the local physicochemical environment influences the pKa of amino acids. This computational work investigates the impact of the protonation state of key residues on the interactions between PCABs and the gastric H,K-ATPase. The study focusses on Glu343, Glu795, and Glu820, from the cation binding cavity, and vonoprazan and tegoprazan; two chemically unrelated PCABs. The results show how protonation states generate changes in interactions, especially with the charged group of inhibitors. Simulations in which Glu343 or Glu795 were protonated showed that the charged secondary amine of vonoprazan could rotate freely, favoring hydrogen bonding with some of the glutamic residues deep within the pocket. In contrast, the positive charge of tegoprazan is located in the benzimidazole ring, a rigid and bulky structure, which moves away from the center of the cavity when Glu343 or Glu795 are protonated. The inspection of the ionization states of amino acids reveals the conformational flexibility of the protonated group of PCABs as pivotal for binding affinity and highlights the importance of considering the protonation state of protein residues before performing any conventional molecular dynamics simulations for drug design.

This work describes a computer study that looks at how different amounts of cholesterol (0%, 25%, and 50%) in cell membranes change the relationship between ATP and the KATP channel. This could explain why pancreatic beta-cells secrete insulin differently. We use computer simulations of molecular dynamics, calculations of binding free energy, and an integrated oscillator model to look at the electrical activity of beta-cells. There is a need for this kind of multiscale approach right now because cholesterol plays a part in metabolic syndrome and early type 2 diabetes. Our results showed that the increase in cholesterol concentration in the cell membrane affects the electrostatic interactions between ATP and the KATP channel, especially with charged residues in the binding site. Cholesterol can influence the properties of a membrane, including its local charge distribution near the channel. This affects the electrostatic environment around the ATP-binding site, increasing  the affinity of ATP for the channel as our results indicated from 0 to 25 and 50% cholesterol (- 141 to - 113 kJ/mol, respectively). Simulating this change in the affinity to ATP of the KATP channels in a model of the electrical activity of the pancreatic beta-cell indicates that even a minimal increase could produce hyperinsulism. The study answers an important research question about how the structure of the membrane affects the function of KATP and, in turn, insulin releases a common feature of metabolic syndrome and early stages of type 2 diabetes.

The complement pathway is one of the most ancient elements of the host's innate response and includes a set of protein effectors that rapidly react against pathogens. The late stages of the complement reaction are broadly categorised into two major outcomes. Firstly, C5a receptors, expressed on membranes of host cells, are activated by C5a to generate pro-inflammatory responses. Secondly, target cells are lysed by a hetero-oligomeric pore known as the membrane attack complex (MAC) that punctures the cellular membrane, causing ion and osmotic flux. Generally, several membrane-bound and soluble inhibitors protect the host membrane from complement damage. This includes inhibitors against the MAC, such as clusterin and CD59. This review addresses the most recent molecular and structural insights behind the activation and modulation of the integral membrane proteins, the C5a receptors (C5aR1 and C5aR2), as well as the regulation of MAC assembly. The second aspect of the review focuses on the molecular basis behind inflammatory diseases that are reflective of failure to regulate the terminal complement effectors. Although each arm is unique in its function, both pathways may share similar outcomes in these diseases. As such, the review outlines potential synergy and crosstalk between C5a receptor activation and MAC-mediated cellular responses.

Magnetobiology studies the effects of magnetic fields on biological systems. In this context, the Magnetobiology Research Group at the University of Caldas has hypothesized that magnetic treatment may influence water flow through cell membranes. To contribute to evaluating this hypothesis, this study analyzes the effect of magnetic fields on water transport through TIP3;1 aquaporins of Zea mays L. Molecular dynamics simulations were performed using a modified version of GROMACS that includes magnetic forces in the Velocity-Verlet algorithm, exposing a TIP3;1 homotetramer embedded in a lipid bilayer to static, uniform magnetic flux densities (B) ranging from 0 to 10 T and oriented along the membrane normal (z-axis). The system was solvated with the SPC/E water model. To assess the impact on water flow, we analyzed the conformational dynamics of TIP3;1, protein-water interactions within the single-file channel, and the osmotic permeability coefficient (pf). Trajectory analysis revealed that the magnetic field alters the average pore radius and increases protein conformational variability. These structural changes affect the intermolecular interactions between the protein and water molecules, influencing water mobility through the channel. Systems exposed to magnetic fields showed up to a threefold increase in pf compared to the control. These findings suggest that magnetic fields can modulate water flow through membranes, supporting a possible mechanism of magnetically influenced water transport in biological systems.

Membrane Attack Complex/Perforin (MACPF) domain proteins are β-pore forming toxins (β-PFTs) involved in the pathogenesis of various organisms. Among them, Perforin-like proteins (PLPs), produced by Plasmodium species, play essential roles in parasite invasion and egress. Due to increasing drug resistance in Plasmodium, PLPs represent promising but underexplored therapeutic targets, largely due to the lack of structural and mechanistic data. This study investigates the binding and pore formation mechanism of the Plasmodium falciparum PLP1 (PfPLP1), which is expressed during the human life cycle of the parasite. We modeled PfPLP1 structure and performed both all-atom and coarse-grained molecular dynamics simulations in soluble and membrane-associated states. PfPLP1 comprises two domains, a canonical MACPF domain and a β-pleated sheet domain- apicomplexan perforin β-domain (APCβ). Initial membrane binding is mediated by cationic residues at the base of the APCβ domain, which interact with the polar headgroups of the lipids from the host cell membrane. We analyzed the membrane-inserted tetrameric form where water molecules were observed to penetrate between the tetramer and the lipid bilayer, initiating pore opening. During this process, lipids reorganize into a toroidal edge to shield their hydrophobic tails, while water mixes with lipid headgroups in a disordered, heterogeneous fashion. Larger oligomeric assemblies show lateral displacement of lipids and a clear tendency to form pore-like structures. This study provides molecular insights into PfPLP1's membrane binding and pore-forming behavior in both monomeric and oligomeric forms. The outcome of this study would be applicable in understanding pore formation mechanism in other PLPs and similar toxins.

Dengue virus, an arbovirus from the genus Flavivirus in the family Flaviviridae, forms a nucleocapsid structure through interactions between its genome and multiple copies of the capsid protein. Experimental studies have confirmed the interaction between the viral capsid protein and lipid droplets, indicating a protein-lipid interaction. Cryo-EM studies show that in immature viruses, the nucleocapsid is located close to the viral membrane. This study uses multiple MD simulations to explore the orientation of the capsid protein relative to the lipid membrane, focusing on how the protein's hydrophobic pocket interacts with the membrane. We also investigated the interaction between the capsid protein and RNA, considering the effects of sequence length and identity. Finally, we construct a model of the lipid-protein-RNA complex, demonstrating that the capsid protein's hydrophobic pocket interacts with the membrane, while the positively charged H4 helix interacts with the negatively charged RNA. This research may identify crucial interactions for immature virus particle formation and provide insights for future therapeutic interventions.

Coronaviruses use the spike protein (spike) to bind to target cells, and fuse the viral envelope with a host lipid membrane. Spike is a large trimeric surface glycoprotein, anchored to the viral membrane (envelope) by a single membrane-spanning polypeptide helix and a short intra-virion domain. In the SARS-CoV-2 virus, the spike is formed by three protomers of 1273 residues, each with two distinct domains separable by enzymatic proteolysis prior to infection. Thus far, enveloped virus surface glycoprotein structures have provided a detailed molecular view of the pre-fusion state, while structures of the post-fusion state have remained incomplete. The determination of the full-length structure of the SARS-CoV-2 spike in the post-fusion state is a landmark in furthering our understanding of the structural pre-requisites for membrane fusion. This perspective analyzes the fusion domain as revealed by the recent structure in the context of conserved sequences across diverse coronaviruses. We highlight the characterization of the membrane-embedded fusion peptide in a helical hairpin topology. This structure is discussed as a re-imagination of the helical hairpin hypothesis for polypeptide insertion into membranes, postulated by Engleman and Steitz over four decades ago.

Pore-forming toxins (PFTs) belong to a class of proteins expressed by bacteria to initiate infections by unregulated pore formation on the plasma membrane of host cells. Although cholesterol is a key sterol motif that promotes toxin activity, the influence of oxysterols, upregulated in senescent cells or in other inflammatory disorders, on lytic activity has not received much attention. Using all-atom molecular dynamics simulations, we study the changes to the sterol binding landscape of membrane-inserted cytolysin A (ClyA), an α -PFT expressed by E. coli, in the presence of tail-oxidized 25-hydroxycholesterol (25-HC) in a palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol:25-HC (70:20:10) membrane. 25-HC was found to entirely replace previously identified cholesterol binding hotspots [PNAS,115 7323-7330] between the membrane-inserted β -tongue motifs with binding lifetimes on the order of microseconds. Although the overall sterol occupancy is lower for the N-terminal helix motif that forms the lining of the water channel, 25-HC binding is less when compared with cholesterol. The presence of the additional OH group on the 25th carbon enhances interactions with polar residues of the β -tongue, increasing 25-HC binding times by several fold when compared with cholesterol. We discuss the implications of this enhanced oxysterol interaction on pore formation of the α family of toxins such as ClyA, in contrast with the cholesterol-dependent cytolysins, where oxysterols have been shown to be detrimental to pore formation.

Insect venom-derived antimicrobial peptides (AMPs) hold significant therapeutic promise, but their application is constrained by mammalian cell toxicity. Toxicity assays are rapid and high-throughput, but screening large peptide libraries remains resource-intensive due to the requirements for peptide synthesis, purification, and testing. Alternatively, molecular dynamics (MD) simulations using mammalian membrane models provide an efficient and robust method for preliminary toxicity prediction. To benchmark the optimal model, two distinct mammalian membrane systems with diverse lipid compositions were evaluated for a set of sixteen toxic and fourteen non-toxic AMP analogs from five distinct insect AMP families, viz. anoplin, polybia, halictine, hyline, and macropin. In this study, a total of 25 µs of MD simulation time was generated. The analysis of MD trajectories, each spanning 500 ns for each of the 30 peptides, revealed significant variations in structural stability and membrane permeability between toxic and non-toxic AMPs, which aligned with the experimental results. Root Mean Square Deviation (RMSD) of the peptides during the last 100 ns of the simulation period successfully distinguished toxic from non-toxic AMPs with 90% accuracy when using realistic membrane models. The well-cited multicomponent mammalian membrane model failed to effectively predict mammalian toxicity. These findings underscore the efficacy of MD simulations in predicting the toxicity of venom-derived AMPs, thereby opening avenues for the accelerated development of safer antimicrobial therapies.

The passage of nystatin through the ergosterol-containing phospholipid bilayer was studied on monovesicular and multivesicular giant unilamellar vesicles (GUVs and MVVs). Phase-contrast optical microscopy was used to examine vesicles composed of a palmitoyl-oleoyl-phosphatidyl-choline (POPC) bilayer with either 15 or 45 mol% ergosterol. Three types of vesicles were analyzed: (i) GUVs, (ii) outer vesicles (outGUVs) of MVVs, and (iii) inner vesicles (inGUVs) of MVVs. The times of their ruptures were determined after their exposure to nystatin at concentrations of 250 and 500 μ M. At both concentrations, the times that the inGUVs spent in the nystatin solution after the rupture of the corresponding outGUVs were significantly shorter than the rupture times of individual GUVs of the similar size. These differences in rupture times demonstrate that the ergosterol-containing POPC membrane is permeable to nystatin.

Research on the auxiliary subunits β (CaVβ) and α2δ (CaVα2δ) of voltage-gated calcium channels has gained increasing interest as novel and unexpected functional interactions for these proteins are discovered. Beyond their classic role as regulators of the channel complex, these subunits participate in the spatiotemporal fine-tuning of the channels and in diverse cellular processes. Currently, multiple studies are investigating the interactions between CaVβ and CaVα2δ with other proteins outside the channel complex, and how these associations affect relevant cellular functions such as cell growth, differentiation, and gene expression beyond their effects on channel activity. The auxiliary subunits have also been observed to associate with components involved in calcium channel biogenesis, a process independent of the direct modulation of channel activity. Furthermore, their involvement in the intracellular transport of other channels and receptors, as well as in nuclear signaling, has been demonstrated. The expression of different variants or isoforms may fulfill specific functions in diverse tissues and developmental stages, even outside the channel complex. Undoubtedly, the study of these proteins as scaffolding or regulatory molecules for other proteins has significantly enriched our understanding of their influence on cell signaling and excitability.

P-type ATPases are active transporter enzymes that maintain ion electrochemical gradients for functions such as muscle contraction, kidney function, digestion, and nerve signalling. The dysfunction of these ATPases has been linked to several neurological, cardiovascular, and metabolic disorders. An electrostatic switch mechanism (ESM) has been hypothesised to be involved in the regulation of the sodium-potassium pump (Na+,K+-ATPase) and the gastric proton pump (H+,K+-ATPase). An analysis of primary and tertiary protein structures suggests a potentially higher prevalence of the ESM in human P-type ATPases than previously thought. Evidence for the ESM was found on the N-terminus for the P1B, and P2C subfamilies of P-type ATPases, and on the C-terminus for the P2A and P5 subfamilies. In the case of the P4 family, evidence for the ESM was found on the N-terminus for some subfamilies and on the C-terminus for others. Evidence for the ESM has been identified in P-type ATPase subfamilies distributed in separate lineages of the phylogenetic tree, suggesting that the ESM evolved multiple times independently.

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.

In this study, we describe the effect of the noble gas, xenon on the electrical properties of tethered lipid bilayer membranes, (tBLMs), including the effect of xenon on the activation energy for electrical conduction through the tBLM. Such studies benefit from the stability of a tethered membrane given the wide range of temperatures that are scanned and the time required for these measurements. The results indicate that xenon increases the activation energy for electrical conduction through bilayers and decreases the average pore size that dominates the electrical conductance of the lipid bilayers at low voltages. Xenon possesses a high affinity for lipid membranes and is a potent general anaesthetic. Its anaesthetic potency is possibly associated with its effects on proteins embedded in the lipid membranes.