Journal of Membrane Biology最新文献

Store-operated calcium entry (SOCE) is a crucial pathway that aids in restoring depleted calcium levels in the endoplasmic reticulum (ER), consequently regulating cellular calcium homeostasis. When calcium stores are low, two key proteins are activated: STIM1, which senses the calcium levels in the ER, and ORAI1, the pore-forming subunit of calcium release-activated calcium (CRAC) channels. ORAI1 is overexpressed in triple-negative breast cancer (TNBC) and regulates the transcription of genes that regulate cancer progression. The complete atomic structure of human ORAI1 (hORAI1) remains unknown, which poses a challenge for developing targeted therapies. This study modeled the closed state of hORAI1, identifying it as a potential target for disrupting calcium influx and oncogenic signaling in TNBC. A pharmacophore hypothesis derived from Mildronate analogues was utilized to screen the COCONUT database for small compounds that stabilize the closed state of hORAI1. Five promising compounds were identified: CNP0006530, CNP0006516, CNP0008628, CNP0002844, and CNP0004972. These compounds exhibited docking scores ranging from - 7.461 to - 5.393 kcal/mol and formed stable interactions with crucial residues, Glu106 and Asp110. This likely aids in stabilizing the closed conformation and inhibiting calcium influx. Molecular dynamics simulations have demonstrated the structural stability and compactness of the lead complexes. Furthermore, principal PCA/FEL analyses have validated their conformational stability within a membrane environment. These findings provide novel insights into the structural gating processes of hORAI1 and emphasize the therapeutic potential of small compounds that target its closed state to inhibit calcium-mediated carcinogenesis in TNBC.

Whole-cell voltage patch clamp studies have shown that in bovine adrenal chromaffin cells held at -70 mV, a single 5-ns, 5 MV/m pulse activates a membrane conductance carried partially by Na⁺ through TRPC4/5 channels and the sodium leak channel (NALCN). Here we used fluorescence imaging with the Na⁺ indicator ING-2 to further investigate Na⁺ influx pathways. A 5-ns, 5 MV pulse elicited a tetrodotoxin-insensitive rise in ING-2 fluorescence that exhibited a similar electric field dependency and response to pulse-pair stimulation as the inward current. Increases in ING-2 fluorescence were partially inhibited by the NALCN inhibitor CP96345, the TRPC4/5 channel inhibitor M084, or the broad spectrum TRP channel inhibitor La³⁺, and fully blocked by the combination of these agents, suggesting involvement also of a La³⁺-sensitive Na⁺ pathway. Cholesterol depletion with methyl-β-cyclodextrin or PIP₂ synthesis inhibition with wortmannin also reduced the response. Full inhibition was achieved with the selective L-type voltage-gated Ca²⁺ channel (VGCC) inhibitors nitrendipine, verapamil, or diltiazem but not the broad-spectrum inhibitor Cd²⁺. Inhibitors of N- and P/Q-type VGCC had no effect. Fluorescence Ca²⁺ imaging in voltage-clamped GCaMP6f-expressing murine chromaffin cells held at -70 mV revealed a nitrendipine-sensitive, Cd²⁺-insensitive increase in intracellular Ca²⁺ that accompanied the inward current. These results provide evidence that nanoelectropulse-induced Na⁺ influx into chromaffin cells involves several Na⁺ influx pathways that are facilitated by Ca²⁺ influx via an L-type-like Ca²⁺ channel associated with cholesterol-rich membrane domains. Investigating such membrane effects is essential for developing nanosecond electric pulse technologies for stimulating and/or modulating excitable cells.

Permeabilization of biomembranes by polycationic peptides is known to depend on the membrane potential, although the exact mechanism of this process is not yet completely defined, to be effectively controlled. We quantified peptide-assisted permeabilization of red blood cells (RBCs) using a custom system that delivered microsecond bipulses configured as bimonopolar (BMP; same polarity) or bipolar (BP; opposite polarity), varying the inter-monopulse interval ([Formula: see text]). In RBC suspensions, bulk light transmittance at 650 nm showed that BMP yielded higher permeabilization effect than BP at short [Formula: see text], whereas lengthening [Formula: see text] mitigated bipolar cancellation and increased BP responses. Membrane surface charge modulation by moderate concentrations of deoxycholate and spermine increased and decreased, respectively, membrane permeabilization effects of polycationic peptides. Complementary measurements in planar lipid bilayers (BLMs) under an applied command voltage ([Formula: see text]) with polarity alternation showed remarkable conduction at negative bias, essentially decreased in the presence of 1 mM Mg²⁺. Infrared thermometry over RBC suspension revealed modest heating (≈ 2.5-6 °C), equal for BMP and BP applications, indicating an electrical rather than thermal origin for waveform effects. Finally, the designed electroporation protocols allow controlled short-time permeabilization of cell membrane that might be useful for biotechnological applications and therapeutic delivery.

Aggressive cancer cells such as pancreatic cells exhibit an enhanced metastatic phenotype that involves cell migration and invasion. Cellular membrane deformation is a key process implicit in cell movement. This implicates a link between altered lipid metabolism during cancer progression and modulated membrane properties and hence associated functions. One of the key factors underlying the aggressiveness of pancreatic cancer is the presence of the highest percentage of hypoxia, which further adds to the lipid metabolic reprogramming. The subsequent effect of hypoxia-induced lipidome changes on membrane properties governing cell movement was investigated in this work using a combination of cell biology, microscopy, and spectroscopy. Our findings revealed that hypoxia induces distinct lipidome signatures in a cell-line-dependent fashion, which in turn differentially modulates the cell membrane stiffness. The correlation of cell stiffness with other membrane properties and the actin cytoskeleton shows a random correlation indicating that hypoxic stress distinctly regulates specific membrane attributes governing cellular functioning and should be consulted for the development of effective treatments for pancreatic cancer.

The growing antimicrobial resistance presents a challenge in developing new potent drugs, but this effort is hindered by a lack of information regarding how these new drugs would behave in biomembranes. Surfactants are considered mimetic models for biomembranes and can be used to study drug-membrane interactions. In this study, we used two well-known surfactants-cationic cetyltrimethylammonium bromide and anionic sodium dodecyl sulfate-as model membranes to investigate their interaction with the antimicrobial drug ofloxacin (OFL). These interactions were studied using volumetric and acoustic methods over the temperature range of 293.15-323.15 K to determine the apparent molar volume, isentropic compressibility, apparent molar compressibility, acoustic impedance, relative association, and intermolecular free length. Furthermore, UV-Vis spectroscopy and cyclic voltammetry were employed to evaluate the binding constants and free energies of the drug-surfactant systems. These results provide key molecular insights into the thermodynamics of OFL partitioning and its binding mechanisms with amphiphilic assemblies. Such mechanistic understanding is crucial for the rational design of antibiotic delivery systems, facilitating precise control over drug loading and release dynamics in surfactant-based formulations.

Chlamydia trachomatis is an obligate intracellular Gram-negative pathogen that causes sexually transmitted infections (STIs) and trachoma. Current interventions are limited due to the widespread nature of asymptomatic infections, and the absence of a licensed vaccine exacerbates the challenge. In this study, we predicted outer membrane β-barrel (OMBB) proteins and designed a multi-epitope vaccine (MEV) construct using identified proteins. We employed a consensus-based computational framework on the C. trachomatis D/UW-3/CX proteome and identified 17 OMBB proteins, including well-known Pmp family members and MOMP. Eight OMBB proteins were computationally characterized, showing significant structural homology with known outer membrane proteins from other bacteria. Sequence-based annotation tools were used to determine their putative functions. B-cell and T-cell epitopes were predicted from the selected proteins. The MEV construct was designed using four cytotoxic T-lymphocyte (CTL) epitopes and 29 helper T-lymphocyte (HTL) epitopes from six OMBB proteins, which were conserved across 106 C. trachomatis serovars. To enhance its immunogenicity, the vaccine was supplemented with the Cholera toxin B subunit and PADRE sequence at the N-terminus. The MEV construct, of length 780 amino acids, was predicted to be antigenic, non-allergenic, non-toxic, and soluble. Secondary structure analysis revealed 95% random coils. A three-dimensional structural model of the MEV was generated and subsequently validated. Molecular docking between MEV and toll-like receptor 4 (TLR4) revealed strong and stable binding interactions. The MEV-TLR4 complex was found to be structurally compact and stable using molecular dynamics simulation. Immune simulation of the MEV construct elicited a strong immune response. This study highlights OMBB proteins as promising immunogenic targets and presents a computationally designed MEV candidate for C. trachomatis infection.

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.

Yeasts are able to tolerate different environmental conditions, including stress situations. Given their broad applications in the food industry, their ability to adapt to stressful conditions is an active area of research. Lipid composition of the yeast membrane is affected by environmental stress, and thus, the regulation of the membrane biophysical properties under such conditions may be a key point for yeast adaptation. Although Saccharomyces cerevisiae is highly tolerant to ethanol, its growth is inhibited when this alcohol accumulates in the medium. Therefore, we studied the effect of ethanol on yeast membranes using the fluorescent probe Laurdan, which is sensitive to water dipolar relaxation. Three strains were used: a laboratory strain of S. cerevisiae (BY4741), a mutant that lacks ergosterol (erg6 $\Delta$ ), and a commercial baker's yeast. At low ethanol levels, the emission signal of the probe remained constant for all strains. For ethanol proportions higher than 20% (v/v), at which cells are no longer viable, the signal changed abruptly, indicating an increase in solvent dipolar relaxation. We further studied BY4741 yeasts acclimated to high ethanol levels and found that water was more ordered in these membranes than in BY4741 grown in the absence of ethanol. We propose that water structure and membrane hydration are key for yeast viability in the presence of ethanol, and that studying the biophysical properties of membranes could be useful to identify yeast strains with a high tolerance to ethanol.

Fatty acid Transport Protein 3 (FATP3) is a single-pass transmembrane protein implicated in the uptake and intracellular transport of long-chain fatty acids, yet the molecular contribution of its transmembrane domain (TMD) remains poorly defined. Here, we establish an efficient and reproducible strategy for heterologous expression, purification, and in vitro reconstitution of FATP3-TMD. FATP3-TMD was over-expressed in Escherichia coli as a TrpLE fusion, liberated by cyanogen-bromide cleavage and polished by one-step reverse-phase HPLC, yielding milligram quantities of highly pure peptide. ¹H-¹⁵N HSQC spectroscopy revealed a well-folded FATP3-TMD in both Fos-choline-14 micelles and DMPC/DHPC bicelles. Strikingly, titration with docosahexaenoic acid (DHA) induced residue-specific chemical-shift perturbations exclusively in bicelles. These data demonstrate that a bilayer-like lipid context is essential for functional recognition of ω-3 fatty acids by the FATP3-TMD and provide a robust platform for mechanistic dissection of FATP3 mediated lipid transport.