Search Results (171)

The alarming rise of antibiotic resistance in Gram-negative bacteria has emerged as a major global health challenge. A key factor contributing to this crisis is the low permeability of the bacterial outer membrane, which acts as a barrier that prevents antibiotics from entering the cell. Protein channels embedded in this outer membrane selectively regulate the influx of hydrophilic compounds, including antibiotics. To combat antibiotic resistance, understanding the molecular mechanisms governing antibiotic permeability through bacterial membrane channels is crucial. This knowledge is key towards elucidating their roles in studing antibiotic resistance. By compiling and analysing the flux data from multiple electrophysiological reversal potential experimental studies, which involves measuring zero-current potentials and the corresponding single-channel conductance, we can calculate the flux of charged antibiotics/compounds across different Gram-negative bacterial outer membrane channels. Through this comprehensive synthesis, this review aims to advance our understanding and stimulate discussions about the physicochemical factors influencing the flux of antibiotics through bacterial membrane protein channels, ultimately enhancing our knowledge in this area. Full article

The Danshen terpenoid cryptotanshinone (CPT) is gaining enormous interest in light of its various outstanding biological activities. Among those, CPT has been shown to interact with cell membranes and, for instance, to have antibacterial activity. Several works have shown that CPT alone, or in combination with other drugs, can effectively act as an antibiotic against various infectious bacteria. Some authors have related the mechanism underlying this action to CPT–membrane interaction. This work shows that CPT readily partitions into phosphatidylcholine membranes, but there is a limiting capacity of accommodation of ca. 1 mol CPT to 3 mol phospholipid. The addition of CPT to unilamellar liposomes composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) causes membrane permeabilization, as shown by fluorescent probe leakage. This process has been kinetically studied, as well as its modulation by incorporation of phosphatidylethanolamine or phosphatidylglycerol, as a model for pathogenic cell membranes. The thermotropic behavior of 1,2-dimyristoylphosphatidylcholine (DMPC) model membranes is weakly affected by CPT, but the terpenoid causes significant dehydration of the polar region of the bilayer and weak disordering of the acyl chain palisade, as observed in Fourier-transform infrared spectroscopy (FTIR) results. Small-angle X-ray scattering (SAXS) shows that CPT increases DMPC bilayer thickness, which could be due to localization near the phospholipid/water interface. Molecular dynamics (MD) simulations show that the lateral diffusion coefficient of the phospholipid increases with the presence of CPT. CPT extends from the polar head region to the center of the bilayer, being centered between the carbonyl groups and the unsaturated region of the POPC, where there is greater overlap. Interestingly, the free energy profiles of a water molecule crossing the lipid membrane show that the POPC membrane becomes more permeable in the presence of CPT. In summary, our results show that CPT perturbs the physicochemical properties of the phospholipid membrane and compromises its barrier function, which could be of relevance to explain part of its antimicrobial or anticancer activities. Full article

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR–D2R, GABAA–D5R, and FGFR1–5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor–receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor–receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor–receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000–2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor–receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor–receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson’s disease. Full article

Membrane protein integrase (MPIase), an endogenous glycolipid in Escherichia coli (E. coli) membranes, is essential for membrane protein insertion in E. coli. We have examined Sec-independent membrane protein insertion mechanisms facilitated by MPIase using physicochemical analytical techniques, namely solid-state nuclear magnetic resonance, fluorescence measurements, and surface plasmon resonance. In this review, we outline the physicochemical characteristics of membranes that may affect membrane insertion of proteins. Subsequently, we introduce our results verifying the effects of membrane lipids on insertion and estimate the impact of MPIase. Although MPIase is a minor component of E. coli membranes, it regulates insertion by altering the physicochemical properties of the membrane. In addition, MPIase promotes insertion by interacting with substrate proteins. We propose comprehensive mechanisms for the membrane insertion of proteins involving MPIase, which provide a physicochemical basis for understanding the roles of glycolipids in protein translocation. Full article

The Gram-negative bacterium Helicobacter pylori is a very successful pathogen, one of the most commonly identified causes of bacterial infections in humans worldwide. H. pylori produces several virulence factors that contribute to its persistence in the hostile host habitat and to its pathogenicity. The most extensively studied are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA). VacA is present in almost all H. pylori strains. As a secreted multifunctional toxin, it assists bacterial colonization, survival, and proliferation during long-lasting infections. To exert its effect on gastric epithelium and other cell types, VacA undergoes several modifications and crosses multiple membrane barriers. Once inside the gastric epithelial cell, VacA disrupts many cellular-signaling pathways and processes, leading mainly to changes in the efflux of various ions, the depolarization of membrane potential, and perturbations in endocytic trafficking and mitochondrial function. The most notable effect of VacA is the formation of vacuole-like structures, which may lead to apoptosis. This review focuses on the processes involved in VacA secretion, processing, and entry into host cells, with a particular emphasis on the interaction of the mature toxin with host membranes and the formation of transmembrane pores. Full article

The calcium ion (Ca²⁺) has been linked to type 2 diabetes mellitus (T2DM), although the role of Ca²⁺ in this disorder is the subject of intense investigation. Serum Ca²⁺ dyshomeostasis is associated with the development of insulin resistance, reduced insulin sensitivity, and impaired glucose tolerance. However, the molecular mechanisms involving Ca²⁺ ions in pancreatic β-cell loss and subsequently in T2DM remain poorly understood. Implicated in the decline in β-cell functions are aggregates of human islet amyloid polypeptide (hIAPP), a small peptide secreted by β-cells that shows a strong tendency to self-aggregate into β-sheet-rich aggregates that evolve toward the formation of amyloid deposits and mature fibrils. The soluble oligomers of hIAPP can permeabilize the cell membrane by interacting with bilayer lipids. Our study aimed to evaluate the effect of Ca²⁺ on the ability of the peptide to incorporate and form ion channels in zwitterionic planar lipid membranes (PLMs) composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) and on the aggregation process of hIAPP molecules in solution. Our results may help to clarify the link between Ca²⁺ ions, hIAPP peptide, and consequently the pathophysiology of T2DM. Full article

The lack of information on structural basis where proteins are involved, as well as the biomineralization processes of different systems such as bones, diatom frustules, and eggshells, have intrigued scientists from different fields for decades. This scientific curiosity has led to the use of methodologies that help understand the mechanism involved in the formation of these complex structures. Therefore, this work focuses on the use of eggshell membranes from different species of ratites (emu and ostrich) and reptiles (two species of crocodiles) as a model to differentiate biocalcification and biosilicification by introducing calcium phosphate or silica inside the membrane fiber mantles. We performed this to obtain information about the process of eggshell formation as well as the changes that occur in the membrane during crystal formation. In order to identify and understand the early processes leading to the formation of the microstructures present in the eggshell, we decided to carry out the synthesis of silica-carbonate of calcium, barium, and strontium called biomorph in the presence of intramineral proteins. This was carried out to evaluate the influence of these proteins on the formation of specific structures. We found that the proteins on untreated membranes, present a structural growth similar to those observed in the inner part of the eggshell, while in treated membranes, the structures formed present a high similarity with those observed in the outer and intermediate part of the eggshell. Finally, a topographic and molecular analysis of the biomorphs and membranes was performed by scanning electron microscopy (SEM), Raman and Fourier-transform Infrared (FTIR) spectroscopies. Full article

Composite flat membranes were prepared using a dry uniaxial pressing process. The effect of the sintering temperature (850–950 °C) and smectite proportion (10–50 wt.%) on membrane properties, such as microstructure, mechanical strength, water permeability, and treatment performances, was explored. It was observed that increasing the sintering temperature and adding higher amounts of smectite increased the mechanical strength and shrinkage. Therefore, 850 °C was chosen as the optimum sintering temperature because the composite membranes had a very low shrinkage that did not exceed 5% with high mechanical strength, above 23 MPa. The study of smectite addition (10–50 wt.%) showed that the pore size and water permeability were significantly reduced from 0.98 to 0.75 µm and from 623 to 371 L·h⁻¹·m⁻²·bar⁻¹, respectively. Furthermore, the application of the used membranes in the treatment of indigo blue (IB) solutions exhibited an almost total turbidity removal. While the removal of color and COD decreased from 95% to 76%, respectively, they decreased from 95% to 52% when the amount of smectite increased. To verify the treated water’s low toxicity, a germination test was performed. It has been shown that the total germination of linseed grains irrigated by M_S10-Z90 membrane permeate was identical to that irrigated with distilled water. Finally, based on its promising properties, its excellent separation efficiency, and its low energy consumption, the M_S10-Z90 (10 wt.% smectite and 90 wt.% zeolite) sintered at 850 °C could be recommended for the treatment of colored industrial wastewater. Full article

Antimicrobial peptides are key components of the immune system. These peptides affect the membrane in various ways; some form nano-sized pores, while others only produce minor defects. Since these peptides are increasingly important in developing antimicrobial drugs, understanding the mechanism of their interactions with lipid bilayers is critical. Here, using atomic force microscopy (AFM), we investigated the effect of a synthetic hybrid peptide, CM15, on the membrane surface comprising E. coli polar lipid extract. Direct imaging of supported lipid bilayers exposed to various concentrations of the peptide revealed significant membrane remodeling. We found that CM15 interacts with supported lipid bilayers and forms membrane-spanning defects very quickly. It is found that CM15 is capable of remodeling both leaflets of the bilayer. For lower CM15 concentrations, punctate void-like defects were observed, some of which re-sealed themselves as a function of time. However, for CM15 concentrations higher than 5 µM, the defects on the bilayers became so widespread that they disrupted the membrane integrity completely. This work enhances the understanding of CM15 interactions with the bacterial lipid bilayer. Full article

The mechanical effects of membrane compositional inhomogeneities are analyzed in a process analogous to neck formation in cellular membranes. We cast on the Canham–Helfrich model of fluid membranes with both the spontaneous curvature and the surface tension being non-homogeneous functions along the cell membrane. The inhomogeneous distribution of necking forces is determined by the equilibrium mechanical equations and the boundary conditions as considered in the axisymmetric setting compatible with the necking process. To establish the role played by mechanical inhomogeneity, we focus on the catenoid, a surface of zero mean curvature. Analytic solutions are shown to exist for the spontaneous curvature and the constrictive forces in terms of the border radii. Our theoretical analysis shows that the inhomogeneous distribution of spontaneous curvature in a mosaic-like neck constrictional forces potentially contributes to the membrane scission under minimized work in living cells. Full article

The transient receptor potential vanilloid type 1 (TRPV1) is a multimodal receptor which responds to various stimuli, including capsaicin, protons, and heat. Recent advances in cryo-electron microscopy have revealed the structures of TRPV1. However, due to the large size of TRPV1 and its structural complexity, the detailed process of channel gating has not been well documented. In this study, we applied the diffracted X-ray tracking (DXT) technique to analyze the intracellular domain dynamics of the TRPV1 protein. DXT enables the capture of intramolecular motion through the analysis of trajectories of Laue spots generated from attached gold nanocrystals. Diffraction data were recorded at two different frame rates: 100 μs/frame and 12.5 ms/frame. The data from the 100 μs/frame recording were further divided into two groups based on the moving speed, using the lifetime filtering technique, and they were analyzed separately. Capsaicin increased the slope angle of the MSD curve of the C-terminus in 100 μs/frame recording, which accompanied a shifting of the rotational bias toward the counterclockwise direction, as viewed from the cytoplasmic side. This capsaicin-induced fluctuation was not observed in the 12.5 ms/frame recording, indicating that it is a high-frequency fluctuation. An intrinsiccounterclockwise twisting motion was observed in various speed components at the N-terminus, regardless of the capsaicin administration. Additionally, the competitive inhibitor AMG9810 induced a clockwise twisting motion, which is the opposite direction to capsaicin. These findings contribute to our understanding of the activation mechanisms of the TRPV1 channel. Full article

G-protein coupled receptors (GPCRs) are versatile signaling proteins that regulate key physiological processes in response to a wide variety of extracellular stimuli. The last decade has seen a revolution in the structural biology of clinically important GPCRs. Indeed, the improvement in molecular and biochemical methods to study GPCRs and their transducer complexes, together with advances in cryo-electron microscopy, NMR development, and progress in molecular dynamic simulations, have led to a better understanding of their regulation by ligands of different efficacy and bias. This has also renewed a great interest in GPCR drug discovery, such as finding biased ligands that can either promote or not promote specific regulations. In this review, we focus on two therapeutically relevant GPCR targets, the V2 vasopressin receptor (V2R) and the mu-opioid receptor (µOR), to shed light on the recent structural biology studies and show the impact of this integrative approach on the determination of new potential clinical effective compounds. Full article

Multidrug resistance (MDR) proteins belonging to the ATP-Binding Cassette (ABC) transporter group play a crucial role in the export of cytotoxic drugs across cell membranes. These proteins are particularly fascinating due to their ability to confer drug resistance, which subsequently leads to the failure of therapeutic interventions and hinders successful treatments. One key mechanism by which multidrug resistance (MDR) proteins carry out their transport function is through alternating access. This mechanism involves intricate conformational changes that enable the binding and transport of substrates across cellular membranes. In this extensive review, we provide an overview of ABC transporters, including their classifications and structural similarities. We focus specifically on well-known mammalian multidrug resistance proteins such as MRP1 and Pgp (MDR1), as well as bacterial counterparts such as Sav1866 and lipid flippase MsbA. By exploring the structural and functional features of these MDR proteins, we shed light on the roles of their nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) in the transport process. Notably, while the structures of NBDs in prokaryotic ABC proteins, such as Sav1866, MsbA, and mammalian Pgp, are identical, MRP1 exhibits distinct characteristics in its NBDs. Our review also emphasizes the importance of two ATP molecules for the formation of an interface between the two binding sites of NBD domains across all these transporters. ATP hydrolysis occurs following substrate transport and is vital for recycling the transporters in subsequent cycles of substrate transportation. Specifically, among the studied transporters, only NBD2 in MRP1 possesses the ability to hydrolyze ATP, while both NBDs of Pgp, Sav1866, and MsbA are capable of carrying out this reaction. Furthermore, we highlight recent advancements in the study of MDR proteins and the alternating access mechanism. We discuss the experimental and computational approaches utilized to investigate the structure and dynamics of MDR proteins, providing valuable insights into their conformational changes and substrate transport. This review not only contributes to an enhanced understanding of multidrug resistance proteins but also holds immense potential for guiding future research and facilitating the development of effective strategies to overcome multidrug resistance, thus improving therapeutic interventions. Full article

Non-genetic photostimulation, which allows for control over cellular activity via the use of cell-targeting phototransducers, is widely used nowadays to study and modulate/restore biological functions. This approach relies on non-covalent interactions between the phototransducer and the cell membrane, thus implying that cell conditions and membrane status can dictate the effectiveness of the method. For instance, although immortalized cell lines are traditionally used in photostimulation experiments, it has been demonstrated that the number of passages they undergo is correlated to the worsening of cell conditions. In principle, this could impact cell responsivity against exogenous stressors, including photostimulation. However, these aspects have usually been neglected in previous experiments. In this work, we investigated whether cell passages could affect membrane properties (such as polarity and fluidity). We applied optical spectroscopy and electrophysiological measurements in two different biological models: (i) an epithelial immortalized cell line (HEK-293T cells) and (ii) liposomes. Different numbers of cell passages were compared to a different morphology in the liposome membrane. We demonstrated that cell membranes show a significant decrease in ordered domains upon increasing the passage number. Furthermore, we observed that cell responsivity against external stressors is markedly different between aged and non-aged cells. Firstly, we noted that the thermal-disordering effect that is usually observed in membranes is more evident in aged cells than in non-aged ones. We then set up a photostimulation experiment by using a membrane-targeted azobenzene as a phototransducer (Ziapin2). As an example of a functional consequence of such a condition, we showed that the rate of isomerization of an intramembrane molecular transducer is significantly impaired in aged cells. The reduction in the photoisomerization rate translates in cells with a sustained reduction of the Ziapin2-related hyperpolarization of the membrane potential and an overall increase in the molecule fluorescence. Overall, our results suggest that membrane stimulation strongly depends on membrane order, highlighting the importance of cell passage during the characterization of the stimulation tools. This study can shine light on the correlation between aging and the development of diseases driven by membrane degradation as well as on the different cell responsivities against external stressors, such as temperature and photostimulation. Full article