Biochimica et biophysica acta. Biomembranes最新文献

The natural transformation system of the thermophilic bacterium Thermus thermophilus comprises at least 16 competence proteins. Recently we found that the outer membrane (OM) competence protein PilW interacts with the secretin channel, which guides type IV pili (T4P) and potential DNA transporter pseudopili through the OM. Here we have used biochemical techniques to study the interactions of cytoplasmic, inner membrane (IM) and OM components of the DNA transporter in T. thermophilus. We report that PilW is part of a heteropolymeric complex comprising of the cytoplasmic PilM protein, IM proteins PilN, PilO, PilC and the secretin PilQ. Co-purification studies revealed that PilO directly interacts with PilW. In vitro affinity co-purification studies using His-tagged PilC led to the detection of PilC-, PilW-, PilN- and PilO-containing complexes. PilO was identified as direct interaction partner of the polytopic IM protein PilC. PilC was also found to directly interact with the cytoplasmic T4P disassembly ATPase PilT1 thereby triggering PilT1 ATPase activity. This, together with the detection of heteropolymeric PilC complexes which contain PilT1 and the pilins PilA2, PilA4 and PilA5 is in line with the hypothesis that PilC connects the depolymerization ATPase to the base of the pili possibly allowing energy transduction for disassembly of the pilins.

This work is devoted to the study of the combined effects of applied magnetic field and MNPs on the electrical characteristics of bilayer lipid membranes. We present results of the study of electrical parameters of azolectin membranes in a static inhomogeneous magnetic field at the one-sided addition of positively charged quasi-spherical superparamagnetic magnetite nanoparticles with a diameter of about 4 nm. The magnet was located at different distances from the membrane, and the magnetic field attracted the nanoparticles to the membrane surface with different strengths. We observed three pronounced effects that depended on the external magnetic field. Firstly, after addition of nanoparticles in a magnetic field, the conductance of the membranes increased. A smooth increase in conductance was accompanied in some cases by the appearance of current jumps, which can be associated with the formation of through pores with a radius of no more than 1 nm. The conductance increased with increasing magnetic field gradient. Secondly, at zero command voltage, a negative current through the membrane was observed. Although our experiments did not allow us to unambiguously determine which particles create this current, we believe that this current is associated with the penetration of particles through the membrane. This effect intensified with increasing magnetic field gradient. Thirdly, we observed a sharp change in the nonlinear dependence of capacitance on voltage associated both with the change in the surface potential of the azolectin membrane and with the effect of MNP binding to the membrane surface on the apparent membrane capacitance.

The combined application of electric fields and ultrasonic waves has shown promise in controlling cell membrane permeability, potentially resulting in synergistic effects that can be explored in the biotechnology industry. However, further clarification on how these processes interact is still needed. The objective of the present study was to investigate the atomic-scale effects of these processes on a DPPC lipid bilayer using molecular dynamics simulations. For higher electric fields, capable of independently forming pores, the application of an ultrasonic wave in the absence of cavitation yielded no additional effects on pore formation. However, for lower electric fields, the reduction in bilayer thickness induced by the shock wave catalyzed the electroporation process, effectively shortening the mean path that water molecules must traverse to form pores. When cavitation was considered, synergistic effects were evident only if the wave alone was able to generate pores through the formation of a water nanojet. In these cases, sonoporation acted as a mean to focus the electroporation effects on the initial pore formed by the nanojet. This study contributes to a better understanding of the synergy between electric fields and ultrasonic waves and to an optimal selection of processing parameters in practical applications of these processes.

The extracellular environment is sensed by receptors in the plasma membrane. Some of these receptors initiate cytoplasmic signaling cascades involving phosphorylation: the addition of a phosphate group to a specific amino acid, such as tyrosine, in a protein. Receptor Tyrosine Kinases (RTKs) are one large class of membrane receptors that can directly initiate signaling cascades through their intracellular kinase domains, which both catalyze tyrosine phosphorylation and get phosphorylated. In the first step of signaling, the ligands stabilize phosphorylation-competent RTK dimers and oligomers, which leads to the phosphorylation of specific tyrosine residues in the activation loop of the kinases. Here we discuss quantitative measurements of tyrosine phosphorylation efficiencies for RTKs, described by the “transducer function”. The transducer function links the phosphorylation (the response) and the binding of the activating ligand to the receptor (the stimulus). We overview a methodology that allows such measurements in direct response to ligand binding. We discuss experiments which demonstrate that EGF is a partial agonist, and that two tyrosines in the intracellular domain of EGFR, Y1068 and Y1173, are differentially phosphorylated in the EGF-bound EGFR dimers.

Cytokine receptor-like factor 2 (CRLF2) and interleukin-7 receptor α (IL-7Rα) form a receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation consisting of the substitution of five amino acids (SLLLL) in the transmembrane domain of CRLF2 with three amino acids, including glutamic acid, isoleucine, and methionine (insEIM), which has been identified in acute lymphocytic leukemia, causes the TSLP-independent dimerization with IL-7Rα and activation. However, the dimerization mechanism remains unclear. In this study, we examined the involvement of the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα in TSLP-independent activation. HEK293 cells were transfected with vectors encoding CRLF2 and IL-7Rα, or their mutants, in which the amino acid of the transmembrane domain was replaced with alanine. STAT5 phosphorylation was detected using western blotting, and receptor dimerization was analyzed using the NanoBiT assay. The substitution of glutamic acid within the insEIM mutation for alanine failed to cause the STAT5 phosphorylation in the absence of TSLP. Moreover, the alanine substation of the specific leucine residues in the transmembrane domains of both CRLF2 and IL-7Rα abrogated the TSLP-independent signal transduction and dimerization. The mutation of IL-7Rα W264 partially reduced the phosphorylation of STAT5 without affecting receptor dimerization. These results suggest that the amino acids in the transmembrane domains of EIM CRLF2 and IL-7Rα play at least three possible functions: interaction through hydrogen bonds, hydrophobic interaction, and signal transduction. Our findings contribute to a better understanding of the function of the transmembrane domains of cytokine receptors in their dimerization and signal transduction.

Cell membranes are responsible for a range of biological processes that require interactions between lipids and proteins. While the effects of lipids on proteins are becoming better understood, our knowledge of how protein conformational changes influence membrane dynamics remains rudimentary. Here, we performed experiments and computer simulations to study the dynamic response of a lipid membrane to changes in the conformational state of pH-low insertion peptide (pHLIP), which transitions from a surface-associated (SA) state at neutral or basic pH to a transmembrane (TM) α-helix under acidic conditions. Our results show that TM-pHLIP significantly slows down membrane thickness fluctuations due to an increase in effective membrane viscosity. Our findings suggest a possible membrane regulatory mechanism, where the TM helix affects lipid chain conformations, and subsequently alters membrane fluctuations and viscosity.

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.

Huntington's Disease (HD) is caused by an abnormal expansion of the polyglutamine (polyQ) domain within the first exon of the huntingtin protein (htt). This expansion promotes disease-related htt aggregation into amyloid fibrils and the formation of proteinaceous inclusion bodies within neurons. Fibril formation is a complex heterogenous process involving an array of aggregate species such as oligomers, protofibrils, and fibrils. In HD, structural abnormalities of membranes of several organelles develop. In particular, the accumulation of htt fibrils near the endoplasmic reticulum (ER) impinges upon the membrane, resulting in ER damage, altered dynamics, and leakage of Ca²⁺. Here, the aggregation of htt at a bilayer interface assembled from ER-derived liposomes was investigated, and fibril formation directly on these membranes was enhanced. Based on these observations, simplified model systems were used to investigate mechanisms associated with htt aggregation on ER membranes. As the ER-derived liposome fractions contained residual Ca²⁺_, the role of divalent cations was also investigated. In the absence of lipids, divalent cations had minimal impact on htt structure and aggregation. However, the presence of Ca²⁺ or Mg²⁺ played a key role in promoting fibril formation on lipid membranes despite reduced htt insertion into and association with lipid interfaces, suggesting that the ability of divalent cations to promote fibril formation on membranes is mediated by induced changes to the lipid membrane physicochemical properties. With enhanced concentrations of intracellular calcium being a hallmark of HD, the ability of divalent cations to influence htt aggregation at lipid membranes may play a role in aggregation events that lead to organelle abnormalities associated with disease.

The molecular structures of the various intrinsic lipids in membranes regulate lipid-protein interactions. These different lipid structures with unique volumes produce different lipid molecular packing stresses/lateral stresses in lipid membranes. Most studies examining lipid packing effects have used phosphatidylcholine and phosphatidylethanolamine (PE), which are the main phospholipids of eukaryotic cell membranes. In contrast, Gram-negative or Gram-positive bacterial membranes are composed primarily of phosphatidylglycerol (PG) and PE, and the physical and thermodynamic properties of each acyl chain in PG at the molecular level remain unresolved. In this study, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG, 16:0–18:1 PG) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (PAPG, 16:0–20:4 PG) to prepare lipid bilayers (liposome) with the rod-type fluorescence probe DPH. We measured the lipid packing conditions by determining the rotational freedom of DPH in POPG or PAPG bilayers. Furthermore, we investigated the effect of different monoacyl chains on a K⁺ channel (KcsA) structure when embedded in POPG or PAPG membranes. The results revealed that differences in the number of double bonds and carbon chain length in the monoacyl chain at sn-2 affected the physicochemical properties of the membrane and the structure and orientation of KcsA.

A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.