Biochimica et biophysica acta. Biomembranes最新文献

The overexpression of G-protein-coupled receptors (GPCRs) remains one of the biggest hurdles for structural studies of these proteins. To date, the most usually applied system for this task is the insect cell/baculovirus expression system. A drawback of this system, however, is the accumulation of protein that is resistant to solubilization with the commonly used mild detergent DoDecylMaltoside (DDM). In addition, poor surface expression is often observed. In this study, it is shown how an earlier AcMNPV 39K promoter, can express receptors that are found primarily on the cell membrane, as revealed by confocal microscopy, and the protein can be solubilized to a higher degree by DDM in a less aggregation-prone form, as monitored by fluorescence size-exclusion chromatography. In addition, a strong effect on the yield was observed when the AcMNPV gp67 signal sequence was used. The documentation of the 39K promoter as an improvement over the frequently used polyhedrin promoter, along with the effect of the gp67 signal sequence are important steps toward ultimately improving the expression in terms of total functional yield, while also shedding light on the nature of the process of overproduction of membrane proteins, in particular, GPCRs.

Unlike homogeneous liposomes, phase-separated liposomes have the potential to be attractive soft materials because they exhibit different properties for each phase. In this study, phase separation was observed when liposomes were prepared using 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol. The pH of the DOTAP-rich phase was evaluated using a coumarin derivative, and measurements showed that DOTAP molecules accumulated hydroxyl ions (OH^-) in the DOTAP-rich phase. Such accumulation of OH^- was not observed when homogeneous DSPC liposomes were used. The difference in local concentration of OH^- in each phase was applied to prepare hollow silica particles with large pores. The OH^- promotes the polymerization of tetraethyl orthosilicate (TEOS). Therefore, TEOS polymerized preferentially in the DOTAP-rich phase, whereas a silica membrane barely formed in the DSPC-rich phase.

The acquisition of ferrous iron (Fe²⁺) is crucial for the survival of many pathogenic bacteria living within acidic and/or anoxic conditions such as Vibrio cholerae, the causative agent of the disease cholera. Bacterial pathogens utilize iron as a cofactor to drive essential metabolic processes, and the primary prokaryotic Fe²⁺ acquisition mechanism is the ferrous iron transport (Feo) system. In V. cholerae, the Feo system comprises two cytosolic proteins (FeoA, FeoC) and a complex, polytopic transmembrane protein (FeoB) that is regulated by an N-terminal soluble domain (NFeoB) with promiscuous NTPase activity. While the soluble components of the Feo system have been frequently studied, very few reports exist on the intact membrane protein FeoB. Moreover, FeoB has been characterize almost exclusively in detergent micelles that can cause protein misfolding, disrupt protein oligomerization, and even dramatically alter protein function. As many of these characteristics of FeoB remain unclear, there is a critical need to characterize FeoB in a more native-like lipid environment. To address this unmet need, we employ styrene-maleic acid (SMA) copolymers to isolate and to characterize V. cholerae FeoB (VcFeoB) encapsulated by a styrene-maleic acid lipid particle (SMALP). In this work, we describe the development of a workflow for the expression and the purification of VcFeoB in a SMALP. Leveraging mass photometry, we explore the oligomerization of FeoB in a lipid bilayer and show that the VcFeoB-SMALP is mostly monomeric, consistent with our previous oligomerization observations in surfo. Finally, we characterize the NTPase activity of VcFeoB in the SMALP and in a detergent (DDM), revealing higher NTPase activity in the presence of the lipid bilayer. When taken together, this report represents the first characterization of any FeoB in a native-like lipid bilayer and provides a viable approach for the future structural characterization of FeoB.

Tetraspanins are four-transmembrane proteins that play fundamental roles in the immune system by enabling processes like migration, proliferation, signaling and protein trafficking. While the importance of cell surface tetraspanins has been established, the function of intracellular tetraspanins is less well understood. Here, we investigated the role of tetraspanin 3 (Tspan3) in lymphocytes. Tspan3 expression was low in T cells and high in B cells which increased during B cell activation. Tspan3 localized to late endosomal structures where it colocalized with major histocompatibility complex II (MHCII) in the MHCII compartment. There, inhibiting the formation of intraluminal vesicles (ILVs) showed that Tspan3 localization was not affected in contrast to its homologue CD63. Using a peptide-pulldown approach, we identified that Tspan3 interacts with AP2 via its C-terminus that harbors a YXXΦ-based sorting motif. Interestingly, mutating this motif did not impair Tspan3 localization. Instead, leucine 246 was required for its intracellular localization, identifying an unrecognized leucine-based sorting motif responsible for Tspan3 localization to MHCII compartment in B cells. Taken together, we report a new sorting motif for Tspan3 to MHCII compartments in human B cells.

The intestinal barrier, held together by epithelial cells and intercellular tight junction (TJ) proteins, prevents the penetration of microbial pathogens. Concurrently, intestinal epithelial cells secrete antimicrobial peptides, including cathelicidin. Cathelicidin has direct antibacterial and immunomodulatory functions, although its role in intestinal integrity remains elusive. In this study, we demonstrate that direct stimulation of human colonic epithelial (T84) cells with human cathelicidin, LL-37, resulted in a rapid and transient increase in epithelial cell permeability. This increased permeability was associated with the TJ proteins occludin and claudin-2 degradation, mediated by these specific proteins' endocytosis and lysosomal degradation. While murine cathelicidin (CRAMP) failed to modify T84 cell permeability, LL-37 degraded TJ proteins in murine rectal epithelial (CMT-93) cells. The stimulus of (CMT-93) cells with LL-37 aggravated the cell permeability and furthered TJ degradation provoked by the intestinal pathogen, attaching/effacing (A/E) Citrobacter rodentium (C. rodentium). The number of C. rodentium that colonized CMT-93 cells was not severely impacted by the presence of LL-37. While a temporary disruption of tight junctions by LL-37 may lead to a 'leaky gut,' this study demonstrates that LL-37 increases epithelial cell permeability by degrading TJ proteins occludin and claudin-2 through endocytosis and lysosomal degradation. These immunomodulatory actions occurring at concentrations lower than those microbicidal uncover a new guise for cathelicidin modulating the epithelial barrier against A/E pathogens. Recognizing native cathelicidin's functions in a specified disease setting (e.g., colitis) will help establish it as an anti-infectious immunomodulator.

Inorganic polyphosphate (polyP) is a polymer that consists of a series of orthophosphates connected by high-energy phosphoanhydride bonds, like those found in ATP. In mammalian mitochondria, polyP has been linked to the activation of the mitochondrial permeability transition pore (mPTP). However, the details of this process are not completely understood. The activation of mPTP by polyP may involve the regulation of bioenergetics, Ca²⁺ buffering, or direct involvement in mPTP channel formation. In this study, using refractive index imaging techniques, we examined mPTP induction in both wild-type (WT) SH-SY5Y cells, and mutant SH-SY5Y cells expressing either mitochondrially targeted exopolyphosphatase (MitoPPX), which depletes polyP by breaking free terminal phosphoanhydride bonds; or endopolyphosphatase (MitoPPN), which cleaves internal phosphoanhydride bonds and thus can target polyP pool with protected terminal groups. Upon treating the cells with the calcium ionophore ferutinin, the influx of Ca²⁺ triggered mitochondrial membrane depolarization and permeabilization in both WT and MitoPPX cells indicating activation of mPTP. However, in MitoPPN cells, mitochondrial depolarization occurred without mPTP activation. Based on these findings we propose the possibility that activation of mPTP is not linked to the pool of free polyP. This supports the hypothesis that polyP is either an important structural component of the mPTP channel or associated with other macromolecular complexes involved in mPTP induction.

The Solanum tuberosum (common potato) plant specific insert (StPSI) is an antimicrobial protein domain that exhibits membrane-disrupting and membrane-fusing activity upon dimerization at acidic pH, activity proposed to involve electrostatic attraction and membrane anchoring mediated by specific positively-charged and conserved tryptophan residues, respectively. This study is the first to employ an in silico mutagenesis approach to clarify the structure-function relationship of a plant specific insert (PSI), where ten rationally-mutated StPSI variants were investigated using all-atom and coarse-grained molecular dynamics. The tryptophan (W) residue at position 18 (W18) of wild-type StPSI was predicted to confer structural flexibility to the dimer and mediate a partial separation of the assembled monomers upon bilayer contact, while residues including W77 and the lysine (K) residue at position 83 (K83) were predicted to stabilize secondary structure and influence association with the model membrane. Mechanisms predicted to influence StPSI-membrane association included the partial separation of assembled monomers on the bilayer surface, formation of a specific salt bridge, and membrane anchoring of hinge 2 residues. The findings suggested that the structure-function relationship of StPSI involved several mechanisms that may each be modulated by specific key residues, insights that may support efforts to develop PSI with tailored membrane association for novel applications in plant biotechnology and crop protection.

Acylation is a common method used to modify antimicrobial peptides to enhance their effectiveness. It increases the interactions between the peptide and the bacterial cell membranes. However, acylation can also reduce the selectivity of the peptides by making them more active on eukaryotic membranes, which can lead to unintended toxicity. This study examines the potential of using in silico tools to evaluate the interaction and selectivity of the antimicrobial peptide CIDEM-501 when acylated with decanoic acid at the N-terminus, compared to the non-acylated counterpart. Circular dichroism, microdilution, and hemolysis assays were used to determine the peptide's secondary structure, antimicrobial activity, and selectivity to validate the theoretical predictions. The acylated peptide showed a more stable interaction with the bacterial membrane by inserting the acyl chain into the membrane's hydrophobic core, which led to tighter adsorption and a greater buried surface area. Additionally, it significantly altered membrane order more than the non-acylated counterpart, suggesting superior antimicrobial potential. Finally, in vitro activity assays confirmed theoretical predictions, showing that the acylated peptide had lower Minimum Inhibitory Concentration (MIC) values than the non-acylated peptide. Neither peptide showed significant hemolytic activity at their MIC. The computational techniques used in this study displayed strong predictive capability and helped to elucidate the interaction between the peptide and the membranes.

In this work, the effects of L- Arginine (L-Arg) insertion on saturated and unsaturated lipid membranes were assessed by fluorescence spectroscopy, dynamic light scattering (DLS) and monolayer measurements. The studied systems were composed by DPPC, 16:0 DietherPC, 16:1 Δ9-CisPC, DPPC:Chol, 16:1 Δ9-CisPC:Chol, and 16:1 Δ9-CisPC:DPPC in the presence of increasing concentrations of L-Arg. The information obtained using fluorescence spectral Laurdan properties indicates that L- Arg produces a decrease in the polarizability of saturated lipids congruent with the increase in vesicle size and area per lipid. However, in unsaturated lipids, the polarizability increases without significant changes in size and area per lipid denoting a different mechanism of insertion. The two opposite effects can be modulated by the saturated and unsaturated ratio and are independent of carbonyl groups. This modulation is damped by cholesterol. The differences in the L-Arg insertion can be explained considering that in the presence of the double bond, L-Arg decreases the organized water in the lipid matrix without expanding the bilayer. Instead, in saturated lipid membranes, L-Arg inserts into the acyl chains dragging water and expanding the membrane area.