Biochimica et biophysica acta. Biomembranes最新文献

Kalata B1 (kB1), a naturally occurring cyclotide has been shown experimentally to bind lipid membranes that contain phosphatidylethanolamine (PE) phospholipids. Here, molecular dynamics simulations were used to explore its interaction with two phospholipids, palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), and a heterogeneous membrane comprising POPC/POPE (90:10), to understand the basis for the selectivity of kB1 towards PE phospholipids. The simulations showed that in the presence of only 10 % POPE lipid, kB1 forms a stable binding complex with membrane bilayers. An ionic interaction between the E7 carboxylate group of kB1 and the ammonium group of PE headgroups consistently initiates binding of kB1 to the membrane. Additionally, stable noncovalent interactions such as hydrogen bonding (E7, T8, V10, G11, T13 and N15), cation–π (W23), and CH–π (W23) interactions between specific residues of kB1 and the lipid membrane play an important role in stabilizing the binding. These findings are consistent with a structure-activity relationship study on kB1 where lysine mutagenesis on the bioactive and hydrophobic faces of the peptide abolished membrane-dependent bioactivities. In summary, our simulations suggest the importance of residue E7 (in the bioactive face) in enabling kB1 to recognize and bind selectively to PE-containing phospholipids bilayers through ionic and hydrogen bonding interactions, and of W23 (in the hydrophobic face) for the association and insertion of kB1 into the lipid bilayer through cation–π and CH–π interactions. Overall, this work enhances our understanding of the molecular basis of the membrane binding and bioactivity of this prototypic cyclotide.

To address the global problem of bacterial antibiotic resistance, antimicrobial peptides (AMPs) are considered promising therapeutic candidates due to their broad-spectrum and membrane-lytic activity. As preferential interactions with bacteria are crucial, it is equally important to investigate and understand their impact on eukaryotic cells. In this study, we employed ¹⁹F solid-state nuclear magnetic resonance (ssNMR) as a novel approach to examine the interaction of AMPs with whole red blood cells (RBCs). We used RBC ghosts (devoid of hemoglobin) and developed a protocol to label their lipid membranes with palmitic acid (PA) monofluorinated at carbon positions 4, 8, or 14 on the acyl chain, allowing us to probe different locations in model and intact RBC ghost membranes. Our work revealed that changes in the ¹⁹F chemical shift anisotropy, monitored through a CF bond order parameter (S_CF), can provide insights into lipid bilayer dynamics. This information was also obtained using magic-angle spinning ¹⁹F ssNMR spectra with and without ¹H decoupling, by studying alterations in the second spectral moment (M₂) as well as the ¹⁹F isotropic chemical shift, linewidth, T₁, and T₂ relaxation times. The appearance of an additional isotropic peak with a smaller chemical shift anisotropy, a narrower linewidth, and a shorter T_1, induced by the AMP caerin 1.1, supports the presence of high-curvature regions in RBCs indicative of pore formation, analogous to its antimicrobial mechanism. In summary, the straightforward incorporation of monofluorinated FAs and rapid signal acquisition offer promising avenues for the study of whole cells using ¹⁹F ssNMR.

NK-2 is an antimicrobial peptide derived from helices 3 and 4 of the pore-forming protein of natural killer cells, NK-lysin. It has potent activities against Gram-negative and Gram-positive bacteria, fungi and protozoan parasites without being toxic to healthy human cells. In biophysical assays its membrane activities were found to require phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), lipids which dominate the composition of bacterial membranes. Here the structure and activities of NK-2 in binary mixtures of different PE/PG composition were investigated. CD spectroscopy reveals that a threshold concentration of 50 % PG is needed for efficient membrane association of NK-2 concomitant with a random coil – helix transition. Association with PE occurs but is qualitatively different when compared to PG membranes. Oriented solid-state NMR spectroscopy of NK-2 specifically labelled with ¹⁵N indicates that the NK-2 helices are oriented parallel to the PG bilayer surface. Upon reduction of the PG content to 20 mol% interactions are weaker and/or an in average more tilted orientation is observed. Fluorescence spectroscopy of differently labelled lipids is in agreement of an interfacial localisation of both helices where the C-terminal end is in a less hydrophobic environment. By inserting into the membrane interface and interacting differently with PE and PG the peptides probably induce high curvature strain which result in membrane openings and rupture.

This work describes the electrochemical studies on the interactions between V57G mutant of human cystatin C (hCC V57G) and membrane bilayer immobilized on the surface of a gold electrode. The electrode was modified with 6-mercaptohexan-1-ol (MCH) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). DMPC was used as a membrane mimetic for monitoring electrochemical changes resulting from the interactions between the functionalized electrode surface and human cystatin C. The interactions between the modified electrode and hCC V57G were investigated by cyclic voltammetry and electrochemical impedance spectroscopy in a phosphate buffered saline (PBS) containing Fe(CN)₆^3−/4- as a redox probe. The electrochemical measurements confirm that fabricated electrode is sensitive to hCC V57G at the concentration of 1 × 10⁻¹⁴ M. The incubation studies carried out at higher concentrations resulted in insignificant changes observed in cyclic voltammetry and electrochemical impedance spectroscopy measurements. The calculated values of surface coverage θ_R confirm that the electrode is equally covered at higher concentrations of hCC V57G. Measurements of wettability and surface free energy made it possible to determine the influence of individual structural elements of the modified gold electrode on its properties, and thus allowed to understand the nature of the interactions. Contact angle values confirmed the results obtained during electrochemical measurements, indicating the sensitivity of the electrode towards hCC V57G at the concentration of 1 × 10⁻¹⁴ M. In addition, the XPS spectra confirmed the successful anchoring of hCC V57G to the DMPC-functionalized surface.

All tetraspanins have four transmembrane domains (TMs). The large extracellular loop (LEL) that connects the third and fourth TMs contains multiple secondary structures together with the family's signature Cys-Cys-Gly motif. These intriguing membrane proteins are involved in diverse and incompletely understood cellular processes including cell adhesion, tissue differentiation, immune cell maturation and host-parasite interactions. Here we present a classification system that accurately describes the position of each amino acid within its primary sequence based on both sequence and topological conservation of the TMs and LEL. This builds on the numbering systems that have been used in the G protein-coupled receptor (GPCR) field for nearly three decades and which have aided the understanding of GPCR structure/activity relationships and ligand interactions. The high-resolution structures of the tetraspanins CD81, CD9, CD53 and Tspan15 were used to validate the structural relevance of our new tetraspanin classification system. Modelling of all tetraspanin LELs highlighted flexibility in LEL disulfide bonding across the family and suggests that the structural arrangement of tetraspanin LELs is more complex than previously thought. We therefore propose a new subfamily naming system that addresses this added complexity and facilitates the systematic classification of human tetraspanins, shedding light on all structural motifs within the family. We anticipate that our universal tetraspanin classification system will enable progress in defining how sequence and structure inform function.

With the increasing prevalence of multidrug resistant (MDR) bacteria, there is a need to design synthetic antimicrobial peptides (AMPs) that are effective and selective for bacteria, i.e. non-toxic to mammalian cells. One design strategy, namely the use of tryptophan- and arginine-rich AMPs, is rooted in the study of natural AMPs that are composed mainly of these amino acids, such as lactoferricin, tritrpticin, and puroindoline. A number of important studies on these AMPs by the Vogel group are reviewed here. More recent work on W-/R-rich peptides is also presented. The examples show that these peptides represent anti-infectives with great potential.

S-palmitoylation is a dynamic lipid-based protein post-translational modification facilitated by a family of protein acyltransferases (PATs) commonly known as DHHC-PATs or DHHCs. It is the only lipid modification that is reversible, and this very fact uniquely qualifies it for therapeutic interventions through the development of DHHC inhibitors. Herein, we report that 4″-alkyl ether lipophilic derivatives of EGCG can effectively inhibit protein S-palmitoylation in vitro. With the help of metabolic labeling followed by copper(I)-catalyzed azide-alkyne cycloaddition Click reaction, we demonstrate that 4″-C₁₄ EGCG and 4″-C₁₆ EGCG markedly inhibited S-palmitoylation in various mammalian cells including HEK 293T, HeLa, and MCF-7 using both in gel fluorescence as well as confocal microscopy. Further, these EGCG derivatives were able to attenuate the S-palmitoylation to the basal level in DHHC3-overexpressed cells, suggesting that they are plausibly targeting DHHCs. Confocal microscopy data qualitatively reflected spatial and temporal distribution of S-palmitoylated proteins in different sub-cellular compartments and the inhibitory effects of 4″-C₁₄ EGCG and 4″-C₁₆ EGCG were clearly observed in the native cellular environment. Our findings were further substantiated by in silico analysis which revealed promising binding affinity and interactions of 4″-C₁₄ EGCG and 4″-C₁₆ EGCG with key amino acid residues present in the hydrophobic cleft of the DHHC20 enzyme. We also demonstrated the successful inhibition of S-palmitoylation of GAPDH by 4″-C₁₆ EGCG. Taken together, our in vitro and in silico data strongly suggest that 4″-C₁₄ EGCG and 4″-C₁₆ EGCG can act as potent inhibitors for S-palmitoylation and can be employed as a complementary tool to investigate S-palmitoylation.

Novel terminally perfluorobutyl group-containing ether-linked phosphatidylcholines with different alkyl chain lengths (di-O-F4-Cn-PCs, n = 14,16 and 18) were developed as possible materials for stable liposomes aiming at applications of structural and functional analyses of membrane proteins. Differential scanning calorimetric investigations of the thermotropic transition of hydrated di-O-F4-Cn-PC bilayers demonstrated that the transition temperature of every di-O-F4-Cn-PC decreases by ~20 °C compared to their corresponding non-fluorinated PCs, di-O-Cn-PCs. With the elongation of the hydrophobic chain, on the other hand, the transition enthalpy (ΔH) and entropy (ΔS) increased in a linear manner. Comparison of ΔH and ΔS values against the net hydrocarbon chain length between di-O-F4-Cn-PCs and di-O-Cn-PCs strongly suggests that in the thermotropic transition of the di-O-F4-Cn-PC membrane, the perfluorobutyl segments undergo very limited structural changes; therefore, the hydrocarbon segments are mainly responsible for the phase transition.

Background

OCTN1 belongs to the SLC22 family, which includes transporters for cationic, zwitterionic, and anionic substrates. OCTN1 function and role in cells are still poorly understood. Not only cations, such as TEA, but also zwitterions, such as carnitine and ergothioneine, figure among transported molecules.

Methods

In this work, we carried out transport assays measuring [¹⁴C]-TEA and [³H]-Carnitine in proteoliposomes reconstituted with the recombinant human OCTN1 in the presence of Na⁺ or other cations. The homology model of OCTN1 was built using the structure of OCT3 as a template for docking analysis.

Results

TEA and carnitine did not inhibit each other. Moreover, carnitine uptake was not affected by the presence of Na⁺ and TEBA, whereas TEA was strongly inhibited by both compounds. Computational data revealed that TEA, Na⁺, and carnitine can interact with E381 in the OCTN1 substrate site. Differently from TEA, in the presence of Na⁺, carnitine is still able to interact with the binding site via R469.

Conclusions

The lack of mutual inhibition of the two prototype substrates, the different effect of Na⁺ and TEBA on their transport reaction, together with the computational analysis supports the existence of two transport pathways for cations and zwitterions.

General significance

The results shed new light on the transport mechanisms of OCTN1, helping to get further insights into the structure/function relationships. The described results correlate well with previous and very recent findings on the polyspecificity of the OCT group of transporters belonging to the same family.

The endoplasmic reticulum acts as a protein quality control center where a range of chaperones and foldases facilitates protein folding. IRE1 is a sensory transmembrane protein that transduces signals of proteotoxic stress by forming clusters and activating a cellular program called the unfolded protein response (UPR). Recently, membrane thickness variation due to membrane compositional changes have been shown to drive IRE1 cluster formation, activating the UPR even in the absence of proteotoxic stress. Here, we demonstrate a direct relationship between bilayer tension and UPR activation based on IRE1 dimer stability. The stability of the IRE1 dimer in a (50%DOPC-50%POPC) membrane at different applied bilayer tensions was analyzed via molecular dynamics simulations. The potential of mean force for IRE1 dimerization predicts a higher concentration of IRE1 dimers for both tensed and compressed ER membranes. This study shows that IRE1 may be a mechanosensitive membrane protein and establishes a direct biophysical relationship between bilayer tension and UPR activation.