10 research outputs found

    Lipid-Protein Interactions Are Unique Fingerprints for Membrane Proteins

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    Cell membranes contain hundreds of different proteins and lipids in an asymmetric arrangement. Our current understanding of the detailed organization of cell membranes remains rather elusive, because of the challenge to study fluctuating nanoscale assemblies of lipids and proteins with the required spatiotemporal resolution. Here, we use molecular dynamics simulations to characterize the lipid environment of 10 different membrane proteins. To provide a realistic lipid environment, the proteins are embedded in a model plasma membrane, where more than 60 lipid species are represented, asymmetrically distributed between the leaflets. The simulations detail how each protein modulates its local lipid environment in a unique way, through enrichment or depletion of specific lipid components, resulting in thickness and curvature gradients. Our results provide a molecular glimpse of the complexity of lipid-protein interactions, with potentially far-reaching implications for our understanding of the overall organization of real cell membranes

    The Structural Dynamics of Soluble and Membrane Proteins Explored through Molecular Simulations

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    Hydrophobic drug/toxin binding sites in voltage-dependent K+ and Na+ channels

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    In the Na(v)channel family the lipophilic drugs/toxins binding sites and the presence of fenestrations in the channel pore wall are well defined and categorized. No such classification exists in the much larger K(v)channel family, although certain lipophilic compounds seem to deviate from binding to well-known hydrophilic binding sites. By mapping different compound binding sites onto 3D structures of Kv channels, there appear to be three distinct lipid-exposed binding sites preserved in K(v)channels: the front and back side of the pore domain, and S2-S3/S3-S4 clefts. One or a combination of these sites is most likely the orthologous equivalent of neurotoxin site 5 in Na(v)channels. This review describes the different lipophilic binding sites and location of pore wall fenestrations within the K(v)channel family and compares it to the knowledge of Na(v)channels

    Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels

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    Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (NaV), potassium (KV), calcium (CaV), and proton (HV) channels, as well as calcium-activated potassium (KCa), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1: The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2: The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3: The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4: The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5: The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels.Funding Agencies|Swedish Research Council; Swedish Brain Foundation; Swedish Society for Medical Research; Swedish Heart-Lung Foundation</p

    Investigating the Antibacterial and Immunomodulatory Properties of Lactobacillus acidophilus Postbiotics

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    Probiotics are nonpathogenic microorganisms that have been extensively studied for their ability to prevent various infectious, gastrointestinal, and autoimmune diseases. The mechanisms underlying these probiotic effects have not been elucidated. However, we and other researchers have evidence suggesting that probiotic bacteria secrete metabolites that are antimicrobial and anti-inflammatory. As such, we developed a methodology to collect the secreted metabolites from a probiotic bacterium, Lactobacillus acidophilus, and tested this cell free filtrate (CFF) both in vitro and in vivo. Using this CFF, we have demonstrated that L. acidophilus secretes a molecule(s) that has specific bactericidal activity against the opportunistic pathogen, Pseudomonas aeruginosa. Additionally, we have shown that the CFF inhibits P. aeruginosa biofilm formation, an important virulence factor that contributes to P. aeruginosa’s antibiotic resistance. Importantly, our data show that the CFF eradicates 20 h and 48 h established biofilm. We hypothesized that the CFF could modulate pro-inflammatory mediator secretion. To investigate the role of the CFF on tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) production, we measured TNF-α and IL-8 production from THP-1 monocytes and macrophages. Interestingly, our data show that the CFF could stimulate or reduce inflammatory mediator release, depending on the concentration of CFF and whether the cells had been prestimulated with LPS. To elucidate the mechanism underlying the immunomodulatory activity of the CFF, we conducted luciferase assays using a NF-ÎșB reporter THP-1 cell line. Here, we provide evidence that the CFF modulates inflammatory mediators at least in part by regulating NF-ÎșB activation. In addition, we tested the effects of the CFF in a mouse model of P. aeruginosa wound infection. The CFF cleared P. aeruginosa bacterial load and reduced plasma inflammatory mediator production by 5 days. To investigate the bioactive molecule within the CFF, we performed sequential fractionation steps and LCMS to identify and characterize the bioactive molecule, which has a similar mass to charge ratio (m/z) as known polyunsaturated fatty acids (PUFAs) and modified fatty acids. Taken together, our findings provide evidence that L. acidophilus secretes a bioactive molecule that has bactericidal, anti-biofilm, and immunomodulatory activities

    Comprendre l’impermĂ©abilitĂ© cutanĂ©e : Ă©tude spectroscopique de mĂ©langes modĂšles de la phase lipidique du stratum corneum

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    Le stratum corneum (SC), la couche la plus externe de l'Ă©piderme des mammifĂšres, agit comme une barriĂšre dictant le taux d'absorption des molĂ©cules exogĂšnes Ă  travers la peau et empĂȘchant la perte d'eau du corps. Le SC est principalement composĂ© de cellules mortes aplaties et entiĂšrement kĂ©ratinisĂ©es (cornĂ©ocytes), noyĂ©es dans une matrice lipidique constituĂ©e fondamentalement de cĂ©ramides, d'acides gras libres et de cholestĂ©rol. La diffusion transdermique des molĂ©cules est principalement liĂ©e aux lipides intercellulaires qui forment des membranes multilamellaires en phase solide. L'objectif principal de cette thĂšse est de mieux comprendre la relation entre la structure de la matrice lipidique et l'impermĂ©abilitĂ© de la peau. La phase lipidique du SC comprend plus de 400 lipides diffĂ©rents. En raison de cette composition trĂšs complexe, des membranes modĂšles sont souvent utilisĂ©es pour l'Ă©tude des propriĂ©tĂ©s physico-chimiques des membranes, du comportement des lipides et de la permĂ©abilitĂ© de diffĂ©rents produits chimiques. Nous avons d'abord dĂ©terminĂ© comment la longueur des chaĂźnes acyles d’acides gras libres influence le comportement de phase d'une matrice lipidique composĂ©e de cĂ©ramide NS24, d'acide lignocĂ©rique (FFA24) ou palmitique (FFA16) et de cholestĂ©rol. Les propriĂ©tĂ©s structurales des membranes ont Ă©tĂ© examinĂ©es par 2H RMN et par spectroscopie infrarouge. Cette Ă©tude a montrĂ© que le comportement de phase de ces mĂ©langes ternaires est fortement affectĂ© par la longueur de l’acide gras. Nous avons trouvĂ© que l'acide lignocĂ©rique avec le cĂ©ramide NS24 et le cholestĂ©rol conduit Ă  la formation d'un mĂ©lange plus homogĂšne que celui qui inclut l'acide palmitique. De plus, le mĂ©lange ternaire contenant de l'acide lignocĂ©rique a montrĂ© une transition de la phase solide vers la phase gel lorsqu'il a Ă©tĂ© chauffĂ© au-dessus de 37 °C, une caractĂ©ristique inhabituelle pour ce type de membranes modĂšles. La combinaison de lipides ordonnĂ©s et membranes homogĂšnes est proposĂ©e comme un Ă©lĂ©ment critique pour l’impermĂ©abilitĂ© du SC. DeuxiĂšmement, nous avons Ă©tudiĂ© un mĂ©lange plus complexe contenant du cholestĂ©rol, une sĂ©rie d'acides gras libres variant de 16 Ă  24 atomes de carbone et deux types de cĂ©ramides : le cĂ©ramide NS24 et le cĂ©ramide EOS. Ce dernier est considĂ©rĂ© comme un composant clĂ© pour la formation de la phase de pĂ©riodicitĂ© longue dans les membranes du SC native et modĂšles. Les rĂ©sultats de spectroscopie 2H RMN, infrarouge, et Raman ont montrĂ© que l'acide gras libre et la chaĂźne acyle du cĂ©ramide NS24 restent en phase solide Ă  la tempĂ©rature physiologique tandis que la chaĂźne olĂ©ate du cĂ©ramide EOS entraĂźne la formation de domaines hautement dĂ©sordonnĂ©s. Ces nanogouttelettes restent Ă  l'Ă©tat liquide jusqu'Ă  -30 °C. La contrainte stĂ©rique imposĂ©e par la matrice lipidique cristalline est proposĂ©e ĂȘtre Ă  l’origine de la difficultĂ© de cristallisation des chaĂźnes olĂ©ate des cĂ©ramides EOS. Cette dĂ©couverte modifie substantiellement la description structurale du SC et propose un nouveau rĂŽle physiologique du cĂ©ramide EOS, ce lipide Ă©tant un puissant modulateur de l'Ă©quilibre solide/liquide du SC. Ces travaux conduisent Ă  rĂ©examiner le mĂ©canisme prĂ©sentement proposĂ© pour expliquer la permĂ©abilitĂ© du SC, ainsi que l’effet d’agents transdermiques. Finalement, nous avons Ă©tudiĂ© l'interaction d’acides gras Ă  trĂšs longue chaĂźne avec des membranes de 1-palmitoyl-2-olĂ©oyl-sn-glycĂ©ro-3-phosphocholine (POPC) afin de dĂ©terminer comment s’adaptent ces acides gras aux contraintes spatiales. Trois acides gras diffĂ©rents avec une chaĂźne acyle variant de 16 Ă  24 atomes de carbone ont Ă©tĂ© utilisĂ©s : l’acide palmitique (FFA16), arachidique (FFA20) et lignocĂ©rique (FFA24). L'Ă©paisseur d’un feuillet d’une bicouche de POPC correspond Ă  la longueur de la partie hydrophobe de FFA16, et donc infĂ©rieure Ă  la longueur de FFA20 et FFA24. La façon dont ces acides gras s'adaptent Ă  la bicouche de POPC a Ă©tĂ© Ă©tudiĂ©e par 2H RMN et par simulations de dynamique molĂ©culaire. Nous avons trouvĂ© que la partie infĂ©rieure de la chaĂźne acyle de FFA24 protonĂ© est dĂ©sordonnĂ©e d’une façon similaire Ă  ce qui a Ă©tĂ© observĂ© pour la chaĂźne olĂ©ate du cĂ©ramide EOS, sa chaĂźne acyle interagit avec la partie la plus fluide du feuillet opposĂ©. Cette interdigitation de la fin de la chaĂźne acyle provoque un deuxiĂšme plateau observĂ© dans les profils d’ordre (SC-D), une caractĂ©ristique qui est inhabituelle dans les systĂšmes lipidiques. Dans ce cas, le groupe carboxyle protonĂ© de FFA24 Ă©tait situĂ© lĂ©gĂšrement sous la tĂȘte polaire de la POPC. La dĂ©protonation du FFA24 dĂ©place la molĂ©cule vers l'interface aqueuse. Cette translation diminue la contrainte spatiale, augmente l'ordre de la chaĂźne acyle et entraĂźne la disparition du plateau correspondant au bout de la chaĂźne dĂ©sordonnĂ©e. Les rĂ©sultats prĂ©sentĂ©s dans cette thĂšse contribuent Ă  mieux comprendre comment la structure de la matrice lipidique du SC dicte l'impermĂ©abilitĂ© de la peau. Nous avons montrĂ© des preuves spectroscopiques du comportement de phase de certains lipides importants du SC et suggĂ©rĂ© un nouveau mĂ©canisme pour la rĂ©gulation de la diffusion transdermique des molĂ©cules.The stratum corneum (SC), the outermost layer of mammal epidermis, acts as a barrier dictating the rate of absorption of exogenous molecules through the skin and preventing water loss from the body. SC is mainly composed of flattened and fully keratinized dead cells (corneocytes), embedded in a lipid matrix, which is mostly constituted of ceramides, free fatty acids, and cholesterol. The transdermal diffusion of molecules is mainly related to the intercellular lipids, which form multilamellar membranes in the solid-crystalline phase. The main goal of this thesis is to better understand the relationship between the structure of the lipid matrix and the skin impermeability. SC lipid phase includes more than 400 different lipid species. Due to this very complex composition, model membranes are often used for the study of the physicochemical properties of membranes, the lipid behavior, and of the permeability of different chemicals. First, we determined how the length of the free fatty acid acyl chains influences the phase behavior of a lipid matrix composed of ceramide NS24, lignoceric (FFA24) or palmitic (FFA16) acid, and cholesterol. The structural properties of membranes were examined by 2H NMR and infrared spectroscopy. This study revealed that the phase behavior of these ternary mixtures is strongly affected by the length of the FFA. We found that lignoceric acid led to the formation of a more homogeneous mixture with ceramide NS24 and cholesterol, than the palmitic acid/ceramide NS24/cholesterol mixture. Also, the tertiary mixture containing lignoceric acid showed a transition from solid to gel phase when heated above 37 oC, an unusual feature for this type of model membranes. The combination of ordered lipids and homogeneous membranes is proposed as a critical element for SC impermeability. Second, we studied a more complex mixture containing cholesterol, a series of free fatty acids varying from 16 to 24 carbon atoms, and two types of ceramides: ceramide NS24 and EOS. The latter is considered a key component for the formation of the long periodicity phase in native and model SC membranes. The 2H NMR, infrared and Raman spectroscopy results showed that both the free fatty acid and the ceramide NS24 acyl chain remained in the solid-crystalline phase at physiological temperature while the oleate chain in ceramide EOS led to the formation of highly disordered domains. These liquid nanodrops remained in the liquid state down to -30 °C. The steric constraint imposed by the crystalline lipid matrix is proposed to prevent the crystallization of ceramide EOS oleate chains. This finding modifies the structural description of the SC substantially and proposes a novel physiological role of ceramide EOS as this lipid is a strong modulator of SC solid/liquid balance. The work leads to a re-examination of the mechanism currently proposed to explain the permeability of SC, as well as the effect of transdermal agents. Finally, we studied the interaction of very long-chain fatty acids with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes to determine how these fatty acids adapt to spatial constraints. Three different FFAs with acyl chain varying from 16 to 24 carbon atoms were used: palmitic (FFA16), arachidic (FFA20) and lignoceric (FFA24) acid. The leaflet thickness of a POPC bilayer corresponds to the length of the hydrophobic part of FFA16, and therefore is smaller than the length of FFA20 and FFA24. The way in which these fatty acids structurally adapt in POPC bilayers was study by 2H NMR and molecular dynamics simulations. We found that the lower part of the protonated FFA24 acyl chain was disordered in a manner similar to that observed for the oleate chain of the EOS ceramide, its acyl chain interacts with the more fluid part of the opposite leaflet. This interdigitation of the end of the acyl chain caused a second plateau observed in the order profiles (SC−D), an unusual feature in lipid systems. In this case, the protonated carboxyl group of FFA24 was located slightly below the polar head of the POPC. The deprotonation of the FFA24 shifted the molecule toward the aqueous interphase. This movement reduces the spatial constraints, increases the order of the acyl chain and causes the disappearance of the plateau at the end of the chain. The results presented in this thesis contributed to better understand how the structure of the SC lipid matrix dictates the skin impermeability. We showed spectroscopic evidences of the distinct phase behavior of some of the most important SC lipids. Furthermore, we suggested a novel mechanism for the regulation of transdermal diffusion of molecules

    Uncovering the molecular mechanisms of cardiac ion channels’ regulation by lipids and pore formation in membranes using computer simulations

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    Membranes are complex cellular structures consisting of many different lipid types, a variety of bound proteins, and other molecules. Growing evidence suggests that membranes and lipids play significant bioactive roles in modulating protein function across several cellular processes. Molecular dynamic (MD) simulations have proven to be a valuable method to study lipid organization and membrane protein activity. In this thesis, I used MD simulations to study how lipids regulate two types of membrane proteins: ion channels and pore-forming proteins. Previous simulations and experimental studies showed that polyunsaturated fatty acids (PUFAs) activate KCNQ1 channels while blocking hERG channels. However, some questions regarding how the channel state or PUFA structural properties influence their molecular mechanisms remained unclear. In part of my work, I built a cardiomyocyte membrane model to study the molecular mechanism underlying the interactions between PUFAs and two voltage-gated potassium channels involved in the cardiac action potential: KCNQ1 and hERG. My results revealed that when KCNQ1 voltage sensor domain (VSD) was in the resting state or ‘down’ conformation, the PUFAs established short-lasting interactions that were different from the long-lasting interactions previously observed in the KCNQ1 intermediate state, where the VSD is in the ‘up’ conformation. Additionally, my studies showed that the number of double bonds in the PUFA acyl tail and the size of the polar head regulates their affinity for KCNQ1. Moreover, MD simulations of the hERG channel in the cardiomyocyte membrane unveiled the PUFA interacting site on hERG at the interface between the VSD and the PD in the open and closed states. I anticipate that this detailed molecular understanding of how PUFAs interact with KCNQ1 and hERG will aid in developing future drugs that utilize these mechanisms. As part of this work, I also studied the pore-forming mechanism of the N-terminal peptide StII1-30, derived from the actinoporin StII. My results revealed that this peptide followed a toroidal pore formation mechanism. Additionally, I unveiled the role of curved lipids as cofactors in the formation of toroidal pores. This work has the potential to lead to strategies for the rational use of these peptides as immunotoxins for immunotherapy in cancer tumors. The overall work in this thesis enhances our understanding of lipid-protein interactions in voltage-gated ion channels and the mechanism underlying pore formation by lytic peptides

    Emerging Diversity in Lipid-Protein Interactions

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    Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions
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