Michel Lafleur

Université de Montréal, Montréal, Quebec, Canada

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Publications (104)356.34 Total impact

  • Alexandre Therrien · Michel Lafleur
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    ABSTRACT: Protein- and peptide-induced lipid extraction from membranes is a critical process for many biological events, including reverse cholesterol transport and sperm capacitation. In this work, we examine whether such processes could display specificity for some lipid species. Melittin, the main component of dry bee venom, was used as a model amphipathic α-helical peptide. We specifically determined the modulation of melittin-induced lipid extraction from membranes by the change of the methylation level of phospholipid headgroups. Phosphatidylcholine (PC) bilayers were demethylated either by substitution with phosphatidylethanolamine (PE) or chemically by using mono- and dimethylated PE. It is shown that demethylation reduces the association of melittin with membranes, likely because of the resulting tighter chain packing of the phospholipids, which reduces the capacity of the membranes to accommodate inserted melittin. This reduced binding of the peptide is accompanied by an inhibition of the lipid extraction caused by melittin. We demonstrate that melittin selectively extracts PC from PC/PE membranes. This selectivity is proposed to be a consequence of a PE depletion in the surroundings of bound melittin to minimize disruption of the interphospholipid interactions. The resulting PC-enriched vicinity of melittin would be responsible for the observed formation of PC-enriched lipid/peptide particles resulting from the lipid efflux. These findings reveal that modulating the methylation level of phospholipid headgroups is a simple way to control the specificity of lipid extraction from membranes by peptides/proteins and thereby modulate the lipid composition of the membranes.
    No preview · Article · Jan 2016 · Biophysical Journal
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    Zhong-Kai Cui · Guillaume Bastiat · Michel Lafleur

    Full-text · Dataset · Dec 2015
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    Full-text · Dataset · Dec 2015
  • Adrian Paz Ramos · Michel Lafleur
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    ABSTRACT: The skin, the largest organ of the human body, forms a flexible interface between our internal and external environment that protects our organism from exogenous compounds as well as an excessive water loss. The stratum corneum (SC), the outermost layer of mammal epidermis, is mainly responsible for the skin impermeability. The SC is formed by corneocytes embedded in a lipid matrix, which is mostly constituted by ceramides (Cer), free fatty acids (FFA) and cholesterol (Chol), organized in two coexisting crystalline lamellar phases. This arrangement of lipids is crucial for the skin barrier function. The aim of this paper is to determine the impact of FFA chain length on the phase behaviour of SC model lipid membranes, using solid-state deuterium NMR and IR spectroscopy. We studied ternary mixtures of N-lignoceroyl-D-erythro-sphingosine (Cer24), cholesterol, and palmitic (FFA16) or lignoceric (FFA24) acid in equimolar ratio. This proportion replicates the lipid composition found in the SC lipid matrix. Our studies revealed that the phase behaviour of Cer24/FFA/Chol ternary mixtures is deeply affected by the length of the FFA. We found the formation of phase-separated crystalline lipid domains when using palmitic acid while the use of lignoceric acid results in a more homogeneous mixture. In addition, it was observed that mixtures with lignoceric acid form a gel phase, a very unusual feature for SC model mixtures.
    No preview · Article · Oct 2015 · Langmuir
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    ABSTRACT: Binder of sperm (BSP) proteins are ubiquitous among mammals and have been extensively investigated over the last three decades. They were first characterized in bull seminal plasma and have now been identified in more than 15 different mammalian species where they represent a superfamily. In addition to sharing a common structure, BSP proteins share many characteristics. They are expressed by seminal vesicles and epididymides, interact with similar ligands and bind to the outer leaflet of sperm membranes via an interaction with choline phospholipids. In addition to playing a major role in sperm capacitation, they are implicated as molecular chaperones in sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume and possibly in the interaction between sperm and oocytes, making them crucial multifunctional proteins. Furthermore, BSP proteins can bind to egg yolk low-density lipoproteins and milk components, an interaction important for the protection of sperm during semen preservation in liquid or frozen state. Our current knowledge of BSP proteins strongly emphasizes their fundamental importance in male fertility and in the optimization of semen preservation techniques. Much work is still ahead in order to fully understand all the mysteries of BSP proteins.
    Full-text · Article · Sep 2015 · Cell and Tissue Research
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    ABSTRACT: Background Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general. Methods Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting. Results Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, β-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1). Conclusions These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.
    Full-text · Article · Aug 2015 · Reproductive Biology and Endocrinology
  • Alexandre Therrien · Michel Lafleur

    No preview · Article · Jan 2015 · Biophysical Journal
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    ABSTRACT: We created novel non-phospholipid photosensitive liposomes from a mixture of a monoacylated azobenzene amphiphile (AzoC10N+), and cholesterol sulfate (Schol). This system belongs to the family of sterol-enriched non-phospholipid liposomes that were shown to form stable large unilamellar vesicles (LUVs) with enhanced impermeability. Fluid bilayers were successfully prepared from AzoC10N+/Schol (25/75, mol/mol) mixtures and LUVs could be derived at room temperature using standard extrusion methods. The isomerization process of the bilayer-inserted AzoC10N+ was characterized. Leakage from these liposomes could be induced by the photoconversion of the AzoC10N+ from its trans to its cis form. This photo-controlled release is obtained from fluid liposomes, contrasting with phospholipid-based azo-containing liposomes, which are generally required to be in the gel phase in order to be photosensitive. It is proposed that the very high conformational order of the monoalkylated amphiphile and the tight packing of the hydrophobic core of the AzoC10N+/Schol liposomes make them responsive to the presence of the bulky cis azo isomer. Interestingly, the liposome impermeability could be fully restored by the photoisomerization of the cis form back to the trans form, providing a sharp on-and-off control of payload release. In addition, these non-phospholipid liposomes display a very limited passive release. Therefore, it is shown that AzoC10N+/Schol LUVs can be used as nanocontainers, whose content can be released by light in a controlled and switchable manner.
    Full-text · Article · Aug 2014 · Langmuir
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    ABSTRACT: Hypothesis: Liposomes made of single-chain amphiphiles and a large amount of sterols display several advantages including a limited permeability. In the present paper, we examine the possibility to prepare such non-phospholipid liposomes with interfacial polyethylene glycol (PEG) in order to improve their circulation in the blood stream. Cholesterol (Chol) was chosen as the PEG anchor. Experiments: The phase behavior of mixtures of palmitic acid (PA) and cholesterol including various proportions of PEGylated cholesterol (PEG-Chol) was characterized. In conditions leading to the formation of fluid bilayers, properties of the resulting liposomes were assessed. Findings: Up to 20 mol% of PEGylated cholesterol could be introduced without significant perturbations in fluid bilayers made of PA and cholesterol. With 10 mol% PEG-Chol, PA/Chol/PEG-Chol liposomes showed a very limited permeability to calcein and doxorubicin. Doxorubicin could be actively loaded in PA/Chol/PEG-Chol liposomes with a high drug loading efficiency and a high drug to lipid ratio. Pharmaco-kinetic experiments in rats indicated that interfacial PEG reduced the clearance of PA/Chol liposomes compared to the naked ones. However the lifetime of these non-phospholipid liposomes in the blood circulation was considerably shorter than that observed for control PEGylated phospholipid liposomes, a phenomenon associated with the negative interfacial charge of the PA/Chol/PEG-Chol liposomes.
    Full-text · Article · Aug 2014 · Journal of Colloid and Interface Science
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    ABSTRACT: Despite the fact that palmitic acid (PA) and cholesterol (Chol) do not form fluid bilayers once hydrated individually, giant unilamellar vesicles (GUVs) were formed from a mixture of palmitic acid and cholesterol, 30/70 mol/mol. These free-floating GUVs were stable over weeks, did not aggregate and were shown to be highly stable in alkaline pH compared to conventional phospholipid-based GUVs. Acidic pH-triggered payload release from the GUVs was associated with the protonation state of palmitic acid that dictated the mixing lipid properties, thus affecting the stability of the fluid lamellar phase. The successful formation of PA-Chol GUVs reveals the possibility to create monoalkylated amphiphile-based GUVs with distinct pH stability/sensitivity.
    Full-text · Article · Jul 2014 · Soft Matter
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    Michel Lafleur · Alexandre Therrien

    Preview · Article · Jan 2014 · Biophysical Journal
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    Zhong-Kai Cui · Michel Lafleur
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    ABSTRACT: Typically, single-chain amphiphiles and sterols do not form fluid lamellar phases once hydrated individually. Most of the single-chain amphiphiles form actually micelles in aqueous environments, while sterols display a very limited solubility in water. However, under certain conditions, mixtures of single-chain amphiphiles and sterols lead to the formation of stable fluid bilayers. Over the past decade, several of these systems leading to fluid lamellar self-assemblies have been identified and this article reviews the current knowledge relative to these non-phospholipid bilayers made of single-chain amphiphiles and sterols. It presents an integrated view about the molecular features that are required for their stability, the properties they share, and the origin of these characteristics. It was also shown that these lamellar systems could lead to the formation of unilamellar vesicles, similar to phospholipid based liposomes. These vesicles display distinct properties that make them potentially appealing for technological applications; they display a limited permeability, they are stable, they are formed with molecules that are relatively chemically inert (and relatively cheap), and they can be readily functionalized. The features of these distinct liposomes and their technological applications are reviewed. Finally, the putative biological implications of these non-phospholipid fluid bilayers are also discussed.
    Full-text · Article · Oct 2013 · Colloids and surfaces B: Biointerfaces
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    ABSTRACT: Cetylpyridinium chloride (CPC) is a surfactant that binds strongly to bacteria and bacterial biofilms. In this study, fluorescence-based techniques were used to determine the penetration and adhesion of CPC when it was introduced in liposomes. In spite of a reduced adhesion as compared to pure CPC micelles, CPC-containing liposomes adhered significantly to the biofilms of Streptococcus mutans. In contrast, no binding was observed for liposomes that were composed of phosphatidylcholine-cholesterol. The influence of the charge of the liposome on its adhesion to biofilms was studied using cholesterol (Chol) and cholesterol sulfate (Schol). In spite of similar binding to the biofilms, positively charged CPC/Chol liposomes were located mainly in the core of the biofilm microcolonies, whereas the negatively charged CPC/Schol liposomes were mainly concentrated at their periphery. This effect may be attributed to the different availability of the CPC head group. In summary, this work demonstrates the high potential for tailoring drug nanovectors by modulating sterol selection in order to selectively target and bind biofilms.
    Full-text · Article · Jul 2013 · Biofouling
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    ABSTRACT: The skin acts mainly as a protective barrier from the external environment, thanks to the stratum corneum which is the outermost layer of the skin. As in vitro tests on skin are essential to elaborate new drugs, the development of skin models closer to reality becomes essential. It is now possible to produce in vitro human skin substitutes through tissue engineering by using the self-assembly method developed by the Laboratoire d'Organogénèse Expérimentale. In the present work, infrared microspectroscopy imaging analyses were performed to get in-depth morpho-spectral characterization of the three characteristic layers of human skin substitutes and normal human skin, namely the stratum corneum, living epidermis, and dermis. An infrared spectral analysis of the skin is a powerful tool to gain information on the order and conformation of the lipid chains and the secondary structure of proteins. On one hand, the symmetric stretching mode of the lipid methylene groups (2,850 cm(-1)) is sensitive to the acyl chain conformational order. The evolution profile of the frequency of this vibrational mode throughout the epidermis suggests that lipids in the stratum corneum are more ordered than those in the living epidermis. On the other hand, the frequencies of the infrared components underneath the envelop of the amide I band provide information about the overall protein conformation. The analysis of this mode establishes that the proteins essentially adopt an α-helix conformation in the epidermis, probably associated with the presence of keratin, while modifications of the protein content are observed in the dermis (extracellular matrix made of collagen). Finally, the lipid organization, as well as the protein composition in the different layers, is similar for human skin substitutes and normal human skin, confirming that the substitutes reproduce essential features of real skin and are appropriate biomimetics.
    Full-text · Article · Jun 2013 · Analytical and Bioanalytical Chemistry
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    ABSTRACT: The bovine seminal plasma contains phosphocholine (PC)-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10-15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.
    Preview · Article · Aug 2012 · Biochimica et Biophysica Acta
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    Zhong-Kai Cui · Anne Bouisse · Nicolas Cottenye · Michel Lafleur
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    ABSTRACT: It has been shown that mixtures of monoalkylated amphiphiles and sterols can form liquid-ordered (lo) lamellar phases. These bilayers can be extruded using conventional methods to obtain large unilamellar vesicles (LUVs) that have very low permeability and a specific response to a given stimulus. For example, pH variations can trigger the release from LUVs formed with palmitic acid and sterols. In the present work, the possibility to form non phospholipid liposomes with mixtures of stearylamine (SA) and cholesterol (Chol) was investigated. The phase behavior of these mixtures was characterized by differential scanning calorimetry, infrared, and (2)H NMR spectroscopy. It is found that this particular mixture can form a lo lamellar phase that is pH-sensitive as the system undergoes a transition from a lo phase to a solid state when pH is increased from 5.5 to 12. LUVs have been successfully extruded from equimolar SA/Chol mixtures. Release experiments as a function of time revealed the relatively low permeability of these systems. The fact that the stability of these liposomes is pH dependent implies that these LUVs display an interesting potential as new cationic carriers for pH-triggered release. This is the first report of non phospholipid liposomes with high sterol content combining an overall positive charge and pH-sensitivity.
    Full-text · Article · Aug 2012 · Langmuir
  • Zhong-Kai Cui · Michel Lafleur

    No preview · Conference Paper · Mar 2012
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    ABSTRACT: Ethanol is used in a variety of topical products. It is known to enhance the permeability of the skin by altering the ability of the stratum corneum (SC) intercellular membranes to form an effective barrier. In addition, ethanol and other alcohols are key components of antiseptic gels currently used for hand wash. Using infrared and deuterium NMR spectroscopy as well as calorimetry, we have investigated the effect of ethanol on a model membrane composed of lipids representing the three classes of SC lipids, an equimolar mixture of N-palmitoylsphingosine (ceramide), palmitic acid and cholesterol. Ethanol is found to influence the membrane in a dose dependent manner, disrupting packing and increasing lipid motion at low concentrations and selectively extracting lipids at moderate concentrations.
    Full-text · Article · Feb 2012 · Biochimica et Biophysica Acta
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    Gustavo Carbajal · Zhong-Kai Cui · Michel Lafleur
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    ABSTRACT: We demonstrate that it is possible to form non-phospholipid fluid bilayers in aqueous milieu with a mixture of palmitic acid (PA), cholesterol (Chol), and cholesterol sulfate (Schol) in a molar proportion of 30/28/42. These self-assemblies are shown to be bilayers in the liquid ordered phase. They are stable between pH 5 and 9. Over this pH range, the protonation/deprotonation of PA carboxylic group is observed but this change does not appear to alter the stability of these bilayers, a behavior contrasting with that observed for binary mixtures of PA/Chol, and PA/Schol. The multilamellar dispersions formed spontaneously from the PA/Chol/Schol mixture could be successfully extruded to form Large Unilamellar Vesicles (LUVs). These LUVs show interesting permeability properties, linked with their high sterol content. These non-phospholipid liposomes can sustain a pH gradient (pHinternal 8/pHexternal 6) 100 times longer than LUVs made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol, with a molar ratio of 60/40. Moreover, the non-phospholipid LUVs are shown to protect ascorbic acid from an oxidizing environment (1 mM iron(III)). Once entrapped in liposomes, ascorbic acid displays a degradation rate similar to that obtained in the absence of iron(III). These results show the possibility to form novel nanocontainers from a mixture of a monoalkylated amphiphile and sterols, with a good pH stability and showing interesting permeability properties.
    Full-text · Article · Jan 2012 · Science China-Chemistry
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    ABSTRACT: Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.
    No preview · Article · May 2011 · Biology of Reproduction

Publication Stats

3k Citations
356.34 Total Impact Points

Institutions

  • 1993-2015
    • Université de Montréal
      • Department of Chemistry
      Montréal, Quebec, Canada
  • 1999-2014
    • Université du Québec à Montréal
      • Department of Chemistry
      Montréal, Quebec, Canada
  • 1996
    • Princeton University
      • Department of Physics
      Princeton, New Jersey, United States
  • 1989-1990
    • University of British Columbia - Vancouver
      Vancouver, British Columbia, Canada
    • Université du Québec
      Québec, Quebec, Canada