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Parkinson's disease (PD) is a major neurodegenerative disorder which exhibits many of the characteristics of a pandemic. Current therapeutic strategies are centered on the dopaminergic system, with limited efficacy, so that a treatment that has a direct impact on the underlying disease pathogenesis is urgently needed. Although α-synuclein is a privileged target for such therapies, this protein has been in the past wrongly considered as exclusively intracellular, so that the impact of paracrine neurotoxicity mechanisms in PD have been largely ignored. In this article we review the data showing that lipid rafts act as plasma membrane machineries for the formation of α-synuclein pore-like oligomers which trigger an increase of intracellular Ca2+. This Ca2+ influx is responsible for a self-sustained cascade of neurotoxic events, including mitochondrial oxidative stress, tau phosphorylation, Ca2+ release from the endoplasmic reticulum, Lewy body formation, and extracellular release of α-synuclein in exosomes. The first step of this cascade is the binding of α-synuclein to lipid raft gangliosides, suggesting that PD should be considered as both a proteinopathy and a ganglioside membrane disorder lipidopathy. Accordingly, blocking α-synuclein-ganglioside interactions should annihilate the whole neurotoxic cascade and stop disease progression. A pipeline of anti-oligomer molecules is under development, among which an in-silico designed synthetic peptide AmyP53 which is the first drug targeting gangliosides and thus able to prevent the formation of α-synuclein oligomers and all downstream neurotoxicity. These new therapeutic avenues challenge the current symptomatic approaches by finally targeting the root cause of PD through a long-awaited paradigm shift.

Gangliosides are anionic lipids that form condensed membrane clusters (lipid rafts) and exert major regulatory functions on a wide range of proteins. In this review, we propose a new view of the structural features of gangliosides with special emphasis on emerging properties associated with protein binding modes. We analyze the different possibilities of molecular associations of gangliosides in lipid rafts and the role of cholesterol in this organization. We are particularly interested in amide groups of N-acetylated sugars which make it possible to neutralize the negative charge of the carboxylate group of sialic acids. We refer to this effect as "NH trick" and we demonstrate that it is operative in GM1, GD1a, GD1b and GT1b gangliosides. The NH trick is key to understand the different topologies adopted by gangliosides (chalice-like at the edge of lipid rafts, condensed clusters in central areas) and their impact on protein binding. We define three major types of ganglioside-binding domains (GBDs): α-helical, loop shaped, and large flat surface. We describe the mode of interaction of each GBD with typical reference proteins: synaptotagmin, 5HT1A receptor, cholera and botulinum toxins, HIV-1 surface envelope glycoprotein gp120, SARS-CoV-2 spike protein, cellular prion protein, Alzheimer's β-amyloid peptide and Parkinson's disease associated α-synuclein. We discuss the common mechanisms and peculiarities of protein binding to gangliosides in the light of physiological and pathological conditions. We anticipate that innovative ganglioside-based therapies will soon show an exponential growth for the treatment of cancer, microbial infections, and neurodegenerative diseases.

The recently emerging SARS-CoV-2 variant omicron displays an unusual association of 30 mutations, 3 deletions, and 1 insertion. To analyze the impact of this atypic mutational landscape, we constructed a complete structure of the omicron spike protein. Compared with the delta variant, the receptor-binding domain (RBD) of omicron has an increased electrostatic surface potential, but a decreased affinity for the ACE-2 receptor. The N-terminal domain (NTD) has both a decreased surface potential and a lower affinity for lipid rafts. The omicron variant is predicted to be less fusogenic and thus less pathogenic than delta, due to a geometric reorganization of the S1-S2 cleavage site. Overall, these virological parameters suggest that omicron does not have a significant infectivity advantage over the delta variant. However, in omicron, neutralizing epitopes are greatly affected, suggesting that current vaccines will probably confer little protection against this variant. In conclusion, the puzzling mutational pattern of the omicron variant combines contradictory properties which may either decrease (virological properties) or increase (immunological escape/facilitation) the transmission of this variant in the human population. This Janus-like phenotype may explain some conflicting reports on the initial assessment of omicron and provide new insights about the molecular mechanisms controlling its dissemination and pathogenesis worldwide.

Fil: Azzaz, Fodil. Institut national de la santé et de la recherche médicale; Francia Fil: Azzaz, Fodil. Aix-Marseille Université; Francia Fil: Chahinian, Henri. Institut national de la santé et de la recherche médicale; Francia Fil: Chahinian, Henri. Aix-Marseille Université; Francia Fil: Yahi, Nouara. Institut national de la santé et de la recherche médicale; Francia Fil: Yahi, Nouara. Aix-Marseille Université; Francia. Fil: Di Scala, Coralie. University of Helsinki. Neuroscience Center; Finlandia Fil: Baier, Carlos J. Universidad Nacional del Sur. Instituto de Ciencias Biológicas y Biomédicas del Sur. Laboratorio de Toxicología; Argentina Fil: Baier, Carlos J. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Barrantes, Francisco José. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Laboratorio de Neurobiología Molecular; Argentina Fil: Barrantes, Francisco José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Abstract: Cholesterol binding to proteins is a dynamic process that involves a combination of geometric, biochemical, and biophysical principles. These properties can be viewed as basic rules which govern any kind of molecular interactions. Nevertheless, cholesterol displays unique features that have made cholesterol recognition motifs in proteins remarkably convergent upon biological evolution. Consequently, simple algorithms based on consensus amino acid sequences (e.g., CARC and CRAC) have been developed to predict the presence of such cholesterol-binding motifs in proteins. The intrinsic weakness of this approach is that CARC and CRAC are both based on a linear (1D) sequence motif, whereas cholesterol-binding sites have a three-dimensional (3D) structure. This issue is discussed in detail in this chapter. We then analyze the performance of these algorithms in the light of structural data obtained by X-ray diffraction and cryoelectron microscopy of membrane proteins, and structure-function studies based on site-directed mutagenesis. Our study not only confirms the overall reliability of CARC and CRAC algorithms but also reveals new clues that could bring forth new ideas on cholesterol recognition motifs in the 3D structure of transmembrane proteins.

Infection-enhancing antibodies may limit the efficiency of Covid-19 vaccines. We analyzed the evolution ofneutralizing and facilitating epitopes in 1,860,489 SARS-CoV-2 genomes stored in the Los Alamos databasefrom June to November 2021. The structural dynamics of these epitopes was determined by molecular modelingof the spike protein on a representative panel of SARS-CoV-2 variants. D614, which belongs to an antibody-dependent-enhancement (ADE) epitope common to SARS-CoV-1 and SARS-CoV-2, has mutated to D614G in2020, which could explain why ADE has not been detected following mass vaccination. A second epitopelocated in the N-terminal domain (NTD), specific of SARS-CoV-2, is highly conserved among most variants. Incontrast, the neutralizing epitope of the NTD showed extensive variations in SARS-CoV-2 variants. The balancebetween facilitating and neutralizing antibodies is in favor of neutralization for the Wuhan strain, alpha and betavariants, but not for gamma, delta, lambda, and mu. The recently emerging omicron variant is atypic as itsmutational profiles affects both neutralization and ADE epitopes. Overall, our data reveal that the evolution ofSARS-CoV-2 has dramatically affected the ADE/neutralization balance. Future vaccines should consider thesefindings to design new formulations adapted to SARS-CoV-2 variants and lacking ADE epitopes in the spikeprotein.

Objectives. The efficiency of Covid-19 vaccination is determined by cellular and humoral immune responses, and for the latter, by the balance between neutralizing and infection-enhancing antibodies. Here we analyzed the evolution of neutralizing and facilitating epitopes in the spike protein among SARS-CoV-2 variants. Methods. Amino acid alignments were performed on 929,203 spike sequences over the 4 last months. Molecular modeling studies of the N-terminal domain (NTD) and rod-like regions of the spike protein were performed on a representative panel of SARS-CoV-2 variants that were structurally compared with the original Wuhan strain. Results. D614, which belongs to an antibody-dependent-enhancement (ADE) epitope common to SARS-CoV-1 and SARS-CoV-2, has rapidly mutated to D614G in the first months of 2020, explaining why ADE has not been detected following mass vaccination. We show that this epitope is conformationally linked to the main ADE epitope of the SARS-CoV-2 NTD which is highly conserved among most variants. In contrast, the neutralizing epitope of the NTD showed extensive variations in SARS-CoV-2 variants. Conclusions. This molecular epidemiology study coupled with structural analysis of the spike protein indicates that the balance between facilitating and neutralizing antibodies in vaccinated people is in favor of neutralization for the Wuhan strain, alpha and beta variants, but not for gamma, delta, lambda and mu. The evolution of SARS-CoV-2 has dramatically affected the ADE/neutralization balance which is nowadays in favor of ADE. Future vaccines should consider these data to design new formulations adapted to SARS-CoV-2 variants and lacking ADE epitopes in the spike protein.

Objectives. The efficiency of Covid-19 vaccination is determined by cellular and humoral immune responses, and for the latter, by the balance between neutralizing and infection-enhancing antibodies. Here we analyzed the evolution of neutralizing and facilitating epitopes in the spike protein among SARS-CoV-2 variants. Methods. Amino acid alignments were performed on 929,203 spike sequences over the 4 last months. Molecular modeling studies of the N-terminal domain (NTD) and rod-like regions of the spike protein were performed on a representative panel of SARS-CoV-2 variants that were structurally compared with the original Wuhan strain. Results. D614, which belongs to an antibody-dependent-enhancement (ADE) epitope common to SARS-CoV-1 and SARS-CoV-2, has rapidly mutated to D614G in the first months of 2020, explaining why ADE has not been detected following mass vaccination. We show that this epitope is conformationally linked to the main ADE epitope of the SARS-CoV-2 NTD which is highly conserved among most variants. In contrast, the neutralizing epitope of the NTD showed extensive variations in SARS-CoV-2 variants. Conclusions. This molecular epidemiology study coupled with structural analysis of the spike protein indicates that the balance between facilitating and neutralizing antibodies in vaccinated people is in favor of neutralization for the Wuhan strain,alpha and beta variants, but not for gamma, delta, lambda and mu. The evolution of SARS-CoV-2 has dramatically affected the ADE/neutralization balance which is nowadays in favor of ADE. Future vaccines should consider these data to design new formulations adapted to SARS-CoV-2 variants and lacking ADE epitopes in the spike protein.

Background. Since the beginning of the COVID-19 pandemic, several SARS-CoV-2 variants have sequentially emerged. In France, most cases were due to spike D641G-harbouring viruses that descended initially from the Wuhan strain, then by variant of B.1.160 lineage we called Marseille-4 since the summer of 2020, which was followed by the alpha (UK) and beta (South African) variants in early 2021, then delta (Indian) now.Methods and Findings. We determined the neutralizing antibody (nAb) titres in sera from convalescent individuals previously infected by these 4 major local variants and from vaccine recipients to the original Wuhan strain and 9 variants, including two recent circulating delta (Indian) isolates. The results show high inter-individual heterogeneity in nAbs, especially according to the variant tested. Unexpectedly, the major variations among nAbs are based on the genotype responsible for the infection. Patients previously infected with the beta and B.1.160 variants had the lowest nAb titres. We show that this heterogeneity is well explained by spike protein mutants modelling using in silico approaches. The highest titres were observed in patients vaccinated with the Pfizer/BioNTech COVID-19 vaccine, even against the delta variant.Conclusions. Immunity acquired naturally after infection is highly dependent on the infecting variant and unexpectedly mRNA-based vaccine efficacy is shown to be often better than natural immunity in eliciting neutralizing antibodies.

Since the beginning of the COVID-19 pandemic, several SARS-CoV-2 variants have sequentially emerged. In France, most cases were due to spike D641G-harbouring viruses that descended initially from the Wuhan strain, then by variant of B.1.160 lineage we called Marseille-4 since the summer of 2020, which was followed by the alpha (UK) and beta (South African) variants in early 2021. We determined the neutralizing antibody (nAb) titres in sera from convalescent individuals previously infected by these 4 major local variants and from vaccine recipients to the original Wuhan strain and 9 variants, including two recent circulating delta (Indian) isolates. The results show high interindividual heterogeneity in nAbs, especially according to the variant tested. Unexpectedly, the major variations among nAbs are based on the genotype responsible for the infection. Patients previously infected with the beta and B.1.160 variants had the lowest nAb titres. The highest titres were observed in patients vaccinated with the Pfizer/BioNTech COVID-19 vaccine, even with antibodies against the delta variant.

Antibody dependent enhancement (ADE) of infection is a safety concern for vaccine strategies. In a recent publication, Li et al. (Cell 184 :4203-4219, 2021) have reported that infection-enhancing antibodies directed against the N-terminal domain (NTD) of the SARS-CoV-2 spike protein facilitate virus infection in vitro, but not in vivo. However, this study was performed with the original Wuhan/D614G strain. Since the Covid-19 pandemic is now dominated with Delta variants, we analyzed the interaction of facilitating antibodies with the NTD of these variants. Using molecular modeling approaches, we show that enhancing antibodies have a higher affinity for Delta variants than for Wuhan/D614G NTDs. We show that enhancing antibodies reinforce the binding of the spike trimer to the host cell membrane by clamping the NTD to lipid raft microdomains. This stabilizing mechanism may facilitate the conformational change that induces the demasking of the receptor binding domain. As the NTD is also targeted by neutralizing antibodies, our data suggest that the balance between neutralizing and facilitating antibodies in vaccinated individuals is in favor of neutralization for the original Wuhan/D614G strain. However, in the case of the Delta variant, neutralizing antibodies have a decreased affinity for the spike protein, whereas facilitating antibodies display a strikingly increased affinity. Thus, ADE may be a concern for people receiving vaccines based on the original Wuhan strain spike sequence (either mRNA or viral vectors). Under these circumstances, second generation vaccines with spike protein formulations lacking structurally-conserved ADE-related epitopes should be considered.

Covid-19 is an infectious respiratory disease due to a coronavirus named SARS-CoV-2. A critical step of the infection cycle is the binding of the virus spike S protein to the cellular ACE-2 receptor. This interaction involves a receptor binding domain (RBD) located at the center of the S trimer, whereas the lateral N-terminal domain (NTD) displays a flat ganglioside binding site that enables the virus to bind to lipid rafts of the plasma membrane, where the ACE-2 receptor resides. S protein binding to lipid rafts can be blocked by hydroxychloroquine, which binds to gangliosides, and by azithromycin, which binds to the NTD. Based on these data, we identified the NTD of SARS-CoV-2 as a promising target for both therapeutic and vaccine strategies, a notion later supported by the discovery, in convalescent Covid-19 patients, of a neutralizing antibody (4A8) that selectively binds to the NTD. The 4A8 epitope overlaps the ganglioside binding domain, denying any access of the virus to lipid rafts when the antibody is bound to the S protein. Thus, our data explain why antibody binding to the tip of the NTD results in SARS-CoV-2 neutralization. The high level of conservation of the ganglioside binding domain of SARS-CoV-2 (100% identity in 584 of 600 isolates analyzed worldwide) offers unique opportunities for innovative vaccine/therapeutic strategies., Highlights • Gangliosides in lipid rafts play a key role in SARS-CoV-2 infection. • The N-terminal domain (NTD) of the virus spike protein binds to gangliosides. • Hydroxychloroquine and azithromycin prevent NTD-gangliosides interactions. • Neutralizing antibodies isolated from Covid-19 patients are directed against the NTD. • The NTD is a promising target for therapeutic and vaccine strategies against Covid-19.

International audience; The recent emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) is responsible for a worldwide pandemic. Given the global health emergency, drug repositioning is the most reliable option to design an efficient therapy for infected patients without delay. The first step of the viral replication cycle [i.e. attachment to the surface of respiratory cells, mediated by the spike (S) viral protein] offers several potential therapeutic targets. The S protein uses the angiotension-converting enzyme-2 (ACE-2) receptor for entry, but also sialic acids linked to host cell surface gangliosides. Using a combination of structural and molecular modelling approaches, this study showed that chloroquine (CLQ), one of the drugs currently under investigation for SARS-CoV-2 treatment, binds sialic acids and gangliosides with high affinity. A new type of ganglioside-binding domain at the tip of the N-terminal domain of the SARS-CoV-2 S protein was identified. This domain (111-158), which is fully conserved among clinical isolates worldwide, may improve attachment of the virus to lipid rafts and facilitate contact with the ACE-2 receptor. This study showed that, in the presence of CLQ [or its more active derivative, hydroxychloroquine (CLQ-OH)], the viral S protein is no longer able to bind gangliosides. The identification of this new mechanism of action of CLQ and CLQ-OH supports the use of these repositioned drugs to cure patients infected with SARS-CoV-2. The in-silico approaches used in this study might also be used to assess the efficiency of a broad range of repositioned and/or innovative drug candidates before clinical evaluation.

After three decades of false hopes and failures, a pipeline of therapeutic drugs that target the actual root cause of Alzheimer's disease (AD) is now available. Challenging the old paradigm that focused on β‐amyloid peptide (Aβ) aggregation in amyloid plaques, these compounds are designed to prevent the neurotoxicity of Aβ oligomers that form Ca(2+) permeable pores in the membranes of brain cells. By triggering an intracellular Ca(2+) overdose, Aβ oligomers induce a cascade of neurotoxic events including oxidative stress, tau hyperphosphorylation, and neuronal loss. Targeting any post‐Ca(2+) entry steps (e.g., tau) will not address the root cause of the disease. Thus, preventing Aβ oligomers formation and/or blocking their toxicity is by essence the best approach to stop any progression of AD. Three categories of anti‐oligomer compounds are already available: antibodies, synthetic peptides, and small drugs. Independent in silico‐based designs of a peptide (AmyP53) and a monoclonal antibody (PMN310) converged to identify a histidine motif (H13/H14) that is critical for oligomer neutralization. This “histidine trick” can be viewed as the Achilles' heel of Aβ in the fight against AD. Moreover, lipid rafts and especially gangliosides play a critical role in the formation and toxicity of Aβ oligomers. Recognizing AD as a membrane disorder and gangliosides as the key anti‐oligomer targets will provide innovative opportunities to find an efficient cure. A “full efficient” solution would also need to be affordable to anyone, as the number of patients has been following an exponential increase, affecting every part of the globe.

Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways.

International audience; Growing evidence supports a role for brain gangliosides in the ă pathogenesis of neurodegenerative diseases including Alzheimer's and ă Parkinson's. Recently we deciphered the ganglioside-recognition code ă controlling specific ganglioside binding to Alzheimer's beta-amyloid (A ă beta 1-42) peptide and Parkinson's disease-associated protein ă alpha-synuclein. Cracking this code allowed us to engineer a short ă chimeric A beta/alpha-synuclein peptide that recognizes all brain ă gangliosides. Here we show that ganglioside-deprived neural cells do no ă longer sustain the formation of zinc-sensitive amyloid pore channels ă induced by either A beta 1-42 or alpha-synuclein, as assessed by ă single-cell Ca2+ fluorescence microscopy. Thus, amyloid channel ă formation, now considered a key step in neurodegeneration, is a ă ganglioside-dependent process. Nanomolar concentrations of chimeric ă peptide competitively inhibited amyloid pore formation induced by A beta ă 1-42 or alpha-synudein in cultured neural cells. Moreover, this peptide ă abrogated the intracellular calcium increases induced by ă Parkinson's-associated mutant forms of alpha-synuclein (A30P, E46K and ă A53T). The chimeric peptide also prevented the deleterious effects of A ă beta 1-42 on synaptic vesicle trafficking and decreased the A beta ă 1-42-induced impairment of spontaneous activity in rat hippocampal ă slices. Taken together, these data show that the chimeric peptide has ă broad anti-amyloid pore activity, suggesting that a common therapeutic ă strategy based on the prevention of amyloid-ganglioside interactions is ă a reachable goal for both Alzheimer's and Parkinson's diseases. (C) 2015 ă Elsevier B.V. All rights reserved.

Anandamide is a lipid neurotransmitter derived from arachidonic acid, a polyunsaturated fatty acid. The chemical differences between anandamide and arachidonic acid result in a slightly enhanced solubility in water and absence of an ionisable group for the neurotransmitter compared with the fatty acid. In this review, we first analyze the conformational flexibility of anandamide in aqueous and membrane phases. We next study the interaction of the neurotransmitter with membrane lipids and discuss the molecular basis of the unexpected selectivity of anandamide for cholesterol and ceramide from among other membrane lipids. We show that cholesterol behaves as a binding partner for anandamide, and that following an initial interaction mediated by the establishment of a hydrogen bond, anandamide is attracted towards the membrane interior, where it forms a molecular complex with cholesterol after a functional conformation adaptation to the apolar membrane milieu. The complex is then directed to the anandamide cannabinoid receptor (CB1) which displays a high affinity binding pocket for anandamide. We propose that cholesterol may regulate the entry and exit of anandamide in and out of CB1 by interacting with low affinity cholesterol recognition sites (CARC and CRAC) located in transmembrane helices. The mirror topology of cholesterol binding sites in the seventh transmembrane domain is consistent with the delivery, extraction and flip-flop of anandamide through a coordinated cholesterol-dependent mechanism. The binding of anandamide to ceramide illustrates another key function of membrane lipids which may occur independently of protein receptors. Interestingly, ceramide forms a tight complex with anandamide which blocks the degradation pathway of both lipids and could be exploited for anti-cancer therapies. Fil: Di Scala, Coralie. Institut de Neurobiologiede la Mediterranee; Francia Fil: Fantini, Jacques. Aix VMarseille Université; Francia Fil: Yahi, Nouara. Aix VMarseille Université; Francia Fil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Chahinian, Henri. Aix VMarseille Université; Francia

Alzheimer β-amyloid (Aβ) peptides can self-organize into oligomeric ion channels with high neurotoxicity potential. Cholesterol is believed to play a key role in this process, but the molecular mechanisms linking cholesterol and amyloid channel formation have so far remained elusive. Here, we show that the short Aβ22-35 peptide, which encompasses the cholesterol-binding domain of Aβ, induces a specific increase of Ca(2+) levels in neural cells. This effect is neither observed in calcium-free medium nor in cholesterol-depleted cells, and is inhibited by zinc, a blocker of amyloid channel activity. Double mutations V24G/K28G and N27R/K28R in Aβ22-35 modify cholesterol binding and abrogate channel formation. Molecular dynamic simulations suggest that cholesterol induces a tilted α-helical topology of Aβ22-35. This facilitates the establishment of an inter-peptide hydrogen bond network involving Asn-27 and Lys-28, a key step in the octamerization of Aβ22-35 which proceeds gradually until the formation of a perfect annular channel in a phosphatidylcholine membrane. Overall, these data give mechanistic insights into the role of cholesterol in amyloid channel formation, opening up new therapeutic options for Alzheimer's disease. Aβ22-35 peptide, which encompasses the cholesterol binding domain of Aβ, induces a specific increase of Ca(2+) level in neural cells. Double mutations V24G/K28G and N27R/K28R modify cholesterol binding and abrogate channels formation. Molecular dynamic simulations suggest that cholesterol induces a tilted α-helical peptide topology facilitating the formation of annular octameric channels, as schematically shown in the graphic (with a hydrogen bond shown in green for two vicinal peptides). Overall, the data give insights into the role of cholesterol in amyloid channel formation and open up new therapeutic options for Alzheimer's disease.

Gastric lipase is active under acidic conditions and shows optimum activity on insoluble triglycerides at pH 4. The present results show that gastric lipase also acts in solution on vinyl butyrate, with an optimum activity above pH 7, which suggests that gastric lipase is able to hydrolyze ester bonds via the classical mechanism of serine hydrolases. These results support previous structural studies in which the catalytic triad of gastric lipase was reported to show no specific features. The optimum activity of gastric lipase shifted toward lower pH values, however, when the vinyl butyrate concentration was greater than the solubility limit. Experiments performed with long-chain triglycerides showed that gastric lipase binds optimally to the oil-water interface at low pH values. To study the effects of the pH on the adsorption step independently from substrate hydrolysis, gastric lipase adsorption on solid hydrophobic surfaces was monitored by total internal reflection fluorescence (TIRF), as well as using a quartz crystal microbalance. Both techniques showed a pH-dependent reversible gastric lipase adsorption process, which was optimum at pH 5 (Kd = 6.5 nM). Lipase adsorption and desorption constants (ka = 147,860 M(-1) s(-1) and kd = 139 x 10(-4) s(-1) at pH 6) were estimated from TIRF experiments. These results indicate that the optimum activity of gastric lipase at acidic pH is only "apparent" and results from the fact that lipase adsorption at lipid-water interfaces is the pH-dependent limiting step in the overall process of insoluble substrate hydrolysis. This specific kinetic feature of interfacial enzymology should be taken into account when studying any soluble enzyme acting on an insoluble substrate.

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245–1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.

Hormone-sensitive lipase (HSL) is thought to contribute importantly to the mobilization of fatty acids from the triacylglycerols (TAGs) stored in adipocytes, providing the main source of energy in mammals. To investigate the HSL substrate specificity more closely, we systematically assessed the lipolytic activity of recombinant human HSL on solutions and emulsions of various vinyl esters and TAG substrates, using the pH-stat assay technique. Recombinant human HSL activity on solutions of partly soluble vinyl esters or TAG was found to range from 35 to 90% of the maximum activity measured with the same substrates in the emulsified state. The possible existence of a lipid-water interface due to the formation of small aggregates of vinyl esters or TAG in solution may account for the HSL activity observed below the solubility limit of the substrate. Recombinant human HSL also hydrolyzes insoluble medium- and long-chain acylglycerols such as trioctanoylglycerol, dioleoylglycerol, and olive oil, and can therefore be classified as a true lipase. Preincubation of the recombinant HSL with a serine esterase inhibitor such as diethyl p-nitrophenyl phosphate in 1:100 molar excess leads to complete HSL inhibition within 15 min. This result indicates that the catalytic serine of HSL is highly reactive and that it is readily accessible. Similar behavior was also observed with lipases with no lid domain covering their active site, or with a deletion in the lid domain. The 3-D structure of HSL, which still remains to be determined, may therefore lack the lid domain known to exist in various other lipases.

Human pancreatic lipase (HPL) consists of two functional domains: an N-terminal catalytic domain (N-HPL), and a β-sandwich C-terminal domain (C-HPL) involved in the binding process between HPL and colipase. Structural similarities between C-HPL and C2 domains have suggested another function in lipase-lipid interactions. C2 domains occur in a wide range of proteins involved in phospholipid interactions, in signal transduction (phosphoinositide-specific phospholipase C, protein kinase C, cytosolic phospholipase A2), membrane traffic (synaptotagmin I, rabphilin), and membrane disruption (perforin). In this paper, we review experiments supporting an absolute requirement of C-HPL for interfacial binding of HPL. N-HPL and C-HPL were produced as individual polypeptides, and their partitioning between the water phase and a lipid interface was assessed using trioctanoin emulsions or the monomolecular film technique. Using monoclonal antibodies and site-directed mutagenesis, the contribution to lipase-lipid interactions of an exposed hydrophobic loop (β5') present in C-HPL was investigated. The β5'loop is located on the same side of HPL as the hydrophobic loops surrounding the active site, and has the same topology as the CBR3 loop found in C2 domains. Based on structural homology between C-HPL and C2 domains, a model of HPL orientation at a lipid interface is proposed.

Brain cholesterol plays a critical role in Alzheimer's disease and other neurodegenerative diseases. The molecular mechanisms linking cholesterol to neurotoxicity have remained elusive for a long time, but recent data have allowed the identification of functional cholesterol-binding domains in several amyloidogenic proteins involved in neurodegenerative diseases, including Alzheimer's disease. In this review, we analyze the cholesterol binding properties of β-amyloid (Aβ) peptides and the impact of these interactions on amyloid pore formation. We show that although the cholesterol-binding domains of Aβ peptides and of transmembrane precursor C99 are partially overlapping, they involve distinct amino acid residues, so that cholesterol has a greater affinity for Aβ than for C99. Synthetic 22-35 and 25-35 fragments of Aβ retained the ability of the full-length peptide 1-42 to bind cholesterol and to form zinc-sensitive, calcium-permeable amyloid pores in cultured neural cells. Studies with mutant peptides allowed the identification of key residues involved in cholesterol binding and channel formation. Cholesterol promoted the insertion of Aβ in the plasma membrane, induced α-helical structuration, and forced the peptide to adopt a tilted topology that favored the oligomerization process. Bexarotene, an amphipathic drug currently considered as a potential candidate medication for the treatment of neurodegenerative diseases, competed with cholesterol for binding to Aβ and prevented oligomeric channel formation. These studies indicate that it is possible to prevent the generation of neurotoxic oligomers by targeting the cholesterol-binding domain of Aβ peptides. This original strategy could be used for the treatment of Alzheimer's and other neurodegenerative diseases that involve cholesterol-dependent toxic oligomers.

The anticancer drug bexarotene has been shown to restore cognitive functions in animal models of Alzheimer's disease, but its exact mechanism of action remains elusive. In the present report, we have used a combination of molecular, physicochemical, and cellular approaches to elucidate the mechanisms underlying the anti-Alzheimer properties of bexarotene in neural cells. First of all, we noticed that bexarotene shares a structural analogy with cholesterol. We showed that cholesterol and bexarotene compete for the same binding site in the C-terminal region of Alzheimer's β-amyloid peptide 1-42 (Aβ1-42). This common bexarotene/cholesterol binding domain was characterized as a linear motif encompassing amino acid residues 25-35 of Aβ1-42. Because cholesterol is involved in the oligomerization of Alzheimer's β-amyloid peptides into neurotoxic amyloid channels, we studied the capability of bexarotene to interfere with this process. We showed that nanomolar concentrations of bexarotene efficiently prevented the cholesterol-dependent increase of calcium fluxes induced by β-amyloid peptides Aβ1-42 and Aβ25-35 in SH-SY5Y cells, suggesting a direct effect of the drug on amyloid channel formation. Molecular dynamics simulations gave structural insights into the role of cholesterol in amyloid channel formation and explained the inhibitory effect of bexarotene. Because it is the first drug that can both inhibit the binding of cholesterol to β-amyloid peptides and prevent calcium-permeable amyloid pore formation in the plasma membrane of neural cells, bexarotene might be considered as the prototype of a new class of anti-Alzheimer compounds. The experimental approach developed herein can be used as a screening strategy to identify such compounds.

Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.

An extracellular lipase, active on water-insoluble triacylglycerols, has been isolated from Penicillium cyclopium. The purified enzyme has a molecular mass of 29 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis. It hydrolyzes emulsions of tributyrin, trioctanoin, and olive oil at the same rate as pancreatic lipase and shows very low activity against partial acylglycerols (monooctanoin and dioctanoin) and methyl esters. It is stable at 35 degrees C for 60 min and has maximal activity in a pH range of 8-10. Hydrolysis of triacylglycerols by P. cyclopium lipase is inhibited by detergents such as Triton X-100. Comparison of the sequence of the 20 first amino acid residues of P. cyclopium triacylglycerol lipase with other Penicillium lipases indicates a high homology with previously characterized lipases produced by P. expansum and P. solitum which are enzymes of comparable size and substrate specificity. Conversely, homology between P. cyclopium lipase and P. simplicissimum lipase, a nonspecific lipolytic enzyme, is low. Penicillium cyclopium triacylglycerol lipase shows no homology with P. camembertii lipase which is specific to monoacylglycerol and diacylglycerol.

Alzheimer's β-amyloid (Aβ) peptides can self-organize into amyloid pores that may induce acute neurotoxic effects in brain cells. Membrane cholesterol, which regulates Aβ production and oligomerization, plays a key role in this process. Although several data suggested that cholesterol could bind to Aβ peptides, the molecular mechanisms underlying cholesterol/Aβ interactions are mostly unknown. On the basis of docking studies, we identified the linear fragment 22-35 of Aβ as a potential cholesterol-binding domain. This domain consists of an atypical concatenation of polar/apolar amino acid residues that was not previously found in cholesterol-binding motifs. Using the Langmuir film balance technique, we showed that synthetic peptides Aβ17-40 and Aβ22-35, but not Aβ1-16, could efficiently penetrate into cholesterol monolayers. The interaction between Aβ22-35 and cholesterol was fully saturable and lipid-specific. Single-point mutations of Val-24 and Lys-28 in Aβ22-35 prevented cholesterol binding, whereas mutations at residues 29, 33, and 34 had little to no effect. These data were consistent with the in silico identification of Val-24 and Lys-28 as critical residues for cholesterol binding. We conclude that the linear fragment 22-35 of Aβ is a functional cholesterol-binding domain that could promote the insertion of β-amyloid peptides or amyloid pore formation in cholesterol-rich membrane domains.

International audience; Alpha-synuclein (alpha-syn) is an amyloidogenic protein that plays a key role in the pathogenesis of Parkinson's disease (PD). The ability of alpha-syn oligomers to form ionic channels is postulated as a channelopathy mechanism in human brain. Here we identified a ganglioside-binding domain in alpha-syn (fragment 34-50), which includes the mutation site 46 linked to a familial form of PD (E46K). We show that this fragment is structurally related to the common glycosphingolipid-binding domain (GBD) shared by various microbial and amyloid proteins, including Alzheimer's beta-amyloid peptide. alpha-Syn GBD interacts with several glycosphingolipids but has a marked preference for GM3, a minor brain ganglioside whose expression increases with aging. The alpha-syn mutant E46K has a stronger affinity for GM3 than the wild-type protein, and the interaction is inhibited by 3'-sialyllactose (the glycone part of GM3). Alanine substitutions of Lys34 and Tyr39 in synthetic GBD peptides resulted in limited interaction with GM3, demonstrating the critical role of these residues in GM3 recognition. When incubated with reconstituted phosphatidylcholine bilayers, the E46K protein formed channels that are five times less conductive than those formed by wild-type alpha-syn, exhibit a higher selectivity for cations, and present an asymmetrical response to voltage and nonstop single-channel activity. This E46K-associated channelopathy was no longer observed when GM3 was present in phosphatidylcholine bilayers. This corrective effect was highly specific for GM3, since it was not obtained with the major brain ganglioside GM1 but was still detected in bilayer membranes containing both GM3 and GM1. Moreover, synthetic GBD peptides prevented the interaction of alpha-syn proteins with GM3, thus abolishing the regulatory effects of GM3 on alpha-syn-mediated channel formation. Overall, these data show that GM3 can specifically regulate alpha-syn-induced channel formation and raise the intriguing possibility that this minor brain ganglioside could play a key protective role in the pathogenesis of PD.

International audience; To differentiate esterases from lipases at the structure-function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116-123 in bBAL is deleted in rLE This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase-lipid adsorption complex at Interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site. (C) 2010 Elsevier B.V All rights reserved

International audience; Background: Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide.Methodology/Principal Findings: Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide.Conclusions/Significance: Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could be an important component of the anandamide transport machinery. Finally, this study provides a mechanistic explanation for the key regulatory activity played by membrane cholesterol in the responsiveness of cells to anandamide.

THL is a potent inhibitor of pancreatic (PPL) and gastric (HGL, RGL) lipases. Inactivation occurs preferentially at the oil/water interface (method B, C). In the aqueous phase (method A), the inhibition of HGL was accelerated by the presence of bile salts. C12:0-TNB, a disulfide reagent, specifically inactivates gastric lipases and had no effect on the pancreatic lipase (in the presence of bile salts) whatever the method used. The capacity of THL and C12:0-TNB to inactivate lipases using Methods B and C was found to depend directly upon the interfacial area of the system used. Consequently, inactivation can be reduced or prevented by further addition of a water-insoluble substrate which reduces the surface density of inactivator molecules. With a heterogeneous system of this kind, typical of lipolysis, the use of a classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (Km, KI, Vmax) are only apparent values.