Nicolas Garmy

Identification of a common sphingolipid-binding domain in Alzheimer, prion, and HIV-1 proteins.

The V3 loop of the human immunodeficiency virus (HIV)-1 surface envelope glycoprotein gp120 is a sphingolipid-binding domain mediating the attachment of HIV-1 to plasma membrane microdomains (rafts). Sphingolipid-induced conformational changes in gp120 are required for HIV-1 fusion. Galactosylceramide and sphingomyelin have been detected in highly purified preparations of prion rods, suggesting that the prion protein (PrP) may interact with selected sphingolipids. Moreover, a major conformational transition of the Alzheimer β-amyloid peptide has been observed upon interaction with sphingolipid-containing membranes. Structure similarity searches with the combinatorial extension method revealed the presence of a V3-like domain in the human prion protein PrP and in the Alzheimer β-amyloid peptide. In each case, synthetic peptides derived from the predicted V3-like domain were found to interact with monomolecular films of galactosylceramide and sphingomyelin at the air-water interface. The V3-like domain of PrP is a disulfide-linked loop (Cys179–Cys214) that includes the E200K mutation site associated with familial Creutzfeldt-Jakob disease. This mutation abrogated sphingomyelin recognition. The identification of a common sphingolipid-binding motif in gp120, PrP, and β-amyloid peptide underscores the role of lipid rafts in the pathogenesis of HIV-1, Alzheimer, and prion diseases and may provide new therapeutic strategies.

Interaction of Alzheimer’s β-Amyloid Peptides with Cholesterol: Mechanistic Insights into Amyloid Pore Formation

Brain cholesterol plays a critical role in Alzheimers 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 Alzheimers 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 Alzheimers and other neurodegenerative diseases that involve cholesterol-dependent toxic oligomers.

Cholesterol accelerates the binding of Alzheimer's β-amyloid peptide to ganglioside GM1 through a universal hydrogen-bond-dependent sterol tuning of glycolipid conformation

Age-related alterations of membrane lipids in brain cell membranes together with high blood cholesterol are considered as major risk factors for Alzheimers disease. Yet the molecular mechanisms by which these factors increase Alzheimers risk are mostly unknown. In lipid raft domains of the plasma membrane, neurotoxic Alzheimers beta-amyloid (Abeta) peptides interact with both cholesterol and ganglioside GM1. Recent data also suggested that cholesterol could stimulate the binding of Abeta to GM1 through conformational modulation of the ganglioside headgroup. Here we used a combination of physicochemical and molecular modeling approaches to decipher the mechanisms of cholesterol-assisted binding of Abeta to GM1. With the aim of decoupling the effect of cholesterol on GM1 from direct Abeta-cholesterol interactions, we designed a minimal peptide (Abeta5-16) containing the GM1-binding domain but lacking the amino acid residues involved in cholesterol recognition. Using the Langmuir technique, we showed that cholesterol (but not phosphatidylcholine or sphingomyelin) significantly accelerates the interaction of Abeta5-16 with GM1. Molecular dynamics simulations suggested that Abeta5-16 interacts with a cholesterol-stabilized dimer of GM1. The main structural effect of cholesterol is to establish a hydrogen-bond between its own OH group and the glycosidic-bond linking ceramide to the glycone part of GM1, thereby inducing a tilt in the glycolipid headgroup. This fine conformational tuning stabilizes the active conformation of the GM1 dimer whose headgroups, oriented in two opposite directions, form a chalice-shaped receptacle for Abeta. These data give new mechanistic insights into the stimulatory effect of cholesterol on Abeta/GM1 interactions. They also support the emerging concept that cholesterol is a universal modulator of protein-glycolipid interactions in the broader context of membrane recognition processes.

The virotoxin model of HIV-1 enteropathy: Involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line

BACKGROUND Malabsorption and diarrhea are common, serious problems in AIDS patients, and are in part due to the incompletely understood entity HIV enteropathy. Our prior in vitro work has shown that increased transepithelial permeability and glucose malabsorption, similar to HIV enteropathy, are caused by HIV surface protein gp120, although the mechanism remains unclear. RESULTS We studied the effects of HIV surface protein gp120 on the differentiated intestinal cell line HT-29-D4, specifically the effects on microtubules, transepithelial resistance, and sodium glucose cotransport. gp120 induced extensive microtubule depolymerization, an 80% decrease in transepithelial resistance, and a 70% decrease in sodium-dependent glucose transport, changes closely paralleling those of HIV enteropathy. The effects on transepithelial resistance were used to study potential inhibitors. Neutralizing antibodies to GPR15/Bob but not to CXCR4 (the coreceptor allowing infection with these HIV strains) inhibited these effects. Antibodies to galactosylceramide (GalCer) and a synthetic analog of GalCer also inhibited the gp120-induced changes, suggesting the involvement of GalCer-enriched lipid rafts in gp120 binding to intestinal epithelial cells. CONCLUSION We conclude that direct HIV infection and gp120-induced cytopathic effects are distinct phenomena. While in vivo confirmation is needed to prove this, gp120 could be a virotoxin significantly contributing to HIV enteropathy.

Biochemical identification of a linear cholesterol-binding domain within Alzheimer's β amyloid peptide.

Alzheimers 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.

The minimal amyloid-forming fragment of the islet amyloid polypeptide is a glycolipid-binding domain

Several proteins that interact with cell surface glycolipids share a common fold with a solvent‐exposed aromatic residue that stacks onto a sugar ring of the glycolipid (CH–π stacking interaction). Stacking interactions between aromatic residues (π–π stacking) also play a pivotal role in the assembly process, including many cases of amyloid fibril formation. We found a structural similarity between a typical glycolipid‐binding domain (the V3 loop of HIV‐1 gp120) and the minimal amyloid‐forming fragment of the human islet amyloid polypeptide, i.e. the octapeptide core module NFGAILSS. In a monolayer assay at the air–water interface, the NFGAILSS peptide specifically interacted with the glycolipid lactosylceramide. The interaction appears to require an aromatic residue, as NLGAILSS was poorly recognized by lactosylceramide, whereas NYGAILSS behaved like NFGAILSS. In addition, we observed that the full‐length human islet amyloid polypeptide (1–37) did interact with a monolayer of lactosylceramide, and that the glycolipid film significantly affected the aggregation process of the peptide. As glycolipid–V3 interactions are efficiently inhibited by suramin, a polyaromatic compound, we investigated the effects of suramin on amyloid formation by human islet amyloid polypeptide. We found that suramin inhibited amyloid fibril formation at low concentrations, but dramatically stimulated the process at high concentrations. Taken togther, our results indicate that the minimal amyloid‐forming fragment of human islet amyloid polypeptide is a glycolipid‐binding domain, and provide further experimental support for the role of aromatic π–π and CH–π stacking interactions in the molecular control of the amyloidogenesis process.

Bexarotene Blocks Calcium-Permeable Ion Channels Formed by Neurotoxic Alzheimer’s β-Amyloid Peptides

The anticancer drug bexarotene has been shown to restore cognitive functions in animal models of Alzheimers 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 Alzheimers 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 Alzheimers 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.

Cellular isoform of the prion protein PrPc in human intestinal cell lines: genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts.

The cellular isoform of the normal prion protein PrPc, encoded by the PRNP gene, is expressed in human intestinal epithelial cells where it may represent a potential target for infectious prions. We have sequenced the PRNP gene in Caco‐2 and HT‐29 parental and clonal cell lines, and found that these cells have a distinct polymorphism at codon 129. HT‐29 cells are homozygous Met/Met, whereas Caco‐2 cells are heterozygous Met/Val. The 129Val variant was also detected in Caco‐2 mRNAs. Real‐time PCR quantifications revealed that PrPc mRNAs were more expressed in HT‐29 cells than in Caco‐2 cells. These data were confirmed by studying the expression of PrPc in plasma membranes and lipid rafts prepared from these cells. Overall, these results may be important in view of using human intestinal cell lines Caco‐2 and HT‐29 as cellular in vitro models to study the initial steps of prion propagation after oral inoculation.

Erratum to: Cellular isoform of the prion protein PrPc in human intestinalcell lines: Genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts [30 (6) 559–567]

In the above article page 563, line 24 an error was made in the text and it should have read as below: We state that ‘the enterocytic differentiation of HT-29-D4 cells was associated with a significant decrease (7.61 fold) of prominin mRNA abundance’. One should read ‘increase’ instead of ‘decrease’. Indeed, from the data shown in Table 2, one can check that the relative abundance of prominin mRNAs in differentiated vs. undifferentiated HT-29-D4 cells is 0.16 and 0.021, respectively. Thus, differentiated HT-29-D4 Gal cells actually express 7.61 more prominin mRNAs than their undifferentiated HT-29-D4 Glc counterparts (0.16 divided by 0.021). This does not change anything to the message of our article which is focused on the expression of PrP in intestinal cell lines.