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A molecular model was built for human lecithin:cholesterol acyltransferase (LCAT) based upon the structural homology between this enzyme and lipases (Peelman et al. 1998. Prot. Sci. 7: 585–597). We proposed that LCAT belongs to the α/β hydrolase fold family, and that the central domain of LCAT consists of a mixed seven-stranded β-pleated sheet with four α-helices and loops linking the β-strands. The catalytic triad of LCAT was identified as Asp345 and His377, as well as Ser181. This model is used here for the interpretation of the structural defects linked to the point mutations identified in LCAT, which cause either familial LCAT deficiency (FLD) or fish-eye disease (FED). We show that these mutations occur in separate domains of the 3D structure of the enzyme. Most mutations causing familial LCAT deficiency are either clustered in the vicinity of the catalytic triad or affect conserved structural elements in LCAT. Most mutations causing fish-eye disease are localized on the outer hydrophilic surface of the amphipathic helical segments. These mutations affect only minimally the overall structure of the enzyme, but are likely to impair the interaction of the enzyme with its co-factor and/or substrate.—Peelman, F., J-L. Verschelde, B. Vanloo, C. Ampe, C. Labeur, J. Tavernier, J. Vanderkerckhove, and M. Rosseneu. Effects of natural mutations in lecithin:cholesterol acyltransferase on the enzyme structure and activity. J. Lipid Res. 1999. 40: 59–69.

The structure, composition, and physico-chemical properties of lipid-protein complexes generated between dimyristoylphosphatidylcholine (DPMC) and the CNBr fragments of human apoA-I were studied. The fragments were separated by high performance liquid chromatography and purified on a reversed-phase column. The complexes with DMPC were isolated on a Superose column; their dimensions were obtained by gradient gel electrophoresis and by electron microscopy. The secondary structure of the protein in the complexes was studied both by circular dichroism and by attenuated total reflection infrared spectroscopy. The fragments 1 and 4 of apoA-I, containing, respectively, two and three amphipathic helices, recombined with the phospholipid to generate discoidal particles with sizes similar to that of apoA-I- and apoA-II-DMPC complexes. The infrared measurements indicated that in all complexes the apolipoprotein helical segments were oriented parallel to the phospholipid acyl chains and that the protein was located around the edges of the discs. Computer modelling of the complexes based on energy minimization techniques proposed a model for these particles in agreement with the dimensions measured experimentally. In conclusion, we propose that apoA-I and its longest CNBr fragments are able to generate discoidal particles with DMPC, with apolipoprotein helical segments oriented parallel to the acyl chains of the phospholipids.

Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp)2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds.-Bertier, L., Boucherie, C., Zwaenepoel, O., Vanloo, B., Van Troys, M., Van Audenhove, I., Gettemans, J. Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion., (© FASEB.)

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.

Introduction: Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit., Methods: We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading., Results: With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS)., Conclusions: CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.

Nanobodies or VHHs are single domain antigen binding fragments derived from heavy-chain antibodies naturally occurring in species of the Camelidae. Due to their ease of cloning, high solubility and intrinsic stability, they can be produced at low cost. Their small size, combined with high affinity and antigen specificity, enables recognition of a broad range of structural (undruggable) proteins and enzymes alike. Focusing on two actin binding proteins, gelsolin and CapG, we summarize a general protocol for the generation, cloning and production of nanobodies. Furthermore, we describe multiple ways to characterize antigen-nanobody binding in more detail and we shed light on some applications with recombinant nanobodies. The use of nanobodies as intrabodies is clarified through several case studies revealing new cytoskeletal protein properties and testifying to the utility of nanobodies as intracellular bona fide protein inhibitors. Moreover, as nanobodies can traverse the plasma membrane of eukaryotic cells by means of the enteropathogenic E. coli type III protein secretion system, we show that in this promising way of nanobody delivery, actin pedestal formation can be affected following nanobody injection., (Copyright © 2013 Wiley Periodicals, Inc.)

The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus. We reported previously that this motif was necessary for YAP2 localization in the nucleus and for promoting cell detachment and apoptosis. In the present study, we show that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2. The endogenous ZO-2 and YAP2 proteins co-localize in the nucleus. We also found that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP2, and that this activity of ZO-2 is PDZ-domain-dependent. The present paper is the first report on a PDZ-based nuclear translocation mechanism. Moreover, since the Hippo pathway acts as a tumour suppressor pathway, the YAP2-ZO-2 complex could represent a target for cancer therapy.

L-plastin, a conserved modular F-actin bundling protein, is ectopically expressed in tumor cells and contributes to cell malignancy and invasion. The underlying molecular mechanisms involved remain unclear, in part, because specific inhibitors of L-plastin are lacking. We used recombinant alpaca-derived L-plastin single-domain antibodies (nanobodies) as effector of L-plastin function in cells.

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P (2)) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.

We showed earlier that human beta-thymosin 15 (Tb15) is up-regulated in prostate cancer, confirming studies from others that propagated Tb15 as a prostate cancer biomarker. In this first report on mouse Tb15, we show that, unlike in humans, four Tb15-like isoforms are present in mouse. We used phylogenetic analysis of deuterostome beta-thymosins to show that these four new isoforms cluster within the vertebrate Tb15-clade. Intriguingly, one of these mouse beta-thymosins, Tb15r, consists of two beta-thymosin domains. The existence of such a repeat beta-thymosin is so far unique in vertebrates, though common in lower eukaryotes. Biochemical data indicate that Tb15r potently sequesters actin. In a cellular context, Tb15r behaves as a bona fide beta-thymosin, lowering central stress fibre content. We reveal that a complex genomic organization underlies Tb15r expression: Tb15r results from read-through transcription and alternative splicing of two tandem duplicated mouse Tb15 genes. Transcript profiling of all mouse beta-thymosin isoforms (Tb15s, Tb4 and Tb10) reveals that two isoform switches occur between embryonic and adult tissues, and indicates Tb15r as the major mouse Tb15 isoform in adult cells. Tb15r is present also in mouse prostate cancer cell lines. This insight into the mouse Tb15 family is fundamental for future studies on Tb15 in mouse (prostate) cancer models.

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG. In digitonin-permeabilized cells, neither GTPgammaS nor the GTP hydrolysis-deficient Ran mutant RanQ69L affect transit of CapG to the nucleus in the presence of cytosol. Obstruction of nucleoporins prevents nuclear transport of CapG, and we show that CapG binds to nucleoporin62. In addition, CapG interacts with NTF2, associates with Ran and is furthermore able to bind the NTF2-Ran complex. NTF2-Ran interaction is required for CapG nuclear import. This is corroborated by a NTF2 mutant with reduced affinity for Ran and a Ran mutant that does not bind NTF2, both of which prevent CapG import. Thus, a ubiquitously expressed protein shuttles to the nucleus through direct association with NTF2 and Ran. The role of NTF2 may therefore not be solely confined to sustaining the Ran gradient in cells.

Syntenin is a tandem PDZ protein that has recently been shown to be overexpressed in several cancer cells and tissues, and that might play an active role in tumor cell invasion and metastasis. Here we show that overexpression of the tandem PDZ domains of syntenin in non-invasive cells is necessary and sufficient to stimulate these cells to invade a collagen I matrix, and this effect can be regulated by ligand binding to the PDZ domains. Furthermore, we show that syntenin-induced invasion requires signaling through ras, rho and PI3K/MAPK signaling pathways and involves changes in cell-cell adhesion. Inversely, when we used RNA interference to inhibit syntenin expression in different invasive cancer cell lines, we observed a drastically decreased ability of these cells to migrate and invade into collagen type I or Matrigel. RNAi-treated cells also show increased cell aggregation, indicating that syntenin is important for cell-cell adhesion in epithelial cells. Together, these results suggest that downregulation of syntenin by RNA interference could provide a means of inhibiting tumor invasion and possibly metastasis in different cancers, and point to syntenin as a potential cancer biomarker and drug target.

Lysophosphatidic acid is a pleiotropic lipid signaling molecule that evokes a broad array of cellular responses including proliferation, tumor cell invasion, neurite retraction, cytoskeletal rearrangements and smooth muscle contraction. Generally, lysophosphatidic acid triggers physiological responses through interaction with specific plasma membrane receptors called LPA 1-4. There is, however, increasing evidence in support of intracellular proteins that interact with LPA. We employed Affigel-immobilized LPA to isolate cytoplasmic proteins that interact with this lysophospholipid. Among the proteins retained by this affinity matrix, pyruvate kinase, clathrin heavy chain and heat shock protein 70 (Hsp70) were identified by mass spectrometry. Isothermal titration calorimetry showed that pyruvate kinase contains one binding site for LPA (Ka approx. 10(6) M(-1)). Furthermore, LPA dissociates enzymatically active pyruvate-kinase tetramers into less active dimers, and is maximally active at concentrations close to its critical micelle concentration. These effects were not mimicked by other lysophospholipids. Co-immunoprecipitation experiments showed that pyruvate kinase interacts with clathrin, and confocal imaging revealed co-localization between clathrin and pyruvate kinase in the perinuclear region of cells. Our data suggest that pyruvate kinase partly exists in complex with clathrin in subcellular membranous areas, and that locally increased LPA levels can trigger inactivation of the metabolic enzyme.

Cationic polymers, such as poly-l-lysine (pLL) and polyethyleneimine (pEI), are receiving growing attention as vectors for gene therapy. They form polyelectrolyte complexes with DNA, resulting in a reduced size of the DNA and an enhanced stability toward nucleases. The major disadvantages of using both polymers for in vivo purposes are their cytotoxicity and, in the case of pEI, the fact that it's not biodegradable. In this work, we investigated the interaction between a series of cationic, glutamic acid based polymers and red blood cells. The MTT test was used to investigate the cytotoxicity of the complexes. The ability of the polymers to stabilize DNA toward nucleases was investigated. Transfection studies were carried out on Cos-1 cells. The results from the haemolysis studies, the haemagglutination studies, and the MTT assay show that the polymers are substantially less toxic than pLL and pEI. The polymers are able to protect the DNA from digestion by DNase I. The transfection studies show that the polymer-DNA complexes are capable of transfecting cells, most of them with poor efficiency compared to pEI-DNA complexes.

We report here the physicochemical and biological evaluation of a series of polymethacrylates with side groups of different pK(a) values, such as tertiary amines, pyridine groups, acid functions and imidazole groups as synthetic vectors for gene delivery. The ability of the different polymers to condense DNA was studied by ethidium bromide exclusion tests and agarose gel electrophoresis. The results show that all polymers are able to condense DNA. Both the molecular weight and the chemical composition of the polymers have an influence on the DNA condensation process. Furthermore, the biological properties of the polymer-DNA complexes were investigated, including their haemolytic activity, cytotoxicity and in vitro transfection efficiency. Complexes based on polymers containing only tertiary amines, have a transfection efficiency similar to that of poly(ethyleneimine) (PEI). Polymers containing pyridine groups have a reduced transfection efficiency compared to polymers containing tertiary amines. Introduction of imidazole groups or acid functions results in a loss of the transfection efficiency of the corresponding complexes with DNA. In general, the viability of cells incubated with complexes based on the polymethacrylates is higher than with PEI. Polymers with high transfection efficiency induce erythrocyte lysis.

An alignment of the mammalian ABCA transporters enabled the identification of sequence segments, specific to the ABCA subfamily, which were used as queries to search for eukaryotic and prokaryotic homologues. Thirty-seven eukaryotic half and full-length transporters were found, and a close relationship with prokaryotic subfamily 7 transporters was detected. Each half of the ABCA full-transporters is predicted to comprise a membrane-spanning domain (MSD) composed of six helices and a large extracellular loop, followed by a nucleotide-binding domain (NBD) and a conserved cytoplasmic 80-residue sequence, which might have a regulatory function. The topology predicted for the ABCA transporters was compared to the crystal structures of the MsbA and BtuCD bacterial transporters. The alignment of the MSD and NBD domains provided an estimate of the degree of residue conservation in the cytoplasmic, extracellular and transmembrane domains of the ABCA transporter subfamily. The phylogenic tree of eukaryotic ABCA transporters based upon the NBD sequences, consists of three major clades, corresponding to the half-transporter single NBDs and to the full-transporter NBDls and NBD2s. A phylogenic tree of prokaryotic transporters and the eukaryotic ABCA transporters confirmed the evolutionary relationship between prokaryotic subfamily 7 transporters and eukaryotic ABCA half and full-transporters.

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.

Penetratin is a 16-amino-acid peptide, derived from the homeodomain of antennapedia, a Drosophila transcription factor, which can be used as a vector for the intracellular delivery of peptides or oligonucleotides. To study the relative importance of the Trp residues in the wild-type penetratin peptide (RQIKIWFQNRRMKWKK) two analogues, the W48F (RQIKIFFQNRRMKWKK) and the W56F (RQI KIWFQNRRMKFKK) variant peptides were synthesized. Binding of the three peptide variants to different lipid vesicles was investigated by fluorescence. Intrinsic Trp fluorescence emission showed a decrease in quantum yield and a blue shift of the maximal emission wavelength upon interaction of the peptides with negatively charged phosphatidylserine, while no changes were recorded with neutral phosphatidylcholine vesicles. Upon binding to phosphatidylcholine vesicles containing 20% (w/w) phosphatidylserine the fluorescence blue shift induced by the W56F-penetratin variant was larger than for the W48F-penetratin. Incorporation of cholesterol into the negatively charged lipid bilayer significantly decreased the binding affinity of the peptides. The Trp mean lifetime of the three peptides decreased upon binding to negatively charged phospholipids, and the Trp residues were shielded from acrylamide and iodide quenching. CD measurements indicated that the peptides are random in buffer, and become alpha helical upon association with negatively charged mixed phosphatidylcholine/phosphatidylserine vesicles, but not with phosphatidylcholine vesicles. These data show that wild-type penetratin and the two analogues interact with negatively charged phospholipids, and that this is accompanied by a conformational change from random to alpha helical structure, and a deeper insertion of W48 compared to W56, into the lipid bilayer.

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.

Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.

The microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) belong to the vitellogenin (VTG) family of lipid transfer proteins. MTP is essential for the intracellular assembly and secretion of apoB-containing lipoproteins, the key intravascular lipid transport proteins in vertebrates. We report the predicted three-dimensional structure of the C-terminal lipid binding cavity of MTP, modeled on the crystal structure of the lamprey VTG gene product, lipovitellin. The cavity in MTP resembles those found in the intracellular lipid-binding proteins and bactericidal/permeability-increasing protein. Two conserved helices, designated A and B, at the entrance to the MTP cavity mediate lipid acquisition and binding. Helix A (amino acids 725-736) interacts with membranes in a manner similar to viral fusion peptides. Mutation of helix A blocks the interaction of MTP with phospholipid vesicles containing triglyceride and impairs triglyceride binding. Mutations of helix B (amino acids 781-786) and of N780Y, which causes abetalipoproteinemia, have no impact on the interaction of MTP with phospholipid vesicles but impair triglyceride binding. We propose that insertion of helix A into lipid membranes is necessary for the acquisition of neutral lipids and that helix B is required for their transfer to the lipid binding cavity of MTP.

In this study, we investigated how the nature of the phospholipid head group and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin cholesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebutanoyl)-phosphatidylcholine, 1, 2-bis-(1-pyrenebutanoyl)-phosphatidylethanolamine and 1, 2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monomers or incorporated into small unilamellar vesicles consisting of dipalmitoylphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled phospholipids was determined both by fluorescence and by high performance liquid chromatography. V(max) and K(m) were calculated for the different substrates. The data show that V(max) is 10- to 30-fold higher for the hydrolysis of monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine (PE) and the phosphatidylalcohols, while K(m) values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholine ether vesicles, we observed a 4- to 10-fold increase of V(max) for PE and the phosphatidylalcohols, and no significant change for K(m). V(max) for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity on monomeric PC and PE. These data suggest that the activity of LCAT is determined both by the molecular structure of the phospholipid and by its macromolecular properties. The LCAT activity on monomeric substrates decreases as: phosphatidylcholine&z. Gt;phosphatidylethanolamine congruent withphosphatidylpropanol congruent withphosphatidylethanol congruent withphosphatidylethyleneglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospholipid head group.

In order to test the hypothesis that fish-eye disease (FED) is due to a deficient activation of lecithin:cholesterol acyltransferase (LCAT) by its co-factor apolipoprotein (apo) A-I, we overexpressed the natural mutants T123I, N131D, N391S, and other engineered mutants in Cos-1 cells. Esterase activity was measured on a monomeric phospholipid enelogue, phospholipase A(2) activity was measured on reconstituted high density lipoprotein (HDL), and acyltransferase activity was measured both on rHDL and on low density lipoprotein (LDL). The natural FED mutants have decreased phospholipase A(2) activity on rHDL, which accounts for the decreased acyltransferase activity previously reported. All mutants engineered at positions 131 and 391 had decreased esterase activity on a monomeric substrate and decreased acyltransferase activity on LDL. In contrast, mutations at position 123 preserved these activities and specifically decreased phospholipase A(2) and acyltransferase activites on rHDL. Mutations of hydrophilic residues in amphipathic helices alpha 3;-4 and alpha His to an alanine did not affect the mutants' activity on rHDL. Based upon the 3D model built for human LCAT, we designed a new mutant F382A, which had a biochemical phenotype similar to the natural T123I FED mutant. These data suggest that residues T123 and F382, located N-terminal of helices alpha 3-4 and alpha His, contribute specifically to the interaction of LCAT with HDL and possibly with its co-factor apoA-I. Residues N131 and N391 seem critical for the optimal orientation of the two amphipathic helices necessary for the recognition of a lipoprotein substrate by the enzyme.

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of action of fusogenic peptides, this hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion. To assess the role of the hydrophobicity gradient upon the fusogenic activity, two of these fusogenic peptides and several variants were synthesized. The LCAT-(57-70) peptide, which is part of the sequence of the lipolytic enzyme lecithin cholesterol acyltransferase, forms stable beta-sheets in lipids, while the apolipoprotein A-II (53-70) peptide remains predominantly helical in membranes. The variant peptides were designed through amino acid permutations, to be either parallel, perpendicular, or to retain an oblique orientation relative to the lipid-water interface. Peptide-induced vesicle fusion was monitored by lipid-mixing experiments, using fluorescent probes, the extent of peptide-lipid association, the conformation of lipid-associated peptides and their orientation in lipids, were studied by Fourier Transformed Infrared Spectroscopy. A comparison of the properties of the wild-type and variant peptides shows that the hydrophobicity gradient, which determines the orientation of helical peptides in lipids and their fusogenic activity, further influences the secondary structure and lipid binding capacity of these peptides.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system., (Copyright 1999 Academic Press.)

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues 50-74 form an interfacial recognition site and this hypothesis was tested by site-directed mutagenesis. The (delta56-68) deletion mutant had no activity on any substrate. Substitution of W61 with F, Y, L or G suggested that an aromatic residue is required for full enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid substrate. The decreased activity of the single R52A and K53A mutants on HDL and LDL and the severer effect of the double mutation suggested that these conserved residues contribute to the folding of the LCAT lid. The membrane-destabilizing properties of the LCAT 56-68 helical segment were demonstrated using the corresponding synthetic peptide. An M65N-N66M substitution decreased both the fusogenic properties of the peptide and the activity of the mutant enzyme on all substrates. These results suggest that the putative interfacial recognition domain of LCAT plays an important role in regulating the interaction of the enzyme with its organized lipoprotein substrates.

A molecular model was built for human lecithin:cholesterol acyltransferase (LCAT) based upon the structural homology between this enzyme and lipases (Peelman et al. 1998. Prot. Sci. 7: 585-597). We proposed that LCAT belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of a mixed seven-stranded beta-pleated sheet with four alpha-helices and loops linking the beta-strands. The catalytic triad of LCAT was identified as Asp345 and His377, as well as Ser181. This model is used here for the interpretation of the structural defects linked to the point mutations identified in LCAT, which cause either familial LCAT deficiency (FLD) or fish-eye disease (FED). We show that these mutations occur in separate domains of the 3D structure of the enzyme. Most mutations causing familial LCAT deficiency are either clustered in the vicinity of the catalytic triad or affect conserved structural elements in LCAT. Most mutations causing fish-eye disease are localized on the outer hydrophilic surface of the amphipathic helical segments. These mutations affect only minimally the overall structure of the enzyme, but are likely to impair the interaction of the enzyme with its co-factor and/or substrate.

This study investigates the suitability of the trimeric apolipoprotein (apo)AI(145-183) peptide that we recently described, to serve as a model to probe the relationship between apoAI structure and function. Three copies of the apoAI(145-183) unit, composed each of two amphipathic alpha-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102-140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)-apoAI complexes. The DMPC-trimeric-apoAI(145-183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149-186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145-183) bind specifically to HeLa cells with comparable affinity to the DMPC apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145-183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145-183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102-140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102-140) construct had low affinity for lipid compared with the (145-183) construct. After association with lipids, it was a poor competitor of DMPC-apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure-function relationship of apoAI.

A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167-171]. Due to an asymmetric distribution of hydrophobic residues along the axis of the alpha-helix, such peptides adopt an oblique orientation which can destabilise membranes or lipid cores. Variants of these oblique peptides, designed to have an homogeneous distribution of hydrophobic and hydrophilic residues along the helical axis, are classified as regular amphipathic peptides. These peptides are expected to lie parallel to the polar/apolar interface with their hydrophobic residues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was identified at residues 56-68 in the sequence of the lecithin-cholesterol acyltransferase (LCAT), enzyme. This peptide is predicted to penetrate a lipid bilayer at an angle of 40 degrees through its more hydrophobic C-terminal end and thereby induce the destabilisation of a membrane or a lipid core. The LCAT-(56-68) wild-type peptide was synthesised together with the LCAT-(56-68, 0 degrees) variant, in which the hydrophobicity gradient was abolished through residue permutations. In two other variants, designed to keep their oblique orientation, the W61 residue was shifted either towards the more hydrophilic N-terminal at residue 57, or to position 68 at the hydrophobic C-terminal end of the peptide. Peptide-induced vesicle fusion was demonstrated by fluorescence measurements using pyrene-labeled vesicles and by monitoring of vesicle size by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emission of the peptides. Fluorescence polarisation measurements, using diphenyl hexatriene, were carried out to follow changes in the lipid fluidity. The LCAT-(56-68) wild-type peptide and the two oblique variants, induced fusion of unilamellar dimyristoylglycerophosphocholine vesicles. Tryptophan fluorescence emission measurements showed a 12-14 nm blue shift upon addition of the wild-type peptide and of the W61-->68 variant to lipids, whereas the fluorescence of the W61-->57 variant did not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as predicted by the theoretical calculations. In contrast, the 0 degrees variant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid transition temperature was decreased after addition of the wild-type, the W61-->68 and W61-->57 fusogenic peptides, whereas the opposite effect was observed with the 0 degrees variant. The behaviour of the wild-type and variant LCAT-(56-68) peptides stresses the contribution of the hydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter consequently influences the structural modifications occurring to lipids upon association with amphipathic peptides.

A novel broad-spectrum antimicrobial peptide produced by vegetable-associated Enterococcus mundtii was purified and characterized, and designated mundticin. To our knowledge, this is the first report on bacteriocin production by this organism. The elucidation of the full primary amino acid sequence of mundticin (KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATGGAAGWSK) revealed that this antimicrobial peptide belongs to the class IIa bacteriocins of lactic acid bacteria which share a highly conserved N-terminal 'YGNGV' motif. Data obtained by computer modelling indicated an oblique orientation of the alpha-helical regions of mundticin and homologous class IIa bacteriocins at a hydrophobic-hydrophilic interface, which may play a role in the destabilization of phospholipid bilayers. The average mass of mundticin, as determined by electron spray mass spectrometry, was found to be 4287.21+/-0.59 Da. With respect to its biological activity, mundticin was shown to inhibit the growth of Listeria monocytogenes, Clostridium botulinum and a variety of lactic acid bacteria. Moreover, it was demonstrated to have a bactericidal effect on L. monocytogenes as a result of the dissipation of the membrane potential, and a loss of intracellular ATP in absence of ATP leakage. Its good solubility in water, and its stability over a wide pH and temperature range indicate the potential of this broad spectrum bacteriocin as a natural preservation agent for foods.

The displacement of apolipoprotein (apo) A-I by apo A-II is a major event in the remodeling of high density lipoproteins (HDL). In the present study, we investigated the displacement of apo A-I both from native and reconstituted HDL (rHDL) by either apo A-II or by the C-terminal helical peptide (i.e. residues 53-70). We studied the remodeling process of the original particles, the changes in size and composition and in their lecithin:cholesterol acyltransferase (LCAT) activating properties. Using gel filtration, we show that, at low apo A-II/AI ratios, the initial lipid apolipoprotein complex containing 2 mol apo A-I is remodeled into a mixed complex containing apo A-I and apo A-II, involving the displacement of one apo A-I by apo A-II. Upon addition of a larger amount of apo A-II, the rHDL particles become more heterogeneous and of larger size. Immunoblotting of the particles separated by non denaturing gradient gel electrophoresis shows that most of the apo A-I remains associated with the largest particles. The LCAT activation properties of the remodeled complexes decrease upon addition of either apo A-II or its C-terminal helix. This decrease is more pronounced when rHDL are incubated with the apo A-II C-terminal helix than with native apo A-II, as VmaX decreases from 28 to 16 and 7 nmol cholesteryl ester/ml per h respectively, whereas Km remains unchanged. The displacement of apo A-I observed with rHDL also occurred with native HDL particles as demonstrated by two-dimensional gel electrophoresis, using pyrene-phospholipid labeled HDL. Displacement of apo A-I generates pre-beta1 migrating particles containing apo A-I and phospholipids. We therefore propose that apo A-II has a dual effect on the role of HDL in reverse cholesterol transport: displacement of apo A-I from rHDL results in a negative control of the LCAT activity, while generation of pre-beta1 migrating particles enhances the formation of potential acceptors of cellular cholesterol.

To assess the functional properties of apolipoprotein (apo) AII and to investigate the mechanism leading to the displacement of apo AI from native and reconstituted high-density lipoproteins (HDL and r-HDL) by apo AII, wild-type and variant apo AII peptides were synthesized. The wild-type peptides, residues 53-70 and 58-70, correspond to the C-terminal helix of apo AII and are predicted to insert at a tilted angle into a lipid bilayer. We demonstrate that both the apo AII-(53-70) peptide, and to a lesser extent the apo AII-(58-70) peptide are able to induce fusion of unilamellar lipid vesicles together with membrane leakage, and to displace apo AI from HDL and r-HDL. Two variants of the apo AII-(53-70)-wild-type (WT) peptide, designed either to be parallel to the water/lipid interface [apo AII-(53-70)-0 degrees] or to retain an oblique orientation [apo AII-(53-70)-30 degrees], were synthesized in order to test the influence of the obliquity on their fusogenic properties and ability to displace apo AI from HDL. The parallel variant did not bind lipids, due to its self-association properties. However, the apo AII-(53-70)-30 degrees variant was fusogenic and promoted the displacement of apo AI from HDL. Moreover, the extent of fusion of the apo AII-(53-70)-WT, apo AII-(58-70)-WT and apo AII-(53-70)-30 degrees peptides was related to the alpha-helical content of the lipid-bound peptides measured by infrared spectroscopy. Infrared measurements using polarized light also confirmed the oblique orientation of the helical component of the three peptides. In native and r-HDL, the tilted insertion of the C-terminal helix of apo AII resulting in a partial destabilization of the HDL external lipid layer might contribute to the displacement of apo AI by apo AII.

The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three-dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta-strands connected by four alpha-helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site-directed mutagenesis for the localization of the active site residues. The wild-type enzyme and mutants were expressed in Cos-1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomeric substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential "lid" domain at residues 50-74 of LCAT might be involved in the enzyme-substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three-dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish-eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.

The prion protein (PrPC) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease in the human, where PrPC is converted into an altered form (termed PrPSc). PrPSc is highly resistant towards proteolytic degradation and accumulates in the central nervous system of affected individuals. By analogy with the pathological events occuring during the development of Alzheimer's disease, controverses still exist regarding the relationship between amyloidogenesis, prion aggregation and neuronal loss. To unravel the mechanism of PrP neurotoxicity and understand the interaction of PrP with cellular membranes, a series of natural and variant peptides spanning residues 118 to 135 of PrP was synthesized. The potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. According to computer modeling calculations, the 120 to 133 domain of PrP is predicted to be a tilted lipid-associating peptide, and to insert in a oblique way into a lipid bilayer through its N-terminal end. In addition to amyloidogenic properties exhibited in vitro by these peptides, peptide-induced vesicle fusion was demonstrated by several techniques, including lipid- and core-mixing assays. Elongation of the 120 to 133 peptide towards the N- and C-terminal ends of the PrP sequence showed that the 118 to 135 PrP peptide has maximal fusogenic properties, while the variant peptides had no effect. Due to their high hydrophobicity, all peptides tested were able to interact with liposomes to induce leakage of encapsulated calcein. We demonstrate also that the propensity of the peptides to fold as an alpha-helix increases their fusogenic activity, thus accounting for the maximal fusogenic activity of the most stable helix at residues 118 to 135. These data suggest that, by analogy with the C-terminal domain of the beta-amyloid peptide, the fusogenic properties exhibited by the prion peptides might contribute to the neurotoxicity of these peptides by destabilizing cellular membranes., (Copyright 1997 Academic Press Limited.)

The 154-171 segment of the human lecithin-cholesterol acyltransferase (LCAT) enzyme was identified as the most stable amphipathic helix in the LCAT sequence. Its mean hydrophobicity, hydrophobic moment and its orientation at a lipid/water interface are similar to those of some of the helical repeats of apolipoprotein A-IV and E. This domain was therefore proposed as a candidate peptide accounting for the association between LCAT and its lipid substrate. To investigate this hypothesis we synthesized the LCAT-(154-171)-peptide, two variants containing the natural Y156N and R158C mutations and a variant with increased hydrophobicity through Y156I, L160I, L163I and Y171W substitutions. The structural and lipid-binding properties of these synthetic peptides were investigated by turbidity, fluorescence, electron microscopy and circular dichroism. The wild-type peptide, the R158C variant in its dimeric form, as well as the more hydrophobic peptide, associated with phospholipids, whereas the Y156N and the R158C variant in its monomeric form did not. However, only the complexes generated with the hydrophobic variant were stable enough to resist dissociation during gel filtration. The wild-type peptide and hydrophobic variant formed discoidal complexes with dimyristoylglycerophosphocholine (Myr2GroPCho) as shown by negative staining electron microscopy. Comparison of the properties of the wild-type and hydrophobic variant LCAT-(154-171)-peptide stresses the contribution of the hydrophobic face of the amphipathic helix to the formation and stabilization of the peptide/lipid complexes. This is further confirmed by the decreased affinity of the Y156N variant peptide for lipids, as this mutation decreased the mean hydrophobicity of the hydrophobic face of the amphipathic helix. These results support the hypothesis that the 154-171 segment of LCAT might be involved in the interaction of the enzyme with its lipid substrate and suggest that the decreased activity of the Y156N natural LCAT mutant might result from a decreased affinity of this mutant for lipids.

The complexes of individual human plasma apolipoproteins (apo) A-I, E and A-II with dipalmitoylphosphatidylcholine (DPPC) in the absence or in the presence of cholesterol (Chol) were prepared with initial DPPC/Chol/protein weight ratio as 3:0.15:1. ApoA-I/DPPC/Chol complexes with different protein content (initial DPPC/apoA-I weight ratios were changed from 10.5:1 to 2.6:1) but with a fixed initial DPPC/Chol weight ratio of 20:1 were also prepared. The complexes were isolated by gel-filtration and characterized by size and composition. ApoA-I- and apoA-II-complexes had the same size (80-84 A) and the complexes became more heterogeneous upon Chol inclusion; apoE-complexes were larger (97-100 A) and more homogeneous and Chol addition had no effect on their hydrodynamic properties. Chol seems to be excluded partially in the following manner for isolated complexes with different apo's: A-II > E > A-I. The possible existence of two lipid regions in the complexes differing in lipid dynamics - the lipid shell in the vicinity of apolipoprotein (boundary lipid) opposite to the remaining part of the lipid bilayer - has been studied by absorbance and fluorescence spectroscopy with cis-parinaric acid (cis-PA) and trans-parinaric acid (trans-PA) embedded into the complexes. Their application is based on a strong preference of trans-PA for solid lipid while cis-PA distributes more equally between co-existing fluid and solid lipid regions (Sklar et al. (1979) Biochemistry 18, 1707-1716). (1) For apoA-I-complexes, the partition of cis-PA between water and lipid phase at temperatures below and above the transition temperature of DPPC (T(t)) was insensitive to Chol and temperature, while partition of trans-PA into the lipid phase of Chol-containing complex was increased at high temperature and decreased at low temperature. These results seem to be related to trans-PA redistribution between Chol-rich and protein-rich lipid domains, the latter being more disordered at T < T(t) and more immobilized at T > T(t) compared to the bulk bilayer; cis-PA localizes preferentially in boundary lipid. This hypothesis was directly confirmed by measurements of energy transfer between apoA-I tryptophanyls and probe molecules. (2) The relative response of trans-PA fluorescence intensity to temperature-induced phase transition of DPPC in apoA-I/DPPC/Chol complexes was decreased as a function of apolipoprotein content in a non-monotonic fashion with a transition midpoint at a mol ratio DPPC/A-I of 250:1, probably indicating two different modes of apolipoprotein/DPPC interaction in different sized complexes. (3) The comparative study of lipid dynamics in apoA-I-, apoE- and apoA-II-containing complexes with temperature response to phospholipid phase transition with fluorescence parameters such as intensity and anisotropy of cis-PA and trans-PA revealed the presence of boundary lipid in all three complexes without Chol. In contrast to apoA-I-containing complexes, in apoA-II/DPPC/Chol complexes, trans-PA seems to move preferentially into boundary lipid and cis-PA to distribute between two different regions probably as a result of more ordering action induced by apoA-II compared to apoA-I on the nearest phospholipid molecules in Chol-containing complexes; the apoE action on trans-PA and cis-PA distribution could be intermediate. Based on these results, the degree of Chol exclusion from the boundary lipid region for complexes with different apo's increasing in the order A-II > E > A-I can be suggested. Different Chol distributions between two lipid regions in the complexes seems not to be a function of complex size, but rather is an inherent property of the particular apolipoprotein molecule.

Amphipathic helical peptides represent the lipid-binding units of the soluble plasma apolipoproteins. Several synthetic peptide analogues have been designed to mimic such structures and have been used to unravel some of the mechanisms involved in the physiological function of the apolipoproteins, including lipid binding, LCAT activation, and enhancement of cholesterol efflux from lipid-laden cells. A series of novel synthetic peptides, named ID peptides, was modeled on the basis of the structural properties common to the amphipathic helices of apolipoprotein (apo) A-I. In these new peptides, however, the segregation between hydrophobic and hydrophilic faces of the helices is more pronounced than in apoA-I, so that the surface of the hydrophobic and hydrophilic faces of the amphipathic helices is equal. Moreover, there are fewer negatively charged residues in the center of the hydrophilic face of the helical peptides. Most charged amino acids are located along the edge of the helix and are susceptible to forming salt bridges with residues of an antiparallel helix, such as around a discoidal phospholipid/peptide complex. The physicochemical characteristics of these peptides and their complexes with phospholipids were compared with those of the 18A peptide and its lipid/peptide complex. All ID peptides bind dimyristoylphosphatidylcholine vesicles more rapidly than the 18A peptide to yield discoidal peptide/phospholipid complexes of comparable size. The alpha-helical content of the lipid-free ID peptides is close to that of the 18A peptide and increases slightly on lipid binding. The stability of the ID and 18A peptides and of the phospholipid/peptide complexes against guanidinium hydrochloride denaturation is higher than that of lipid-free and lipid-bound apoA-I. LCAT activation by the 18A/phospholipid/cholesterol complexes equals that of apoA-I/ phospholipid/cholesterol complexes, whereas none of the ID peptides tested is able to activate LCAT to a significant extent. Incubation of the peptide/phospholipid complexes with lipid-laden macrophages induces cellular cholesterol efflux and incorporation of cholesterol into the complexes. The cholesterol efflux capacity of the peptide/phospholipid complexes is comparable among the peptides and higher than that of apoprotein/phospholipid complexes. In conclusion, although the amphipathicity of the new peptides is higher than that of the 18A model peptide, the lack of LCAT activation by the ID peptides suggests that an enhanced segregation of the hydrophobic and hydrophilic residues, equal magnitude of hydrophobic and hydrophilic faces of the helix, and the absence of negatively charged residues in the central part of the hydrophilic face might account for the lack of LCAT activity of these peptides. These parameters do not affect the capacity of the peptide/phospholipid complexes to promote cellular cholesterol efflux.

To model the common structural unit in the system of reverse cholesterol transport, we studied the composition, structure, and physicochemical properties of complexes generated between dipalmitoylphosphatidylcholine (DPPC) or palmitoyllinoleoylphosphatidylcholine (PLPC) and apoE3 in the absence and in the presence of cholesterol (Chol); the data were compared with similar experiments using apoA-I, the major proteins of high-density lipoproteins. The conformation and organization of lipid-binding domains of apoE3 within the complexes were calculated by computer modeling. The transition temperatures of DPPC within discoidal complexes with mean diameters of 116 A (GGE) or 148 A (EM) were higher for complexes versus liposomes both in the absence and in the presence of Chol. Association of apoE3 with DPPC resulted in a more structured state of the apolipoprotein molecule versus the soluble apolipoprotein; this state was characterized by parallel orientation of alpha-helixes of apoE3 and DPPC acyl chains. Substrate efficiency of the apoE3-PLPC-Chol complexes in the lecithin-cholesterol acyltransferase (LCAT) reaction expressed as Vmax/Km was 0.5 mole cholesteryl esters/h per 1 microM. The transformation of discoidal apoE3-DPPC-Chol complexes into spherical particles was induced by LCAT and accumulation of cholesteryl esters was approximately 62% of the total cholesterol. Parallel orientation of phospholipid acyl chains with helical segments disappeared in these particles. Discoidal apoE3-DPPC complexes incorporated unesterified cholesterol released from Chol-loaded J774 macrophages. The data support the concept that association of apoE3 and apoA-I with phospholipids is qualitatively similar due to similar orientation of helical repeats in the C-terminal domains of apoE3 and apoA-I.

C-terminal fragments of the Alzheimer amyloid peptide (amino acids 29-40 and 29-42) have physico-chemical properties related to those of the fusion peptides of viral proteins and they are able to induce the fusion of liposomes in vitro. We proposed that these properties could mediate a direct interaction of the amyloid peptide with cell membranes and account for part of the cytotoxicity of the amyloid peptide. In view of the epidemiologic and biochemical linkages between the pathology of Alzheimer's disease and apolipoprotein E (apoE) polymorphism, we examined the potential interaction between the three common apoE isoforms and the C-terminal fragments of the amyloid peptide. We show that, at low concentration, only apoE2 and apoE3 are potent inhibitors of the amyloid peptide fusogenic and aggregational properties, whereas the apoE4 isoform has no effect. We further show that the protective effect of apoE is mediated by the formation of stable apoE/amyloid peptide complexes, as determined by tryptophan emission fluorescence measurements and by gel electrophoresis. The interaction specificity between apoE2 and apoE3 and the amyloid fragments is demonstrated here, since other apolipoproteins (e.g. apolipoprotein A-I and A-II), with similar amphipathic structures, do not interact with the amyloid C-terminal fragments. Finally, we show that, reciprocally, the amyloid peptide can interact directly with the apoE2 and apoE3 isoforms to decrease or perturb their normal association with lipids. These data suggest that the 29-40 and 29-42 domains of the amyloid peptide could be critical for the amyloid-apoE interaction, and that apoE2 and apoE3 isoforms, but not apoE4, could play a protective role against the formation of amyloid aggregates and/or against their interaction with cellular membranes.

A series of natural peptides and mutants, derived from the Alzheimer beta-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29-40 and 29-42) and a central domain (13-28) of the beta-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire beta-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29-40 and 29-42 beta-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29-42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 beta-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the alpha-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the alpha-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29-40 and 29-42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the beta-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.

Amphipathic helical repeats are considered as the structural units of numerous apolipoproteins and have been described as being responsible for the interaction of apolipoproteins with phospholipids in high-density lipoproteins (HDL). Furthermore, apolipoproteins, and especially apolipoprotein AI (apoAI), are involved in various biological functions of these circulating particles in plasma. Studies with synthetic peptides corresponding to domains of the apoAI sequence have however shown that short 39-residue fragments do not interact strongly enough with phospholipids to generate particles that correctly mimic the physico-chemical properties of HDL reconstituted with native apoAI [Vanloo, B., Demoor, L., Boutillon, C., Lins, L., Baert, J., Fruchart, J. C., Tartar, A. & Rosseneu, M. (1995) Association of synthetic peptide fragments of human apolipoprotein A-I with phospholipids, J. Lipid Res. 36, 1686-1696.]. Here we show that synthetic branched multimeric peptides, often used as carriers for the design of synthetic vaccines (multiple-antigen peptides), can be used to mimic the physiochemical properties of apoAI in HDL. This type of molecule is obtained by using a small core matrix of Lys residues bearing radially branched synthetic peptides as dendritic arms. We compared the lipid-binding capacities and the structural properties of a linear peptide corresponding to residues 145-183 of apoAI [apoAI-(145-183)-peptide] with those of two multimeric peptides consisting respectively of three [trimeric apoAI-(145-183)] and four copies [tetrameric apoAI-(145-183)] of the selected sequence, branched on a covalent core matrix. This paper provides evidence for the increased abilities of the multimeric peptides to associate with phospholipids compared with the short linear peptides. Moreover, the trimeric apoAI-(145-183) peptide was most efficient in mimicking the physico-chemical and structural properties of native apoAI in reconstituted HDL. As tools adequate to unravel the structure/function relationship of separate apolipoprotein domains are still missing, these multimeric peptides might constitute an alternative approach to linear peptides which are poor mimetics and to protein mutants which are difficult to produce and only provide information about the total sequence.

Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(delta Ala190-Gln243) with dimyristoylphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding. The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A-I(delta Leu44-Leu126), 6.1 nm for apo A-I(delta Glu139-Leu170), and 6.5 nm for apo A-I(delta Ala190-Gln243)] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(delta Leu44-Leu126), 6 per apo A-I(delta Glu139-Leu170), and 4 per apo A-I(delta Ala190-Gln243) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)