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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 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.

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 α 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 α 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 α/β 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 α/β 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. —Peelman, F., B. Vanloo, J-L. Verschelde, C. Labeur, H. Caster, J. Taveirne, A. Verhee, N. Duverger, J. Vandekerckhove, J. Tavernier, and M. Rosseneu. Effect of mutations of N- and C-terminal charged residues on the activity of LCAT. J. Lipid Res. 2001. 42: 471–479.

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., This work was supported by grants from the League against Cancer (Stichting tegen Kanker, Belgium), Ghent University (BOF-GOA), the Research Foundation-Flanders (FWO-Vlaanderen), the Interuniversity Attraction Poles program (IUAP), the Spanish Ministry of Science and Innovation, (BIO2011-26689), the Autonomous Community of Madrid (S2010-BMD-2312), “la Caixa” Foundation, the Fund for Scientific Spearheads of Ghent University Hospital and NKP-29, and the Flanders Institute for Biotechnology (VIB). This work further received funding from the European Community’s Health 7thFramework Programme (PRIME-XS project, grant agreement number 262067). JB is supported by the Agency for Innovation by Science and Technology in Flanders (IWT-Vlaanderen). FI is a Postdoctoral Fellow of the Research Foundation-Flanders (FWO-Vlaanderen). DRG is supported by a contract of “Personal Investigador de Apoyo” from the Autonomous Community of Madrid. SC is supported by SonoDrugs (FP7 programme) and Ghent University.

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.

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 K-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. fl 1998 Elsevier Science B.V. All rights reserved.

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 formdid 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 (Myr2GraPCho) 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.

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 trypto-phan 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 β-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, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) of the β-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 β-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 β-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 β-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the α-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 α-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 β-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.

The sequences of the plasma apolipoproteins have a high degree of internal homology as they contain several 22-mer internal repeats. These amphipathic helical repeats are considered as the structural and functional units of this class of proteins. We proposed that the 22-mer repeats of the plasma apolipoproteins consist of 17-mer helical segments separated by extended beta-strands comprising five amino acid residues with a proline in the center of this segment. These beta-strand segments help reverse the orientation of the consecutive helices of apoA-I, A-IV, and E in a discoidal apolipoprotein-phospholipid complex. In order to support this hypothesis, we synthesized apoA-I fragments consisting of, respectively, one putative helix (residues 166-183), one helix plus a beta-strand (residues 161-183), and a pair of helices separated by a beta-strand (residues 145-183). The structural and lipid-binding properties of these peptides were investigated by turbidity, fluorescence, binding studies with unilamellar phospholipid vesicles, electron microscopy, and circular dichroism measurements. Our data show that one single putative helical segment or one helical segment plus one extended beta-strand do not form stable complexes with phospholipids. The addition of a second adjacent helix has no influence on the lipid affinity of the apoA-I 145-183 peptide compared to the shorter segments but substantially improves the stability of the complexes. The helical content of the peptide increases upon lipid association as observed with apoA-I. The complexes generated with the apoA-I 145-183 peptide appear as discoidal particles by negative staining electron microscopy, with heterogeneous sizes ranging between 250 and 450 A. The relative orientation of the peptide and the phospholipid is the same as in a DMPC/apoA-I complex as the helices are oriented parallel to the acyl chains of the phospholipid. However, the stability of these complexes is significantly lower than that of the corresponding DMPC/apoA-I complexes. The transition temperature, fluidity, and cooperativity of the phospholipid bilayer are only weakly affected by the association with the apoA-I 145-183 peptide. These data suggest that a pair of helical peptides linked through a beta-strand associates more tightly with lipids and can form discoidal lipid-peptide complexes, than a single helix. A comparison with the properties of native apoA-I suggests, however, that the cooperativity between pairs of helices in native apoA-I further contributes to strengthen the lipid-protein association.

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.

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.

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.

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.

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.

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 lipidprotein 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 Vmax because the apparent Michaelis-Menten constant Km 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 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 A2 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 A2 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 A2 and acyltransferase activites on rHDL. Mutations of hydrophilic residues in amphipathic helices α 3–4 and α 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 α 3–4 and α 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. —Vanloo, B., F. Peelman, K. Deschuymere, J. Taveirne, A. Verhee, C. Gouyette, C. Labeur, J. Vandekerckhove, J. Tavernier, and M. Rosseneu. Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease. J. Lipid Res. 2000. 41: 752–761.

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 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.

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.

We have expressed recombinant human apolipoprotein A-II (apoA-II) in Escherichia coli, as a fusion protein with Schistosoma japonicum glutathione-S-transferase (GST). The GST-AII fusion protein was recovered by affinity chromatography using glutathione as a ligand. After thrombin cleavage and removal of the GST carrier, recombinant apoA-II was obtained in a highly purified form and was exclusively composed of dimeric apoA-II. Kinetics of association to dimyristoylglycerophosphocholine (Myr2GroPCho) vesicles showed that recombinant apoA-II exhibited the same pattern of association as human plasma apoA-II. Electron microscopic analysis of the complexes showed a typical pattern of rouleaux, characteristic of stacked discs, with a diameter similar to that determined by gradient-gel electrophoresis. Circular dichroism measurements showed that the alpha-helical content of both plasma and recombinant apoA-II increased similarly when the proteins associated with Myr2GroPCho vesicles, at the expense of a random-coil structure. Lipid-bound apoA-II consisted of 70-72% alpha helices, suggesting the presence of three 18-residue alpha helices/apoA-II monomer. Cross-linking experiments indicated that Myr2GroPCho complexes contained two molecules dimeric apoA-II/vesicle. Recombinant apoA-II was as efficient as plasma apoA-II in associating with HDL subclasses, and in displacing apoA-I from dipalmitoylglycerophosphocholine/cholesterol/apoA-I complexes, most likely due to its highly ordered secondary structure when associated with Myr2GroPCho vesicles. These findings demonstrate that recombinant apoA-II exhibits the same structural and functional properties as human plasma apoA-II. Thus, the expression system utilized is appropriate to produce mutagenized forms to further structure/function analysis.

mAb4E4, a murine monoclonal antibody that is specific for acetylated LDL and malondialdehyde-treated LDL, binds specifically to modified LDL present in human atherosclerotic lesions. It is directed against an epitope that is poorly exposed in delipidated and solubilized apolipoprotein B-100 from modified LDL. mAb4E4, as well as its F(ab')2 and Fab fragments, enhanced the uptake of both acetylated LDL and malondialdehyde-treated LDL by THP-1-derived macrophages resulting in a sixfold increase of cytoplasmic cholesteryl ester levels. The increased uptake of modified LDL/mAb4E4 complexes did not occur via the Fc receptor and did not depend on aggregation of modified LDL particles. However, their uptake was inhibited by blocking the scavenger receptors with fucoidin or by downregulation of receptor expression with endotoxins or interferon-gamma, indicating that their uptake is mediated via these receptors. Thus, generation of autoimmune antibodies against modified LDL and subsequent endocytosis of soluble modified LDL/antibody complexes via scavenger receptors may enhance foam cell generation. This mechanism may contribute to the progression of atherosclerotic lesions.