Molecular modeling of the amphipathic helices of the plasma apolipoproteins.

In this paper we propose a classification of the amphipathic helical repeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential around the helical segments. The repeats were identified using a new autocorrelation matrix, based upon similarities of hydrophobic and hydrophilic properties of the amino acid residues within the apolipoprotein sequences. The helices were constructed by molecular modeling, the molecular hydrophobicity potential was calculated, and isopotential contour lines drawn around the helices yielded a three-dimensional visualization of the hydrophobicity potential. Two classes of apolipoproteins could be differentiated by comparing the hydrophobic angles obtained by projection of the isopotential contour lines on a plane perpendicular to the long axis of the helix. The isopotential contour lines around apo AI, AIV, and E are more hydrophilic than hydrophobic, whereas they are of similar intensity for apo AII, CI, and CIII. In both cases discoidal lipid-protein complexes are generated, with the amphipathic helices around the edge of the lipid core. The long axis of the helices is oriented parallel to the phospholipid acyl chains and the hydrophilic side of the helix toward the aqueous phase. As a result of the differences in hydrophobicity potential, the contact between the hydrophobic side of the helices and the phospholipid acyl chains is larger for apo AII, CI, and CIII than for the other apolipoproteins. This might account for the greater stability of the discoidal complexes generated between phospholipids and these apoproteins.     

The tandem PDZ domains of syntenin promote cell invasion

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.     

Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

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.     

Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease

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.     

Evaluation of the secondary structure of apo B-100 in low-density lipoprotein (LDL) by infrared spectroscopy

The secondary structure of the apo B-100 protein present in human low density lipoprotein has been investigated by transmission and attenuated total reflection infrared spectroscopy. The amount of beta-sheet (41%) is significantly higher than that determined by CD spectroscopy in the present study (12%) and elsewhere (15-16%). The high percentage of beta-sheet structure in apo B-100 supports the importance of such segments in maintaining the lipid-protein assembly in LDL. Polarized infrared spectroscopy indicates that the beta-sheet component of apo B-100 adopts a preferential orientation with respect to the phospholipid monolayer surrounding the LDL, whereas no such orientation is observed for the other secondary structure components.     

Lysophosphatidic acid affinity chromatography reveals pyruvate kinase as a specific LPA-binding protein

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.     

Contribution of the hydrophobicity gradient to the secondary structure and activity of fusogenic peptides

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.