Abstract:The toxicity of the nonaggregated amyloid ␤-peptide (1-40) [A␤(1-40)] on the viability of rat cortical neurons in primary culture was investigated. We demonstrated that low concentrations of A␤ peptide, in a nonfibrillar form, induced a time-and dose-dependent apoptotic cell death, including DNA condensation and fragmentation. We compared the neurotoxicity of the A␤(1-40) peptide with those of several A␤-peptide domains, comprising the membrane-destabilizing C-terminal domain of A␤ peptide (e.g., amino acids 29 -40 and 29 -42). These peptides reproduced the effects of the (1-40) peptide, whereas mutant nonfusogenic A␤ peptides and the central region of the A␤ peptide (e.g., amino acids 13-28) had no effect on cell viability. We further demonstrated that the neurotoxicity of the nonaggregated A␤ peptide paralleled a rapid and stable interaction between the A␤ peptide and the plasma membrane of neurons, preceding apoptosis and DNA fragmentation. By contrast, the peptide in a fibrillar form induced a rapid and dramatic neuronal death mainly through a necrotic pathway, under our conditions. Taken together, our results suggest that A␤ induces neuronal cell death by either apoptosis and necrosis and that an interaction between the nonfibrillar C-terminal domain of the A␤ peptide and the plasma membrane of cortical neurons might represent an early event in a cascade leading to neurodegeneration. Key Words: Alzheimer's disease -Amyloid ␤-peptideApoptosis-Fusogenic peptides-Neurotoxicity-Cortical primary neurons.

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 α/β hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta‐strands connected by four α‐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 monomelic 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.

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Abstract:Growing evidence indicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease, although its exact role remains unclear. We previously demonstrated that ␤-amyloid peptide (A␤) displays membrane-destabilizing properties and that only apoE2 and E3 isoforms inhibit these properties. In this study, we clearly demonstrate that the carboxy-terminal lipid-binding domain of apoE (e.g., residues 200 -299) is responsible for the A␤-binding activity of apoE and that this interaction involves pairs of apoE amphipathic ␣-helices. We further demonstrate that A␤ is able to inhibit the association of the C-terminal domain of apoE with lipids due to the formation of A␤/apoE complexes resistant to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the contrary, the amino-terminal receptor-binding domain of apoE (e.g., residues 129 -169) is not able to form stable complexes with A␤. These data extend our understanding of human apoE-dependent binding of A␤ by involving the C-terminal domain of apoE in the efficient formation of apoE/A␤ complex. Key Words: Apolipoprotein E-␤-Amyloid peptide-Peptide/peptide interaction-Alzheimer's disease.