Role of ?- Amyloid Structure and residue accessibility in cell interactions associated with Alzheimer's disease
Creators: name: Ramos, Irina
Publisher: name: Maryland Shared Open Access Repository
Year: 2009
Subjects: Alzheimer's disease
Beta Amyloid
Fluorescence Ressonance Energy Transfer FRET
Mass Spectrometry
Neurotoxicity
Protein Structure

description: Beta- amyloid peptide ( A?) is the major protein constituent found in senile  plaques in Alzheimer’s disease ( AD). A? is able to bind in the cell membrane to a  variety of biomolecules, including lipids and protein, but the sequence of biological  events that leads to cell death is not well understood. Its extracellular accumulation  is believed to be related with neurodegeneration and its toxicity related to its  structure or aggregation state.  The purpose of this study was to characterize A? structure; first, at the residue  level to identify which residues have an important role on peptide’s tertiary  structure, followed by the examination of its interactions with the cell membrane,  with residue level resolution.  An approach based on chemical modification of primary amines and mass  spectrometric ( MS) detection was used to identify residues on A? peptide that were  exposed or buried upon changes in peptide structure associated with aggregation.  Tandem mass spectrometry and limited proteolysis were used to identify the  location of the modified primary amines within the A? sequence. Results indicate  that the N- terminus was the most accessible primary amine in the fibril, followed by  lysine 28, then lysine 16. A kinetic analysis of the data was then performed to  quantify differences in accessibility between these modification sites. We estimated  equilibrium unfolding constants for each modified site of the peptide, and  determined that the unfolding constant for the N- terminus was ~ 100 times greater  than that for K28, which was ~ 6 times greater than that for K16.  The interaction between A? and the cells is followed by a cascade of biological  responses not well characterized. In order to target cell membrane proteins  preferred for A? binding, fluorescent techniques ( FRET and FCS) were used to  measure proximities and elucidate conformational changes in A? structure upon  interactions. Our study compared different cell membrane receptors and we found  that A? bound to cell surface integrins within 6nm. In addition, while A? still bound  to the surface when lysines on the A? structure were modified, A? no longer bound  near integrins. Finally, employing FCS, we analyzed the A? aggregation process in the  absence and presence of cells in order to identify structures with more propensity  to interact with cells. In the presence of the cells, the concentration of soluble  species associated with A? fibrils decreases ~ 10 times, suggesting that soluble  oligomers leave the solution to interact with the cells.  This work establishes an approach to examine A? peptide structure and cell  interactions at the molecular level. It is a first step in designing novel therapies for  prevention of A? structural transitions and/ or cell interactions associated with  neurotoxicity in Alzheimer’s disease.