Role of ?- Amyloid Structure and residue accessibility in cell interactions associated with Alzheimer's disease
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Date
2009-01-01
Type of Work
Department
Chemical, Biochemical & Environmental Engineering
Program
Engineering, Chemical and Biochemical
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Access limited to the UMBC community. Item may possibly be obtained via Interlibrary Loan through a local library, pending author/copyright holder's permission.
Abstract
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.