ReviewAmyloid-β fibrillogenesis: Structural insight and therapeutic intervention
Introduction
The amyloidoses are protein-misfolding diseases characterized by the conversion of peptides or proteins from a soluble state into insoluble, β-sheet rich, fibrillar aggregates termed amyloid fibrils (Stefani and Dobson, 2003). The deposition of proteins, either extracellularly as plaques or intracellularly as inclusions, is a characteristic of approximately 20 amyloid disorders, including those which affect the brain or peripheral tissue. Although the proteins that comprise these deposits do not share sequence homology, amyloid fibrils formed by different proteins share biophysical properties and, possibly, a common pathogenic mechanism (Kayed et al., 2003). The major constituent of amyloid in Alzheimer's disease is Aβ, which is produced after proteolytic cleavage of the amyloid precursor protein to form peptides of predominantly 40–42 amino acid in length.
Section snippets
Structural analysis of amyloid fibrils
The biophysical definition of amyloid includes filaments of any polypeptide with a diameter of approximately 10 nm, which have a cross-β-sheet structure, and are commonly identified by birefringence under polarized light upon binding Congo red (Eanes and Glenner, 1968, Goedert and Spillantini, 2006). Electron microscopy (EM) studies have revealed that amyloid fibrils are typically long, straight, and unbranched, ranging from 0.1 to 10 μm in length (Serpell et al., 2000). Early studies using
Amyloid aggregation
The aggregation of Aβ and its assembly into fibrils does not occur in a linear fashion; rather, distinct aggregation intermediates or oligomers are formed, which either give rise to fibrils (termed on-pathway) or do not (termed off-pathway; Fig. 2; Wetzel, 2006). Regardless of which aggregation pathway is utilized, all Aβ species equilibrate into an array characterized by size, not the number of Aβ peptides within each assembly. Elucidation of the mechanisms involved in aggregation, including
Surface-facilitated assembly of Aβ peptides on synthetic or inorganic templates
A number of studies have characterized the assembly of Aβ fibrils in solution using thioflavin T fluorescence, and studied their morphology by EM, atomic force microscopy (AFM), or fluorescence microscopy of fibril assembly on solid surfaces (Stine et al., 1996, Antzutkin, 2004, Ban et al., 2006, Karsai et al., 2006). Peptide concentration, changes in Aβ primary sequence, pH, and interactions with other elements such as other proteins and lipid species can influence the formation of fibers
Therapeutic strategies aimed at inhibiting fibrillogenesis
The characteristics of nucleation-dependent growth can be used to direct pharmacologic approaches to inhibit fibrillogenesis. To illustrate this point we will utilize a few inhibitor molecules that have been reported in the literature to have distinct mechanisms of action and direct effects on Aβ aggregation, rather than detail a list of all reported inhibitors.
Novel screening tools for fibrillogenesis inhibitors
Common screening tools for fibrillogenesis inhibitors include analyses of fibril formation through thioflavin or Congo red binding, and turbidity; analysis of morphology by EM and AFM; spectroscopic techniques such as circular dichroism and NMR; and biochemical readouts including Western and dot blots, and cell culture viability assays (Kirschner et al., 2008). However, novel techniques have emerged which permit detailed analyses of the mechanism of action and affinity of inhibitors, as well as
Conclusion
Structural insights into the conformational changes associated with aggregation and assembly of monomers into oligomers, protofibrils, and fibrils have provided a number of targets for therapeutic intervention. Knowledge of key residues important for β-sheet formation and stabilization of fibrils permits rational design of fibrillogenesis inhibitors. Many of these compounds, including peptide-based inhibitors and small molecules, have demonstrated effectiveness in vitro and in vivo at
Acknowledgments
The authors acknowledge support from the Ontario Alzheimer's Society (J.M.), Canadian Institutes of Health Research (K.A.D. and J.M.), Alzheimer Society of Canada (J.E.S.), and Natural Science and Engineering Research Council of Canada (J.M.).
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