Inhibition of β-Amyloid Fibrillization by Directed Evolution of a β-Sheet Presenting Miniature Protein

Abstract

We describe the directed evolution of a miniature beta-sheet protein for targeting beta-amyloid oligomers implicated in Alzheimer's disease. Circular dichroism spectroscopy, thermal denaturation experiments, and immunoglobulin binding assays established that our beta-amyloid-targeted miniature protein, TJ10, presents a well-folded thermostable beta-sheet. TJ10 was found to prevent beta-amyloid fibrillization at stoichiometric concentrations and was also an effective inhibitor at substoichiometric concentrations. Thus our results provide a new and potent beta-sheet chemical template for effectively targeting beta-amyloid while also demonstrating a general strategy for targeting proteins implicated in other amyloidogenic diseases.

Stabilization of a β-hairpin in monomeric Alzheimer's amyloid-β peptide inhibits amyloid formation

“…The selection of alanine or proline at position 24 can be rationalized Short peptides, which are homologous to A␤ but contain proline residues as ␤-sheet blockers, have been developed as fibrillogenesis inhibitors (16). The aim of this and related peptide-and protein-based approaches that target A␤ aggregation is to bind the hydrophobic part of A␤ by exploiting the same intermolecular interactions formed in A␤ self-assembly, e.g., ␤-sheet backbone hydrogen bonds, and to consequently disrupt the potential for further ␤-sheet extension (15)(16)(17)29). Although no structure of a ␤-sheet breaker peptide in complex with A␤ has been reported, certain aspects of the concept appear to be reflected in the Z A␤3 :A␤(1-40) interaction: The Z A␤3 ␤-strands cap the edges of the A␤(1-40) ␤-sheet; the strands are short and terminated on their C-terminal side by a proline and on the N-terminal side by a sequence region selected to have little propensity for either ␣-or ␤-structure, resulting in an inability to serve as a template for further ␤-sheet extension.…”

Inhibition of Amyloid Formation

“…192 Several groups have subsequently employed combinatorial libraries to select short peptides that block amyloid formation and, in some cases, stabilize non-amyloid aggregates. [193][194][195][196][197] (Phage display and related techniques are also commonly used to select binding proteins, as discussed in other sections of this article. )…”

Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster Maturation

“…Amyloid-␤(1-40) Preparation-A␤(1-40) fibrils were formed as described previously (23,46). Briefly, 250 l of a caspase activity buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 1.5% sucrose, 10 mM DTT, 0.1% CHAPS) was added to the lyophilized solid to 50 M. The peptide was agitated at 37°C for 4 -6 h. …”