Short Peptides as Inhibitors of Amyloid Aggregation

Abstract

The misfolding and aggregation of proteins into amyloid has been linked to a variety of age-related diseases. Aggregation of proteins, such as Aβ in Alzheimer's disease and Islet Amyloid Polypeptide (IAPP, amylin) in type 2 diabetes, appears to lead to the formation of toxic assemblies. These assemblies range in size from small oligomers (2-8 proteins) to large fibrils (thousands of proteins). It remains unclear how these amyloidogenic proteins misfold and form toxic species, but growing evidence suggests that inhibiting the aggregation of these proteins could slow, if not prevent altogether, the progression of these diseases. We describe the use of small peptides (<43 amino acids) as inhibitors of amyloid-based aggregation. These peptides, often short complementary segments of the amyloid proteins, can be useful (i) for identifying the aggregation-prone regions of the amyloid proteins (ii) as models for drug discovery and (iii) as potential therapeutic agents themselves.

Keywords: ABeta; Amylin; Amyloid Inhibition; IAPP; Peptide Libraries.

Fig. (1)

(A) Amyloidogenic peptides, such as Aβ42 and IAPP self-assemble, ultimately leading to the formation of amyloid. (B) When linked to an amyloid peptide, EGFP does not fold or fluoresce. The aggregation of the amyloid peptides prevents proper folding and fluorescence of EGFP. (C) Inhibitors that prevent the self-assembly of the amyloid peptides allow the EGFP to fold and fluoresce.

Fig. (2)

Amino Acid Sequences of Aβ42 and Library 1 and Library 2 Peptides. The amino acid sequence for Aβ42 is shown. Library 1 was constructed to have mixtures of amino acids in the bold-faced positions. Degenerate gene construction: Codon ANT encodes for an equal mixture of I, T, N and S. Codon GNT encodes for an equal mixture of V, A, D and G. Codon NTN encodes for a mixture of F, L, I, M and V.

Fig. (3)

Oligo overlap and extension: Single-stranded DNA oligos were designed having complementary 3′ overhang regions (dashed lines). When mixed, the complementary regions anneal and act as templates for Klenow Fragment catalyzed DNA synthesis. Nucleotides not involved in annealing (solid lines) can be explicitly designed base by base or combinatorially varied.

Fig. (4)

Construction and selection of peptide libraries. Cotransformed cells were plated on nitrocellulose absorbed on LB plates containing Ampicillin and Streptomycin. After incubation, resulting colonies are transferred to LB plates containing IPTG. Green-colored colonies are selected and analyzed.