Resveratrol inhibits the formation of multiple-layered β-sheet oligomers of the human islet amyloid polypeptide segment 22-27

Abstract

The abnormal self-assembly of a number of proteins or peptides is a hallmark of >20 amyloidogenic diseases. Recent studies suggest that the pathology of amyloidogenesis can be attributed primarily to cytotoxic, soluble, intermediate oligomeric species rather than to mature amyloid fibrils. Despite the lack of available structural information regarding these transient species, many therapeutic efforts have focused on inhibiting the formation of these aggregates. One of the most successful approaches has been to use small molecules, many of which have been found to inhibit toxic species with high efficacy. A significant issue that remains to be resolved is the mechanism underlying the inhibitory effects of these molecules. In this article, we present extensive replica-exchange molecular dynamics simulations to study the early aggregation of the human islet amyloid polypeptide segment 22-27 in the presence and absence of the small-molecule inhibitor resveratrol. The simulations indicate that aggregation of these peptides was hindered by resveratrol via a mechanism of blocking the lateral growth of a single-layered β-sheet oligomer (rather than preventing growth by elongation along the fibril axis). Intersheet side-chain stacking, especially stacking of the aromatic rings, was blocked by the presence of resveratrol molecules, and the overall aggregation level was reduced.

Figure 1

(A) Molecular geometry of trans-resveratrol. All carbon atoms are distinguished by sequential numbers (black), and oxygen atoms on three hydroxyl groups are labeled with red numbers. (B) Cartoon representation of seven strands of hIAPP_22–27. A preformed parallel-β-sheet tetramer (red online) at center is surrounded by three APMs (blue online) placed at least 10 Å away from the tetramer.

Figure 2

Time evolution of the β-content of PBPs and APMs in the absence and presence of resveratrol. β-content is the ratio of the number of residues in β secondary structure to the total residue number for each species, 24 for PBP and 18 for APM. Each data point on the curves is an average over 10,000 time frames (10 ns). The secondary structures of peptides were assessed by the DSSP algorithm (45). All data are from the ensemble of T = 315 K.

Figure 3

(A) Species distribution of β-sheet aggregates. (B) Species distribution of collapsed aggregates. See text for definition of β-sheet aggregates and collapsed aggregates. Aggregates with very small populations are excluded from the figures. All data are from the ensemble of T = 315 K.

Figure 4

Representative structures of aggregates with double β-sheets. Percentages in parentheses show the size of the first-ranked cluster in the overall aggregates with certain size. The stacking side chains are indicated by red balls and sticks. Peptide backbones are shown as ribbons, yellow for oligomers and green for monomers. Resveratrol molecules are shown in black, line representation. All six aggregates in the absence of resveratrol (A–F) show a multilayer arrangement. (G–I) Three aggregates in the presence of resveratrol. Except in the case of 4.2 (H), there are no side-chain contacts between the two β sheets.

Figure 5

Temperature-dependent features of peptide aggregation. (A) Proportion of peptide monomers, the ratio of the number of monomers to the total number of peptide strands. (B) β-content. (C) β-content contributions from PBPs and APMs. (D) Histogram statistics of survival ratios of the preformed hydrogen bonds. Calculations of A–C are based on data from the last 300 ns of simulations. Error bars are obtained from the standard deviation of three average values from nonoverlapping time blocks: 200–300 ns, 300–400 ns, and 400–500 ns. The survival ratio is the ratio of the number of hydrogen bonds maintained in the PBP to the total number of 15 in the intact PBP. The histogram in D is based on a subset of data from the last 300 ns of simulations.

Figure 6

Six binding sites of resveratrol molecules on β-sheet hIAPP_22–27 pentamers (left), with the representative structures at each binding site (right). Oligomers with size 5 in Fig. 4 were chosen and distribution of small molecules (colored dots) surrounding the β-sheet pentamers (blue) was drawn by superimposing the peptides in the middle. Roughly six binding sites are identified and represented by different colors (hot pink, Site 1; yellow, Site 2; violet, Site 3; green, Site 4; cyan, Site 5; orange, Site 6; white, all other sites). The corresponding representative structures are shown on the right. Arrows point to the longitude direction of the fibril. Binding sites are shown along the direction of fibril growth (A) and perpendicular to the direction of growth (B). For the sake of clarity, sites on the front and back sides of the structures are not shown, corresponding to sites 5 and 6 in A and sites 3 and 4 in B.

Figure 7

Contact maps of heavy atoms on resveratrol (abscissa) and on the residues of peptides (ordinate). The contact intensities have been normalized by dividing the individual largest contact occurrence number (in parentheses next to the color bar). Contacts on residues are divided into four groups: backbones of BSOs (A), side chains of BSOs (B), backbones of monomers (C), and side chains of monomers (D). The atoms of resveratrol are ordered according to spatial proximity in chemical structure rather than sequence atomic numbers (see Fig. 1 for the chemical structure).

Figure 8

Self-assembly of amyloidogenic peptides and suggested inhibitory mechanism of resveratrol. When monomers attempt to integrate into a fibril nucleus, aggregates grow in two directions, by lateral association and longitudinal elongation (path 1). The end product is a multiple-layer fibril. When peptides are incubated with resveratrol, resveratrol is capable of blocking the lateral growth, whereas longitudinal elongation remains nearly intact (path 2). Black and gray are used to distinguish β-strands belonging to different layers. Dashed arrows represent the nascent β-strands in oligomers.