Alzheimer’s disease, Huntington’s disease, and type II diabetes are among the human diseases where the self-assembly of polypeptides into amyloid structures has been associated with significant physiological impairments (1
). Recognizing the need for effective therapeutics to combat these debilitating conditions, much attention has been focused on understanding what drives particular peptide sequences to aggregate. Though the sequences of the peptides associated with these diseases differ considerably from one another, the similarity of their cross-β sheet fibrillar structures suggests a common underlying molecular organization (4
). Probing the primary sequences of these amyloidogenic polypeptides for features that are critical to fibril formation may reveal specific interactions that must be disrupted to prevent aggregation. This knowledge can then be used to direct the design of therapeutics that move beyond symptom management and towards treatments targeted at the misfolding and aggregation pathways that underlie amyloid disorders.
In Alzheimer’s disease (AD), the accumulation and aggregation of the amyloid-beta peptide (Aβ) is posited as triggering a cascade of events that ultimately results in memory loss, dementia, and other neurocognitive deficits. This “amyloid cascade hypothesis” proposes that the path toward disease begins with the release of Aβ from the amyloid precursor protein (APP) (5
). Variable cleavage by secretase enzymes produces Aβ isoforms of various lengths with the 40-residue Aβ40 and the more toxic and highly aggregating 42-residue Aβ42 variants being most prevalent (10
). Once released, the Aβ peptide self-associates into oligomers, which ultimately assemble into the fibrils that comprise the extracellular plaque first observed by Alois Alzheimer over a century ago (12
). It is now believed that early oligomeric intermediates – rather than the later fibrils and plaques – are the toxic species in AD (13
). Therefore, disrupting the aggregation pathway at its earliest stages may represent an effective route toward the development of therapeutics. In order to design targeted inhibitors of aggregation, it is important to understand which features of the primary sequence lead peptides to aggregate in AD and other amyloid disorders.
The π-stacking of aromatic residues has been suggested as a key feature promoting the assembly of polypeptides into amyloid structures. While relatively infrequent in proteins in general, aromatic residues occur frequently in amyloid sequences – a fact suggestive of their potential involvement in the aggregation process (15
). Furthermore, examination of shorter variants of amyloid-forming polypeptides showed that the minimal fragments necessary for aggregation almost always contain aromatic residues. Well-ordered fibrils have been generated from short penta- and tetra-peptides containing phenylalanine (16
), and even a Phe-Phe dipeptide assembles into tubular structures with some similarities to amyloid fibrils (18
The putative role of aromatic-aromatic interactions in aggregation was also suggested by an examination of compounds that inhibit fibril formation. Aromatic groups are a frequent feature in these inhibitors. For example, various polyphenols inhibit aggregation in vitro,
and in some cases, provide protective effects in vivo
in animal models of AD and other amyloid diseases (19
). Gazit and coworkers suggested that aromatic groups in these inhibitors prevent amyloid formation by interfering with π-stacking interactions between aromatic side chains (21
). They posited that such stacking interactions provide both energetic and directional contributions toward peptide self-assembly and that disrupting these interactions provides a mechanism for inhibition (15
Initial evidence from scanning mutagenesis experiments supported the premise that aromatic side chains promote amyloidogenesis. Replacement of phenylalanine with alanine diminished fibril formation in fragments of islet amyloid polypeptide (IAPP) and calcitonin, which are both associated with amyloid-based human diseases, (17
). However, the Phe→Ala mutations in those studies not only change the phenylalanine to an aliphatic side chain but also considerably alter the hydrophobicity, size, and β-sheet propensity of the side chains ().
Side-chain properties of Ala, Phe, Ile, and Leu residuesa
Recent mutagenesis studies using more conservative substitutions suggest that aromatic π-stacking interactions may not be critical for fibril formation. For example, Tracz et
al. showed that in IAPP fragments, the replacement of Phe residues with Leu, a residue of similar size and hydrophobicity, did not prevent aggregation into amyloid fibrils (28
). Furthermore, Marek et
al. reported that fibrillization remained possible even when all three aryl residues in IAPP were mutated to Leu in a F15L/F23L/Y37L triple mutant (29
These findings suggest that π-stacking may not be critical for amyloid formation, and point to other driving forces for aggregation. Indeed, the smaller size and lower hydrophobicity of alanine may account for the reduced aggregation of the Phe→Ala mutants of IAPP relative to the wild-type and Phe→Leu peptides. Thus hydrophobic burial, rather than aromaticity, may be the driving force in amyloidogenesis. Consistent with this suggestion, we have previously shown that random mutations of nonpolar side chains in Aβ42 to other nonpolar residues do not prevent aggregation, thereby demonstrating that “generic” hydrophobic interactions may suffice for amyloidogeneis (30
). Our observation that the aggregation rates and morphologies of nonpolar→nonpolar Aβ42 mutants differed from the wild-type peptide, however, suggested that specific steric interactions may guide the precise aggregation pathway (30
More detailed evidence that specific van der Waals packing of nonpolar residues promotes the formation of amyloid structures was provided by the crystallographic studies of Eisenberg and coworkers, who solved the structures of dozens of short peptides derived from various amyloid-forming polypeptides, including Aβ (31
). These peptides all formed “steric zipper” structures of tightly interdigitated β-sheets with closely packed side chains. The precise fit required for steric zipper formation may explain the lag-dependent kinetics of fibril formation: the side chains must adopt the proper rotamers for interdigitation and pack together tightly to exclude solvent. The accompanying decrease in entropy is balanced by enthalpically favorable packing interactions (32
In the current study, we investigate the relative contributions of π-stacking interactions versus generic hydrophobic packing in the aggregation of Aβ by examining variants in which the Phe side chains at positions 19 and 20 are replaced by Ile and Leu residues. Our results show that both the F19L/F20L and F19I/F20I mutants form amyloid fibrils, and do so at higher levels than the wild-type peptide. These findings provide clear evidence that aromatic interactions are not required for the aggregation of Aβ. Distinctions between the Leu and Ile mutants in both their aggregation pathways and fibril morphologies further implicate steric packing as a driving force for amyloidogenesis. Finally, we show that aromatic compounds inhibit not only wild-type Aβ aggregation but also, with similar effectiveness, the aggregation of non-aromatic mutant peptides. Together, these results suggest that steric packing of hydrophobic residues – rather than aromatic π-stacking interactions – promote Aβ aggregation.