The αA66–80 Peptide Interacts with Soluble α-Crystallin and Induces Its Aggregation and Precipitation: A Contribution to Age-Related Cataract Formation

Abstract

Formation of protein aggregates in the aging eye lens has been shown to correlate with progressive accumulation of specific low molecular weight (LMW) peptides derived from crystallins. Prominent among the LMW fragments is αA66-80, a peptide derived from αA-crystallin and present in increased concentrations in the water-insoluble (WIS) nuclear fractions of the aging lens. The αA66-80 peptide has amyloid-like properties and preferentially insolubilizes α-crystallin from soluble lens fractions. However, the specific interactions and mechanisms by which the peptide induces α-crystallin aggregation have not been delineated. To gain insights into the mechanisms of peptide-induced aggregation, we investigated the peptide interactions with α-crystallin by various biochemical approaches. The peptide diminishes α-crystallin chaperone ability and drastically promotes α-crystallin aggregation by formation of insoluble peptide-protein complexes through transient intermediates. Bis-ANS studies suggest that the peptide induces changes in hydrophobicity of α-crystallin that could trigger the formation and growth of aggregates. The peptide-α-crystallin aggregates were found to be resistant to dissociation by high ionic strength, whereas guanidium hydrochloride and urea were effective dissociating agents. We conclude that the αA66-80 peptide forms a hydrophobically driven, stable complex with α-crystallin and reduces its solubility. Using isotope-labeled chemical crosslinking and mass spectrometry, we show that the peptide binds to multiple sites, including the chaperone site, C-terminal extension and subunit interaction sites in αB-crystallin, which may explain the anti-chaperone property of the peptide and the consequential age-related accumulation of aggregated proteins. Thus, the α-crystallin-derived peptide could play a role in the pathogenesis of cataract formation in the aging lens.

The critical role of the central hydrophobic core (residues 71–77) of amyloid-forming αA66-80 peptide in α-crystallin aggregation: a systematic proline replacement study

“…These findings correlate with the previous observation that hydrophobic interactions stabilize αA66-80–α-crystallin complexes and that the peptide increases the surface hydrophobicity of α-crystallin, which in turn drives the aggregation process [19]. …”

“…The αA66-80 peptide binds to multiple sites in α-crystallin, including the chaperone site, the subunit interaction region and the C-terminal extension. The αA66-80 peptide suppresses α-crystallin chaperone activity, decreases the solubility of α-crystallin and increases the surface hydrophobicity in α-crystallin by forming stable non-covalent interactions with α-crystallin [19]. In order to understand the mechanisms of peptide-induced aggregation and to design inhibitors to control peptide-mediated protein precipitation, it is important to identify which amino acids in the primary sequence of the peptide are critical for its aggregation activity.…”

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization

“…In contrast, when a scrambled αA66-80 peptide was used in the assay in place of αA66-80 peptide, there was no loss in α-crystallin chaperone activity toward aggregating ADH, suggesting that the effect of αA66-80 peptide was specific. We demonstrated in earlier studies that control peptides have no effect on the chaperone function of α-crystallin (Kannan et al, 2013) and the effect of αA66-80 peptide appears specific. SDS-PAGE analysis of the precipitate obtained from a separate experiment with a different ratio of peptide and a fixed amount of ADH revealed co-precipitation of αA66-80 peptide and α-crystallin.…”

Using chiral peptide substitutions to probe the structure function relationship of a key residue of Aβ42

“…9 Signal intensity subsequently began to decline, presumably due to formation of insoluble (fibrillary) species. 10 The methylene shortening of the sidechain in framework 2 resulted in a pronounced delay in β-sheet formation, with the molecule still exhibiting substantial random coil characteristics after 2 h of incubation.…”