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The ?A66-80 peptide interacts with soluble ?-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 ?A66-80, a peptide derived from ?A-crystallin and present at higher concentrations in the water-insoluble nuclear fractions of the aging lens. The ?A66-80 peptide has amyloid-like properties and preferentially insolubilizes ?-crystallin from soluble lens fractions. However, the specific interactions and mechanisms by which the peptide induces ?-crystallin aggregation have not been delineated. To gain insight into the mechanisms of peptide-induced aggregation, we investigated the interactions of the peptide with ?-crystallin by various biochemical approaches. The peptide weakens ?-crystallin chaperone ability and drastically promotes ?-crystallin aggregation via the formation of insoluble peptide-protein complexes through transient intermediates. 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid studies suggest that the peptide induces changes in the hydrophobicity of ?-crystallin that could trigger the formation and growth of aggregates. The peptide-?-crystallin aggregates were found to be resistant to dissociation by high ionic strengths, whereas guanidinium hydrochloride and urea were effective dissociating agents. We conclude that the ?A66-80 peptide forms a hydrophobically driven, stable complex with ?-crystallin and reduces its solubility. Using isotope-labeled chemical cross-linking and mass spectrometry, we show that the peptide binds to multiple sites, including the chaperone site, the C-terminal extension, and subunit interaction sites in ?B-crystallin, which may explain the antichaperone property of the peptide and the consequential age-related accumulation of aggregated proteins. Thus, the ?-crystallin-derived peptide could play a role in the pathogenesis of cataract formation in the aging lens.

SUBMITTER: Kannan R

PROVIDER: S-EPMC3796022 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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The αA66-80 peptide interacts with soluble α-crystallin and induces its aggregation and precipitation: a contribution to age-related cataract formation.

Kannan Rama R Santhoshkumar Puttur P Mooney Brian P BP Sharma K Krishna KK

Biochemistry 20130516 21

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 at higher concentrations in the water-insoluble nuclear fractions of the aging lens. The αA66-80 peptide has amyloid-like properties and preferentially insolubilizes α-crystallin from soluble lens fractions. However, th ...[more]

PMID: 23631441

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Project description:Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (μs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Dataset Information

The ?A66-80 peptide interacts with soluble ?-crystallin and induces its aggregation and precipitation: a contribution to age-related cataract formation.

Publications

The αA66-80 peptide interacts with soluble α-crystallin and induces its aggregation and precipitation: a contribution to age-related cataract formation.

Similar Datasets

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