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Effect of ?B-crystallin on protein aggregation in Drosophila.

ABSTRACT: Disorganisation and aggregation of proteins containing expanded polyglutamine (polyQ) repeats, or ectopic expression of ?-synuclein, underlie neurodegenerative diseases including Alzheimer's, Parkinson, Huntington, Creutzfeldt diseases. Small heat-shock proteins, such as ?B-crystallin, act as chaperones to prevent protein aggregation and play a key role in the prevention of such protein disorganisation diseases. In this study, we have explored the potential for chaperone activity of ?B-crystallin to suppress the formation of protein aggregates. We tested the ability of ?B-crystallin to suppress the aggregation of a polyQ protein and ?-synuclein in Drosophila. We found that ?B-crystallin suppresses both the compound eye degeneration induced by polyQ and the ?-synuclein-induced rough eye phenotype. Furthermore, by using histochemical staining we have determined that ?B-crystallin inhibits the aggregation of polyQ in vivo. These data provide a clue for the development of therapeutics for neurodegenerative diseases.

SUBMITTER: Tue NT

PROVIDER: S-EPMC3312385 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Effect of αB-crystallin on protein aggregation in Drosophila.

Tue Nguyen Trong NT Shimaji Kouhei K Tanaka Naoki N Yamaguchi Masamitsu M

Journal of biomedicine & biotechnology 20120307

Disorganisation and aggregation of proteins containing expanded polyglutamine (polyQ) repeats, or ectopic expression of α-synuclein, underlie neurodegenerative diseases including Alzheimer's, Parkinson, Huntington, Creutzfeldt diseases. Small heat-shock proteins, such as αB-crystallin, act as chaperones to prevent protein aggregation and play a key role in the prevention of such protein disorganisation diseases. In this study, we have explored the potential for chaperone activity of αB-crystalli ...[more]

PMID: 22505806

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Project description:BackgroundThe eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human ?S-crystallin (?S-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein ?B-crystallin does not interact with wild-type ?S-crystallin, but does bind its G18V variant. The specific molecular determinants of ?B-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of ?S, ?S-G18A, was created to test the limits of ?B-crystallin selectivity.MethodsMolecular dynamics simulations were used to investigate the structure and dynamics of ?S-G18A. The overall fold of ?S-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between ?B-crystallin and ?S-G18A.Results?S-G18A exhibits minimal structural changes, but has compromised thermal stability relative to ?S-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human ?B-crystallin does not bind the G18A variant, in contrast to previous observations for ?S-G18V, which aggregates at physiological temperature.Conclusions?B-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant.General significanceHuman ?B-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related ?S-G18V variant, but not the function-preserving ?S-G18A variant, which is monomeric at physiological temperature.

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.

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Effect of ?B-crystallin on protein aggregation in Drosophila.

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Effect of αB-crystallin on protein aggregation in Drosophila.

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