Dataset Information

Multiple Aggregation Pathways in Human ?S-Crystallin and Its Aggregation-Prone G18V Variant.

ABSTRACT: Cataract results from the formation of light-scattering precipitates due to point mutations or accumulated damage in the structural crystallins of the eye lens. Although excised cataracts are predominantly amorphous, in vitro studies show that crystallins are capable of adopting a variety of morphologies depending on the preparation method. Here we characterize thermal, pH-dependent, and UV-irradiated aggregates from wild-type human ?S-crystallin (?S-WT) and its aggregation-prone variant, ?S-G18V.Aggregates of ?S-WT and ?S-G18V were prepared under acidic, neutral, and basic pH conditions and held at 25°C or 37°C for 48 hours. UV-induced aggregates were produced by irradiation with a 355-nm laser. Aggregation and fibril formation were monitored via turbidity and thioflavin T (ThT) assays. Aggregates were characterized using intrinsic aromatic fluorescence, powder x-ray diffraction, and mass spectrometry.?S-crystallin aggregates displayed different characteristics depending on the preparation method. ?S-G18V produced a larger amount of detectable aggregates than did ?S-WT and at less-extreme conditions. Aggregates formed under basic and acidic conditions yielded elevated ThT fluorescence; however, aggregates formed at low pH did not produce strongly turbid solutions. UV-induced aggregates produced highly turbid solutions but displayed only moderate ThT fluorescence. X-ray diffraction confirms amyloid character in low-pH samples and UV-irradiated samples, although the relative amounts vary.?S-G18V demonstrates increased aggregation propensity compared to ?S-WT when treated with heat, acid, or UV light. The resulting aggregates differ in their ThT fluorescence and turbidity, suggesting that at least two different aggregation pathways are accessible to both proteins under the conditions tested.

SUBMITTER: Roskamp KW

PROVIDER: S-EPMC5407245 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Multiple Aggregation Pathways in Human γS-Crystallin and Its Aggregation-Prone G18V Variant.

Roskamp Kyle W KW Montelongo David M DM Anorma Chelsea D CD Bandak Diana N DN Chua Janine A JA Malecha Kurtis T KT Martin Rachel W RW

Investigative ophthalmology & visual science 20170401 4

<h4>Purpose</h4>Cataract results from the formation of light-scattering precipitates due to point mutations or accumulated damage in the structural crystallins of the eye lens. Although excised cataracts are predominantly amorphous, in vitro studies show that crystallins are capable of adopting a variety of morphologies depending on the preparation method. Here we characterize thermal, pH-dependent, and UV-irradiated aggregates from wild-type human γS-crystallin (γS-WT) and its aggregation-prone ...[more]

PMID: 28444328

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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.

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.

Dataset Information

Multiple Aggregation Pathways in Human ?S-Crystallin and Its Aggregation-Prone G18V Variant.

Publications

Multiple Aggregation Pathways in Human γS-Crystallin and Its Aggregation-Prone G18V Variant.

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