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R. A. Quinlan, V. Hayes, A. Landsbury, G. Devlin; Structure and Function Analyses of the Disease-Causing Q151x Mutation in Alphab-Crystallin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5025.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the structural and functional consequences of the Q151X mutation in αB-crystallin in comparison with other disease causing mutations affecting the C-terminus
The expression construct (pET23) for αB-crystallin was constructed by a SDM strategy. Protein expression was monitored by SDS-PAGE. Unlike other αB-crystallinproteins expressed using this system the protein did not remain soluble and had to be purified to homogeneity after solubilisation in urea-containing buffers by anion exchange chromatography. The functional integrity of the soluble recombinant protein after a refolding strategy was analysed by a series of assays, including CD, bisANS binding and size exclusion chromatography. In vitro chaperone assays, desmin filament binding assays and electron microscopy using negatively stained samples. The activity was compared to wild type αB-crystallin and aseries of other C-terminal mutations in αB-crystallin linked to human disease, such as 464delCT, 450delA and R120G.
This mutation removes the C-terminal domain of αB-crystallin, which is expected to compromise both its oligomerisation and chaperone activity. We compared this to two other α αB-crystallin mutants (450delA, 464delCT) and also to a series of C-terminal truncations (E164X, E165X, K174X and A171X). We find that the effects of the Q151X mutation were not always as predicted. Specifically, we have found that although the Q151X mutation decreased oligomerisation of αB-crystallin, it increased some chaperone activities and also significantly destabilized αB-crystallin causing it to self-aggregate. This conclusion was supported by our analyses of both the other disease causing mutants and the series of C-terminal truncation constructs of αB-crystallin. The450delA and 464delCT mutants could only be refolded and assayed as a complex with wild type αB-crystallin, which was not the case for Q151X αB-crystallin. From these studies, we conclude that all three disease-causing mutations (450delA, 464delCT and Q151X) in the C-terminal extension destabilise αB-crystallin and increase its tendency to self-aggregate.
We propose that it is the dramatic loss of stability, rather than a catastrophic loss of chaperone activity, that is a major factor in disease development and in support of this hypothesis we show by transient transfection that Q151X αB-crystallin is found mainly in the insoluble fraction of cell extracts due to the formation of cytoplasmic aggregates.
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