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J. Biol. Chem., Vol. 283, Issue 16, 10500-10512, April 18, 2008

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Truncation of B-Crystallin by the Myopathy-causing Q151X Mutation Significantly Destabilizes the Protein Leading to Aggregate Formation in Transfected Cells^*

Victoria H. Hayes, Glyn Devlin, and Roy A. Quinlan¹

From the School of Biological and Biomedical Sciences, South Road Science Site, Durham University, Durham DH1 3LE and the Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

Here we investigate the effects of a myopathy-causing mutation in B-crystallin, Q151X, upon its structure and function. This mutation removes the C-terminal domain of B-crystallin, which is expected to compromise both its oligomerization and chaperone activity. We compared this to two other B-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 oligomerization of B-crystallin and even increased some chaperone activities, it also significantly destabilized B-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 B-crystallin. The 450delA and 464delCT mutants could only be refolded and assayed as a complex with wild type B-crystallin, which was not the case for Q151X B-crystallin. From these studies, we conclude that all three disease-causing mutations (450delA, 464delCT, and Q151X) in the C-terminal extension destabilize B-crystallin and increase its tendency to self-aggregate. We propose that it is this, rather than a catastrophic loss of chaperone activity, which is a major factor in the development of the reported diseases for the three disease-causing mutations studied here. In support of this hypothesis, we show that Q151X B-crystallin is found mainly in the insoluble fraction of cell extracts from transient transfected cells, due to the formation of cytoplasmic aggregates.

Received for publication, August 3, 2007 , and in revised form, January 29, 2008.

^* This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) and ImmunoDiagnostics Systems BBSRC CASE award (to V. H. H.) and National Institutes of Health NINDS Grant P01NS42803 (to R. A. Q.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed. Fax: 44-191-334-1201; E-mail: r.a.quinlan{at}durham.ac.uk.

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