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J. Biol. Chem., Vol. 283, Issue 9, 5801-5814, February 29, 2008

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Mechanism of Small Heat Shock Protein Function in Vivo

A KNOCK-IN MOUSE MODEL DEMONSTRATES THAT THE R49C MUTATION IN A-CRYSTALLIN ENHANCES PROTEIN INSOLUBILITY AND CELL DEATH^*

Jing-hua Xi, Fang Bai, Julia Gross, R. Reid Townsend^¶, A. Sue Menko^||, and Usha P. Andley^**1

From the Departments of Ophthalmology and Visual Sciences, ^**Biochemistry and Molecular Biophysics, Internal Medicine, and ^¶Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^||Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

A-crystallin (Cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents nonspecific aggregation of denaturing proteins. Several point mutations in the A-crystallin gene cause congenital human cataracts by unknown mechanisms. We took a novel approach to investigate the molecular mechanism of cataract formation in vivo by creating gene knock-in mice expressing the arginine 49 to cysteine mutation (R49C) in A-crystallin (A-R49C). This mutation has been linked with autosomal dominant hereditary cataracts in a four-generation Caucasian family. Homologous recombination in embryonic stem cells was performed using a plasmid containing the C to T transition in exon 1 of the cryaa gene. A-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, A-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type littermates did not show these abnormalities. Lens fiber cells of A-R49C homozygous mice displayed an increase in cell death by apoptosis mediated by a 5-fold decrease in phosphorylated Bad, an anti-apoptotic protein, but an increase in Bcl-2 expression. However, proliferation measured by in vivo bromodeoxyuridine labeling did not decline. The A-R49C heterozygous and homozygous knock-in lenses demonstrated an increase in insoluble A-crystallin and B-crystallin and a surprising increase in expression of cytoplasmic -crystallin, whereas no changes in β-crystallin were observed. Co-immunoprecipitation analysis showed increased interaction between A-crystallin and lens substrate proteins in the heterozygous knock-in lenses. To our knowledge this is the first knock-in mouse model for a crystallin mutation causing hereditary human cataract and establishes that A-R49C promotes protein insolubility and cell death in vivo.

Received for publication, October 22, 2007 , and in revised form, November 30, 2007.

^* This work was supported by National Institutes of Health NEI Grant R01EY05681 (to U. P. A.) and Core Grant EY02687. 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 and S2 and Table 1.

¹ To whom correspondence should be addressed: Dept. of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8096, St. Louis, MO 63110. Tel.: 314-362-7167; Fax: 314-362-3638; E-mail: andley{at}vision.wustl.edu.

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