Mechanism of Small Heat Shock Protein Function in Vivo: A KNOCK-IN MOUSE MODEL DEMONSTRATES THAT THE R49C MUTATION IN {alpha}A-CRYSTALLIN ENHANCES PROTEIN INSOLUBILITY AND CELL DEATH

doi:10.1074/jbc.M708704200

Mechanism of Small Heat Shock Protein Function in Vivo

A KNOCK-IN MOUSE MODEL DEMONSTRATES THAT THE R49C MUTATION IN ${alpha}$ 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

${alpha}$ A-crystallin (Cryaa/HSPB4) is a small heat shock protein andmolecular chaperone that prevents nonspecific aggregation ofdenaturing proteins. Several point mutations in the ${alpha}$ A-crystallingene cause congenital human cataracts by unknown mechanisms.We took a novel approach to investigate the molecular mechanismof cataract formation in vivo by creating gene knock-in miceexpressing the arginine 49 to cysteine mutation (R49C) in ${alpha}$ A-crystallin( ${alpha}$ A-R49C). This mutation has been linked with autosomal dominanthereditary cataracts in a four-generation Caucasian family.Homologous recombination in embryonic stem cells was performedusing a plasmid containing the C to T transition in exon 1 ofthe cryaa gene. ${alpha}$ A-R49C heterozygosity led to early cataractscharacterized by nuclear opacities. Unexpectedly, ${alpha}$ A-R49C homozygosityled to small eye phenotype and severe cataracts at birth. Wildtype littermates did not show these abnormalities. Lens fibercells of ${alpha}$ A-R49C homozygous mice displayed an increase in celldeath by apoptosis mediated by a 5-fold decrease in phosphorylatedBad, an anti-apoptotic protein, but an increase in Bcl-2 expression.However, proliferation measured by in vivo bromodeoxyuridinelabeling did not decline. The ${alpha}$ A-R49C heterozygous and homozygousknock-in lenses demonstrated an increase in insoluble ${alpha}$ A-crystallinand ${alpha}$ B-crystallin and a surprising increase in expression ofcytoplasmic ${gamma}$ -crystallin, whereas no changes in β-crystallinwere observed. Co-immunoprecipitation analysis showed increasedinteraction between ${alpha}$ A-crystallin and lens substrate proteinsin the heterozygous knock-in lenses. To our knowledge this isthe first knock-in mouse model for a crystallin mutation causinghereditary human cataract and establishes that ${alpha}$ A-R49C promotesprotein 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 NEIGrant R01EY05681 (to U. P. A.) and Core Grant EY02687. The costsof publication of this article were defrayed in part by thepayment of page charges. This article must therefore be herebymarked "advertisement" in accordance with 18 U.S.C. Section1734 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.

Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Add to Reddit Reddit Add to Technorati Technorati What's this?