18056999

pubmed:Citation

9

alphaA-crystallin (Cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents nonspecific aggregation of denaturing proteins. Several point mutations in the alphaA-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 alphaA-crystallin (alphaA-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. alphaA-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, alphaA-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type littermates did not show these abnormalities. Lens fiber cells of alphaA-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 alphaA-R49C heterozygous and homozygous knock-in lenses demonstrated an increase in insoluble alphaA-crystallin and alphaB-crystallin and a surprising increase in expression of cytoplasmic gamma-crystallin, whereas no changes in beta-crystallin were observed. Co-immunoprecipitation analysis showed increased interaction between alphaA-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 alphaA-R49C promotes protein insolubility and cell death in vivo.

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MEDLINE

0021-9258

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pubmed-meshheading:18056999-Amino Acid Substitution, pubmed-meshheading:18056999-Animals, pubmed-meshheading:18056999-Apoptosis, pubmed-meshheading:18056999-Cataract, pubmed-meshheading:18056999-Cell Death, pubmed-meshheading:18056999-Crystallins, pubmed-meshheading:18056999-Disease Models, Animal, pubmed-meshheading:18056999-Embryonic Stem Cells, pubmed-meshheading:18056999-European Continental Ancestry Group, pubmed-meshheading:18056999-Exons, pubmed-meshheading:18056999-Genetic Diseases, Inborn, pubmed-meshheading:18056999-Heat-Shock Proteins, Small, pubmed-meshheading:18056999-Heterozygote, pubmed-meshheading:18056999-Homozygote, pubmed-meshheading:18056999-Humans, pubmed-meshheading:18056999-Mice, pubmed-meshheading:18056999-Mice, Transgenic, pubmed-meshheading:18056999-Mutation, Missense, pubmed-meshheading:18056999-Phosphorylation, pubmed-meshheading:18056999-Recombination, Genetic, pubmed-meshheading:18056999-Solubility, pubmed-meshheading:18056999-bcl-Associated Death Protein

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.

Journal Article, Research Support, N.I.H., Extramural