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.
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.
Xi
Jing-hua
JH
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Bai
Fang
F
Gross
Julia
J
Townsend
R Reid
RR
Menko
A Sue
AS
Andley
Usha P
UP
D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002386:Cataract / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016923:Cell Death / Q000235:genetics; D003459:Crystallins / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000473:pathology; D005091:Exons / Q000235:genetics; D030342:Genetic Diseases, Inborn; D050888:Heat-Shock Proteins, Small / Q000235:genetics / Q000378:metabolism*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D020125:Mutation, Missense; D010766:Phosphorylation; D011995:Recombination, Genetic / Q000235:genetics; D012995:Solubility; D044465:White People; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism
C494820:Bad protein, mouse; C511246:CRYAA protein, human; D003459:Crystallins; D050888:Heat-Shock Proteins, Small; D051025:bcl-Associated Death Protein