In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin.
αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America; Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States of America.
Usha P
UP
Andley
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.
James P
JP
Malone
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America; Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.
R Reid
RR
Townsend
D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D003459:Crystallins / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D018260:Gelsolin / Q000235:genetics / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000235:genetics / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000378:metabolism*
D003459:Crystallins; D005136:Eye Proteins; D018260:Gelsolin; D007381:Intermediate Filament Proteins; D009124:Muscle Proteins; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; C079098:filensin; D002154:Calpain; C491373:Capn3 protein, mouse