In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin

In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin. Abstract aA-crystallin and aB-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 aA-R49C and aB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old aA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of aA-crystallin; increased association between aA-crystallin and filensin, actin, or creatine kinase B; increased acidification of bB1-crystallin; increased levels of grifin; and an association between bA3/A1-crystallin and aA-crystallin. Homozygous aA-R49C mutant lenses exhibited increased associations between aA-crystallin and bB3-, bA4-, bA2-crystallins, and grifin, whereas levels of bB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, a-enolase, and cytochrome c increased more than 50-fold in homozygous aA-R49C mutant lenses. In aB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several band c-crystallins, and degradation of aA-and aB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal a-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 aA-and aB-crystallin. Copyright: ß 2014 Andley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.