The objective of this proposal is to elucidate the effect of some common posttranslational modifications of alpha-crystallin (alphaA and alphaB) on the molecular chaperone property, i.e., the ability of alpha-crystallin to protect other proteins from denaturation and aggregation. By choosing biologically relevant modifications different domains of alphaA- and alphaB-crystallins will be targeted for in vitro structural modifications which in turn will be correlated with changes in chaperone function. Studying in vitro modification will facilitate the characterization of in vivo modifications that cause a decline in chaperone like property of human alpha-crystallin. This is the first study ever where posttranslational modifications that occur in vivo and that can be inflicted in vitro will be correlated with chaperone activity and chaperone-target protein binding.
The specific aims of this proposal are based on our most recent findings that glycation, oxidation and mixed disulfide formation have inhibitory effects on alpha-crystallin chaperone function and that both aging and diabetes decrease the chaperone property. The following specific aims are hypothesis driven, each designed to test a specific hypothesis: 1) In vitro modifications of calf or young human alpha-crystallin by oxidation with H2O2, by glycation with ascorbic acid and fructose, and by mixed disulfide formation with GSSG will be used to test the hypothesis that such posttranslational modifications will influence the chaperone function as determined by the ability of alpha-crystallin to protect beta- and gamma-crystallin from thermal denaturation and aggregation. In addition, we will study the combined effect of more than one type of modification hoping to show a synergistic effect by two treatments. 2) With both alphaL and alphaH fractions from 1 month to 90 years old human lenses it will be shown whether with increasing age increasing levels of posttranslationally modified alpha-crystallin with altered chaperone function accumulates. 3) Since diabetic lenses are exposed to higher levels of oxidation and glycation than age-matched non-diabetic lenses we will test the possibility that the alpha-crystallin chaperone function is altered even more in diabetic human or rat lenses. 4) We will identify the protein modifications in alphaA- and alphaB-crystallins that will be introduced in vitro or that occur in vivo by mass spectral analysis and correlate with changes in chaperone function including chaperone-target protein binding. These analyses will identify the types of modifications that produce a decrease in the chaperone property. 5) We will investigate the mechanism of the loss of chaperone function due to in vitro modifications or in vivo modifications during aging or diabetes. We will test the hypothesis that posttranslational modifications affect the chaperone-target protein binding leading to reduced chaperone function.
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