INTERACTIONS AND CHAPERONE FUNCTION OF A–CRYSTALLIN TOWARD T5P C–CRYSTALLIN MUTANT

Abstract

Abstract: : Purpose: T5P γC–crystallin mutation is associated with Coppock–like cataract, one of the autosomal dominant congenital cataracts. It is not known why the abundant α–crystallin cannot prevent the mutation related aggregation. Our pervious studies indicate the mutation changes conformation, reduces solubility and stability. It is not known whether these events or loss of interaction with other crystallins causes cataract. It is also not known whether α–crystallin can protect T5P mutant as effectively as wild–type γC–crystallin from heat–induced aggregation. Methods: Human αA–crystallin and W9F mutant as well as γC–crystallin and T5P mutant were cloned. Interactions between αA– and γC–crystallin were studied with fluorescence resonance energy transfer (FRET) and chaperone activity was studied by the ability to suppress heat–induced aggregation of substrate proteins. Conformational changes of substrate proteins were studied by spectroscopic measurements. Results: Chaperone activity measurements show that αA–crystallin could effectively prevent both WT and T5P γC–crystallin from heat–induced aggregation. However, T5P mutant did not show a greater FRET with αA–crystallin than WT γC–crystallin Conclusions: Interactions between αA– and γC–crystallin studied by two–hybrid system, FRET and chaperone activity are different and it appears that loss of interactions with other crystallins is more important than the reduced chaperone protection in Coppock–like cataract.

Keywords: crystallins • protein structure/function • cataract