alpha beta-crystallin selectively targets intermediate filament proteins during thermal stress

PURPOSE. alpha B-Crystallin is a small heat shock protein (sHsp) expressed at high levels in the lens of the eye, where its molecular chaperone functi ons may protect against cataract formation in vivo. The purpose of this stu dy was to identify protein targets for the sHsp alpha B-crystallin in lens cell homogenates during conditions of mild thermal stress. METHODS. The authors report the use of a fusion protein, maltose-binding pr otein alpha B-crystallin (MBP-alpha B), immobilized on amylose resin as a n ovel method for isolating endogenous alpha B-crystallin-binding proteins fr om lens cell homogenates after mild thermal stress. RESULTS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG E) and western immunoblot analyses showed selective interactions in lens ce ll homogenates between MBP-alpha B and endogenous alpha A- and alpha B-crys tallins, the lens-specific intermediate filament proteins phakinin (CP49) a nd filensin (CP115), and vimentin during a mild 20-minute heat shock at 45 degrees C. No interactions were observed with the beta- or gamma-crystallin s, or the cytoskeletal proteins actin, alpha-tubulin, and spectrin, althoug h these proteins were present in lens cell homogenates. In contrast, gamma- crystallin and actin interacted with MBP-alpha B at 45 degrees C only in th eir purified states. The results obtained with MBP-alpha B were confirmed b y immunoprecipitation reactions in which immunoprecipitation of native bovi ne alpha B-crystallin from heat-shocked lens cell homogenates resulted in t he coprecipitation of phakinin and filensin. CONCLUSIONS. In the lens the sHsp alpha B-crystallin may selectively target intermediate filaments for protection against unfolding during conditions of stress.