AlphaB-crystallin selectively targets intermediate filament proteins during thermal stress.
OBJECTIVE
AlphaB-Crystallin is a small heat shock protein (sHsp) expressed at high levels in the lens of the eye, where its molecular chaperone functions may protect against cataract formation in vivo. The purpose of this study was to identify protein targets for the sHsp alphaB-crystallin in lens cell homogenates during conditions of mild thermal stress.


METHODS
The authors report the use of a fusion protein, maltose-binding protein alphaB-crystallin (MBP-alphaB), immobilized on amylose resin as a novel method for isolating endogenous alphaB-crystallin-binding proteins from lens cell homogenates after mild thermal stress.


RESULTS
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western immunoblot analyses showed selective interactions in lens cell homogenates between MBP-alphaB and endogenous alphaA- and alphaB-crystallins, the lens-specific intermediate filament proteins phakinin (CP49) and filensin (CP115), and vimentin during a mild 20-minute heat shock at 45 degrees C. No interactions were observed with the beta- or gamma-crystallins, or the cytoskeletal proteins actin, alpha-tubulin, and spectrin, although these proteins were present in lens cell homogenates. In contrast, gamma-crystallin and actin interacted with MBP-alphaB at 45 degrees C only in their purified states. The results obtained with MBP-alphaB were confirmed by immunoprecipitation reactions in which immunoprecipitation of native bovine alphaB-crystallin from heat-shocked lens cell homogenates resulted in the coprecipitation of phakinin and filensin.


CONCLUSIONS
In the lens the sHsp alphaB-crystallin may selectively target intermediate filaments for protection against unfolding during conditions of stress.
Muchowski
P J
PJ
Department of Biological Structure, University of Washington, Seattle 98195-7420, USA.
Valdez
M M
MM
Clark
J I
JI
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D007908:Lens, Crystalline / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D003459:Crystallins; D005136:Eye Proteins; D006360:Heat-Shock Proteins; D007381:Intermediate Filament Proteins; D011993:Recombinant Fusion Proteins; D014746:Vimentin; C079098:filensin; C084808:phakinin; D008320:Maltose