A Relay Between Calpain and Ubiquitin-Proteasome Pathway in Degradation of A-Crystallin

Abstract

Purpose:: Calpain-mediated C-terminal cleavage of αA-cystallins occurs during aging and diabetic cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in removing the C-terminal truncated αA-cystallins.

Methods:: Recombinant wild-type αA-crystallin and C-terminal truncatedαA_1-168, αA_1-163 and αA_1-162-crystallins were expressed in E. coli and purified to homogeneity. Native and truncated αA-crystallins were labeled with ¹²⁵I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe 4,4’-dianilino-1,1’-binaphthalene-5,5’-disulfonic acid (Bis-ANS) were used to characterize the native and truncated αA-crystallin. The oligomer sizes of these crystallins were determined by multi-angle light scattering.

Results:: Whereas native αA-crystallin was degraded at a moderate rate in both lens fiber lysate and reticulocyte lysate, αA_1-168 crytallin was resistant to degradation. However, αA_1-162-crystallin was degraded at significant higher rates than the native crystalline in both lens epithelial cell and reticulocyte lysates. The degradation of both native and c-terminal truncated αA-crystallin requires ATP and was stimulated by addition of a ubiquitin conjugating enzyme, Ubc4. The degradation was substantially inhibited by the proteasome inhibitor (MG132) and a dominant negative mutant of ubiquitin (K6W-Ub), indicating that at least part of the proteolysis was mediated by the UPP. Spectroscopic analyses of wild-type and C-terminal truncated αA-crystallin revealed that C-terminal truncated αA-crystallin have subtle conformational changes altered surface hydrophobicity and altered oligomer states. These conformational changes may reveal or mask the signal for the ubiquitin-dependent degradation.

Conclusions:: The present data demonstrated that there is a relay between calpains and the UPP in degradation of αA-crystallin. The rapid degradation of αA-1-162 by the UPP may prevent its accumulation in the lens.

Keywords: crystallins • proteolysis • cataract