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Degradation of C-terminal Truncated A-crystallins by the Ubiquitin–Proteasome Pathway

¹From the Laboratory for Nutrition and Vision Research, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts; the ²Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; the ³Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California.

PURPOSE. Calpain-mediated C-terminal cleavage of A-crystallins occurs during aging and cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in degrading C-terminal truncated A-crystallins.

METHODS. Recombinant wild-type (wt) A-crystallin and C-terminal truncated A_1–168-, A_1–163-, and A_1–162-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt and truncated A-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 Bis-ANS were used to characterize the wt and truncated A-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering.

RESULTS. Whereas wt A-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, A_1–168-crystallin was resistant to degradation. The susceptibility of A_1–163-crystallin to degradation was comparable to that of wt A-crystallin. However, A_1–162-crystallin was much more susceptible than wt A-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated A_1–162-crystallins requires adenosine triphosphate (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 wt and C-terminal truncated A-crystallins revealed that C-terminal truncation of A-crystallin resulted in only subtle changes in secondary structures. However, C-terminal truncations resulted in significant changes in surface hydrophobicity and thermal stability. Thus, these conformational changes may reveal or mask the signals for the ubiquitin-dependent degradation.

CONCLUSIONS. The present data demonstrate that C-terminal cleavage of A-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of A_1–162 by the UPP may prevent its accumulation in the lens.