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

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Abstract
Purpose. Calpain-mediated C-terminal cleavage of αA-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 αA-crystallins. methods. Recombinant wild-type (wt) αA-crystallin and C-terminal truncated αA1–168-, αA1–163-, and αA1–162-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt 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 Bis-ANS were used to characterize the wt and truncated αA-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering. results. Whereas wt αA-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, αA1–168-crystallin was resistant to degradation. The susceptibility of αA1–163-crystallin to degradation was comparable to that of wt αA-crystallin. However, αA1–162-crystallin was much more susceptible than wt αA-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated αA1–162-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 αA-crystallins revealed that C-terminal truncation of αA-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 αA-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of αA1–162 by the UPP may prevent its accumulation in the lens.