Oligomerization with wt ?±A- and ?±B-Crystallins Reduces Proteasome-Mediated Degradation of C-Terminally Truncated ?±A-Crystallin

摘要

Purpose.: We previously demonstrated that the ubiquitin-proteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated ?±A-crystallin. The objective of this work was to determine the effects of wt ?±A- and ?±B-crystallins on the degradation of C-terminally truncated ?±A-crystallin (?±A1a??162) and vice versa. Methods.: Recombinant wt ?±A, ?±B, and ?±A1a??162 were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multiangle-light scattering and coprecipitation assays. Protein substrates were labeled with 125I and lens epithelial cell lysates were used as the source of the UPP for degradation assays. Results.: FRET, multiangle light scattering, and coprecipitation assays showed that ?±A1a??162 exchanged subunits with wt ?±A- or wt ?±B- crystallin to form hetero-oligomers. ?±A1a??162 was more susceptible than wt ?±A-crystallin to degradation by the UPP. When mixed with wt ?±A-crystallin at 1:1 or 1:4 (?±A1a??162 : wt) ratios to form hetero-oligomers, the degradation of ?±A1a??162 was significantly decreased. Conversely, formation of hetero-oligomers with ?±A1a??162 enhanced the degradation of wt ?±A-crystallin. The presence of ?±A1a??162, but not wt ?±A-crystallin, decreased the degradation of wt ?±B-crystallin. Conclusions.: ?±A1a??162 forms hetero-oligomers with wt ?±A- and ?±B-crystallins. Oligomerization with wt ?±A- or ?±B-crystallins reduces the susceptibility of ?±A1a??162 to degradation by the UPP. In addition, the presence of ?±A1a??162 in the hetero-oligomers also affects the degradation of wt ?±A- and ?±B-crystallins.