Oligomerization with wt A And B-crystallins Prevents the Degradation of C-terminal Truncated A-crystallins by the Proteasome

Abstract

Purpose: : We previously demonstrated that the ubiquitin-proteasome is general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminal truncated A-crystallin (alpha A 1-162). The objective of this work is to determine the effects of wt A and B-crystallins on the degradation of A_1-162 by the proteasome.

Methods: : Recombinant wt A, B and A _1-162 were expressed in E. coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multiangle-light scattering and co-precipitation assays. The test substrates were labeled with ¹²⁵I and the degradation was performed in the supernatants of lens epithelial cells.

Results: : FRET, multiangle light scattering and co-precipitation assays showed that the A_1-162 exchanged subunits with wt A or wt B cyrstallin oligomers as efficiently as wt A-crystallin. The A_1-162 was more susceptible than wt A crystallin to degradation by the proteasome. When mixed with wt A crystallin at 1:1 or 1:4 ratios to form hetero-oligomers, the degradation of A_1-162 was significantly decreased. In contrast, formation of hetero-oligomers with A_1-162 enhanced the degradation of wt A-crystallin. The presence of the A_1-162, but not wt A-crystallin, decreased the degradation of wt B-crystallin. The apparent inhibition of the degradation of aB-crystallin by the A_1-162 was due to competition between A_1-162 and B-crystallin for the proteasome.

Conclusions: : C-terminal truncated A-crystallin may exist as hetero-oligomers with wt A and B crystallins in the lens. Oligomerization with wt A or B crystalins reduces the susceptibility of the C-terminal truncated A crystallin to degradation by the proteasome. These data suggest that oligomerization with wt A or B-crystallins may mask the degradation signals on the C-terminal truncated A-crystallin and reduce its susceptibility to proteasome-mediated degradation.

Keywords: proteolysis • protein modifications-post translational • protein structure/function