Oligomerization with wt αA- and αB-Crystallins Reduces Proteasome-Mediated Degradation of C-Terminally Truncated αA-Crystallin

Abstract

PURPOSE. We previously demonstrated that the ubiquitinproteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated aA-crystallin. The objective of this work was to determine the effects of wt aAand aB-crystallins on the degradation of C-terminally truncated aA-crystallin (aA ) and vice versa.METHODS. Recombinant wt aA, aB, and aA 1-162 were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multianglelight scattering and coprecipitation assays. Protein substrates were labeled with 125 I 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 aA 1-162 exchanged subunits with wt aA-or wt aB-crystallin to form hetero-oligomers. aA 1-162 was more susceptible than wt aA-crystallin to degradation by the UPP. When mixed with wt aA-crystallin at 1:1 or 1:4 (aA 1-162 : wt) ratios to form hetero-oligomers, the degradation of aA 1-162 was significantly decreased. Conversely, formation of hetero-oligomers with aA 1-162 enhanced the degradation of wt aAcrystallin. The presence of aA 1-162 , but not wt aA-crystallin, decreased the degradation of wt aB-crystallin.CONCLUSIONS. aA 1-162 forms hetero-oligomers with wt aA-and aB-crystallins. Oligomerization with wt aA-or aB-crystallins reduces the susceptibility of aA 1-162 to degradation by the UPP. In addition, the presence of aA 1-162 in the hetero-oligomers also affects the degradation of wt aA-and aB-crystallins. (Invest Ophthalmol Vis Sci. 2012;53:2541-2550) DOI: 10.1167/iovs.11-9147 L ens fiber cells contain high concentrations of crystallins in the cytoplasm. These crystallins provide the lens with a high refractive index and minimize light scattering at the membranecytoplasm interface. The proper packing of these proteins in cells is essential for maintaining lens transparency.1,2 Disruption of the proper arrangement of these crystallins by adverse modifications is causally related to cataractogenesis.3-6 With aging and environmental insults, lens proteins undergo various modifications, such as oxidation, glycation, deamidation, and truncations. [7][8][9][10][11][12][13][14][15][16] These modified or damaged proteins are usually unstable in aqueous solution and tend to aggregate and precipitate. The aggregation and precipitation of damaged/ modified proteins accounts for a large portion of the age-and cataract-related increases in water-insoluble proteins in the lens.17-24 Thus, efficient removal or repair of the damaged or adversely modified proteins before their aggregation and precipitation appears to be essential for lens transparency.The ubiquitin-proteasome pathway (UPP) is an important protein quality control mechanism that selectively recognizes and degrades proteins with abnormal structures. [25][26][27][28][29][30][31] W...

Enhancement of Ubiquitin Conjugation Activity Reduces Intracellular Aggregation of V76D Mutant γD-Crystallin

“…Although the results were obtained using V76D mutant cD-crystallin as a model substrate, the impact of this work is not limited to the clearance of mutant cD-crystallins, but also applies to other forms of misfolded or damaged proteins, because many forms of damaged proteins, including truncated, deamidated, and oxidized crystallins, are selectively degraded by the UPP. 41,43,47,62,[73][74][75][76] This work proved the principle that timely degradation of mutant proteins in living lens cells prevents its accumulation and aggregation. However, these results need to be confirmed in fiber cells in future studies, since the majority of mutant crystallins are in lens fiber cells.…”

The multifaceted nature of αB-crystallin

“…Proteolytic and ubiquitinating enzymes sourced from human lymphatic endothelial cell lysate, reflective of the ubiquitin-proteasome pathway (UPP), were co-incubated with hetero-oligomers formed between αA-c 1-62 and full length αA-c or αB-c. Hetero-oligomerisation of WT-αA-c or WT-αB-c with αA-c 1-62 made the latter more resistant to degradation compared to αA-c 1-62 alone. However, hetero-oligomerisation with αA-c 1-62 also changed the resistance to degradation by the UPP of WT-αA-c and WT-αB-c. WT-αA-c became more susceptible to degradation, whereas WT-αB-c became more resistant (Wu et al 2012). Similarly, the interaction of these αA-c mutants with Hsp27 stimulated the UPP, a response mediated by Hsp27 (Zhang et al 2010).…”

Lens regeneration in humans: using regenerative potential for tissue repairing

“…These results show that the proteome of mammalian regenerative lens mimics a "mature" lens, from this point of view, the regeneration process does not fully simulate the embryonic development. Congenital cataracts caused by gene defect is one of the main causes of lens opacification in infants (19)(20)(21)(22). The proteome of mammalian regenerative lens indicates that in the process of lens regeneration after surgery, even without additional molecular intervention, transparent regenerated lens may be obtained for congenital cataracts caused by abnormal embryonic crystallin (16).…”