Abstract
Purpose: :
The ubiquitin-proteasome pathway (UPP) is an important protein quality control mechanism which selectively degrades abnormal and damaged proteins. The objective of this work is to determine the role of the UPP in degradation of cataract-causing mutant crystallins and to investigate the physiological significance of such degradation.
Methods: :
Recombinant wild type (wt) and mutant αB-crystallins and γC-crystallins were expressed in bacterial and purified to homogeneity. The susceptibility of these crystallins to ubiquitin-mediated degradation was assessed in lens epithelial cell lysate. To investigate the physiological significance of the degradation of these mutant crystallins, wt and mutant αB-crystallins were fused with red fluorescence protein (RFP)and co-expressed with wt or dominant negative mutant ubiquitin in HeLa cells. The intracellular aggregates of the αB-crystallin-RFP fusion proteins were monitored under fluorescent microscope.
Results: :
Wt γC-crystallin was resistant to degradation, but T5P-mutant γC-crystallin was rapidly degraded. Addition of K6W-mutant, but not wt ubiquitin, inhibits the degradation of T5P-mutant γC-crystallin, indicating that the degradation is ubiquitin-dependent. Wt αB-crystallin was readily degraded and addition of K6W-ubiquitin inhibited the degradation. Whereas D140N mutant αB-crystallin was more susceptible than wt αB-crystallin to degradation by the UPP, R120G mutant αB-crystallin was less susceptible to degradation by the UPP. In a cell free system, both R120G and D140N mutant αB-crystallin formed amyloid-like aggregates. However, when expressed in HeLa cells, only R120G mutant αB-crystallin formed perinuclear aggregates. However, when the UPP was impaired by co-expression with K6W-mutant ubiquitin, both R120G and D140N mutant αB-crystallin formed perinuclear aggregates.
Conclusions: :
The data indicate that the UPP degrades some, but not all, mutant crystallins. Failure in degradation of the mutant crystallins results in aggregation of such mutant proteins in the cells. Accumulation and aggregation of damaged proteins is associated with cataract formation, and therefore an active UPP in the lens is essential for preventing the accumulation and aggregation of mutant and other forms of damaged crystallins to maintain the transparency of the lens.
Keywords: proteolysis • crystallins • cataract