Abstract
Abstract: :
Purpose: The UPP is a quality control machinery in various types of cells, including lens epithelial cells. To further determine the quality control function of the UPP in lens fibers, we determined whether components and activities of the UPP are retained during maturation of lens fibers. Methods:Calf lenses were dissected into four different regions: epithelial layer (EL), outer cortex (OC), inner cortex (IC) and inner nucleus (IN). Proteins levels of ubiquitination enzymes (E1 and E2s) and subunits of the 26S proteasome were determined by Western blotting. The activities of E1 and E2 were determined by thiolester assays, and the proteasome activity was determined by the proteasome-dependent degradation of ubiquitin conjugates. Isopeptidases were determined by rates of removal of ubiquitin from preformed ubiquitin-protein conjugates Results: We found that all enzymes, including E1, some E2s, proteasome and isopeptidases, were present along the whole thickness of the lens although there was a decreasing gradient from EL to IN. All the layers of the lens had the capacity to form ubiquitin conjugates using endogenous substrates or alpha-lactalbumin as a model substrate. But this capability were significantly lower in IC and IN regions as compared to EL and OC regions. Supplementation of lens supernatant with specific E2s (Ubc4 or Ubc5) can partially restore the conjugation activiies in IC and IN. Lens fibers from all layers also have the capability to degrade ubiquitin-protein conjugates in an ATP-dependent manner. Conclusion: Lens fibers, including those from lens nucleus, have a fully functional UPP. We hypothesize that the UPP in lens fibers may function as a quality control machinery as we demonstrated in lens epithelial cells.
Keywords: 530 proteolysis • 309 aging