Abstract
Purpose: :
Differentiation in many types of tissues involves the ubiquitin–proteasome pathway. Ubiquitination of substrates is assisted by a series of enzymes including ubiquitin–conjugating (E2) enzyme Ubc4. In the past we observed several unidentified conjugates when Ubc4 was added to lysates of lens cortical tissue in reconstitution assays. The purpose of this study was to identify proteins that are conjugated to ubiquitin when Ubc4 is added to rat lens lysates.
Methods: :
Lenses from 1–month old rat were micro–dissected to obtain anterior and equatorial epithelia, outer cortex fibers, and lens core. Soluble proteins were extracted using buffer containing 50 mM Tris–HCl (pH 7.6) and 1 mM DTT. Ubiquitin–conjugation assays were performed using the above lysates supplemented with 1 mM ATP, 2 µg of (His)6–ubiquitin, and 100 ng of Ubc4 in 20 µl at 37oC for 1 hour. 20 µl of the reaction mixture was loaded on 15% SDS–PAGE and immunoblotted with anti–ubiquitin antibody for the detection of ubiquitin conjugation substrates. The remaining samples were purified on a nickel column using buffer containing 20 mM Tris–HCl (pH 7.6) and 8 M urea. Purified reaction products were separated on a 15% SDS gel and stained with colloidal coomassie G–250. Visible bands were cut and destained. After reduction and alkylation, gel slices were digested with trypsin and the tryptic peptides were analyzed by a LC/MS/MS. A SEQUEST search of the LC/MS/MS data was done using the NCBI non–redundant protein database.
Results: :
Western blot analysis showed increased ubiquitin conjugation activity in lens epithelium and cortex fibers which were supplemented with Ubc4. Five new ubiquitin conjugates were detected when exogenous Ubc4 was added. LC/MS/MS indicated the presence of ubiquitin in every Ubc4–induced conjugate. These conjugates contained beta–crystallin, a component of 40S ribosome, and thimet oligoendopeptidase. Ubiquitination sites on substrates were also determined.
Conclusions: :
Addition of Ubc4 reactivates a complete ubiquitination system, suggestes that Ubc4 is inactivated and/or is rate limiting upon development. These findings confirmed our previous observation that supplementation of Ubc4 may promote conjugate formation in all layers of lens. Further characterization and functional studies of these conjugates will advance our understanding of the role of Ubc4 in lens proliferation and differentiation.
Keywords: differentiation • protein modifications-post translational • proteolysis