Abstract
Purpose: :
Ubiquitin is involved in many cellular functions. Previously we demonstrated that targeted expression of a dominant negative ubiquitin in lens results in defects in lens development and differentiation. The purpose of this work is to investigate the effects of mutant ubiquitin on the lens proteome.
Methods: :
Lenses from wild type and K6W ubiquitin expressing mice were homogenized in 20 mM phosphate buffer containing 1 mM EGTA, pH 7.0. 100 μg of lens protein from each sample was dried and dissolved in 8 M deionized urea, reduced with dithioerythritol (DTT) and alkylated iodoacetamide. After dilution with water, proteins in the sample were digested with trypsin. After desalting, peptides were first separated into 8 fractions using Reliasil cation exchange cartridge. 8 fractions from each digest were analyzed by reversed phase LC/MS/MS. MS.MS spectra were collected in a data-dependent mode. Peptides were identified using SEQUEST with trypsin cleavage specificity. SEQUEST results were filtered to strict peptide and protein false discovery rates, estimated from decoy sequences with additional control for modified peptide errors. Differential expression of lens proteins was quantified by comparing the number of MS/MS spectra of each protein.
Results: :
Morphological characterization showed that low expressers of the K6W-Ub have normal lenses whereas high expressers of K6W-Ub have defects in lens development and cataracts. A total of 1792 proteins were identified in these lenses and 986 of these were reliably quantified and compared between wt and K6W-Ub transgenic mice. When compared with lenses from wt mice, 22 proteins were down-regulated and 31 proteins were up-regulated in lenses of high expressers of K6W-Ub. In contrast, only 24 proteins showed differential expression between lenses from wt mice and lenses from low expressers of K6W-Ub. Among these, 15 were down-regulated and 9 were up-regulated in the low expresser lenses. Consistent with the defects in lens development and differentiation, many of the proteins that were down-regulated in lenses of K6W-Ub high expressers were fiber-specific proteins, such as γ-crystallins, filensin and heat shock response proteins. Many of proteins that are up-regulated in lenses of K6W-Ub high expressers are involved in energy metabolism, signal transduction and proteolysis.
Conclusions: :
Impairment of the ubiquitin system in the lens results in alteration of the proteome. The down-regulation of some fiber-specific proteins is consistent with the defect in lens fiber differentiation that we have observed. Up-regulation of some of lens proteins may be due to reduced degradation of these proteins by mutant ubiquitin or to increased expression in response to impairment of the ubiquitin system.
Keywords: differentiation • proteomics • cataract