Purposes Ubiquitin is involved in many aspects of cellular functions. Our previous experiments demonstrated that targeted expression of a dominant negative ubiquitin in lens results in defects in lens development and differentiation. The purpose of this work was to investigate the effects of mutant ubiquitin on homeostasis of lens proteome.
Methods Lenses from wild type and K6W ubiquitin expressing mice (low expressers and high expressers) were homogenized by sonication. A fraction of the suspension equivalent to 100 ug lens proteins from each sample were then reduced alkylated with iodoacetamide.  After digestion with trypsin, the desalted peptides were analyzed by two dimensional LC-LC/MS/MS.  MS-MS spectra were collected in a data-dependent mode.  Peptides were identified using SEQUEST software and the 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 transgenic mice have normal lenses whereas high expressers of K6W-Ub transgenic mice 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-ubiquitin 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 transgenic mice.  In contrast, only 24 proteins showed differential expression between lenses from wt mice and lenses from low expressers of K6W-Ub transgenic mice.  Consistent with the defects in lens development and differentiation, many of the proteins that were down-regulated in lenses of high expressers of K6W-ubiquitin transgenic lenses were lens fiber-specific proteins, such as γ-crystallins, filensin and heat shock response proteins.  Many of proteins that are up-regulated in lenses of high expressers of K6W-Ub transgenic mice are involved in energy metabolism, signal transduction and proteolysis.
Conclusion Impairment of the ubiquitin system in lens results in alteration in lens proteome.  The down-regulation of some fiber-specific proteins is consistent with the defect of lens fiber differentiation of the transgenic mice.  Up-regulation of some of lens proteins may be due to reduced degradation of these proteins by mutant ubiquitin or due to increased expression in response to impairment of the ubiquitin system.