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Ke Liu, Andrea Caceres, Junmin Peng, Fu Shang, Junyuan Gao, Xiurong Sun, Richard T. Mathias, Allen Taylor; K6W Mutant Ubiquitin Activates Calpain in Lens. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5593.
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© ARVO (1962-2015); The Authors (2016-present)
The Ubiquitin Proteasome System (UPS) regulates a broad array of basic cellular processes including proteolysis, cell cycle and intracellular trafficking of cellular proteins. Perturbing the UPS by expressing K6W ubiquitin in mouse lens delays lens development and results congenital cataract (Caceres, PLoS 2010). The goal of the present study was to determine molecular mechanisms by which the mutant ubiquitin regulates lens development, thus establishing roles for the UPS in proper lens formation.
Expression of K6W-Ubiquitin was directed to the lens using the alpha crystallin promoter. Lenses from transgenic mice expressing either wild type or K6W mutant ubiquitin were lysed and analyzed by LC-MS/MS and western blotting. Changes in protein profiles and cytoskeleton structure were measured. Intracellular calcium concentration in intact lenses from new born mice was measured by injecting the calcium-sensitive dye Fura2.
As revealed by mass spectrometry (spectral counts), protein levels of γ-crystalins, filensin, TDRD7 and caprin2 were decreased, while fodrin (spectrin alpha 2) and vimentin were significantly increased in lens expressing K6W mutant ubiquitin. The changes of levels for these proteins do not appear to be due primarily to alterations in transcription. Interestingly, the increased fodrin and vimentin appear to be fragmented as revealed by western blotting, consistent with enhanced proteolysis in lenses that express K6W ubiquitin. Fodrin, vimentin and filensin are established calpain substrates. Consistent with calpain activation and cleavage of vimentin and fodrin, calcium concentrations are 3-5 times higher in cores of lenses expressing K6W ubiquitin vs. wt lenses. Calpain 3, the most abundant calpain in the lens, is also more degraded in lens expressing K6W ubiquitin, consistent with enhanced autolyses. The degradation of fodrin and vimentin and decrease of filensin may contribute to the destruction of intermediate filament and F-actin cytoskeleton structures in lens cell. The data also suggest that the UPS is required for intercellular junction formation.
Our results provide a proteomics landscape of changes of lens architecture and proteins when the UPS is compromised, confirming a role for a properly functioning ubiquitin pathway in lens development. They also suggest calpain proteolysis as an important effector of lens development.
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