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Lixing W. Reneker, Huiyi Chen; Protein Aggregate Formation and SQSTM1/p62 Induction in the Transgenic Mouse Lens Expressing Dominant Negative Mutant FGFR. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3927.
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Expressing a dominant negative (DN) mutant of FGFR can induce ER stress and cell death in the lens fiber cells. Previously, we showed that ER stress-induced cell death in the DN-FGFR lens is mediated through the activation of calpain and caspase-12. The ER stress-induced changes in the DN-FGFR transgenic lens are further investigated in this study.
Formation of ubiquitin-labeled protein aggregates in the DN-FGFR lens was demonstrated by immunofluorescence. Water-soluble proteins were prepared from newborn wild type (WT) and DN-FGFR lenses for western blot analysis against LC3, a protein involved in lysosome-dependent macroautophagy (autophagy). Induction of sequestosome 1 (SQSTM1)/p62 expression in the lens fiber cells was shown by immunostaining.
Formation of ubiquitinylated protein aggregates in the fiber cells of DN-FGFR lens was initially detected by immunofluorescence at embryonic day 14.5 (E14.5). The protein aggregates accumulated as lens development progressed, suggesting that the ubiquitin-proteosome system (UPS) was ineffective to dispose the unfolded/misfolded mutant transgenic proteins. Western blot analysis showed that LC-I, the cytosolic form of LC3 was present in the WT lens. In contrast, the membrane bound form, LC3-II, which specifically associated with autophagosome membrane, was the main form of LC3 in the DN-FGFR lens. This result indicated that autophagy system was activated in the transgenic lens. SQSTM1/p62 is known to couple ubiquitinylated protein aggregates to autophagy for clearance in other systems. In the DN-FGFR lens, SQSTM1/p62 expression was induced in the lens fiber cells at E16.5 and its immunofluorescence was colocalized with ubiquitinylated protein aggregates.
The UPS in the transgenic lens fiber cells is insufficient to dispose the unfolded/misfolded mutant proteins and, as a result, the compensatory autophagy was activated for clearance of the ubiquitinylated protein aggregates. The mechanism coupling the UPS to selective autophagy for degradation of protein aggregates is through the induction of SQSTM1/62.
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