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A. Taylor, Q. Liu, F. Shang, A. Cvekl, Y. Yang, E. Wawrousek; Expression of K6W Ubiquitin Confirms Critical Roles for the Ubiquitin Proteasome Pathway in Lens Formation and Function. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2000.
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The ubiquitin proteolytic pathway (UPP) has well documented roles in regulation of (a) protein quality, (b) cell proliferation and differentiation, (c) signaling, (d) intracellular trafficking, etc. Surprisingly, definitions of functions for the UPP in the visual pathway are limited (1). In order to begin to define functions for the UPP in eye tissues, we created a mutant, K6W Ub (2), in which Lysine 6 was replaced by Tryptophan. K6W Ub forms protein-ubiquitin conjugates, but these are poorly recognized by the 26 S proteasome. Thus, high mass conjugates accumulate, rather than being rapidly degraded as occurs with wt Ub.
RGSHis6K6W Ub was expressed in human lens epithelial cells and lens tissues in in vitro culture. A vector in which the alphaA-crystallin promoter, along with its associated DCR1 enhancer drives expression of 8 tandem copies of RGSHis6K6W Ub was synthesized. This was injected into FVB/N mouse oocytes to generate transgenic mice expressing K6W Ub in their lenses. Resulting founder transgenic mice were identified by PCR screening, then their dilated eyes were examined by slit lamp biomicroscopy, under anesthesia.
Expression of K6W Ub resulted in enhanced levels of ubiquitin conjugates in cultured cells, delayed the cell cycle (3), and limited cells' ability to respond to stress (2). All (5/5) of the founder transgenic mice harboring the DCR1/alphaA/(RGSHis6K6W Ub)8 vector show bilateral nuclear cataract.
Taken together the data establish for the first time that a functional UPP is an absolute requirement for proper lens formation, function and/or maintenance. The data are consistent with a requirement for the timely degradation of conjugates and a potential cytotoxicity if they accumulate.1. Shang and Taylor Exp. Eye Res. (2004) 78,1-14.2. Shang, et al. J. Biol. Chem. (2005) 280, 20365-74.3. Liu, et., al. Mol. Vis. (2006) 12, 931-6.
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