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Lydia Ann, Ricardo F Frausto, Evelyn Hanser, Doug Chung, Anthony J Aldave; Characterization of an Alternative ZEB1 Nuclear Localization Signal in Posterior Polymorphous Corneal Dystrophy 3. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4822. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the ability of an alternative nuclear localization signal (aNLS) to mediate nuclear translocation of mutant two-handed zinc-finger E-box binding homeodomain transcription factor 1 (ZEB1) proteins associated with posterior polymorphous corneal dystrophy 3 (PPCD3).
The full-length sequence of the ZEB1 protein was screened for alternative NLS motifs by in silico analysis. Overexpression DNA constructs containing wild type ZEB1 (ZEB1WT) or mutant ZEB1 (ZEB1MU) (representing mutations associated with PPCD3) cDNA and an N-terminal halo-tag sequence were generated. A construct containing ZEB1 cDNA lacking the classical NLS sequence (ZEB1NLSdel), spanning amino acids 892-998, was used as a control. ZEB1 cDNA containing the PPCD3 mutations with or without deletion of the aNLS sequence were generated using site-directed mutagenesis. These cDNAs were overexpressed in Ishikawa cells, a human cell line. Localization of the exogenous halo-tagged ZEB1 proteins was determined by fluorescence immunocytochemistry using an anti-halo antibody and confocal microscopy.
An aNLS motif (Lys-Lys-Arg) was identified at amino acid positions 274-276 by in silico analysis. ZEB1WT protein localized exclusively within the nucleus, while ZEB1NLSdel demonstrated a marked cytoplasmic localization. Similarly, while ZEB1 mutant proteins p.(Arg325*), p.(Glu495fs), and p.(Val526*) containing an intact aNLS displayed nuclear localization, deletion of the aNLS sequence for these proteins resulted in the distinct cytoplasmic localization, while the effect was less pronounced with p.(Val526fs). In contrast, deletion of the alternative NLS motif did not impact the distinct cytoplasmic localization observed with p.(Ser638fs) and p.(Gln884fs) mutants.
ZEB1 possesses a functional alternative NLS and is only active in truncated proteins with no more than between 250-360 amino acids downstream of the aNLS.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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