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Elena Segal, Ronit Heinrich, Shadi Safuri, Ami Aronheim, Naim Shehadeh, Ido Perlman; Mapping protein-protein interactions of Bestrophin1 - a potential insight into the development of Bestrophinopathies. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1754.
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© ARVO (1962-2015); The Authors (2016-present)
Specific mutations in hBest1 gene result in different phenotypes, depending upon the mutation, including juvenile-onset Best Vitelliform Macular Degeneration, adult-onset Vitelliform Macular Dystrophy, Autosomal Dominant Vitreoretinochoroidopathy and Autosomal Recessive Bestrophinopathy. The expression of Best1 is confined to the retinal pigment epithelium (RPE), responsible for phagocytosis of photoreceptors outer segments (POS). We have recently found that mutated Best1, expressed in ARPE-19 cells, alter POS phagocytosis compared to the wild type Best1 in a manner depending upon the mutation. We hypothesize that Best1 modulates POS phagocytosis via protein-protein interactions with regulatory proteins
The Ras recruitment system (Broder et al., 1998) is based on the ability of Ras mutant to be localized to the plasma membrane through the interaction between two proteins. Best1 is fused to Ras whereas human RPE cDNA library is fused to v-Src membrane localization signal. Ras membrane localization via protein-protein interaction results in the complementation of a yeast temperature sensitive mutant strain in the Ras guanyl nucleotide exchange factor, Cdc25-2 (Aronheim, 2001a and b). The screening is performed as follows: Best1 is prepared on Met425 plasmid whereas the library is constructed on galactose inducible plasmid. The Met425 promoter is repressed in the presence of methionine, thus Best1 expression is induced when cells are grown on a medium lacking methionine. The library expression is induced in medium containing galactose and repressed in the presence of glucose. In order to induce the expression of Best1 and library, cells are grown on galactose medium lacking methionine. The candidate clones are further analyzed by DNA extraction and sequencing and screened with 4 different Best1 mutations
Mapping protein-protein interactions of wild type Best1 and RPE library resulted in 13 candidates, from which two DNA sequences analyzed by BLAST were consistent with 2 different proteins that may potentially interact with Best1. Those 2 proteins failed to interact with mutated Best1; Arg47His and Arg200X mutations
Our findings suggest that Best1 may interact with 2 different proteins, previously reported to regulate cell signaling, and that those interactions may be altered by mutations, thus contributing to disease manifestation
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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