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Arnold Salazar, Peipei Pan, Nora Blanca Caberoy; Tubby and Estrogen Related Receptor Beta (Esrrβ) Participate in Oxidative Pathway in the Retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5440.
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© ARVO (1962-2015); The Authors (2016-present)
Mutation in the Tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanisms. Tubby was identified as a MerTK ligand for phagocytosis in the RPE, and was shown to play a role in ciliogenesis. However, its poorly defined protein interaction networks hinder delineation of its pathological mechanisms. Using ORF phage display for protein-protein interactions, we identified putative Tubby-binding proteins, including Esrrβ. Esrrβ is a nuclear receptor associated with cell survival in low-oxygen environments, pluripotency in embryonic stem cells, regulation of energy metabolism, and oxidative stress. Mutation in Esrrβ causes hearing impairment, dental decay, and rotator cuff disease. Its role in retinal cell physiology has not been established. This study aims to characterize Tubby and Esrrβ interaction to elucidate their roles in retinal homeostasis and disease pathogenesis.
The binding specificity of Tubby to Esrrβ was analyzed by protein pull-down assay. The interaction of Tubby and Esrrβ was demonstrated in vivo using co-immunoprecipitation and FRET analyses. The expression of Esrrβ in the retina was characterized by reverse transcription-PCR and Western blot. Co-localization of Tubby and Esrrβ in the retina was examined through immunohistochemistry. Promoter binding and transcriptional activation assays were also done to determine the effect of Tubby in Esrrβ expression.
Tubby and Esrrβ interacts in vitro and in vivo and the interaction is mediated through Tubby N-terminal domain. Promoter binding and transcriptional activation studies showed that Tubby directly binds to Esrrβ response elements and activates its transcription. Esrrβ is expressed in human testis, breast, kidney, endometrium, and prostate. This study further showed its expression in the retina.
The results revealed that Tubby not only interacts with the Esrrβ protein but also regulates Esrrβ expression. Tubby was demonstrated as a putative membrane-bound, G protein-activated transcription factor although its target gene(s) has not been reported. To our knowledge, this is the first to describe a gene targeted for Tubby regulation. Esrrβ has been shown to be involved in oxidative stress. The interaction and direct regulation of Esrrβ by Tubby open up the possibility of a potential role of Tubby protein in oxidative stress in the retina.
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