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Arnold Salazar, Josue Portillo, Nora Blanca Caberoy; Tubby Participates in a Wide-variety of Pathways to Promote and Maintain Retinal Homeostasis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4372.
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© ARVO (1962-2015); The Authors (2016-present)
Spontaneous Tubby mutation in mice causes Retinitis pigmentosa, hearing loss and adult-onset obesity. However, delineation of its pathological mechanisms is hindered by its poorly defined protein interaction networks. The purpose of this study is to elucidate its binding partners to help define its underlying molecular mechanisms in the maintenance of retinal homeostasis and disease development.
Tubby-binding proteins were identified by open reading frame (ORF) phage display for protein-protein interactions. ORF phage display cDNA library of mouse eye was incubated with immobilized Tubby protein on ELISA plates, washed, eluted, amplified and reselected. A total of three rounds of affinity selection were performed. Enriched individual phage clones were re-verified for their binding activity to Tubby and identified by sequencing. Their binding specificity to Tubby and other proteins in the same family were analyzed. Identified Tubby-binding proteins were independently validated by co-immunoprecipitation and protein pull-down assays. The expression of the proteins in the retina and other tissues were characterized by reverse transcription-PCR (RT-PCR) using gene specific primers.
Several new Tubby-binding proteins were efficiently identified, including creatine kinase, estrogen related receptor beta (Esrrb), p53 apoptosis-associated target (Perp), Unc-5 homolog A (Unc5a) Abl-interactor 1 (Abl-1), ADP ribosylation factor-like 2 (Arl2), lectin galactose binding soluble proteins 4 and 6 (Lgals4 and 6), and pleckstrin homology-like domain family A, member 1 (Phlda1). These proteins exhibited different binding specificities to Tubby and other proteins in the same family. These proteins participate in signal transduction, actin polymerization, cytoskeletal remodeling, stratified epithelial adhesion, cell cycle progression, mitosis, microtubule formation, induction of photoreceptor genes, and apoptosis.
These data revealed that Tubby has multiple binding partners suggesting multiple roles in a wide-variety of pathways. The elucidation of these new binding partners will facilitate the delineation of its functional roles and disease mechanisms.
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