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Nora Caberoy, Gabriela Alvarado, Josephine Nocillado; Identification and characterization of Tubby C-terminal binding proteins by ORF phage display. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4196.
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Tubby mice with a spontaneous deletion mutation of C-terminal 44 amino acids exhibit progressive retinal and cochlear degeneration, and adult-onset obesity with undefined mechanisms. Tubby belongs to a well-characterized tubby protein family with four members (tubby, tubby-like protein 1, 2 and 3; or Tulps), which share a highly conserved C-terminal “tubby domain” of ~260 amino acids. Identification of Tubby C-terminal binding proteins will help define its underlying molecular mechanisms in the maintenance of retinal homeostasis and/or delineate its role in obesity. The purpose of this study is to define the role of the highly conserved C-terminal domain of Tubby by identifying its C-terminal binding proteins by a new technology of open reading frame (ORF) phage display.
Tubby-C terminal binding proteins were screened by ORF phage display as a new technology for protein-protein interactions. The interactions of tubby and its binding partners were verified by co-immunoprecipitation and protein pull-down assays. The expression of the proteins in the retina were characterized by reverse transcription-PCR (RT-PCR), in situ hybridization (ISH) or immunohistochemistry.
ORF phage display screening with purified tubby C-terminal domain (Tubby-C) as bait identified 11 putative Tubby C-binding proteins. The interactions of tubby and its binding partners were independently verified by co-immunoprecipation and protein pull-down assays. Proteins with positive binding to Tubby-C were further characterized for their distribution in the retina.
The results showed that ORF phage display is a powerful technology of functional proteomics for identification of binding proteins. The data demonstrated that Tubby-C has a wide range of binding specificities in the eye and may regulate a number of pathways during retinal homeostasis. Detailed characterization of Tubby and its binding partners will provide an in-depth understanding of its role in ocular physiology and disease pathogenesis.
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