Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Keywords: 660 proteins encoded by disease genes •
696 retinal degenerations: hereditary •
658 protein purification and characterization