Abstract
Purpose :
Tubby mice with a spontaneous deletion of the C-terminal 44 amino acids exhibit progressive retinal and cochlear degeneration, and adult-onset obesity with undefined mechanisms. Using Open Reading Frame (ORF) phage display technology for protein-protein interactions, we identified TDAG51 (also known as pleckstrin homology-like domain family A member 1, PHLDA1) as a putative tubby-binding protein. The purpose of this study is to characterize the association of Tubby with TDAG51 and define their roles in Tubby-mediated disease pathogenesis.
Methods :
TDAG51 was identified as a tubby-binding protein by ORF phage display. Expression of TDAG51 in mouse eye and brain was verified by reverse transcription-PCR. Tubby-TDAG51 interaction was independently verified by in-vitro assays, e.g. co-immunoprecipitation and protein pull-down assays. The in vivo direct interactions between TDAG51 and Tubby proteins was characterized by fluorescence resonance energy transfer (FRET) analysis. Colocalization of tubby and TDAG51 in mouse retina was characterized by immunohistochemical analysis.
Results :
TDAG51 is expressed in the mouse retina and brain. It exhibited different binding specificities to Tubby and other proteins in the same family. Protein pull-down assay demonstrated that it binds specifically to Tubby N-terminal. TDAG51 was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death and is used as prognostic marker for breast cancer diagnosis. Its role in retinal homeostasis is not well established. Studies on subcellular localization and distribution of TDAG51 using immunohistochemical methods showed that tubby and TDAG51 colocalized in the retina. Studies on TDAG51 expression in tubby KO mice using RT-PCR and western blotting are currently underway.
Conclusions :
The data revealed that TDAG51 specifically recognizes Tubby N-terminal domain. The Tubby family of proteins are highly homologous in their C-terminal region, but diverse in the N-terminal half. Mutations in members of the family result to distinct disease profiles. Thus, their divergent N-termini may hold a key to elucidate the molecular mechanisms for different clinical manifestations. Understanding Tubby-TDAG51 interaction will allow us to understand the role of tubby in retinal cell physiology and disease pathogenesis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.