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Josue Portillo, Arnold Salazar, Nora Blanca Caberoy; Characterizing Tubby and PHLDA1 Interaction in the Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4373.
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© ARVO (1962-2015); The Authors (2016-present)
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 pleckstrin homology-like domain family A member 1, PHLDA1; also known as TDAG51) as a putative tubby-binding protein. The purpose of this study is to characterize the association of Tubby with PHLDA1 and define their roles in Tubby-mediated disease pathogenesis.
PHLDA1 was identified as a tubby-binding protein by ORF phage display. Expression of PHLDA1 in mouse retina and brain was verified by reverse transcription-PCR (RT-PCR). Tubby-PHLDA1 interaction was independently verified by co-immunoprecipitation and protein pull-down assays.
PHLDA1 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. PHLDA1 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 the cellular localization and distribution of PHLDA1 are currently underway.
The data revealed that PHLDA1 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-PHLDA1 interaction will allow us to understand the role of tubby in retinal cell physiology and disease pathogenesis.
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