Purchase this article with an account.
Barbara Czub, Amna Shah, Giovanna Alfano, Lourdes Valdés Sánchez, Christina Chakarova, Shomi Bhattacharya; Identification of Interacting Protein Partners of TOPORS in the Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6105.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disease characterised by loss of rod photoreceptors. Historically genes linked to RP were associated with rod-specific functions. Recently a novel class of ubiquitously expressed causative genes emerged including splicing factor genes and TOPORS. To date studies show TOPORS is expressed in all tested human tissues including retina and the protein localises to several cellular compartments. However, mutations in TOPORS only cause RP (no systemic symptoms); this may be due to retina-specific protein interactions, which are perturbed as a result of mutations. This work aims to understand why mutations in this ubiquitously expressed gene cause a retina-only disease by identifying interacting partners of TOPORS from human retina.
Human retinal cDNA library was constructed from total retinal cDNA directly in Y187 S.cerevisiae yeast strain by homologous recombination in-frame with GAL4 AD. Retina-specificity was validated by sequencing. Bait plasmids (full-length TOPORS and deletion constructs) were cloned in-frame with GAL4 BD. The library was screened for protein interacting partners of TOPORS using MatchmakerTM Gold Yeast Two-Hybrid (Y2H) System.
Library validation identified several retina-specific genes including RHO (NM_000539). Over 107 cDNA clones were subsequently screened, leading to isolation of 53 potential interactions. The identified peptides were prioritised for further evaluation and re-tested in yeast leading to identification of three candidates: A brain prostaglandinD2 synthase (PTGDS; NM_000954) highly expressed in the retina, previously suggested to play a role in retinal homeostasis; regulatory subunit 4 of the 26S protease (PSMC1; NM_002802) conferring substrate specificity to the proteasome complex during degradation of ubiquitinated proteins; and a soluble fragment of integral membrane protein 2B (ITM2B; NM_0219999) previously linked to neurodegenerative disorders.
The Y2H screen identified potential interacting partners of TOPORS and, following validation experiments, three distinct proteins were selected for further studies. Defining their roles will help elucidate the functions of TOPORS as well as better understand the photoreceptor cell biology. The outcomes could help explain how mutations in TOPORS result only in RP despite its ubiquitous expression and multifunctional character.
This PDF is available to Subscribers Only