Identification of interacting protein partners of TOPORS in the retina

• Main Author: Czub, B.
• Other Authors: Bhattacharya, S. S.
• Published: University College London (University of London) 2015
• Subjects: 617.7
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.666806

Retinitis pigmentosa (RP, MIM#268000) is a heterogeneous disease characterised by loss of rod photoreceptors and pigment deposits in the retina. Historically, genes linked to RP were associated with rod-specific functions. Recently, a novel class of ubiquitously expressed causative genes has emerged including splicing factor genes and TOPORS (NM_005802). To date, studies show TOPORS is expressed in all tested human tissues, including the retina. However, mutations in this ubiquitously expressed gene only cause RP without any systemic symptoms. The purpose of this work was to understand why mutations in TOPORS, which encodes a multifunctional protein, cause a retina-only disease by identifying protein interacting partner(s) of TOPORS, using a yeast-two hybrid (Y2H) screen. In case the interacting partner(s) turn out to be retina specific, it may explain the retina-only phenotype. Human retinal cDNA library was constructed from total retinal cDNA directly in the Y187 Saccharomyces cerevisiae yeast strain. Retina-specificity of the cDNA library was validated by sequencing, leading to identification of several retina-specific genes, including rhodopsin (RHO; NM_000539). The library was screened for protein interacting partners of TOPORS, using MatchmakerTM Gold Yeast Two-Hybrid System (Clontech, CA, USA). Over 10^7 cDNA clones were screened, leading to isolation of 53 potential interactions. The identified interacting partners were prioritised for further evaluation, based on literature and database searches, and re-tested in yeast leading to identification of three candidates for further functional studies: a soluble fragment of integral membrane protein 2B (ITM2B; NM_0219999), previously linked to neurodegenerative disorders, and more recently associated with an inherited retinal dystrophy; a brain prostaglandin D2 synthase (PTGDS; NM_000954), highly expressed in the retina, previously suggested to play a role in retinal homeostasis; a regulatory subunit 4 of the 26 S protease (PSMC1; NM_002802), conferring substrate specificity to the proteasome complex during degradation of ubiquitinated proteins. The outcomes suggest several scenarios for why mutations in TOPORS result only in RP; however, further studies are essential to elucidate the role of TOPORS and its interacting partners in the aetiology of this debilitating disease.