June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Identification of Interacting Protein Partners of TOPORS in the Retina
Author Affiliations & Notes
  • Barbara Czub
    Genetics, UCL Institute of Ophthalmology, London, United Kingdom
  • Amna Shah
    Genetics, UCL Institute of Ophthalmology, London, United Kingdom
  • Giovanna Alfano
    Genetics, UCL Institute of Ophthalmology, London, United Kingdom
  • Lourdes Valdés Sánchez
    Cellular Therapy and Regenerative Medicine, Andalusian Centre for Molecular Biology and Regenerative Medicine (CABIMER), Seville, Spain
  • Christina Chakarova
    Genetics, UCL Institute of Ophthalmology, London, United Kingdom
  • Shomi Bhattacharya
    Genetics, UCL Institute of Ophthalmology, London, United Kingdom
    Cellular Therapy and Regenerative Medicine, Andalusian Centre for Molecular Biology and Regenerative Medicine (CABIMER), Seville, Spain
  • Footnotes
    Commercial Relationships Barbara Czub, None; Amna Shah, None; Giovanna Alfano, None; Lourdes Valdés Sánchez, None; Christina Chakarova, None; Shomi Bhattacharya, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 6105. doi:
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      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.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

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.

 
Methods
 

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.

 
Results
 

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.

 
Conclusions
 

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.

  
Keywords: 659 protein structure/function • 660 proteins encoded by disease genes • 688 retina  
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