Abstract
Purpose::
Our original study identified association of optineurin (OPTN) mutations with adult onset primary open-angle glaucoma. We recently showed expression and co-localization of OPTN with its known interacting proteins of RAB8, Myosin VI, FOS, Huntingtin and metabotropic glutamate receptor 1a in various ocular tissues. This study aimed to identify novel OPTN interacting partners (OPTN-IPs) and their cellular pathways through which OPTN mutations lead to glaucoma.
Methods::
Isolated RNA from human trabecular meshwork primary cell line was used for cDNA synthesis. The library was constructed by cloning of the cDNA in ProQuest Two-Hybrid prey vector pEXP-AD502 (InVitrogen) containing GAL4 DNA activation domain. The bait plasmid was constructed by cloning of OPTN full-length cDNA in pDEST32 vector containing GAL4 DNA binding domain.
Results::
We have identified 7 putative positive colonies that present interaction phenotypes and were able to activate 3 reporter genes of HIS3, URA3 and lacZ. Bait and prey plasmids were recovered from yeast colonies for further characterization. Sequencing of bait plasmids confirmed the specificity and absence of undesired mutations. Partial sequencing of prey colonies has identified 4 potentially new OPTN-IPs. Further validation and characterization of these colonies by retransformation of yeast cells, full-length sequencing of prey plasmids and confirmation of interaction by co-immunoprecipitation and pull-down assay is underway. These 4 new candidates consist of an interacting protein to TNF-alpha, an activator for RAB-like small GTPases, a subunit of RNA splicing factor machinery and a kinase protein.
Conclusions::
The HTM cDNA library is a valuable tool for identification of genes with ocular functions. Association of the new putative OPTN-IPs with TNF-alpha and RAB GTPase pathways is in agreement with earlier findings that OPTN is a TNF-alpha inducible protein and it is an interacting partner of RAB. The genes for these 4 new OPTN-IPs are potential candidates for the glaucoma phenotype. Outcome of this study will facilitate mapping of OPTN interacting domains, identification of bridging proteins and their disease-related pathways. This study may help to assign functions to uncharacterized proteins, to understand the composition of protein complexes and may lead to identification and validation of novel therapeutic targets.
Keywords: protein purification and characterization • proteins encoded by disease genes • anterior segment