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Generation and Characterization of a Stable Inducible O‐GlcNAcase Cell Line
Author(s) -
Maduka Austin,
Groves Jennifer,
Zachara Natasha
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.717.12
Subject(s) - cell culture , interactome , microbiology and biotechnology , cardioprotection , chemistry , cytoplasm , cell , ubiquitin , biochemistry , biology , gene , medicine , genetics , ischemia
O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification that is added and removed from nuclear, cytoplasmic, and mitochondrial proteins by the O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA), respectively. Suggesting that O‐GlcNAc plays a role in cell survival, elevated O‐GlcNAcylation protects cells from many stressors including myocardial ischemia reperfusion injury. To target O‐GlcNAc as a potential cardioprotective therapeutic, we need a better understanding of the mechanisms by which O‐GlcNAc modulates protein function and how OGT and OGA are regulated during times of injury. Recent studies in our laboratory aim to understand the regulation of OGA by its oxidative stress‐dependent binding partners. As existing antibodies do not effectively purify OGA, the goal of this project is to generate and characterize U2OS (human osteosarcoma) and H9C2 (rat myoblast) stable cell lines overexpressing wild type or catalytically dead His 6 ‐tagged OGA. As constitutive overexpression of OGA is toxic, protein expression will be controlled using a tetracycline‐on system (Invitrogen, pT‐Rex). First, we will determine the expression, localization, and activity of His 6 ‐OGA in these cell lines. Ultimately, we will perform a large‐scale isolation of His 6 ‐OGA from oxidatively stressed cells to validate OGA's interactome. These cells lines will be vital for performing physiological studies that will provide molecular insight into the regulation of OGA and the role of O‐GlcNAc in cell survival and cardioprotection.