UBC Theses and Dissertations
Effects of eukaryotic initiation factor 2A upregulation on protein synthesis in insulin producing cells Rahardjo, Amanda Karunaputri
Death and dysfunction of insulin producing pancreatic β-cells is a major feature of diabetes. Recently, it was discovered that the eukaryotic initiation factor 2A (EIF2A) – an unconventional translation factor – was uniquely abundant in β-cells compared to other cell types. Past studies using lentiviral transduction to overexpress EIF2A in MIN6 cells experiencing ER stress resulted in increased β-cell survival and reversed the downregulation of global translation that normally occurs as part of the unfolded protein response (UPR), as it seeks to alleviate stress. In this study, we used MIN6 cells and human islets transduced with adenoviruses to induce EIF2A overexpression. Amino acid radiolabeling was used to measure nascent global translation, in combination with immunoprecipitation to isolate and measure proinsulin synthesis. Western blotting was used to quantify changes in steady state protein levels under stressed and unstressed conditions. In MIN6 cells, adenovirus-induced overexpression of GFP-tagged EIF2A did not result in restoration of translation as previously seen, and proinsulin synthesis did not appear to be affected separately. However, EIF2A overexpression appeared to improve proinsulin-insulin conversion under stress. In human islets, regardless of EIF2A overexpression, thapsigargin treatment did not induce a significant repression of translation, nor were we able to measure proinsulin synthesis in any condition using our methods. Western blotting showed that EIF2B subunits were affected differentially by EIF2A overexpression. EIF2B5, which contains the guanine exchange factor catalytic site, was upregulated in EIF2A overexpressing cells in both stressed and unstressed conditions. EIF2B3, which is responsible for GTP binding, was upregulated in EIF2A-overexpressing cells only under stress. The EIF2B2 subunit, which is part of the core that mediates interactions with EIF2S1, showed no trend towards upregulation by EIF2A overexpression. This suggests that EIF2A’s effects on translation may indeed be mediated through EIF2B, but consequently the exact mechanism by which this occurs, and whether or not there is a specific link to insulin synthesis, remain questions in need of further study.
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