Analysis of the Potential Post-Translational Regulation of Zebrafish Homologs of GADD34 and CReP via Ubiquitin-Mediated Turnover
Faculty Mentor Name
Lisa Wrischnik (University of the Pacific, Department of Biological Sciences)
Research or Creativity Area
Natural Sciences
Abstract
The disruption of protein homeostasis caused by several cellular stressors leads to the accumulation of unfolded proteins and activation of the Unfolded Protein Response (UPR). Under stress, the UPR functions to restore cellular balance by reducing protein synthesis via phosphorylating eIF2α, a key initiation factor required for translation. Phosphorylated eIF2α results in global attenuation of protein synthesis, allowing time to clear misfolded proteins. eIF2α phosphatases, which consist of Protein Phosphatase 1 and regulated subunits such as GADD34 and CReP, work together to dephosphorylate eIF2α to reset the cell. Prior studies have demonstrated that human GADD34 (hGADD) is transcriptionally induced in response to stress whereas human CReP (hCReP) is constitutively expressed, but both proteins are downregulated through ubiquitin-mediated degradation. However, it is unknown whether the zebrafish homologs of these proteins, zCReP and zGADD, are similarly regulated.
To compare zGADD and zCReP turnover between species, each human and fish gene was cloned into vectors placing a GFP tag on either the N- or C-terminus of the GADD or CReP protein, because the Ub-mediated turnover of hGADD is known to be blocked by an N-terminal tag. HEK293 cells were transfected with equal concentrations of vectors expressing the GFP-tagged proteins. Transfected cells were treated with cyclohexamide (CHX) to block translation elongation, which allows the monitoring of protein degradation over time. Cell lysate was collected at different times after CHX addition and levels of GADD or CReP in the lysates were analyzed through Western blotting.
This study aimed to determine whether zebrafish homologs of the GADD34 and CReP genes exhibit similar regulatory patterns under normal conditions, with the goal of eventually examining if turnover changes during UPR. The findings will help to evaluate the suitability of zebrafish as a model organism for studying stress-response pathways and provide insight into the conservation of UPR across species.
Analysis of the Potential Post-Translational Regulation of Zebrafish Homologs of GADD34 and CReP via Ubiquitin-Mediated Turnover
The disruption of protein homeostasis caused by several cellular stressors leads to the accumulation of unfolded proteins and activation of the Unfolded Protein Response (UPR). Under stress, the UPR functions to restore cellular balance by reducing protein synthesis via phosphorylating eIF2α, a key initiation factor required for translation. Phosphorylated eIF2α results in global attenuation of protein synthesis, allowing time to clear misfolded proteins. eIF2α phosphatases, which consist of Protein Phosphatase 1 and regulated subunits such as GADD34 and CReP, work together to dephosphorylate eIF2α to reset the cell. Prior studies have demonstrated that human GADD34 (hGADD) is transcriptionally induced in response to stress whereas human CReP (hCReP) is constitutively expressed, but both proteins are downregulated through ubiquitin-mediated degradation. However, it is unknown whether the zebrafish homologs of these proteins, zCReP and zGADD, are similarly regulated.
To compare zGADD and zCReP turnover between species, each human and fish gene was cloned into vectors placing a GFP tag on either the N- or C-terminus of the GADD or CReP protein, because the Ub-mediated turnover of hGADD is known to be blocked by an N-terminal tag. HEK293 cells were transfected with equal concentrations of vectors expressing the GFP-tagged proteins. Transfected cells were treated with cyclohexamide (CHX) to block translation elongation, which allows the monitoring of protein degradation over time. Cell lysate was collected at different times after CHX addition and levels of GADD or CReP in the lysates were analyzed through Western blotting.
This study aimed to determine whether zebrafish homologs of the GADD34 and CReP genes exhibit similar regulatory patterns under normal conditions, with the goal of eventually examining if turnover changes during UPR. The findings will help to evaluate the suitability of zebrafish as a model organism for studying stress-response pathways and provide insight into the conservation of UPR across species.
