Honors College Thesis


Impact of Unfolded Protein Response, USP14, and NEDD8 Inhibitors Upon Antigen Presentation in SaI/Ak and 4T1 Tumor Cell Lines Public Deposited

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  • Antigen presentation is a mechanism used by cells to present peptides on the cell surface. Peptides, derived from both viral or self-proteins are presented on the cell surface by MHC Class I molecules to be recognized and detected by cytotoxic T cells. T cells comprise the defense system to protect cells from infection by intracellular pathogens and oncogenic transformation. T cells are constantly scanning the surface of cells for specific peptides, a signal for the T cell to kill the infected or transformed cell. The consequences of antigen presentation are important for a variety of clinical diseases. Erroneous detection by the T cell can result in labeling a healthy cell as diseased and lead to the development of an autoimmune disease. Antigen presentation can also be used in tumor immunotherapy. By manipulating a tumor cell, it can cause the presentation of own tumor peptides to be recognized and targeted by the T cell. Three pathways that may assist in antigen presentation are the Unfolded Protein Response Pathway, USP14 Pathway and Nedd8 Pathway. Each pathway interacts with diverse aspects of antigen presentation. The Unfolded Protein Response combats unfolded proteins floating within the ER by using two sensor proteins, IRE1-alpha and PERK. USP14 pathway targets proteins for degradation by the proteasome. Nedd8 pathway binds and modified proteins called cullins, thereby, signaling cell division and protein degradation. The goal of this research study is to analyze the total MHC Class I levels at the cell surface after treatment with inhibitor. The three mechanisms, Unfolded Protein Response, USP14 or Nedd8, are tested and analyzed by treating murine tumor cell lines, SaI/Ak and 4T1, with inhibitor. Before treatment with an inhibitor, the cells undergo an acid wash procedure to denature existing MHC Class I levels at the cell surface, allowing us to measure recovery of MHC class I levels. Following six hours of incubation with inhibitor, cells are stained with antibodies specific to MHC class I molecules and analyzed via flow cytometry. Our results conclude that unfolded protein response inhibitors slightly decrease MHC Class I levels in SaI/Ak cells, and do not change MHC Class I Levels in 4T1 cells. Overall, USP14 inhibitors had no effect in SaI/Ak and 4T1 cell lines. MLN-4924, the inhibitor of Nedd8 conjugation, slightly decreases MHC Class I levels in SaI/Ak cell line, but not in 4T1 cell line. Key Words: Antigen presentation, Nedd8, USP14, Unfolded Protein Response
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