| Summary: | 碩士 === 國立臺灣大學 === 免疫學研究所 === 106 === Interleukin-15 (IL-15) is a member of the four α-helix bundle cytokine family, which is best known to support the proliferation and survival of memory CD8+ T cells, natural killer (NK) cells, NKT cells, and intestinal intraepithelial lymphocytes (IELs). IL-15 also functions as a proinflammatory cytokine that promotes pathophysiologic state for human inflammatory diseases such as rheumatoid arthritis, psoriasis, and coeliac disease, etc. Previous study has shown that IL-15ΔE7, one variant of IL-15 generated by alternative splicing, has immune modulatory function in skin, and the effect is not caused by the lack of IL-15. However, the mechanism underlying this modulatory effect is still unknown. To find the possible mechanism underlying the IL-15ΔE7 modulation, in this thesis, I used (PS)2 (Protein Structure Prediction Server) web portal to predict the protein structure of IL-15ΔE7 and comparatively analyzed the interactions between IL-15 receptor subunits with IL-15 versus IL-15ΔE7 by PyMOL software. The results from structural analysis revealed that IL-15ΔE7 might lose its binding to IL-15Rα because it lost key glutamate residues that were responsible for receptor interaction. While the binding with IL-2/IL-15Rβ might be preserved, impaired interaction with IL-2/IL-15Rγc caused by protein conformational change could severely impede the bioactivity for IL-15ΔE7 as was well known for IL-15. Aided by the capability to generate recombinant IL-15ΔE7 protein in better quality using yeast recombinant system, the functional effects of IL-15ΔE7 on IL-15-mediated responses in IL-2/IL-15-dependent HT-2 cell line were further investigated to confirm the information derived from structural analysis. In consistent with the previous study, I demonstrated that IL-15ΔE7 treatment at saturating doses did not activate the phosphorylation of STAT5 (IL-2/IL-15Rγc signaling) and ERK1/2 (IL-2/IL-15Rβ signaling) as were induced by IL-15 in HT-2 cell line. Also, cotreatment and pretreatment experiments showed that IL-15ΔE7 had no antagonistic effect on IL-15-mediated signaling transduction. Variants of many immunologically important molecules such as IL-2, IL-4, IL-6, IL-15, generated by alternative splicing, are believed to be natural inhibitors of cytokine signaling, essentially acting as dominant negative forms of the cytokine that compete with the full-length cytokine for receptor binding. However, whether IL-15ΔE7 can also act as a competitive inhibitor of IL-15 function is unknown. To test this possibility, a “modified” ligand competition assay was performed by cotreating HT-2 cell line with fixed concentration of IL-15 (around EC50) and 10-fold serial titrated concentrations of IL-15ΔE7. IL-15-mediated responses of cell proliferation and downstream gene expression (cell cycle regulation gene Cdk1 and anti-apoptotic gene Bcl2) were measured by real-time PCR analysis. The results showed that cotreatment of IL-15ΔE7 did not block Cdk1 and Bcl2 expression even at 100 times of concentration than IL-15, which suggested that IL-15ΔE7 might not compete IL-15 for IL-15 receptor binding. Since the structural analysis have predicted the change in IL-15 receptor binding of IL-15ΔE7, it is worth to further measure the binding constant of IL-15ΔE7 to each IL-15 receptor subunits, which helps to better design the experiments and to understand the molecular mechanism of IL-15ΔE7 biological function. Also, whether IL-15ΔE7 transduces differential signaling through IL-15 receptors or that may have interfering effect on IL-15 remain to be elucidated.
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