Functional and Biochemical Characterization of a T Cell-Associated Anti-apoptotic Protein, GIMAP6
| Autor: | Ching-Huang Ho, 何慶凰 |
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| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 GTPases of immunity-associated proteins (GIMAPs) were first identified in angiosperms plants in responding to bacterial infections. Later, GIMAP genes were reported to express in lymphomyeloid cells that are well conserved among vertebrates. GIMAPs are expressed in lymphocytes and regulate survival/death signaling and cell development within the immune system. In a knockout mice study, deletion of Gimap1 resulted in a severe reduction in peripheral T cells numbers, and the subsequent studies further suggested that Gimap1 is essential for the survival of both activated and naïve peripheral B cells. Peripheral T cells from Gimap4-deficient mice have revealed that mouse Gimap4 acts as an accelerator of apoptosis in T cells. In 2015, a study reported that human GIMAP4 affects T cell differentiation via the regulation of interferon-γ secretion. In cell-based assays, human GIMAP5 and mouse Gimap8 were reported to act as apoptosis inhibitors. Previously we discovered that the RNA expression of the GIMAPs is uniformly lower in human lung tumor tissue samples, compared to that in the adjacent normal tissues. GIMAP6 showed the highest normal/tumor expression ratio in the lung cancer paired samples, and this attracted our attention. In this study, we have focused on GIMAP6 and set out to investigate its function in T cells and to characterize its biochemical properties. We found that human GIMAP6 is expressed primarily in T cell lines. By sorting human peripheral blood mononuclear cells and performing quantitative RT-PCR, GIMAP6 was found to be expressed in CD3+ cells. In Jurkat cells that had been knocked down for GIMAP6, treatment with hydrogen peroxide, FasL or okadaic acid significantly increased cell death/apoptosis. Exogenous expression of GMAP6 protected Huh-7 cells from apoptosis, suggesting that GIMAP6 is an anti-apoptotic protein. Furthermore, knockdown of GIMAP6 not only accelerated T cell activation, but also rendered Jurkat cells sensitive to apoptosis under PMA/ionomycin treatment conditions. It is speculated that in the GIMAP6 knockdown cells, accelerated T cell activation initiates and enhances the "activation-induced cell death" response, and finally, results in an increment of apoptosis. Using this experimental system, we also observed a down-regulation of p65 phosphorylation (ser536) in GIMAP6 knockdown cells, indicating that GIMAP6 might display anti-apoptotic function through NF-B activation. The conclusion from the study on cultured T cells was corroborated by the analysis of primary CD3+ T cells, showing that specific knockdown of GIMAP6 led to enhancement of PMA/ionomycin-mediated activation signals. To characterize the biochemical properties of GIMAP6, we purified the recombinant GIMAP6 to homogeneity and revealed that GIMAP6 had ATPase as well as GTPase activity. We further demonstrated that the hydrolysis activity of GIMAP6 was not essential for its anti-apoptotic function in Huh-7 cells. Combining the expression data, biochemical properties, and cellular features, it is concluded that by controlling cell death and the activation of the T cells, GIMAP6 plays a role as a T cell preserver in the immune system. This feature of GIMAP6 might be manipulated to protect effector T cells in response to the invasion of pathogens, thereby enhancing the anti-infection efficiency in the immune system. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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