| Summary: | 碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 90 === LIGHT is a member of TNF superfamily and binds to LTbR, HVEM and DcR3. LIGHT is expressed on activated T cells and serves as a costimulatory factor to enhance IFN-g production by activated T cells. In cojunction with IFN-g, LIGHT induces cell death via apoptosis. However, the underlying mechanism for LIGHT/IFN-g mediated apoptosis is not illustrated yet.
In order to investigate the role of LTbR in LIGHT-mediated apoptosis, we designed a LIGHT mutant that retained its binding ability to LTbR but not to HVEM. A three-dimensional model of LIGHT based on the crystallographic struncture of LT-a and TNFR1 complex was generated to predict residues that are likely involoved in receptor binding. One mutant, LIGHT-R228E, of which 228th arginine is replaced by glutamic acid, exhibits specific binding to LTR and fails to bind with HVEM. Receptor neutralization assay and surface plasmon resonance analysis were performed to elucidate the specific binding ability of LIGHT-R228E to LTbR, HVEM, and DcR3 respectively. The lost of LIGHT-R228E’s binding to HVEM was further confirmed by its inactivity to trigger T cell proliferation. This result further confirm previous hypothesis that the apoptotic ability of LIGHT is via LTbR, and HVEM is dispensible for LIGHT-mediated apoptosis.
Moreover, caspase-3 activation, decreased mitochondria membrane potential, and release of cytochrome c and Smac/DIABLO from mitochondria were also observed in LIGHT/IFN-g or LIGHT-R228E/IFN-g-mediated apoptosis in Hep3BT2 cells, indicating the involvement of mitochondria pathway in LIGHT-mediated apoptosis. However, over-expression of non-caspase-3-cleavable Bcl-2 cannot fully rescue Hep3BT2 cells from apoptosis. Based on this study, we concluded that both mitochondria dependent and independent pathways are involved in LIGHT-mediated apoptosis.
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