Human Glutamylcysteine Synthetase Protects HEK293 Cells Against UV-induced Cell Death Through Inhibition Of C-Jun NH₂-terminal Kinase

An intact death pathway is required for successful embryonic development and the maintenance of normal tissue homeostasis. At the molecular level, apoptosis is tightly regulated. Either insufficient or excessive apoptosis can lead to diseases such as tumors, autoimmune disorders or neurodegenerative conditions. So apoptosis has become a major research area in the biomedical sciences. The research focused on the signaling pathways that trigger apoptosis and the modulation of cell death pathways may provide promising new therapies for a number of diseases.We studied the effect of glutamylcysteine synthetase-one of the glutathione synthetases-on the UV-induced HEK293 cell death. The experiment is divided into two part as followed: 1. The cloning and expression of glutathione synthetasesThe cDNA encoding GCLC or GSS was obtained from human cDNA library by PCR, and inserted respectively into the multiple clone sites of expression plasmids. So the aim of this experiment is to construct recombinant plasmids and lay a primary foundation for the further reseach of GCLC involved in UV-induced apoptotic signal regulation. The constructed prokaryotic expression recombinant plasmids were induced respectively by IPTG for the high-level expression of target proteins. The expression of these proteins is used for the preparation of monoclonal antibodies. So we can monitor efficiently the endogenous or exogenous expression changes of these proteins.2. Human Glutamylcysteine synthetase protects HEK293 cells against UV-induced cell deathHuman glutamylcysteine synthetase is the rate-limiting enzyme for glutathione synthesis. Glutamylcysteine ligase catalytic subunit (GCLC) possesses all the catalytic activity. UV irradiation (UV-C, 30 J/m2) induced cell death in HEK293 cells, but the morphological changes were inhibited significantly by expression of GCLC. MTS assay and flow cytometry results also indicated that GCLC and JNK1APF expression enhanced cellular resistance to UV irradiation. Western blotting showed that irradiation strongly activated the c-Jun NH2-terminal kinases (JNKs) and caspase-3 as well as p38 in HEK293 cells. Interestingly, existing data show that GCLC blocks UV induced JNK1 phosphorylation but does not affect p38 phosphorylation. Therefore, over-expression of GCLC protected HEK293 cells against UV irradiation-induced cell death by inhibiting the phosphorylation and activation of JNK1, concomitantly with the inhibition of caspase-3 activation and p21WAF1"luciferase activity downstream of JNK.