Relationship between interaction ability of thioredoxin with apoptosis signal-regulating kinase-1 and amino acid sites of thioredoxin

AIM:To investigate the relationship between the interaction ability of thioredoxin (Trx) with apoptosis signal-regulating kinase-1 (ASK1) and the amino acid sites of Trx with which ASK1 interacts. METHODS: WT-Trx-His tag (TrxA, wild-type Trx) plasmids, hTrx-Y49F-His tag (TrxB, mutating the Trx tyrosine 49 to phenylalanine) mutant plasmids and ASK1-HA tag (ASK1) plasmids were constructed, and plasmids of TrxA or TrxB with ASK1 plasmids were co-transfected into HEK 293A cells using HD transfection reagent. Twenty hours after transfection, cells were harvested for evaluation of apoptosis and caspase-3 activity or further treated with hydrogen peroxide at different concentrations (0, 0.5, 1, 2 mmol/L) for an additional 20 min for evaluation of the interaction between Trx and ASK1. Transfection efficiency was evaluated by Western blot. Interaction of Trx with ASK1 was tested by co-immunoprecipitation. Apoptosis was tested by TUNEL, and caspase-3 activity was detected by ELISA. RESULTS: ASK1, TrxA and TrxB were effectively transfected into the cells. ASK1 transfection increased the apoptosis and caspase-3 activity of the cells. TrxA and ASK1 co-transfection attenuated the apoptosis and caspase-3 activity promoted by ASK1 transfection. Compared with TrxA and ASK1 co-transfection, TrxB and ASK1 co-transfection increased the interaction of Trx with ASK1 and further decreased the apoptosis and caspase-3 activity induced by ASK1 transfection. In addition, after pretreatment with H2O2, ASK1 is more susceptible to interact with TrxB than TrxA. CONCLUSION: Our study demonstrates that mutating the 49 tyrosine of Trx to phenylalanine promotes the interaction ability between Trx and ASK1 and, therefore, enhances the anti-apoptosis ability of Trx. Our present study further indicates that the interaction ability of Trx with ASK1 is involved in the amino acid sites of Trx with which ASK1 interacts.