Abstract P047: A Novel Antiapoptotic Function of Thioredoxin-1 Is Mediated by Interaction with Caspase 3
Thioredoxin-1 (Trx-1) is one of the most important redox regulators in endothelial cells (EC). It is an oxidoreductase with anti-oxidative and anti-apoptotic properties. In EC not only the active site cysteines 32 and 35 but also the cysteine 69 are required for its anti-apoptotic function. Trx-1 has several interaction partners, but this list is far from complete. The underlying molecular mechanisms in apoptosis protection by Trx-1 are not completely understood. The most important apoptosis executor protein in EC is Caspase 3. Therefore, the aim of this study was to investigate whether Trx-1 interferes with Caspase 3 function. We first analyzed if Trx-1 is associated with Caspase 3. Coimmunoprecipitation revealed an interaction between both proteins, which was only found under non-reducing conditions, suggesting involvement of a disulfide bridge. To get insights into the functional relevance of the association between Trx-1 and Caspase 3, we investigated whether Trx-1 can directly interact with the catalytic subunit p17 of Caspase 3. After coexpression of p17 and Trx-1, coimmunoprecipitation experiments under non-reducing conditions revealed that p17 associates with Trx-1. Analysis of Trx-1 mutants demonstrated that the interaction depends on cysteine 32 and/or 69 in Trx-1. To assess the functional relevance of the Trx-1/Caspase 3 interaction in EC, we induced apoptosis by overexpression of p17 and investigated the influence Trx-1 wildtype (wt) and mutants in which single or multiple cysteine residues were replaced by serines thereby preventing disulfide bridge formation. Apoptosis induction by p17 was significantly reduced by Trx-1 wt (p17+lacZ 16.99±1.35% vs. p17+Trxwt 8.60±1.31% apoptotic cells). Interestingly, overexpression of Trx-1 with mutations of cysteines 32 and 69 enhanced apoptosis induction by Caspase 3 p17 (23.42±2.84% apoptotic cells). On the contrary, no increase in apoptosis was observed when only cysteine 32 or 69 were mutated, suggesting that both residues can scavenge p17 via disulfide bridge formation thereby preventing its association with the second Caspase 3 subunit p12 and thus activation of the enzyme. These data suggest that Trx-1 has a novel anti-apoptotic function by binding the apoptosis executing Caspase 3.
- © 2011 by American Heart Association, Inc.