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Thiol switches are important regulators of cellular signaling and are coordinated by several redox enzyme systems including thioredoxins, peroxiredoxins, and glutathione. Thioredoxin-1 (Trx1), in particular, is an important signaling molecule not only in response to redox perturbations, but also in cellular growth, regulation of gene expression, and apoptosis. The active site of this enzyme is a highly conserved C-G-P-C motif and the redox mechanism of Trx1 is rapid which presents a challenge in determining specific substrates. Numerous in vitro approaches have identified Trx1-dependent thiol switches; however, these findings may not be physiologically relevant and little is known about Trx1 interactions in vivo. In order to identify Trx1 targets in vivo, we generated a transgenic mouse with inducible expression of a mutant Trx1 transgene to stabilize intermolecular disulfides with protein substrates. Expression of the Trx1 “substrate trap” transgene did not interfere with endogenous thioredoxin or glutathione systems in brain, heart, lung, liver, and kidney. Following immunoprecipitation and proteomic analysis, we identified 41 homeostatic Trx1 interactions in perinatal lung, including previously described Trx1 substrates such as members of the peroxiredoxin family and collapsin response mediator protein 2. Using perinatal hyperoxia as a model of oxidative injury, we found 17 oxygen-induced interactions which included several cytoskeletal proteins which may be important to alveolar development. The data herein validates this novel mouse model for identification of tissue- and cell-specific Trx1-dependent pathways that regulate physiological signals in response to redox perturbations.Characterization of a cre-inducible transgenic mouse to identify Trx1 substrates.Detection of previously known and novel Trx1 redox-dependent interactions in vivo.Dynamic Trx1 substrates that occur in lung during perinatal hyperoxic injury.