Structural Interaction of Thioredoxin (Trx) and Thioredoxin-Interacting Protein (Txnip)

Thioredoxin (Trx) is a ubiquitous antioxidant protein that can reduce disulfides through two redox-active cysteines, Cys32 and Cys35. Thioredoxin interacting protein (Txnip) binds to thioredoxin and can inhibit the antioxidant activity of thioredoxin. Recent studies have also demonstrated that Txnip regulates glucose metabolism. Thus, Txnip could represent an important link between oxidative stress and metabolism. However, direct evidence for the interaction of Txnip and thioredoxin has not yet been shown. Initial studies to isolate recombinant Txnip in E. coli were hindered by the lack of purity and aggregation of the Txnip preparation. The aims of this study were to i) improve the methods for purifying recombinant Txnip and ii) test whether binding of Txnip to thioredoxin could disaggregate the recombinant Txnip. We demonstrated that Txnip could be purified to near homogeneity by inducing with IPTG at 4°C rather than at 37°C. Mutations have been introduced into Trx by replacing Cys35 and Cys73 with serines to eliminate aggregation and to facilitate formations of stable complexes of Txnip and Trx. We then incubated the purified Txnip protein with recombinant thioredoxin-GST and quantified the molecular size by dynamic light scattering studies. The DLS results indicate that addition of Trx does not abolish the aggregation of Txnip. However, purification of recombinant Txnip in E. coli will now allow us to attempt crystallization and thus may provide key insight into the important association of insulin resistance with oxidative stress.