Regulation of the thioredoxin system in Saccharomyces cerevisiae.
The thioredoxin system consisting of thioredoxin (Trx), thioredoxin reductase and NADPH plays a significant role in a large number of redox-dependent processes such as DNA synthesis and anti-oxidant defense. Elevated levels of this system have been associated with a number of diseases including cancer and HIV. Understanding the regulation of this network from a systems perspective is therefore essential. However, contradictory descriptions of thioredoxin as both an enzyme and redox couple have stifled the adoption of systems biology approaches within the field. Using kinetic modeling, this discrepancy was resolved by proposing that saturation of Trx activity could be due to the saturation of the Trx redox cycle which consequently allowed development of the first computational models of the thioredoxin system in Jurkat T-cells and Escherichia coli. While these models successfully described the network properties of the thioredoxin system in these organisms, further confirmatory studies were required before this modeling approach could be generally accepted. The aim of this study was to utilize computational and molecular methods to confirm or reject this proposed mechanism for thioredoxin activity. To determine if there is any difference in the kinetic models obtained when thioredoxin was modeled as an enzyme or as a redox couple, representative core models were developed. The data showed that when modeling Trx as a redox couple, the system was able to achieve steady state, there was a re-distribution of Trx into its oxidized form and, thioredoxin reductase affected the rates within the system. On the other hand, when Trx was modeled as an enzyme, the system could not reach a steady state, Trx remained in the reduced form and thioredoxin reductase concentration had no effect on the rates within the system. As these properties could be directly tested invitro, we sought to directly confirm which model was correct. The thioredoxin system from Saccharomyces cerevisiae was cloned, expressed and purified and substrate saturation curves were generated using insulin as a model substrate. The data showed that the system reached steady state and with increasing concentrations of insulin, the system saturated with a progressive re-distribution of the thioredoxin moiety into its oxidized form. Further, increasing the thioredoxin reductase concentration increased the flux through the system. Collectively, the results obtained through invitro analyses provided unambiguous support for the thioredoxin redox couple model. These results will enable the construction of a complete computational model of the yeast thioredoxin system and provide a basis for the analysis of this network in a number of pathologies.