Browsing by Author "Photolo, Mampolelo M."

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The dynamics of thioredoxin-dependent reactions.
(2014) Photolo, Mampolelo M.; Pillay, Che Sobashkar.
The thioredoxin system, comprising thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, is present in most organisms from prokaryotes to eukaryotes. The system plays a central role in the redox regulation of several key cellular processes including DNA synthesis, apoptosis, glycolysis and redox signal transduction and changes in this system are associated with the progression of a number of diseases including certain cancers, malaria and HIV. Understanding the regulation of this network from a systems perspective is therefore essential. Our lab developed the first computational model of the Escherichia coli thioredoxin system and analyzed this system using mathematical and core models. In contrast to the commonly held assumption of the thioredoxin network as an electrical circuit with no cross-talk, our analysis showed this system displayed ultrasensitivity, adaptability and was interconnected via the thioredoxin redox cycle. In this study, a model of the Saccharomyces cerevisiae thioredoxin system was developed and computational modeling showed that an increase in concentration of one of the substrates in the thioredoxin system could decrease the flux of another thioredoxin oxidation reaction in a concentration-dependent manner. To complement these computational analyses, yeast thioredoxin reductase and thioredoxin were cloned, expressed and purified. An in vitro kinetic assay using insulin as a substrate and immunoglobin Y (IgY) as a competing substrate was subsequently undertaken. Our results showed that in some cases an increase in IgY concentration affected the rate of insulin reduction as measured by turbidity at 650 nm confirming the computational model's prediction. However, unexpectedly, with an increase in IgY concentration, there was a decrease in apparent absorbance at 650 nm at longer time points. These in silico and in vitro analyses shed light on how the thioredoxin system connects seemingly unrelated parts of metabolism into an integrated network, but additional experiments are required in order to improve the kinetic analyses in this study.