Modelling and analysis of peroxiredoxin kinetics for systems biology applications.
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Date
2015
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Abstract
Oxidative stress, caused by reactive oxygen species (ROS) such as hydrogen
peroxide, can have harmful effects on important cellular components and processes which
can lead to cell death. Cells have evolved extensive protein and non-protein antioxidant
molecules to deal with hydrogen peroxide but it is now clear that hydrogen peroxide is also
important signal molecule. It is not fully understood how cells maintain the balance between
hydrogen peroxide detoxification and signal transduction. Peroxiredoxins are a ubiquitous
family of antioxidant proteins that are the primary reductants of hydrogen peroxide and
appear to be key molecules in mediating this balance. Using catalytic cysteines,
peroxiredoxins reduce hydrogen peroxide and other ROS and in turn are reduced by
thioredoxin and thioredoxin reductase. This coupled set of reactions collectively constitute
the peroxiredoxin system and its precise role in redox signalling could be established using
systems biology studies. However, there are some discrepancies on how peroxiredoxins
should be described in these studies as three distinct kinetic models have been proposed for
peroxiredoxin activity: the ping-pong enzyme, redox couple monomer and redox couple
homodimer models. Further, different rate constants for hydrogen peroxide reduction by
peroxiredoxins have been reported using steady state and competition assays and it is not
clear which of these parameters should be used in computational models. In order to resolve
these discrepancies, the three proposed peroxiredoxin kinetic models were simulated with
core parameters and showed different responses to parameter changes. Computational
modelling with in vitro datasets confirmed this result and also showed that many of the
reported peroxiredoxin kinetic parameters have limited predictive value. Thus, the kinetic
models for peroxiredoxin activity cannot be used interchangeably and computational models
based on the reported peroxiredoxin kinetic parameters for hydrogen peroxide reduction
should be viewed with caution. To confirm this result, the cytosolic peroxiredoxin thiolspecific
antioxidant 1 (TSA1) from Saccharomyces cerevisiae was cloned, expressed and
purified for in vitro analysis of this system. Data fitting of the peroxiredoxin kinetic models
determined parameters that were able to predict independent datasets with increasing
thioredoxin and peroxiredoxin concentrations using the ping-pong enzyme and redox couple
monomer models but the redox couple homodimer model was unable to fit these datasets. A
complex flux control pattern was also determined for the fitted models and whole system
fitting to in vitro datasets is proposed to be a more accurate method for parameter
determination for the peroxiredoxin system kinetic assays.
Description
Master of Science in Genetics.
Keywords
Peroxiredoxins., Peroxidase., Systems biology., Peroxidation., Theses -- Genetics.