Identification of residues in human peroxiredoxin I for their role in maintaining the redox potential in cells

Abstract

newline vii newlineABSTRACT newlinePeroxiredoxins (Prxs) are antioxidant proteins which are involved in cellular defense against the reactive oxygen species (ROS) and reactive nitrogen species (RNS). All the Prxs contain a N-terminal conserved Peroxidatic Cys (CP-SH) residue that reacts with peroxides to form CP-SOH (sulfenic acid). Humans have six peroxiredoxins hPrxI-VI, out of which hPrxI and hPrxII belongs to typical 2-cys class. hPrxI and hPrxII are 91% homologous in their amino acid sequences including their catalytic cysteine residues (C52, C173 in hPrxI and C51, C172 in hPrxII). It is well established that hPrxI and hPrxII are dependent on these catalytic residues to carry out peroxidase and chaperone activity. But despite this, PrxI is shown to behave differently in its peroxidase and chaperone activity. It has been suggested that mammalian typical 2-Cys Prxs act like floodgates i.e. they buffer the low levels of H2O2, hence protect signaling proteins such as protein tyrosine phosphatase (PTP) from oxidation and upon hyperoxidation, they also permit the accumulation of H2O2 which then activates H2O2 signal transduction. Hyperoxidized Prxs enhance the stability of the Prx oligomers, and thus increase the signaling and chaperone activities that help in the repair of oxidative stress induced damage, for example hyperoxidized Prxs interact with Hsp70, Hsp104 and aggregated misfolded proteins and help in the disaggregation of the misfolded proteins. It has been shown that hPrxI and not hPrxII, could complement the defects induced in tsa1and#916; mutant suggesting that human hPrxI is an orthologue of yeast Tsa1. This shows the difference in the behaviour of hPrxI and hPrxII, despite having 91% homologues in their amino acid sequence. Previously, using yeast as a model system we have shown that how intracellular redox state of hPrxs represents their ability to overcome oxidative stress. In this study we wanted to dissect the role of different conserved residues in hPrxI for overcoming oxidative and nitrosative stress using the same model system. So, to check the exact role of all these Cysteine residues in hPrxI through Mutations we used yeast as a model system. We found that all the catalytic mutants including hPrxIC52S and hPrxIC173S were not able to overcome nitrosative stress but interestingly they were able to counter oxidative stress. Whereas Cys83 seems to be involved in the chaperone acivity of hPrxI as hPrxIC83T was unable to form oligomers even with the intact catalytic newlineviii newlinecysteines. These results suggest that in hPrxI, Cys52 is crucial to counter nitrosative newlinestress and Cys83 plays a critical role in the chaperone activity. newlineKeywords: Human peroxiredoxins, catalytic residues, yeast, redox stress, chaperone newlineactivity.