Biochemical Characterization And Preliminary Genetic Analysis Of Peroxiredoxins Of Sulfolobus Islandicus REY15A

All Aerobic organisms are confronted with the problem of "How to maintain intracellular redox homeostasis".ROS?reactive oxygen species?,which include hydrogen peroxide,superoxide anion free radicals,hydroxyl radicals and singlet oxygen,are responsible for excessive oxidative stress in cells,causing damage to biological macromolecules?proteins.DNA and Lipids?and in turn affecting cell growth.The level of ROS in cells is influenced not only by metabolism,but also by the cells' microenvironment.Enzymes known to clear ROS include Catalase.Peroxiredoxins?Prxs?.Glutathione Peroxidase,and some metal-dependent enzymes.However.Prxs are more sensitive and responsive to peroxide because of-thier unique catalytic center structure.Therefore,they play an important role in maintaining the redox homeostasis in the cells.Compared with other proteins that can reduce ROS.the study of Prxs started relatively late,and mainly focused on eukaryotes,especially mammals.However.whether the Prxs in Sulfolobus islandicus REY15A,a kind of thermophilic and acidophilic Archaea.have special properties and functions is unknown.Therefore this project is to characterize and in vitro functionally analyze Prxs of S.islandicus REY 5A".First,we identified seven annotated Prxs in S.islandicus REY15A by bioinformatics analysis.They are SiRe₀067.SiRe₀346.SiRe₀725.SiRe₀977.SiRe 1643.SiRe₂162 and SiRe₂592.SiRe₁643 and SiRe 2162 belong to Prxs-like families,which are not the focus of our study.We analyzed the conservativeness of the remaining 5 Prxs in Archaea and predicted their active sites.By sequence alignment.SiRe₀067,SiRe₀346.SiRe₀725 and SiRe₂592 were found to be conserved in Archaea.and their catalytic sites match the sequence of PXXXT/SXXCP?Cysteine is the catalytic active site?.Then.the wild-type and activity deficient mutant Prxs were expressed and purified in E.coli or Sulfolobus.We identified the in vitro properties of these Prxs.Using hydrogen peroxide and tert-butyl peroxide as substrates,we determined whether the five Prxs had peroxidase activity.It was found that SiRe₀346.SiRe₀067 and SiRe₂592 can react with hydrogen peroxide and tert-butyl Peroxide.C42 is the catalytic site of SiRe 0067.and C45/50 are the catalytic site of SiRe 2592.Using many kinds of DNA as substrates,the ability of these proteins to bind to DNA and the factors influencing their binding to DNA were identified.We found that SiRe 0067 and SiRe 0067-C42S can bind to DNA.and the redox state of SiRe 0067 can affect its DNA binding ability.We want to know whether these Prxs protect DNA from hydroxyl radical,and found that SiRe 0067,SiRe 0346,SiRe 0725 and SiRe 2592 can protect DNA from hydroxyl radical to some extent and SiRe 0346 is the most protective.Next,we analyzed whether the overexpression of these five Prxs had an effect on cell growth.We found that only SiRe₀067's overexpression could slow down cell growth and the other Prxs' overexpression had no significant effect on cell growth.To determine whether the overexpression of Prxs protects cells from oxidative stress when cells are subjected to oxidative stress,we found that SiRe 0346 exhibits a strong protective effect against oxidative stress.Finally,a histidine-tag was added to the C terminal of the Prxs in situ,.and Western-Blot was used to further analyze the difference in the intracellular Prxs content under normal conditions and the changes in the expression level of Prxs when cells were subjected to oxidative stress.In in vitro experiments.SiRe 0346 showed highly efficient activity to reduce hydrogen peroxide,tent-butyl peroxide and hydroxyl radical.Further,SiRe 0346 can improve the ability of cells to resist oxidative stress to some extent.Therefore.SiRe 0346 is considered to be the main Prxs to maintain the redox homeostasis in S.islandicus REY15A.and this result corresponds to that in Sulfolobus solfalaricus P2 after UV treatment,the transcription level of SS02121?SiRe 0346's homologous protein?doubled.In vitro,SiRe 0067 and SiRe 2592 also showed a certain ability to scavenge peroxide and free radicals,so they are likely to exist as backup proteins in S.islandicus REYI15A.When SiRe 0346 fails to function,they keep cells free from oxidative stress.Considering the unique properties of SiRe 0067.which is the only protein of Prxs that has DNA binding ability and can affect cell growth after overexpression.we speculate that SiRe₀067 may be involved in the intracellular signaling pathway and the oxidation-reduction state of SiRe₀067 can regulate its function.