The typical 2-Cys peroxiredoxins (Prxs) are key antioxidant enzymes in the detoxification of reactive oxygen species including hydrogen peroxide (H2O2). At lower concentrations, H2O2 has also been recognized as an important mediator of cell signaling. In this context, the high cellular concentration and reactivity of Prxs with H2O2 makes them ideally suited to regulate redox-dependent signaling events. Human 2-Cys Prxs can be inactivated, however, through hyperoxidation to the Cys sulfinic acid (Cys-SO2-), a hallmark of many aging- related diseases and cancer. The sensitivity to hyperoxidation and the repair of these Prxs by the enzyme sulfiredoxin (Srx) differs. The mitochondrial PrxIII is the most resistant to hyperoxidation. Surprisingly, few details are available for the structures of the human Prxs when present in different oxidation states (Cys-SH, Cys-S-S-Cys, and Cys-SO2-), and even less is known about the kinetics of hyperoxidation and Srx-mediated repair for this class of enzymes. We have shown that Srx utilizes a novel nucleotide binding motif and sulfur chemistry to reduce the Prx molecule and were able to identify critical kinetic intermediates in the repair of hyperoxidized PrxII by Srx. PrxIII on the other hand exhibits a unique C-terminal sequence in the region that is expected to make direct contact with Srx, based on our crystal structure of the human Srx7PrxI complex. As such, we hypothesize that PrxIII is not only more resistant to hyperoxidation due to its C-terminus and active site differences, but also that it will have a unique interaction with Srx that may influence the repair process. Preliminary studies on the four human, 2-Cys Prxs (PrxI-IV) have confirmed the results obtained in cell culture and have shown that indeed PrxIII is the most resistant to hyperoxidation. In addition, we have generated preliminary crystals for PrxI-IV in different oxidation states and performed comparative kinetics studies for PrxII and PrxIII hyperoxidation by time-resolved mass spectrometry. These analyses have shown for the first time the formation of an intramolecular Cys sulfenamide intermediate in PrxII. Interestingly, PrxIII did not form this species under the same reaction conditions, identifying one potential scenario that may impart sensitivity to hyperoxidation in PrxI, PrxII, and PrxIV and resistance to hyperoxidation in PrxIII. Given that the transgenic expression of PrxIII and Srx results in protection against oxidative stress-induced apoptosis and tissue damage during myocardial infarction, an understanding of the structural and kinetics bases of Prxs catalysis, hyperoxidation, and repair by Srx will be invaluable for the future design of novel treatment strategies using PrxIII and/or Srx variants in gene therapy.
The specific aims of the proposal are to investigate the structural and kinetic determinants of hyperoxidation in human 2-Cys Prxs (Aim I), and to investigate the repair mechanisms of human 2-Cys Prxs by Srx (Aim2).

Public Health Relevance

The inactivation of 2-Cys peroxiredoxins (Prxs) by hydrogen peroxide is a hallmark of many aging-related diseases including cancer, cardiovascular disease, and Alzheimer's disease. Humans possess four Prxs;one of which is unusually resistant to inactivation by hyperoxidation and has been shown to prevent cell death and tissue damage from a heart attack. The purpose of this study is to understand how Prxs become inactivated and how they can be repaired by an enzyme called sulfiredoxin.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072866-09
Application #
8623135
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Barski, Oleg
Project Start
2005-08-01
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
9
Fiscal Year
2014
Total Cost
$347,803
Indirect Cost
$112,801
Name
Wake Forest University Health Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157
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Chen, Xiaofei; Wu, Hanzhi; Park, Chung-Min et al. (2017) Discovery of Heteroaromatic Sulfones As a New Class of Biologically Compatible Thiol-Selective Reagents. ACS Chem Biol 12:2201-2208
Poynton, Rebecca A; Peskin, Alexander V; Haynes, Alexina C et al. (2016) Kinetic analysis of structural influences on the susceptibility of peroxiredoxins 2 and 3 to hyperoxidation. Biochem J 473:411-21
Cunniff, Brian; Newick, Kheng; Nelson, Kimberly J et al. (2015) Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma. PLoS One 10:e0127310
Haynes, Alexina C; Qian, Jiang; Reisz, Julie A et al. (2013) Molecular basis for the resistance of human mitochondrial 2-Cys peroxiredoxin 3 to hyperoxidation. J Biol Chem 288:29714-23
Lowther, W Todd; Haynes, Alexina C (2011) Reduction of cysteine sulfinic acid in eukaryotic, typical 2-Cys peroxiredoxins by sulfiredoxin. Antioxid Redox Signal 15:99-109
Klomsiri, Chananat; Nelson, Kimberly J; Bechtold, Erika et al. (2010) Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins. Methods Enzymol 473:77-94
Jonsson, Thomas J; Johnson, Lynnette C; Lowther, W Todd (2009) Protein engineering of the quaternary sulfiredoxin.peroxiredoxin enzyme.substrate complex reveals the molecular basis for cysteine sulfinic acid phosphorylation. J Biol Chem 284:33305-10
Cox, Andrew G; Pearson, Andree G; Pullar, Juliet M et al. (2009) Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins. Biochem J 421:51-8

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