Hydrogen peroxide is a toxin used by the human immune system to kill infectious organisms, and increasing evidence shows that it is also a common second messenger in eukaryotic signaling. In humans, cytokines, growth factors and toll-like receptors of the innate immune system are thought to signal via hydrogen peroxide. Catalase and glutathione peroxidase have long been viewed as the major enzymes degrading peroxide in cells, however, over the past few years, a distinct, highly abundant family of peroxide- reducing enzymes, peroxiredoxins (Prxs), have moved from relative obscurity to become a major focus of redox biology research. The peroxidase activity of eukaryotic Prxs was overlooked for many years, because those Prxs that are highly expressed in eukaryotes are easily inactivated by peroxide. We have developed expertise in Prx enzymology over more than 15 years of characterizing of Prxs from pathogenic bacteria (e.g. Salmonella typhimurium AhpC). These Prxs are targets for antibiotic development because of the role they play in protecting the bacteria from the human immune system. In 2003, our structural and functional studies on S. typhimurium AhpC led us to discover the structural basis for the sensitivity toward peroxides that is conserved for a subset of Prxs that are highly expressed across all eukarya (this is the basis for the structural hypothesis that underlies the present grant, which dictates that the mobility of proximal secondary structures packing near the active site is a key determinant of the sensitivity of Prxs to overoxidation by peroxides and of the ability of Prxs to act as antioxidants). We further proposed the """"""""floodgate hypothesis"""""""" for how this sensitivity to inactivation would actually be beneficial in organisms where hydrogen peroxide is being used as a signaling molecule, so that the antioxidant properties of the Prxs could be switched off under appropriate conditions to allow for a controlled burst in peroxide levels. Given the importance of Prxs both in pathogen defense and in human cells for combating oxidative stress and for cellular regulation, we propose here to identify the key determinants of sensitivity toward overoxidation and of efficient antioxidant function by investigating the conformational """"""""mobility"""""""" of a few carefully chosen proteins and mutants;relevant rates constants within the catalytic cycle and inactivation pathways for these proteins will also be examined (Aim 1).
In Aim 2, we will identify structural features around the highly conserved active site of Prxs which are important for binding and reduction of distinct hydroperoxide substrates.
In Aim 3, we will determine whether or not the sensitivity of Prxs toward inactivation by peroxides during turnover (the """"""""floodgate"""""""") is critical to modulating the levels of H2O2 generated during cell signaling events through cell-based studies of Prx functions.

Public Health Relevance

Oxidative damage is thought to be important in aging, in the development of cancer and in many degenerative diseases. Moreover, impairments in cell signaling processes controlling proliferation, differentiation and apoptosis are associated with many disease states. An enhanced understanding of Prxs and the roles they play in both cell signaling and antioxidant protection will thus have important implications for the prevention of human diseases. In addition, the role of Prxs in protecting human pathogens against killing by the immune system implicates Prxs as targets for the development of new therapeutic agents to combat infectious diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050389-19
Application #
8269972
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1993-12-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
19
Fiscal Year
2012
Total Cost
$394,793
Indirect Cost
$94,812
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
Buchko, Garry W; Perkins, Arden; Parsonage, Derek et al. (2016) Backbone chemical shift assignments for Xanthomonas campestris peroxiredoxin Q in the reduced and oxidized states: a dramatic change in backbone dynamics. Biomol NMR Assign 10:57-61
Poole, Leslie B; Nelson, Kimberly J (2016) Distribution and Features of the Six Classes of Peroxiredoxins. Mol Cells 39:53-9
Parsonage, Derek; Sheng, Fang; Hirata, Ken et al. (2016) X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action. J Struct Biol 194:180-90
Randall, Lía; Manta, Bruno; Nelson, Kimberly J et al. (2016) Structural changes upon peroxynitrite-mediated nitration of peroxiredoxin 2; nitrated Prx2 resembles its disulfide-oxidized form. Arch Biochem Biophys 590:101-8
Poole, Leslie B (2015) The basics of thiols and cysteines in redox biology and chemistry. Free Radic Biol Med 80:148-57
Parsonage, Derek; Nelson, Kimberly J; Ferrer-Sueta, Gerardo et al. (2015) Dissecting peroxiredoxin catalysis: separating binding, peroxidation, and resolution for a bacterial AhpC. Biochemistry 54:1567-75
Karplus, P Andrew (2015) A primer on peroxiredoxin biochemistry. Free Radic Biol Med 80:183-90
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
Poole, Leslie B; Schöneich, Christian (2015) Introduction: What we do and do not know regarding redox processes of thiols in signaling pathways. Free Radic Biol Med 80:145-7
Perkins, Arden; Nelson, Kimberly J; Parsonage, Derek et al. (2015) Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling. Trends Biochem Sci 40:435-45

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