Post-translational protein modification by S-nitrosylation, the covalent addition of a nitric oxide (NO) group to a Cys thiol to form an S-nitroso-protein (SNO-protein), mediates a large part of the ubiquitous influence of NO on cellular function in mammalian systems, and dysregulated S-nitrosylation has been associated with a broad spectrum of human diseases. Increasing evidence points to essential roles for enzymatically mediated denitrosylation, that is, the removal of the NO group from SNO-proteins, in regulating the levels and dynamics of protein S-nitrosylation, but there has been no systematic identification of denitrosylases or delineation of their substrates. Previously, we used E. coli as a model system to identify an evolutionarily conserved enzymatic mechanism that regulates denitrosylation, S-nitrosoglutathione reductase (GSNOR), which does not act directly on SNO-proteins but regulates protein S-nitrosylation by virtue of the cellular equilibrium between at least some SNO-proteins and S-nitrosoglutathione. More recently, our analysis in E. coli has identified a novel SNO-protein denitrosylase (the first described in microorganisms). In the studies proposed here, we will employ E. coli as a model system to identify systematically denitrosylases, based partly on our finding that a specific transcription factor (TF) is S-nitrosylated and activated under nitrosative stress. The unique regulon that is consequently up-regulated governs cellular SNO-protein levels, at least in part through the induction of denitrosylating activity.
In Aim 1, we will focus on the newly identified denitrosylase and on the dithiol reductase thioredoxin, previously identified by us as a SNO-protein denitrosylase in mammalian cells, and we will employ solid-phase proteomic methods introduced by us to determine the SNO-proteins (induced by nitrosative stress) that serve as substrates of these enzymes. We have found that multiple proteins, including TF itself, are rapidly denitrosylated in cells deficient in all known denitrosylases, and in Aim 2 we will: a) interrogate the SNO-TF interactome and b) establish a biochemical screen for denitrosylase activity, to identify the responsible denitrosylase(s).
In Aim 3, we will screen the components of the regulon that is induced upon S-nitrosylation of TF for novel denitrosylating activities. Thus, these Aims converge on the identification of novel denitrosylases and their substrates. The proposed studies have direct relevance for human pathophysiology, because we have established previously that denitrosylating activates discovered in microorganisms are likely to be highly conserved through phylogeny, and our analysis is thus likely to reveal novel enzymatic activities of broad purview in the analysis of dysregulated S-nitrosylation in human disease. In addition, inasmuch as denitrosylases protect bacteria against the nitrosative stress that is a principal component of mammalian innate immunity, our studies may point to potential therapeutic targets in the treatment of bacterial pathogenesis.

Public Health Relevance

Protein S-nitrosylation, the covalent attachment of a nitric oxide group to a Cys thiol side chain, is a principal mechanism for regulating cellular signaling i mammalian systems, and dysregulated protein S-nitrosylation has been implicated in a broad spectrum of human diseases. Accumulating evidence demonstrates that enzymatic denitrosylation plays essential roles in regulating the levels and dynamics of protein S-nitrosylation, but our understanding of denitrosylating mechanisms remains rudimentary. In the proposed studies, we will utilize the bacterium E. coli as a model system to identify novel denitrosylases and to characterize their substrate specificities, employing a combination of microarray-based, proteomic and biochemical approaches.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Anderson, Vernon
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Case Western Reserve University
Internal Medicine/Medicine
Schools of Medicine
United States
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Rizza, Salvatore; Cardaci, Simone; Montagna, Costanza et al. (2018) S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy. Proc Natl Acad Sci U S A 115:E3388-E3397
Seth, Divya; Hess, Douglas T; Hausladen, Alfred et al. (2018) A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation. Mol Cell 69:451-464.e6
Elphinstone, Robyn E; Besla, Rickvinder; Shikatani, Eric A et al. (2017) S-Nitrosoglutathione Reductase Deficiency Confers Improved Survival and Neurological Outcome in Experimental Cerebral Malaria. Infect Immun 85:
Benhar, Moran; Shytaj, Iart Luca; Stamler, Jonathan S et al. (2016) Dual targeting of the thioredoxin and glutathione systems in cancer and HIV. J Clin Invest 126:1630-9
Seth, Divya; Stamler, Jonathan S (2015) SNOs Differ: Methodological and Biological Implications. Circ Res 117:826-9
Shinozaki, Shohei; Chang, Kyungho; Sakai, Michihiro et al. (2014) Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65. Sci Signal 7:ra106