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Protein denitrosylation: enzymatic mechanisms and cellular functions

Key Points

  • The ubiquitous influence of nitric oxide (NO) in cellular signalling is largely mediated by S-nitrosylation, the covalent attachment of NO to the thiol side chain of Cys residues. Proteins in most or all functional classes are substrates for S-nitrosylation, and a growing body of research shows that aberrant S-nitrosylation is implicated in a wide range of human pathologies.

  • Recent discoveries indicate that the denitrosylation of proteins, which was once considered to be a spontaneous and unregulated event, is catalysed by enzymes in vivo. Denitrosylases might either directly mediate denitrosylation of proteins or govern the equilibrium between protein and low-molecular-weight nitrosothiols (collectively referred to as SNOs).

  • It has become increasingly clear that both S-nitrosylation and denitrosylation are precisely regulated in time and space. In particular, protein denitrosylation can be triggered by the stimulation of multiple classes of cell surface receptors, including members of the tumour necrosis factor family of receptors, G protein-coupled receptors and receptor Tyr kinases.

  • Several denitrosylases have recently been discovered, and two highly conserved enzyme systems in particular, the thioredoxin (Trx) system (which comprises Trx and Trx reductase (TrxR)) and the S-nitrosoglutathione reductase (GSNOR) system (which comprises glutathione (GSH) and GSNOR; GSNOR is also known as GSH-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase (ADH5) and is encoded by human gene ADH5) have been established to be physiologically relevant.

  • Trx proteins and GSNOR regulate the denitrosylation of multiple mammalian proteins and thereby modulate diverse cellular responses, including β-adrenergic receptor signalling, endocytosis, inflammation, angiogenesis and apoptotic cell death.

  • Denitrosylases are ubiquitously expressed in microbes and plants, in which they confer protection from nitrosative stress that is mediated by the host (that is, they serve as virulence factors) and exert profound effects on cellular immunity.

Abstract

S-Nitrosylation, the redox-based modification of Cys thiol side chains by nitric oxide, is a common mechanism in signal transduction. Dysregulated S-nitrosylation contributes to a range of human pathologies. New roles for protein denitrosylation in regulating S-nitrosylation are being revealed. Recently, several denitrosylases — the enzymes that mediate Cys denitrosylation — have been discovered, of which two enzyme systems in particular, the S-nitrosoglutathione reductase and thioredoxin systems, have been shown to be physiologically relevant. These highly conserved enzymes regulate signalling through multiple classes of receptors and influence diverse cellular responses. In addition, they protect from nitrosative stress in microorganisms, mammals and plants, thereby exerting profound effects on host–microbe interactions and innate immunity.

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Figure 1: Mechanisms and consequences of reversible protein S-nitrosylation.
Figure 2: Biochemical mechanisms of protein denitrosylation.
Figure 3: Protein denitrosylation in cellular signalling.
Figure 4: Role of GSNO and GSNOR in hypoxic responses.
Figure 5: Regulation of apoptotic signalling by reversible S-nitrosylation.
Figure 6: Denitrosylases in nitrosative stress and host–microbe interactions.

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Correspondence to Jonathan S. Stamler.

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The authors are inventors on patents that concern protein denitrosylation, and J. Stamler is involved in the development of nitric oxide-based technologies.

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Glossary

G protein-coupled receptor

A cell surface receptor (such as the β-adrenergic and the cholinergic receptors) that possesses seven transmembrane domains and is coupled to G proteins. Typically, the activation of a G protein-coupled receptor produces a second messenger that initiates a signal transduction cascade.

NO synthase

(NOS). Mammals have three nitric oxide (NO) synthases that generate NO from Arg NOS1 (or neuronal NOS), NOS2 (or inducible NOS) and NOS3 (or endothelial NOS) one or more of which reside or can be induced in most or all cell types. NOS orthologues are distributed broadly across phylogeny.

Metalloprotein

A protein that contains a metal ion or ions (including Fe2+, Cu2+ or Zn2+) as a prosthetic group that is coordinated by amino acid side chains.

Nitrosative stress

The dysregulated production and/or metabolism of reactive nitrogen species, which generate nitrosative chemistries that can result in disrupted cellular signalling, injury and death. Oxidative stress is brought about by reactive oxygen species.

S-Nitrosoglutathione

The main non-protein S-nitrosothiol (SNO) in cells, which can be present in micromolar concentrations, and that is in equilibrium with protein SNOs.

Glutathione

A tripeptide composed of Glu, Cys and Gly that is the principal, small-molecular-weight, thiol-containing molecule in the cell.

Nucleophilic

Having an affinity for positive charge. Nucleophilic molecules are electron rich and tend to attack electron-poor molecules or behave as electron donors.

Reactive oxygen species

Reduced derivatives of molecular oxygen that include, in particular, the superoxide radical (O2) and hydrogen peroxide (H2O2), which can have substantial reactivity towards biological macromolecules and towards other reactive small molecules.

Lyase

Typically, an enzyme that breaks a bond without hydrolysis or oxidation.

NADH

The reduced form of nicotinamide adenine dinucleotide. This coenzyme serves as an electron donor for various biochemical reactions.

Caspase

A family of Cys proteases, divided into initiator and effector caspases, that might require proteolytic cleavage to liberate subunits that reconstitute an active caspase heterodimer. All caspases contain a Cys residue at the active site and cleave substrates carboxy-terminal to an Asp residue.

Oxidoreductase

An enzyme that catalyses oxidation–reduction reactions. These entail the transfer of electrons from a substrate that becomes oxidized (electron donor) to a substrate that becomes reduced (electron acceptor).

Death receptor

One of a family of cell surface receptors that mediate cell death upon ligand-induced trimerization. The best-studied members include tumour necrosis factor receptor 1 (TNFR1) and FAS (or CD95), which binds the FAS ligand.

Virulence

The damage caused to the host by a parasite or pathogen, which is measured as a decrease in host fitness.

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Benhar, M., Forrester, M. & Stamler, J. Protein denitrosylation: enzymatic mechanisms and cellular functions. Nat Rev Mol Cell Biol 10, 721–732 (2009). https://doi.org/10.1038/nrm2764

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