Chemical approaches to discovery and study of sources and targets of hydrogen peroxide redox signaling through NADPH oxidase proteins

Abstract

Hydrogen peroxide (H2O2) is a prime member of the reactive oxygen species (ROS) family of molecules produced during normal cell function and in response to various stimuli, but if left unchecked, it can inflict oxidative damage on all types of biological macromolecules and lead to cell death. In this context, a major source of H2O2 for redox signaling purposes is the NADPH oxidase (Nox) family of enzymes, which were classically studied for their roles in phagocytic immune response but have now been found to exist in virtually all mammalian cell types in various isoforms with distinct tissue and subcellular localizations. Downstream of this tightly regulated ROS generation, site-specific, reversible covalent modification of proteins, particularly oxidation of cysteine thiols to sulfenic acids, represents a prominent posttranslational modification akin to phosphorylation as an emerging molecular mechanism for transforming an oxidant signal into a dynamic biological response. We review two complementary types of chemical tools that enable (a) specific detection of H2O2 generated at its sources and (b) mapping of sulfenic acid posttranslational modification targets that mediate its signaling functions, which can be used to study this important chemical signal in biological systems.

Keywords: bioorthogonal chemistry; fluorescent probes; molecular imaging; oxidative stress; posttranslational modifications; reactive oxygen species.

Figure 1

The perceived role of H₂O₂ in biology has evolved from toxin to agent of the immune system to a key component of signaling pathways. Abbreviations: cGMP, cyclic guanosine monophosphate; IL, interleukin; PDGF, platelet-derived growth factor; TNF, tumor necrosis factor.

Figure 2

Strategies for detection of the sources, targets, and downstream consequences of H₂O₂ signaling. Abbreviations: ER, endoplasmic reticulum; Nox, NADPH oxidase.

Figure 3

Aryl boronates are a selective platform for H₂O₂ detection. (a) General oxidation of an aryl boronate by H₂O₂. (b–g) Selected small-molecule boronate-based probes for H₂O₂. Abbreviations: PG1, Peroxy Green 1; PO1, Peroxy Orange 1; NucPE1, Nuclear Peroxy Emerald 1; PF6AM, Peroxyfluor-6 acetoxymethyl ester; PY1ME, Peroxy Yellow 1 Methyl Ester.

Figure 4

Applications of small-molecule boronate probes. (a) Response of PCL1 to increasing amounts of H₂O₂ in live mice (33). (b) PG1 detects H₂O₂ production by neurons stimulated with epidermal growth factor (51). (c) PO1 and APF detect three distinct types of phagosomes that produce primarily H₂O₂ (orange), primarily hROS (green), and both (yellow) (43). (d) PF6AM detects H₂O₂ production by adult hippocampal progenitor cells stimulated with fibroblast growth factor 2 (55). (e) PY1ME detects increased H₂O₂ uptake by cells expressing aquaporin-3 (16). (f) NucPE1 detects nuclear H₂O₂ fluxes in Caenorhabditis elegans (64). Abbreviations: APF, aminophenyl fluorescein; hROS, highly reactive oxygen species; NucPE1, Nuclear Peroxy Emerald 1; PF6AM, Peroxyfluor-6 acetoxymethyl ester; PG1, Peroxy Green 1; PO1, Peroxy Orange 1; PY1ME, Peroxy Yellow 1 Methyl Ester. Modified from References , , , , , and .

Figure 5

Oxidative modifications of cysteine residues by H₂O₂. Abbreviations: GR, glutaredoxin; Grx, glutaredoxin reductase; TR, thioredoxin; Trx, thioredoxin reductase.

Figure 6

Indirect approaches to detect protein sulfenic acids. (a) Modified biotin switch technique adapted to indirectly detect sulfenic acid–modified proteins (85, 86). (b) Loss of reactivity with thiol-modifying reagents, such as BIAM, indirectly monitors cysteine oxidation. (c) ICAT reagents determine the ratio of oxidized cysteine residues (87, 88). Abbreviations: BIAM, biotinylated iodoacetamide; ICAT, isotope-coded affinity tag; NEM, N-ethylmaleimide; ROS, reactive oxygen species.

Figure 7

Dimedone-based probes for chemoselective detection of protein sulfenic acids. (a) Chemoselective reaction to yield a stable thioether adduct. (b) Dimedone-based probes directly conjugated with biotin or fluorescent tags (90, 93, 94, 100, 101). (c) Dimedone-based probes with bioorthogonal handles for subsequent enrichment or detection of sulfenic acid–modified proteins (71, 101, 108, 114, 116, 118). (d) Redox-based probes that target the redox-sensitive catalytic cysteine in protein tyrosine phosphatases through incorporation of a chemical scaffold with high affinity for the enzyme’s active site (107, 124). (e,f) Bioorthogonal reactions for appending tags to dimedone-based probes with handles (109, 110). Abbreviations: BP1, 4-(ethylthio)cyclopentane-1,3-dione; DCP-FL1, fluoresceinamine-5′-N-[3-(2,4-dioxocyclohexyl)propyl)]carbamate; DCP-MCC, 3-(2,4-dioxocyclohexyl)propyl 7-methoxy-2-oxo-2H-chromen-3-ylcarbamate; DCP-Rho1, rhodamine B [4-[3-(2,4-dioxocyclohexyl)propyl]carbamate]piperazine amide.

Figure 8

Methods to directly detect protein sulfenic acids. (a) Direct in situ labeling of sulfenylated proteins using cell-permeable chemoselective reagents. (b) Isotope-coded dimedone 2-iododimedone allows for quantification of sulfenylated proteins (118). (c) Use of ACLs and isotopically labeled DAz-2 to facilitate enrichment and quantification of sulfenylated proteins (119). Abbreviations: ACL, acid-cleavable linker; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 9

Detection of sulfinic acid– and sulfonic acid–modified proteins. (a) Reaction of a sulfinic acid with an aryl nitroso compound to yield a stable cyclic sulfonamide analog (148). (b) Antibodies that recognize the sulfonic acid–modified proteins (146).