Downstream targets and intracellular compartmentalization in Nox signaling

Abstract

Reactive oxygen species (ROS) have become recognized for their role as second messengers in a multitude of physiologic responses. Emerging evidence points to the importance of the NADPH oxidase family of ROS-producing enzymes in mediating redox-sensitive signal transduction. However, a clear paradox exists between the specificity required for signaling and the nature of ROS as both diffusible and highly reactive molecules. We seek to understand the targets and compartmentalization of the NADPH oxidase signaling to determine how NADPH oxidase-derived ROS fit into established signaling paradigms. Herein we review recent data that link cellular NADPH oxidase enzymes to ROS signaling, with a particular focus on the mechanism(s) involved in achieving signaling specificity.

FIG. 1.

NADPH oxidase family members translocate electrons (from NADPH) across a membrane, which results in the formation of ROS (predominantly ). The seven NADPH oxidase family members (Nox 1 to 5 and Duox 1 and 2) share conserved features, including six transmembrane domains, where transmembrane domains III and V contain four heme-binding histidines. The carboxy terminus consists of an FAD-binding domain followed by an NADPH-binding domain. Nox5 includes an additional N-terminal calmodulin-like Ca²⁺-binding domain. Duox 1 and 2 also include peroxidase homology domains.

FIG. 2.

Protein cysteine (Cys) residues are fundamental in ROS signal transduction. (A) Protein Cys residues with the pKa lower than intracellular pH are readily deprotonated, leading to formation of the more-reactive thiolate anion (RS⁻). (B) ROS reaction with thiolate anions leads to formation of sulfenic acid (RSOH), which is highly reactive. (C) Sulfenic acid can form reversible modifications with surrounding thiols, nitrogens, or GSH to form disulfides, sulfenamides, or protein S-glutathionylation, leading to changes in protein function. (D) Reversible Cys modifications are reduced by antioxidant systems such as glutaredoxin (Grx) and thioredoxin (Trx) (E) Sulfenamides can also be further oxidized to sulfinic (RSO₂H) or sulfonic (RSO₃H) acid, which is generally considered irreversible.

FIG. 3.

Protein tyrosine phosphatases (PTPs) are subject to oxidant-induced signaling. (A) In the presence of an electrophile such as H₂O₂, the active-site cysteine (R-S⁻) residue is oxidized to a sulfenic acid (R-SOH) intermediate. (B) This is followed by rapid intraprotein conversion to a cysteine sulfenyl-amide, which produces an active-site conformational change that inhibits substrate binding. (C) The activity of the PTPs is restored by antioxidant/antioxidant systems such as glutathione (GSH)/glutaredoxin (Grx).

FIG. 4.

Nox 4 mediates EGFR activity. (A)Vesiculation of internalized EGFR trafficks phosphorylated EGFR near the endoplasmic reticulum (ER). (B) Nox 4 is localized to the ER in close proximity to PTP1b, thus influencing PTP1b activity. (C) ROS emanating from ER Nox4 are then able to mediate EGFR signaling through deactivation of PTP1b, leading to increased EGFR phosphorylation and recycling of phosphorylated (active) receptor to the plasma membrane.

FIG. 5.

Ras proteins undergo modification of conserved Cys residues. In this paradigm, Cys118 is glutathionylated (S-SG) under oxidizing conditions. Glutathionylation of Cys118 stimulates nucleotide exchange (GDP to GTP), leading to enhanced Ras activation.

FIG. 6.

Nrf2 is under the control of its negative regulator, Keap1. Redox-sensitive cysteine residues in Keap1 are important in Nrf2 association with Keap1. Keap1 forms a complex with Nrf2 that facilitates its targeting by cullin family ubiquitin E3 ligases for ubiquitination and proteasomal degradation. However, oxidation of Cys residues in Keap1 triggers its dissociation from Nrf2, allowing Nrf2 to translocate into the nucleus and activate stress-response genes.