Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling

Abstract

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.

Fig. 1

Cellular signaling pathways regulated by ROS. Reactive oxygen species (ROS) regulate several signaling pathways through interaction with critical signalingmolecules, affecting a variety of cellular processes, such as proliferation, metabolism, differentiation, and survival (apoptosis signal-regulated kinase 1 (ASK1), PI3 kinase (PI3K), protein tyrosine phosphatase (PTP), and Src homology 2 domain-containing (Shc)); antioxidant and anti-inflammatory response (thioredoxin (TRX), redox-factor 1 (Ref-1), and NFE2-like 2 (Nrf-2)); iron homeostasis (iron regulatory protein (IRP)); and DNA damage response (ataxia–telangiectasia mutated (ATM)).

Fig. 2

Activation of ASK kinases in response to oxidative stress. A) Oxidation of thioredoxin (TRX) results in disulfide bond formation between Cys-32 and Cys-35 and subsequent dissociation from ASK1. ASK1 undergoes complete homo-oligomerization and subsequent autophosphorylation at Thr-838 located in the kinase domain. B) Hetero-oligomerization of ASK1 and ASK2 stabilizes ASK2, resulting in 1) the autophosphorylation of ASK2 at Thr-806, and 2) the subsequent phosphorylation of ASK1 at Thr-838 by ASK2.

Fig. 3

Mechanism of ROS-mediated protein tyrosine phosphatase inactivation. Tyrosine kinases, activated by growth factors, cytokines, and hormones, phosphorylate target proteins. Phosphorylation can be reversed by protein tyrosine phosphatases (PTP); ROS inactivates PTP by oxidation of catalytic cysteine residues resulting in the formation of the sulfenic acid (−SOH) intermediate that can form disulfide bonds or sulfenamide residues. Further oxidation of sulfenic acid results in formation of sulfinic (−SO₂H) or sulfonic acid (−SO₃H), which are relatively irreversible.

Fig. 4

Ref1-mediated redox cell signaling. A) The human Ref-1N-terminal region consists of the redox domain (REDOX; residues 1–127) and a 20 amino acid nuclear localization sequence (NLS); the C-terminal region contains the apurinic/apyrimidinic endonuclease domain (APE; residues 162–318). Cysteine-65 and -93 are major redox active sites. Under oxidative stress, Ref-1 translocates into the nucleus by NLS-importin-dependent or -independent pathways where Ref-1 regulates the activity of b-zip transcription factors (bZIP-TF) by redox mechanisms. Oxidized Ref-1 is subject to redox regulation by nuclear translocated thioredoxin (TRX) through the TRX catalytic center (Cys-32 and -35). B) The conserved redox-active cysteine of various human b-zip transcription factors is indicated in red color and labeled with the residue number. In addition to the b-zip family, other transcription factors such as p53, NFκB, and HIF-1α are also regulated by Ref-1 [56].

Fig. 5

Redox regulation of the Nrf2–ARE pathway. ROS oxidation of cysteines (Cys-151, -273 and -288) in the mouse Kelch-like ECH-associated protein-1 (Keap1) results in the release of Nrf2 from the Keap1/cullin-3 E3-ubiquitin ligase (Cul3) complex, preventing Nrf2 degradation. Nrf2 subsequently undergoes nuclear translocation. In the nucleus, a heterodimer of Nrf2 and small Maf members (Maf-F, Maf-G, and Maf-K) binds the antioxidant-responsive element (ARE); oxidation of a b-zip transcriptional repressor of ARE, the human BTB and CNC homolog 1 (Bach1) at Cys-557 and -574 results in cytoplasmic translocation of Bach1, both leading to activation of the ARE.

Fig. 6

Isoforms of p66Shc, p52Shc and p46Shc. Schematic representation of three isoforms produced from the human ShcA gene (CH1 and 2, proline-rich collagen homology domain-1 and 2; CB, cytochrome c binding domain; PTB, phosphotyrosine binding domain; and SH2, Src homology 2 domain); numbers define the regions of different domains according to human p66Shc amino acid sequence; Met-111 and -156 of p66shc are equivalent to the first methionines of p52Shc and p42Shc. ROS-mediated phosphorylation sites are at Ser-36 and -54; Thr-386; pro-apoptotic residues are Cys-59 and Glu-132/133.

Fig. 7

Regulation of IRP–IRE interactions. Under iron rich conditions, IRP1 contains a [4Fe–4S] cluster and is unable to bind to the IRE, though loss of iron from the cluster (destabilization) under iron deficient conditions allows IRP1 to bind to the IRE. IRP2 does not contain a [4Fe–4S] cluster and is degraded by F-box/LRR-repeat protein 5 (FBXL5)-dependent ubiquitination. Iron chelators, nitric oxide (NO), hypoxia, and hydrogen peroxide (H₂O₂) increase IRP/IRE interaction. H₂O₂ destabilizes the [4Fe–4S] cluster of IRP1 and also stabilizes IRP2 protein by preventing FBXL5-dependent ubiquitination. Increasing IRP–IRE interaction in 5′UTR results in translational block of ferritin (Ft), ferroportin (Fpn), aminolevulinic acid synthase-2 (ALAS2), hypoxia inducible factor-2α (HIF-2α), amyloid precursor protein (APP), and NADH dehydrogenase (ubiquinone) Fe–S protein 1 (NDUFS1) genes, but in the 3′UTR it results in mRNA stabilization of transferrin receptor (TfR), divalent metal transporter 1 (DMT1), hydroxyacid oxidase 1 (HAO-1), myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKα), and CDC14 cell division cycle 14 homolog A (CDC14A).

Fig. 8

Schematic of ATM signaling upon oxidative stress and double-strand DNA breaks. Double-strand DNA breaks mediate the phospho-ATM–Chk2 pathway; however, oxidative stress elicits both the phospho-ATM–LKB1 and the ATM–homodimer pathways (ataxia–telangiectasia mutated (ATM); liver kinase B1 (LKB1); AMP activated protein kinase (AMPK); tuberous sclerosis complex 2 (TSC2); mammalian target of rapamycin complex1 (mTORC1)).