Reactive oxygen species, cellular redox systems, and apoptosis

Abstract

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues. At physiological low levels, ROS function as "redox messengers" in intracellular signaling and regulation, whereas excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death. Additionally, various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the death receptor- and the mitochondria-mediated pathways. Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to ROS increases and/or antioxidant decreases, disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein, or DNA. In this review, we focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.

Figure 1. Death receptor-mediated and mitochondrial pathways of cell apoptosis

Death receptor extrinsic pathway. The major death receptor pathways include Fas/FasL, TNF-R1/TNFα, and TRAIL-R1/TRAIL. Binding of ligands to respective receptors activates downstream signaling and the formation of death-inducing signaling complex. Activation of the NF-κB survival pathway enhances transcription of the anti-apoptotic proteins like FLIP_L or MnSOD and apoptosis blockade. At high ROS and failure to activate NF-κB promotes ASK1/JNK activation that triggers cell apoptosis. Death receptor signaling is associated with caspase-8 activation that promotes apoptosis via activation of effector caspases (e.g. caspase-3) or engages mitochondrial apoptotic signaling via truncated Bid, leading to the release of apoptogenic factors such as cytochrome c into the cytosol. ASK1, apoptosis signal-regulating kinase 1; Apaf-1: apoptotic protease-activating factor-1; Bid, BH3-only pro-apoptotic protein; tBid, truncated form of Bid; casp 8, 9, active forms of caspases-8 and -9; cyt c: cytochrome c; FLIP_L: FLICE inhibitory protein; FADD: Fas-associated death domain; FasL: Fas ligand; JNK: c-Jun N-terminal kinase; NF-κB: nuclear transcription factor kappa B; I B/NF-κB: the inactive form of NF-κB associated with its inhibitor; MnSOD: manganese superoxide dismutase; RIP1: receptor-interacting kinase1; ROS: reactive oxygen species; TNF-α: tumor necrosis factor-α; TNFR1: TNF receptor-1; TRAIL: TNF-related apoptosis-inducing ligand; TRAIL-R1: Trail receptor-1; TRADD: TNF receptor-associated death domain; TRAF-2: TNF receptor-associated factor-2. Mitochondrial intrinsic pathway. Various apoptotic stimuli (e.g. ROS) mediate permeabilization of the mitochondrial outer membrane and the release of pro-apoptotic proteins. Within the cytosol, cytochrome c together with Apaf-1, and dATP form the apoptosome complex to which the initiator procaspase-9 is recruited and activated. Caspase-9-catalyzed activation of the effector caspase-3 executes the final steps of apoptosis. Caspase activation is further enhanced through neutralization of caspase inhibitors by apoptogenic proteins like Smac/Diablo and Omi/HtrA2 that are released from the mitochondria. In addition, mitochondrial proteins such as AIF and endoG promote caspase-independent apoptosis through nuclear translocation and mediating genomic DNA fragmentation. Cyt c: cytochrome c; casp 9: activated caspase-9; Apaf-1: apoptotic protease activation factor-1; Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein of low pI; Omi/HtrA2: high temperature requirement A2 serine protease; IAP: inhibitors of apoptosis proteins; AIF: apoptosis inducing factor; endoG, endonuclease G; Bax/Bak, pro-apoptotic proteins.

Figure 2. ROS-induced ASK1/JNK signaling and apoptosis

Reactive oxygen species initiate Trx dissociation from the ASK1-Trx complex, the ASK1 signalosome, through oxidation of Trx redox active site. ASK1 undergoes auto-phosphorylation and covalent binding between its subunits, leading to the formation of “activated signalosome”, and recruitment of tumor necrosis factor receptor-associated factor 2 and 6 to the complex. Activated ASK1 signals downstream JNK activation and induce apoptosis either via mitochondrial signaling or via transcription of AP-1-dependent pro-apoptotic genes. Additionally, ROS-mediated disruption of mitochondrial ASK1/ASK2/Trx2 complex induces cytochrome c release. ROS: reactive oxygen species; TRAF2/6: TNFα receptor-associated factor 2, 6; ASK1, -2: apoptosis signal-regulating kinase 1 and 2; JNK: c-Jun N-terminal kinase, Trx1: thioredoxin 1, reduced form; TrxSS: thioredoxin, oxidized form; Trx2: thioredoxin 2, mitochondrial enzyme; Bak: pro-apoptotic protein; cyt c, cytochrome c; TNFα: tumor necrosis factor alpha; FasL: Fas ligand.

Figure 3. Redox mediation of mitochondria-to-cytosol release of cytochrome c and activation/inactivation of caspase-9

Interaction with mitochondria-specific cardiolipin sequesters cytochrome c in the mitochondrial inter-membrane space. Enhanced mitochondrial generation of H₂O₂ activates cytochrome c peroxidase activity and induces cardiolipin peroxidation and cytochrome c detachment. Oxidized cardiolipin is translocated to the mitochondrial outer membrane, and together with the proapoptotic proteins, Bid and Bax, forms a megapore channel that enables the mitochondria-to-cytosol transit of cytochrome c. Through a nitrosation reaction, the peroxidase activity of cytochrome c is inhibited by NO. Within the cytosol, cytochrome c interacts with Apaf-1 and dATP, forming the apoptosome complex to which pro-caspase-9 is recruited. Proteolytic activation of pro-caspase-9 is mediated by post-translational modification of the catalytic site cysteines through thiol oxidation, nitrosation or glutathiolation. H₂O₂- and GSSG-mediated cysteine oxidation or S-glutathiolation, respectively results in pro-caspase inactivation. NO-mediated S-nitrosation similarly inhibits caspase-9 activation whereas de-nitrosation promotes proenzyme proteolysis and activation. Additionally, GSSG-dependent glutathiolation of active caspase-9 results in direct inhibition of enzyme activity. Apaf-1: apoptotic protease activation factor-1; CL: cardiolipin; CL-OOH: peroxidized cardiolipin; cyt c: cytochrome c; H₂O₂: hydrogen peroxide; NO.: nitric oxide; Bid/Bax: proapoptotic proteins.