Superoxide dismutases: role in redox signaling, vascular function, and diseases

Abstract

Excessive reactive oxygen species Revised abstract, especially superoxide anion (O₂•-), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. Superoxide dismutases (SODs) are the major antioxidant defense systems against (O₂•-), which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation. Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of (O₂•-), H2O2, which may participate in cell signaling. In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer. Given the essential role of SODs in cardiovascular disease, the concept of antioxidant therapies, that is, reinforcement of endogenous antioxidant defenses to more effectively protect against oxidative stress, is of substantial interest. However, the clinical evidence remains controversial. In this review, we will update the role of each SOD in vascular biologies, physiologies, and pathophysiologies such as atherosclerosis, hypertension, and angiogenesis. Because of the importance of metal cofactors in the activity of SODs, we will also discuss how each SOD obtains catalytic metal in the active sites. Finally, we will discuss the development of future SOD-dependent therapeutic strategies.

FIG. 1.

Generation and metabolism of reactive oxygen species (ROS). Superoxide (O₂^•−) is produced by NADPH oxidase, xanthine oxidase, nitric oxide synthase (NOS), lipoxygenase, and mitochondrial enzymes. Superoxide is converted by superoxide dismutase (SOD) to H₂O₂, which, in turn, is reduced to water by catalase, glutathione peroxidases (GPx), and peroxiredoxins (Prx). In the presence of reduced transition metal (Fe²⁺, Cu⁺), H₂O₂ can undergo spontaneous conversion to hydroxyl radical (OH•), or related metal-associated reactive species, which is extremely reactive. Importantly, nitric oxide (NO) can be rapidly inactivated by reaction with O₂^•− and leading to the production of the strong oxidant peroxynitrite (ONOO⁻). Thus, SOD is a first line of defense against toxicity of superoxide anion radicals. The enzyme also participates in cell signaling via regulating ROS (e.g., O₂^•−, H₂O₂) and available NO.

FIG. 2.

Common mechanism of scavenging O₂^•− by SODs. Enzymatic activity of SOD involves alternate reduction and reoxidation of catalytic metal (i.e., Cu or Mn) at the active site of the enzyme. Thus, Cu or Mn will be a key modulator of SOD activity of SOD1/SOD3 or SOD2, respectively.

FIG. 3.

Metal trafficking pathways to SODs in vascular tissue. Various SOD enzymes employ catalytic metal cofactor such as copper (Cu) and manganese (Mn) to carry out the disproportion of superoxide. Under physiological conditions, the level of intracellular free Cu is extraordinarily restricted (192). Thus, once transported by Cu uptake transporter hCTR1, soluble cytosolic Cu carrier proteins termed “Cu chaperones” are required for trafficking Cu to specific Cu-containing enzymes through direct protein–protein interaction (116). Three copper chaperones have been characterized thus far: (i) uncharacterized Cu ligands and various Cu chaperones (Cox1, Cox 2, Cox 11, Cox 17, and Sco1), which deliver Cu to cytochrome c oxidase in the mitochondria (not shown); (ii) CCS (Cu chaperone for SOD1), which delivers copper to SOD1 in the cytosol and mitochondrial intermembrane space (IMS); and (iii) Atox1, which delivers copper to some of the secretory copper enzymes such as extracellular superoxide dismutase (ecSOD, SOD3) via the copper transporter ATP7A (Menkes ATPase, MNK) in the trans-Golgi network. In addition to its chaperone function, Atox1 also function as a Cu-dependent transcription factor for ecSOD and cyclin D1 (102, 103, 105). Thus, full activation of SOD3 requires both Cu chaperone function of Atox1 via ATP7A to obtain catalytic Cu as well as Cu-dependent transcription factor function for its transcriptional regulation (103, 105, 189, 190). Cytosolic concentrations of free Cu are typically maintained at exquisitely low levels (10⁻¹⁸ M) by metal scavenging systems, including metallothioneins (MT) and GSH (142, 192). Yeast genetic studies show that Smf2 p and Mtm1p are involved in Mn delivery to MnSOD, but role of these proteins in mammals remains unclear (44).

FIG. 4.

A schematic alignments of each SOD isoform and Cu chaperones. (1) SOD1 (CuZnSOD) and the SOD3 (ecSOD) active site domain (amino acid residues 96–193) shares about 50% homology, such that all the ligands to Cu and Zn and the arginine in the entrance to the active site in SOD1 can be identified in this domain of SOD3. The distinct region of SOD3, as compared to SOD1, includes (a) an amino-terminal signal peptide, which permits secretion from the cell; (b) an N-linked glycosylation site at Asn-89, which is useful in the separation of SOD3 from SOD1 and greatly increases the solubility of the protein; (c) C-terminal region corresponding to heparin-binding domain has a cluster of positively charged residues. This region is critical for binding to extracellular matrix, such as heparan sulfate proteoglycan. (2) SOD2 (MnSOD) protein is composed of three domains. First, N-terminal mitochondrial signal peptide directs protein synthesized in the cytoplasm to the mitochondria. Second, the active site of SOD2 contains Mn and has the N-terminal helical hairpin and C-terminal alpha/beta domain (241). They show no homology to SOD1 and SOD3, but it is similar to that of the Fe SOD, which is commonly absent from eukaryotes (21). (3) Cu chaperone CCS folds into three functionally distinct protein domains (123, 202). The N-terminal Domain I of CCS bears striking homology to Atox1, including the MXCXXC copper-binding site. The central domain of CCS (domain II) exhibits significant homology with its target of copper delivery, SOD1. Of note, this domain also share the strong homology with the central domain of SOD3, which is a catalytic site of it, such that all four of the zinc binding ligands of SOD1 and SOD3 and three of four histidine copper binding ligands are present in CCS. The C-terminal Domain III of CCS1 is quite small yet is extremely crucial for activating SOD1 in vivo (202). This peptide is highly conserved among CCS molecules from diverse species and includes an invariant CXC motif that can bind copper (202). Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites.

FIG. 5.

Heparin binding affinity patterns of SOD3 (ecSOD) in vivo. (A) The sequence of the heparin-binding domain is present in the full-length type C subunit and confers the heparin-binding affinity to the protein. The truncated type A subunit has no affinity to heparin. Proteolytic processing leading to the appearance of truncated ecSOD in vivo can occur both intracellularly and in the extracellular space (59). (B) The subunit compositions of the two classes of ecSOD and their relative affinities to heparin are shown. In most species, ecSOD exists as a tetramer composed of two disulfide-linked dimers (63, 67). Two of these dimers are held together noncovalently. In vivo, both circulating (class A) and tissue bound (class C) are present, with the tissue bound (class C) being approximately 99% of the total ecSOD. Class B (not shown in the figure) of ecSOD reveals partial C-terminal truncation and medium heparin affinity. Class C of ecSOD consists of all four C-terminal intact subunits (type C) that contains two disulfide bonds linking two pairs of heparin-binding domains together. In contrast, class A of ecSOD consists of all four C-terminal truncated subunits (type A) that do not contain a disulfide bond to link two heparin-binding domains. Truncation of the C-terminal region does not affect the nonconvalent protein–protein interactions stabilizing the tetramer, but affects the heparin binding properties significantly.

FIG. 6.

Role of superoxide in various physiological and pathophysiological functions. SOD has a potential impact on various biological function and pathogenesis by regulating NO signaling, ROS (O₂^•−, H₂O₂) signaling, and mitochondrial function.

FIG. 7.

(A) Role of SOD in redox-sensitive signaling pathways and (B) role of SODs in activation of redox signaling at specific compartments. (A) Production of O₂^•− and its metabolite H₂O₂ lead to activation of redox-sensitive kinases and potentially inactivation of specific phosphatases to modulate redox-sensitive signaling, including hypertrophy, proliferation, and migration. Activation of redox-sensitive transcription factors leads to redox-sensitive changes in expression of proinflammatory genes, such as vascular cellular adhesion molecule 1 (VCAM1), monocyte chemotactic protein 1 (MCP1), and intercellular adhesion molecule 1 (ICAM1). ROS modulate ion channels and, therefore, influence intracellular Ca²⁺ and K¹⁺ concentrations. Finally, ROS can activate matrix metalloproteinases (MMPs). (B) Extracellular H₂O₂ generated by SOD3 (ecSOD) localized at caveolae/lipid rafts via binding to heparan sulfate proteoglycans (HSPGs) promotes VEGF receptor type2 (VEGFR2) signaling linked to angiogenesis via oxidative inactivation of protein tyrosine phosphatases (PTPs; DEP1 and PTP1B) (180); SOD1 is recruited to redox active endosomal surface where it binds to Rac1 to regulate Nox2 activity. Thus, SOD1-Nox2-mediated increase in O₂^•− exits endosomes through chloride channels (ClC3) and SOD1-mediated dismutation of O₂^•− at the endosomal surface produces the localized H₂O₂, thereby promoting redox activation of NF-kB (162); MnSOD (SOD2) localizes in mitochondria matrix. SOD2 (MnSOD) overexpression-induced H₂O₂ induces the tumor suppressor PTEN oxidation, leading to enhanced formation of phosphatidylinositol 3,4,5-triphoshate, resulting in activation of Akt and angiogenesis in vivo (40). Thus, O₂^•− dismutation to H₂O₂ by the three isoforms of SODs contributes to activation of specific redox signaling events at distinct compartments.

FIG. 8.

(A) Role of nitric oxide–superoxide interactions in vascular (endothelial) dysfunction in cardiovascular disease and (B) protective role of SODs in oxidative stress-dependent vascular (endothelial) dysfunction. (A) NO rapidly reacts with O₂^•− generated by ROS-generating enzymes, including NADPH oxidase, xanthine oxidase, and mitochondria, to form peroxynitrite anion (ONOO⁻), which in turn oxidizes various molecules, such as the heme of sGC, lipids, and the endothelial NOS (eNOS) cofactor BH4. This in turn induces uncoupled eNOS to promote further increase in O₂^•−. These consequences will be further enhanced by interaction of ROS-generating enzymes. Both O₂^•− and ONOO⁻ promote mitochondrial dysfunction, thereby increasing mitochondrial ROS production. Mitochondria-derived ROS, which in turn further activates NADPH oxidase, results in increased ROS production and reduced NO bioavailability. Further, either O₂^•− or ONOO⁻ can stimulate other ROS-generating enzymes, such as xanthine oxidase. The loss of bioavailable NO and formation of ONOO⁻ can lead to vascular inflammation, vascular remodeling, altered vascular tone, enhanced vascular permeability, and increased platelet aggregation. These responses are inhibited by SODs. (B) Because of its location, SOD3 (ecSOD) plays a critical role in preventing O₂^•−-mediated destruction of NO• released from the endothelium at the extracellular space, whereas SOD1 preserves NO levels within the endothelium. Thus, SODs regulate endothelial function and NO mediating signaling by inhibiting O₂^•−-mediated inactivation of NO•, thereby increasing bioavailable NO•. Because O₂^•− and NO^• are both radicals and contain unpaired electrons in their outer orbitals, they undergo an extremely rapid, diffusion-limited radical–radical reaction (6.7×10⁹ M⁻¹ s⁻¹, three times faster than the dismutation of O₂^•− by SOD). This reaction leads to the formation of nitrite, nitrate, and, very importantly, the peroxynitrite anion (ONOO⁻), which in turn induces endothelial dysfunction, vascular inflammation, vascular remodeling, altered vascular tone, enhanced vascular permeability, and increased platelet aggregation. Both O₂^•− and ONOO⁻ promote mitochondrial dysfunction, thereby increasing mitochondrial ROS production, reducing NO bioavailability, mitochondrial (mt)DNA damage, and inhibition of mitochondrial enzymes. These events result in endothelial dysfunction.