Superoxide Dismutase 1-3
According to oxygenic theory of evolution, the atmosphere initially contained low concentration of oxygen. The genesis of photosynthetic organisms approximately 2.5 billion years ago initiated the development of plants and other life-forms able to use aerobic metabolism. Reactive oxygen species (ROS), although needed to normal cellular functions, are in aberrant concentrations potentially harmful molecules damaging various cellular structures. Hence, it is probable that already the first living organisms acquired antioxidative defense mechanisms. Superoxide dismutases (SOD) represent a reduction-oxidation (redox) metalloprotein enzyme family, which, according to oxygenic theory of evolution, was connected to the availability of transition metals in the biosphere (Bannister et al. 1991).
There are three members in mammalian superoxide dismutase family: CuZnSOD (SOD1), MnSOD (SOD2), and EC-SOD (SOD3). Superoxide dismutase protein, first reported 1938 by Mann and Keilin, was purified from bovine blood and named as hemocuprein. The same protein was isolated from the liver in 1939, called as hepatocuprein (Mann and Keilin 1939), and from the brain, named as cerebrocuprein (Porter and Folch 1957). McCord and Fridovich discovered at the end of the 1960s the ability of the protein to catalyze dismutase reaction, thus identifying it as an enzyme and naming it as superoxide dismutase (CuZnSOD). Cupper ion located at the active center of the enzyme functions as an electron carrier in the dismutase reaction, whereas zinc participates indirectly the reaction by regulating the three-dimensional conformation of the active center (Tainer et al. 1983). Zimmermann and coworkers published in 1973 the discovery of the second member of SOD family, the mitochondrial SOD (MnSOD, SOD2) (Zimmermann et al. 1973), which was confirmed a few months later by Weisiger and Fridovich (Weisiger and Fridovich 1973). Stefan L. Marklund reported in 1982 the third superoxide dismutase, extracellular superoxide dismutase (EC-SOD, SOD3), demonstrating the ability of the enzyme to bind reversibly to cell membrane proteoglycan structures (Marklund 1982).
SOD1, SOD2, and SOD3 enzymes regulate reduction-oxidation (redox) balance by converting superoxide anion (O2-) to hydrogen peroxide (H2O2) in two half reactions:
In the two-step reaction the oxidized form of the enzyme binds to O2-., reacts with proton, and releases molecular oxygen. In the second half-reaction, the reduced form of the enzyme binds a second O2-. and two protons to synthesize H2O2. Consequently the enzyme returns to oxidized state. Hydrogen peroxide is further processed to water by catalase and a large family of peroxidases. Catalase is able to utilize H2O2 itself as a reductant oxidizing it to molecular oxygen, whereas peroxidases dispose H2O2 by oxidizing a secondary reductant, such as glutathione.
The primary location, and thus also cellular response to dismutase reaction, of each SOD varies. SOD1, which is mainly observed in cytoplasm, has a prominent role in amyotrophic lateral sclerosis (ALS). Mitochondrial SOD2 is essentially involved in cellular senescence and survival. Lack of SOD2 expression causes death of newborn mice and induction of cell cycle arrest with consequent cellular senescence in mouse embryonic fibroblasts (MEF). The extracellular cell membrane-bound SOD3 activates cell survival, antiapoptotic, and growth signal transduction resulting in increased cell proliferation and increased healing of tissue damages.
Secreted SOD3 binds reversibly to cell membrane proteoglycan structures, which brings the enzyme at close proximity with membrane-associated signaling molecules. SOD3 activates several cell membrane tyrosine kinase receptors (RTKs) and their downstream signaling molecules, most importantly RAS-RAF-MAPKK (MEK)-MAPK p44/42 (ERK1/ERK2) signaling pathway. Increased MAPK p44/42 expression stimulates SOD3 expression, thus forming a positive feedback loop. Interestingly, the positive feedback look is controlled at the level of small GTPases and correlates to the SOD3 concentration; high SOD3 concentration inhibits small GTPase RAS, RAC, RHO, and CDC42 activation by downregulating guanine nucleotide exchange factor (GEF) gene expression responsible for GTP loading to small GTPases and by upregulating GTPase-activating protein (GAP) expression that functions opposite to GEF. Data demonstrating increased phosphorylation of RAS upstream signaling molecules RTKs, focal adhesion kinase (FAK), and family of SRC proto-oncogenes at high SOD3 concentrations support this conclusion suggesting that regulation of SOD3-stimulated signal transduction occurs at the level of small GTPases. Moreover, moderately overexpressed SOD3 increases GEF and inhibits GAP gene expression, hence resulting in increased GTP loading to small GTPases and consequent activation of downstream signaling (Laukkanen et al. 2015).
Other important signal transduction molecules and pathways involved in SOD3 signaling include p38 MAPK, which inhibits SOD3 expression (Adachi et al. 2006), although the mechanism of inhibition is not completely characterized. Large G-protein-associated thyroid-stimulating hormone (TSH) receptor activation has been demonstrated to increase expression of SOD3 through cyclic AMP – protein kinase A (cAMP-PKA) pathway and through phospholipase C-calcium 2+ (PLC-Ca2+) signaling inducing cell proliferation (Laatikainen et al. 2010). SOD3 has also shown to increase PI3K-AKT signaling causing inactivation of FOXO3a by phosphorylation and consequent translocation from the nucleus into the cytosol (Laatikainen et al. 2011).
ROS are often connected to tissue damages and pathological conditions although they are essential maintainers of normal cellular functions. Once the concentration of redox enzymes, oxidants, and antioxidants is unbalanced by tissue trauma or pathological condition, excess ROS activates aberrant signal transduction or directly injures cellular macromolecules. O2-. and H2O2 are both secondary messengers targeting unspecifically signaling molecules; hence, the function of SOD enzymes goes beyond the balancing of O2-. and H2O concentrations extending to signal transduction regulation. ROS has been shown to mediate RAS oncogene-induced primary cell senescence (Leikam et al. 2008) and cellular transformation (Mitsushita et al. 2004) highlighting the importance of redox system in defending normal tissue functions and in progression of pathological conditions. Cellular response of ROS depends on their quality, quantity, and cellular location. Each ROS has characteristically different function in cellular environment that further depends on the concentration of a specific ROS, which then may directly interact with macromolecules in the cytoplasm, in mitochondria, or at cell membranes. Correspondingly, cellular response of SOD enzymes correlates to their location and level of expression. Because SODs are difficult drug targets, clarification of the signal transduction pathways could potentially reveal a selective group of molecules mediating the cellular response of the enzymes. Hence, efforts elucidating SOD-stimulated signaling routes could lead to the development of novel therapeutic regimens.
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