Parsing the Dual Roles of Cu/Zn Superoxide Dismutase (Sod1) in Oxidative Stress Protection and Redox Signaling
Montllor Albalate, Claudia
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Superoxide dismutases (SODs) are a highly conserved class of antioxidant enzymes that serve on the frontline of defense against reactive oxygen species (ROS). SODs, which detoxify superoxide radicals (O2•-) by catalyzing their disproportionation into molecular oxygen (O2) and hydrogen peroxide (H2O2), are rather unusual “antioxidant” enzymes in that they catalyze the production of one ROS – H2O2 – while scavenging another – O2•-. While much is known about the necessity for O2•- detoxification, it is less clear what the physiological consequences of SOD-derived H2O2 are. Given that hormetic levels of H2O2 are important for physiological redox signaling and also act as a pro-growth signal, SODs, especially Cu/Zn SOD (Sod1), which accounts for the majority of intracellular SOD activity in eukaryotes and is localized virtually everywhere in the cell except the mitochondrial matrix, may be key to promote various redox signaling pathways. My thesis work has been focused on parsing the dual roles of Sod1 in oxidative stress protection and redox signaling. I have found that the vast majority of Sod1 is dispensable for protection against O2•- toxicity using Saccharomyces cerevisiae and human cell lines as model organisms (Chapter 2 and 3, respectively). However, the bulk of Sod1 is required for proteome-wide H2O2-based redox signaling, including regulation of the of yeast casein kinase (Yck1, Chapter 2), the canonical Wnt signaling pathway (Chapter 3) and a molecular circuit that links O2 availability to the production of NADPH, a key cellular reductant that regenerates thiol-based antioxidant enzymes (Chapter 4). Altogether, my work finally explains the physiological necessity for an “antioxidant” enzyme like Sod1 to produce an oxidant – namely that the H2O2 that Sod1 produces is used to stimulate NADPH-dependent ROS scavenging and redox regulate a large network of metabolic enzymes. In the first part of my thesis work, described in Chapter 2, I discovered that < 1% of the total cellular Sod1 pool is required for protection against superoxide damage in yeast. Superoxide toxicity stems from the oxidative inactivation of 4Fe-4S clusters, resulting in defects in a number of pathways containing Fe/S dependent metabolic enzymes, and toxicity from the iron released from damaged clusters. The iron leads to deleterious redox reactions that oxidatively damage DNA, lipids, and proteins. By profiling cell wide markers of superoxide toxicity, including Fe/S cluster enzyme activity, DNA damage, and membrane fragmentation, in cell lines expressing a regulatable SOD1 promoter, I found that an undetectable (< 1%) amount of Sod1 is sufficient for superoxide resistance in air. Instead, the bulk of Sod1 is required to promote the stabilization of Yck1, a glucose sensing plasma membrane casein kinase previously found to be stabilized in a Sod1-dependent manner. This led me to conclude that the majority of Sod1 plays a more important role as a source for hormetic H2O2 than for scavenging O2•-. In Chapter 3, I extend my findings from yeast to mammalian cells, and found that ~50-80% depletion of Sod1 in human embryonic kidney cells using RNA interference does not result in oxidative stress but does impact casein kinase signaling in the Wnt pathway. The Yck1 homology in humans, CK1, is an integral component of the Wnt signaling pathway, which is necessary for embryonic development and is prooncogenic when hyperactivated. I found that silencing Sod1 does not affect cell proliferation or the expression of a panel of antioxidant enzymes, consistent with bulk Sod1 being dispensable for oxidative stress protection. However, Sod1 silencing resulted in reduced CK1 expression and attenuated Wnt signaling and Wnt-dependent cell proliferation. Thus, as in yeast, bulk Sod1 is dispersible for oxidative stress protection but seemingly required for redox signaling in human cell lines. Having established that bulk Sod1 is dispensible for protection from superoxide toxicity, in the third section of my thesis, described in Chapter 4, I probed the physiological roles of Sod1-derived spatio-temporal bursts of H2O2. As part of this effort, I discovered that an important but previously unknown antioxidant function of Sod1 is to integrate O2 availability to stimulate production of NADPH, a key cellular reductant that regenerates peroxide-scavenging thiol peroxidases and catalases. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the catalytic Cys residue in the glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH), which in turn re-routes carbohydrate flux through the oxidative phase of the pentose phosphate pathway (oxPPP) to increase NADPH. Sod1 senses O2 via O2.- generated from mitochondrial respiration and a NADPH oxidase, Yno1. The oxidation of GAPDH is exclusively dependent on and rate limited by Sod1, suggesting that Sod1 provides a highly localized pool of H2O2 in close proximity to GAPDH, likely via transient interactions between Sod1 and GAPDH. These findings broaden the antioxidant role of Sod1 to include stimulation of NADPH production, which offers more expansive protection against redox stress than just defending against O2.-, which most Sod1 is dispensable for as determined in Chapter 2. Moreover, in collaboration with the laboratory of Matthew Torres, we employed mass spectrometry based redox proteomics approaches to identify cell-wide targets of Sod1-dependent protein oxidation. This analysis revealed that Sod1 is a master regulator of metabolism and the thiol redoxome. Overall, my findings shed light on a broader role for Sod1 that extends beyond just superoxide scavenging as was previously thought. Given that changes in Sod1 expression and activity are a central aspect of the pathogenesis of a number of diseases, including many cancers and neurodegenerative disorders, my thesis work highlights how metabolic rewiring due to Sod1-based redox control may underlie the progression of human disease and may inspire new Sod1-based therapeutics and aid to understand the effectiveness of anti-Sod1 therapeutic interventions in cancer.