THE ROLE OF CU/ZN SUPEROXIDE DISMUTASE IN REDOX SIGNALING

1. THE ROLE OF CU/ZN SUPEROXIDE DISMUTASE IN REDOX SIGNALING Alyson Colin, Dr. AmitReddiSchool of Chemistry and Biochemistry, Georgia Institute of Technology ABSTRACT SOD1 DEPENDENT THIOL OXIDATION SOH DETECTION Cu/Zn Superoxide Dismutase (SOD1) is in the frontline of defense against reactive oxygen species (ROS) by scavenging superoxide radicals, which are produced as a byproduct of normal aerobic metabolism. Indeed, ablation of SOD1 across many different cell types and species, from yeast to man, results in profound oxidative stress. SOD1 is a highly conserved and abundant cellular protein, constituting as much as 1% of total soluble protein in many cell types. However, quite interestingly, <0.1% of SOD1 is actually required to protect against oxidative stress. This begs the question, what is the rationale for having a large excess of SOD1? We propose that a major role for SOD1 in cell biology is to mediate redox-signaling cascades through its ability to disproportionate superoxide into oxygen and hydrogen peroxide, the latter of which has proved to be an important signaling molecule. While there have been a few isolated examples of redox regulation by SOD1 [1], there have not been systematic proteome-wide studies on its impact on peroxide-based regulation of cell signaling and metabolism. Herein we employ a redox proteomics strategy toward identifying SOD1-dependent redox modifications in an effort to characterize the SOD1-redoxome. In fact, our preliminary data suggest multiple protein targets of SOD1-derived peroxide. We anticipate our studies uncovering new and exciting aspects of the redox biology of SOD1 that go well beyond its role in oxidative stress protection. Wild Type sod1D R-S-OH 195 Sulfenic Acid 142 R-S-O2H R-SH Dimedone 96 Sulfinic Acid 71 H2O2 + O2 O2- R-S-O3H H2O2+ O2 O2- 48 33 Sulfonic Acid SOD1 R-S-S-R SOD1 28 Detectable by anti-SOH-Dimedone polyclonal antibody in a Western Blot 22 Disulfide bond REINTRODUCTION OF SOD1 IMPACT & FUTURE WORK These studies are the first to elucidate the SOD1 redox-ome. Our results demonstrate that there are potentially numerous SOD1-dependent sulfenic acid modifications throughout the proteome. Tandem mass spec will be used to identify the proteins of interest, which will allow for follow up experiments to determine their role in redox pathways. Successful completion of these studies will reveal the role of peroxide metabolizing enzymes in redox signaling processes akin to the role of phosphatases and kinases in protein phosphorylation. Further, our work re-conceptualizes the cell biology of SOD1 beyond its canonical function as a ROS scavenger. sod1D + mito-SOD1 sod1D + cyto-SOD2 A functional yeast SOD1 was incorporated into sod1D cells. The expectation was full rescue of growth and complementation of sulfenic acid modifications by western blot. However, that was not the case. sod1D + SOD1 Wild Type sod2D sod1D 195 142 96 71 48 33 28 SOD1 & REDOX SIGNALING 22 12 7.6 Cu/Zn Superoxide Dismutase ACKNOWLEDGMENTS Less than 0.1% of SOD1 is needed to combat oxidative stress. Western blot to detect SOH-Dimedone conjugates 195 142 96 • Professor AmitReddi • The Reddi Lab • Professor Wendy Kelly • Professor Pamela Peralta-Yahya • Professor Matthew Torres • Professor Melissa Kemp 71 Georgia Tech Start-Up Funds Teaching Assistantships 48 33 SOD1 28 2O2˙-+ 2 H+O2 +H2O2 22 Further growth tests support the need of continued use of selective media to maintain plasmid incorporation and expression. • Cu is the catalytic site • Zn is the structural site • Highly conserved • Abundant enzyme 12 7.6 Corson et, al., (1998) What is the purpose for the extreme excess??? Coomassie stain to support equal loading REFERENCES Wild Type sod1D sod2D HYPOTHESIS SOD2 Activity SOD1 Activity (two bands) The remaining SOD1 plays a crucial role in redox signaling pathways through cysteine modifications that dictate downstream effects. • Corson, L.B., Strain, J.J., Culotta, V.C., and Cleveland, D.W. (1998). Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants. Proc. Natl. Acad. Sci. USA 95, 6361–6366. • Reddi, A., Culotta, V.C., (2013). SOD1 Integrates Signals from Oxygen and Glucose to Repress Respiration. Cell. 152, 224–235 • Martínez-Acedo, P., Gupta, V., Carroll, K.S. (2014). Proteomic analysis of peptides tagged with dimedone and related probes. J. Mass Spectrometry. 49,257-265 Theoretical Activity Stain Activity gel using riboflavin to stain and TEMED serving as the electron donor