1. Zinc-deficient SOD in ALS Emily Clark Dr. Joe Beckman Department of Biochemistry/Biophysics

2. Amyotrophic Lateral Sclerosis (ALS) Fatal disease Progressive motor neuron death in the brain and spinal cord 5,000 people diagnosed per year Mutations to superoxide dismutase (SOD) occur in only 2-3% of individuals with ALS Louis Gehrig Disease

3. Cu-Zn Superoxide Dismutase (SOD) 153 amino acid protein Exists as a homodimer, containing one copper and one zinc atom per monomer SOD functions as a superoxide scavenger in cells throughout the body

4. SOD Function Zn2+Cu2+ Superoxide ( O 2 -) O2O2 Hydrogen Peroxide Zn2+ Cu1+ Oxidized SOD Reduced SOD

5. Mutations in SOD Over 100 mutations causing ALS have been discovered in the gene coding for SOD It is not known exactly what about these mutations causes toxicity in cells The toxicity is not due to reduced superoxide scavenging ability Mutant SOD has reduced affinity for binding Zn, leaving a more reactive Cu atom

6. Zn-deficient SOD Hypothesis

7. The Disulfide Bond When SOD loses the Zn atom, the disulfide bond is more easily reduced

8. When the disulfide bond is reduced, is Zn more readily separated from SOD? Reduced +2e-

9. Native Gels - +

10. Experiment 1.Cu, Zn Wt SOD 2.SOD, TCEP 3.SOD, TCEP, EDTA 4.SOD, EDTA 5. Zn(-) Wt SOD 6. SOD, TCEP 7. SOD, TCEP,EDTA 8. SOD, EDTA 1 2 3 4 5 6 7 8

11. Experiment 1 2 3 4 5 6 7 8 1.Cu, Zn Wt SOD 2.SOD, TCEP 3.SOD, TCEP, EDTA 4.SOD, EDTA 5. Zn(-) Wt SOD 6. SOD, TCEP 7. SOD, TCEP,EDTA 8. SOD, EDTA

12. NFL Most abundant neuron filament protein in neurons Essential for neurofilament assembly Binds Zn

13. Expression and Purification of NFL Human plasmids used to express NFL in E. coli

14. Acknowledgments The Beckman Lab HHMI Kevin Ahearn