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Zinc-deficient SOD in ALS

Zinc-deficient SOD in ALS. Emily Clark Dr. Joe Beckman Department of Biochemistry/Biophysics. Amyotrophic Lateral Sclerosis (ALS). Louis Gehrig Disease. Fatal disease Progressive motor neuron death in the brain and spinal cord 5,000 people diagnosed per year

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Zinc-deficient SOD in ALS

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

  2. Amyotrophic Lateral Sclerosis (ALS) Louis Gehrig Disease • 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

  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. SODFunction Superoxide (O2-) Hydrogen Peroxide Superoxide (O2-) O2 Zn2+ Cu2+ Cu1+ Zn2+ 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? +2e- Reduced

  9. Native Gels - +

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

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

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