1. Comparing the Toxicity of Zinc Deficient Superoxide Dismutase (SOD) and the Quad SOD mutant: Implications for Amyotrophic Lateral Sclerosis Jesse Fitzpatrick Dr. Joe Beckman’s Lab The Linus Pauling Institute Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute

2. Amyotrophic Lateral Sclerosis (ALS) ALS is neurodegenerative disease Two Forms of ALS –Sporadic –Familial (fALS) (10-20%) fALS caused by mutant Superoxide Dismutase (SOD) (10- 20% of fALS cases) Mutant SOD becomes toxic (gain of function) Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Superoxide Dismutase (dimer)

3. Wild Type SOD Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute O2-O2- O2O2 Zn Cu 2+ Cu 1+ SOD regeneration Cu, Zn SOD Zn Cu 2+ e-e- O2-O2- H2O2H2O2

4. Mutant SOD Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Zn Cu 2+

5. Copper Toxicity: Mutant SOD Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute O2-O2- O2O2 Cu 2+ Cu 1+ NO ONOO - Peroxynitrite O2-O2- H2O2H2O2 ½ Rate Cellular Reductases Mutant Zn - SOD

6. Summary Zn - Mutant SOD O2-O2- Superoxide NO Nitric Oxide OONO - Peroxynitrite (toxic!) Cu Mouse with ALS “Quad” Mutant *Cu binding site mutated

7. Why do Quad SOD Mice get sick? 1.Cu toxicity may not contribute significantly to ALS symptoms 2.Quad SOD causes ALS because Cu binds to another part of the SOD protein Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute

8. Research Aims 1.Determine whether Quad SOD binds Cu 2.If Quad SOD binds Cu, determine whether it still exhibits deleterious redox behavior

9. Quad SOD Binds Copper Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Metalation Variables –pH –Cu concentration –Folding Agents –Urea Successful Conditions –Combine Quad with Urea (conc. 1.5M) –Dialyze against Tris buffer pH 7.5 with 300uM CuCl 2 –Dialyze against Tris buffer pH 7.5

10. Quad SOD Produces Superoxide Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Mutant Zn - SOD O2-O2- NO Nitric Oxide OONO - Cu NO Sensor NO

11. Effects of Concentration on Toxicity Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Concentration and Zn- SOD Concentration and Quad Conclusions - Quad SOD does not act catalytically and is consumed in the reaction

12. Effects of pH on Toxicity Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Effect of pH on Quad ActivityEffect of pH on Zn- SOD Activity Conclusions - Zn- SOD activity is pH dependent while Quad activity is not - Supports that Cu is binding to the Zn site in the Quad mutant

13. The Big Picture Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Quad SOD binds Cu (possibly at the Zn site) Cu Quad exhibits toxic activity invitro Could explain why transgenic Quad mice develop ALS Allays the dissonance between the Cu toxicity theory and the Quad transgenic mouse study

14. Future Directions Mutate both the copper and zinc binding site and test for Cu binding Investigate the redox differences between Zn- SOD and Cu Quad SOD Analyze invivo Quad SOD from Quad SOD transgenic mice Look for evidence of peroxynitrite damage Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute

15. Acknowledgments Dr. Joe Beckmen Keith Nylin Nathan Lopez Dr. Mark Levi Dr. Keri Trumbull Megan Hirko Kevin Ahern HHMI The Linus Pauling Institute Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute

16. Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute

17. Results Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Cu 2+ His 46 His 48 His 63 His 120 Mutate (Again) Cu 2+ Arg 46 Gln 48 Gly 63 Gly 120 Cu 2+ Quad SOD Cu Binding Site Mouse with ALS

18. What Function does Mutant SOD acquire? Mutant SOD has lower affinity for metals (Zn and Cu) Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute SOD Aggregation Theory Loss of metals leads to SOD aggregation which triggers apoptosis Cu Toxicity Theory Loss of metals cause SOD to function backwards, creating super oxides which indirectly trigger apoptosis

19. What Function Does Mutant SOD Acquire? Mutant SOD has lower affinity for metals (Zn and Cu) Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute SOD Aggregation Theory Loss of metals leads to SOD aggregation which triggers apoptosis Cu Toxicity Theory Loss of metals cause SOD to function backwards, creating superoxides which indirectly trigger apoptosis

20. Consequences of Zn Deficiency Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute 1.Copper becomes more exposed 2.Regeneration of Cu 2+ SOD is decreased 3.Cu 2+ SOD is reduced by cellular reductase 4.Cu 2+ availability decreases while Cu 1+ increases 5.Superoxide concentrations increase 6.Superoxides reacts with NO to produce peroxynitrate 7.Peroxynitrate nitrates tyrosine residues and triggers cell death

21. The Problem with Cu Toxicity Theory Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute His Arg Gly Gln Gly Mouse with ALS Quad Mutant

22. The Problem with Cu Toxicity Theory Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute His Cu 2+ Arg Gly Gln Gly

23. Amyotrophic Lateral Sclerosis (ALS) Symptoms –Neuronal death –Muscle weakness –Paralysis Death occurs in 3-5 years from onset Two Forms of ALS –Sporadic –Familial (2-3% of cases) Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Upper motor neuron Lower motor neuron

24. Familial ALS: The Genetic Link Familial ALS patients have mutations in the Superoxide Dismutase (SOD) protein SOD –Structure Binds a Cu and a Zn cofactors Exists as a homodimer –Normal function Scavenges superoxides such as O 2 - Protects the cell from oxidative stress SOD Mutations do not cause ALS by hindering normal SOD activity SOD Mutations likely result in gain of harmful function Jesse Fitzpatrick (2008) Dr. Beckman’s Lab The Linus Pauling Institute Superoxide Dismutase (dimer)