1. An Investigation of the Interactions Between Zinc-deficient and Copper, Zinc Superoxide Dismutase Katie Meyers Dr. Joe Beckman Department of Biochemistry/Biophysics

2. Amyotrophic Lateral Sclerosis Fatal neurodegenerative disease that is characterized by selective motor neuron death Majority of ALS cases are sporadic but approximately 10% of all cases are familial Of these familial cases, 20% of individuals inherit dominant autosomal mutations in the SOD1 gene SOD1 gene codes for copper, zinc superoxide dismutase (SOD)

3. Superoxide Dismutase (SOD) Small: 153 amino acid protein Exists as a dimer of identical subunits, containing one copper and one zinc atom per monomer SOD functions as a superoxide scavenger in cells throughout the body

4. SOD Function

5. SOD Mutations Over 100 different ALS causing mutations have been discovered dispersed throughout the gene The toxicity of these mutations is not due to reduced superoxide scavenging ability Something about these mutations causes them to become toxic to cells

6. SOD Chemistry Mutant SODs have a reduced affinity for binding zinc, leaving the copper more reactive In Cu, Zn(-) SODs, copper can react with ascorbate and facilitate the transfer of electrons from ascorbate to oxygen, producing superoxide Superoxide reacts with nitric oxide to form peroxynitrite, which can nitrate tyrosine residues and is harmful to motor neurons

8. Zinc Deficiency Wild-type Cu, Zn Protects motor neurons Motor neuron death Mutant Cu, Zn Mutant Cu, Zn(-) Protects motor neurons Rapid motor neuron death

9. Objective  Why does Cu, Zn SOD make Cu, Zn(-) SOD more toxic to motor neurons in vitro? Cu, Zn homodimer 31,808 Da Cu, Zn(-) homodimer 31,732 Da Cu, Zn + Cu, Zn(-) heterodimer 31,780 Da Can subunits exchange between dimers?

10. SOD dimers do exchange their subunits and form heterodimers The exchange is rapid with a half life of 15-20 minutes 0 10 20 30 40 50 60 90 120 12hrs Time, min

11. Why would heterodimers be toxic? Cu, Zn(-) SOD may be able to transfer an electron to Cu, Zn SOD increasing the rate of reduction and resulting in tyrosine nitration The presence of Cu, Zn SOD could slow down the aggregation of Cu, Zn(-) SOD, a protective mechanism, resulting in increased cell damage

12. Does the Presence of Cu, Zn(-) SOD Increase the Rate of Reduction of Cu, Zn SOD? Cu, Zn and Cu, Zn(-) SODs were individually reduced by ascorbate and analyzed using a stopped flow spectrophotometer An equimolar mixture of Cu, Zn and Cu, Zn(-) SOD was reduced by ascorbate and rates of reduction were measured as a function of time

13. Cu, Zn + Cu, Zn(-) Cu, Zn(-)Cu, Zn

14. Rates of Reduction

15. Does Cu, Zn SOD Affect the Aggregation of Cu, Zn(-) SOD? Cu, Zn(-) aggregates as a mechanism to protect motor neurons from apoptosis Analytical ultracentrifugation was used to measure aggregation as a function of absorbance When Cu, Zn(-) is mixed with Cu, Zn SOD, aggregation is severely reduced Interactions increase the toxicity of Cu, Zn SOD

16. Conclusions Cu, Zn and Cu, Zn(-) SODs exchange monomers with a half life of 15-20 minutes The presence of Cu, Zn(-) SOD may cause the Cu, Zn rate of reduction to increase, but further data collection and analysis are necessary Cu, Zn SOD interferes with the aggregation of Cu, Zn(-) SOD, preventing this protective mechanism and increasing the toxicity of Cu, Zn(-) SOD

17. Acknowledgements Dr. Joe Beckman Howard Hughes Medical Institute Linus Pauling Institute Keith Nylin, Blaine Roberts, Val Bomben, Kristine Robinson Kevin Ahern