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An Investigation of the Interactions Between Zinc-deficient and Copper, Zinc Superoxide Dismutase

An Investigation of the Interactions Between Zinc-deficient and Copper, Zinc Superoxide Dismutase. Katie Meyers Dr. Joe Beckman Department of Biochemistry/Biophysics. Amyotrophic Lateral Sclerosis. Fatal neurodegenerative disease that is characterized by selective motor neuron death

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An Investigation of the Interactions Between Zinc-deficient and Copper, Zinc Superoxide Dismutase

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  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

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

  8. Why does Cu, Zn SOD make Cu, Zn(-) SOD more toxic to motor neurons in vitro? Objective Can subunits exchange between dimers? Cu, Zn homodimer31,808 Da Cu, Zn(-) homodimer 31,732 Da Cu, Zn + Cu, Zn(-) heterodimer 31,780 Da

  9. 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

  10. 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

  11. 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

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

  13. Rates of Reduction

  14. 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

  15. 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

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

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