Copper Binding of Mutant Quad SOD1

1. And Its Relationship to Disease Progression of ALS in Transgenic Mice Copper Binding of Mutant Quad SOD1 Nick Classen Dr. Joseph Beckman Linus Pauling Institute HHMI Summer 2011

2. Amyotrophic Lateral Sclerosis • Loss of motor functions (eventually paralysis and loss of lung function) • More than 3,000 Americans are diagnosed with ALS each year, and 3,000 deaths are attributed to ALS every year • Average life span is 2-5 years after diagnosis • Familial (<10%) vs. sporadic (>90%) • Mutations in superoxide dismutase (SOD1) gene were the first known familial link for ALS • Transgenic mice with human mutant sod1 genes develop disease

3. Superoxide Dismutase (SOD1) • The major cytosolic antioxidant within cells • Active site characterized by a Cu ion, which participates in redox reactions of superoxide radicals. The Zn ion adjacent to the Cu stabilizes the structure Cu-Toxicity Hypothesis • Dr. Beckman’s group has shown Zn-deficient SOD1 to activate death cascades in motor neurons due to a gain of function • Mutations weaken structure, and decrease binding affinity for metals, therefore it is possible that they lose Zn more easily than normal SOD1

4. Understanding Metal Binding of SOD1 • Cu and Zn ions are adjacent to each other in the periodic table and have similar binding sites • Each binding site is primarily histidine residues, with the Zn-site containing a negatively charged aspartic acid • Dr. Beckman’s group hypothesizes that the gain of function occurs due to Zn being removed, thus exposing the Cu ion, which is redox active and initiates oxidative stress

5. X X X X The “Quad” Mutant Mouse • This diseased transgenic mouse was developed by Borchelt to better understand the role of Cu in disease progression • All four Cu-binding histidine ligands were mutated to non-Cu-binding residues • Seemed to dismiss the Cu-toxicity hypothesis when a study showed that these mutant proteins lack bound Cu (Wang, Journal of Biological Chemistry, Vol. 282, No. 1, pp345-352, 2007) • However, to remove adventitious metals from their protein extraction, they used 5 mM EDTA, which is strong enough to remove Cu from Quad-SOD1 • It may be possible to keep Cu in Quad-SOD1 by not introducing EDTA

6. Aims of Our Study Goal • To determine whether Cu can bind to Quad-SOD1 in vitro Hypothesis • Copper binds to Quad for one of two reasons: • - Cu binds to Zn site • - A new site is formed on protein Rationale • It is known that Cu is capable of binding to the Zn site • Other Cu binding sites have been observed

7. Obtaining Cu-Bound Quad SOD1 Proteins • Quad-SOD1 is extracted from auto-lysing E. coli containing human Quad gene in a pET3d plasmid • Copper is loaded into the Quad mutants by slightly unfolding the protein with urea in the presence of Cu, and then refolding the protein by dialyzing out the urea • Urea loosens the electrostatic loop, but the -barrel is unaffected • Metalation status is analyzed using mass spectrometry

8. Mass Spectrometry Results • Initial results show small amounts of Cu binding to Quad after in vitro addition • This graph shows the isotopic distribution of all the charge states of metalated Quad-SOD1 from a mass spectrometer assay Apo-Quad-SOD1 63.5 amu 63.5 amu Relative Intensity Mass

9. Conclusions • Initial results from mass spectrometry support the hypothesis that Cu is able to bind to Quad-SOD1 Future Aims • Perfect methods of in vitro Cu-binding analysis of the Quad-SOD1 • Develop methods to explore in vivo binding of Cu to Quad-SOD1 • Develop methods to compare Cu and Zn binding both in vitro and in vivo. • Explore the possibility that Copper Chaperone for SOD1 (CCS) is stabilizing Cu in Quad-SOD1

10. Acknowledgements • I would like to thank: • Joe Beckman • Jared Williams • Nathan Lopez • The HHMI Program • Kevin Ahern