Copper Binding of Mutant Quad SOD1 And Its Relationship to Disease Progression of ALS in Transgenic Mice Nick Classen Dr. Joseph Beckman Linus Pauling Institute HHMI Summer 2011 Hi, my name is Nick Classen, and I am currently working with Dr. Joseph Beckman in the Linus Pauling Institute. My summer project is entitled Copper Binding of Mutant Quad SOD1

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%) First, I’d like to talk a little bit about amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease or ALS. This is a neurodegenerative disease that attacks the motor neurons in the brain and spinal cord, so victims lose motor function over time. Eventually, paralysis and loss of lung function occur. There are approximately 5000 people diagnosed with ALS every year, and the average life span is 2-5 years after diagnosis. Mutations in superoxide dismutase (SOD1) gene were the first known familial link for ALS Transgenic mice with human mutant sod1 genes develop disease

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 Dr. Beckman’s lab focuses primarily on the protein superoxide dismutase, or SOD. SOD is found in the cytosol and removes harmful superoxide radicals. As you can see, each monomer contains a Cu and Zn ions. The active site for this enzyme is next to the Cu ion. SOD functions by removing harmful superoxide radicals from the cell, and turning them into non-toxic hydrogen peroxide. To help illustrate this, I’m going to use a little help from George Lucas. Mutations weaken structure, and decrease binding affinity for metals, therefore it is possible that they lose Zn more easily than normal SOD1

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 As I mentioned before, oxidative stress can occur in the form of peroxynitrite. Peroxynitrite, a very powerful oxidizing agent, is formed from the combination of superoxide with nitric oxide. Peroxynitrite has been linked to numerous diseases due to its ability to nitrate amino acids, especially tyrosines, on proteins. More importantly for our study, and for this presentation, evidence indicates that the formation of peroxynitrite can be catalyzed by mutant SOD1. Nitration of proteins has been shown to be a factor in many diseases. Our lab has observed significantly higher levels of nitration in the motor neurons of ALS models, and more research is being done to understand the implications of this fact. Also, nitration of proteins has been observed in many other diseases including cancers, diabetes, neurodegenerative diseases, cardiovascular diseases, and even in aging. So, mutant-SOD creates a disturbance in the Force.

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) X However, to remove adventitious metals from their protein extraction, they used 5 mM EDTA, which is strong enough to remove Cu from Quad-SOD1 The so called “quad” mutant SOD1 contains four mutations within the Cu binding site, yet animals with these mutations still develop ALS. One previous study, (Wang, 2006), used Cu isotopes to show that Cu was unable to bind to quad-SOD1, thus, by all indications, dismantling the Cu-toxicity theory. However, we suspect that their methods led to skewed results, and improper conclusions. First, they used EDTA and SDS in their sample preparations and during assays. EDTA has a relatively high binding affinity for Cu and SDS is capable of denaturing weaker proteins, potentially even this quad-mutant. Also, they did not check for Zn binding during their study, which could have revealed important information about denaturation. It may be possible to keep Cu in Quad-SOD1 by not introducing EDTA

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 Make bigger It is known that Cu is capable of binding to the Zn site Other Cu binding sites have been observed

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 We can test metal loss from SOD using competition assays. These work by inserting a compound, known as chelators, with a known binding affinity for Cu, that also absorbs light at a specific wavelength, into a solution containing SOD. As Cu is removed from SOD, it binds to the chelator, and therefore we see absorbance increase. This graph is an example of adding BCS to a sample of Zn-deficient SOD. As you can see, the absorbance (which is the y-axis) increases over time (the x-axis) meaning that SOD is slowly losing its Cu to BCS. Urea loosens the electrostatic loop, but the -barrel is unaffected Metalation status is analyzed using mass spectrometry

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

Conclusions Future Aims 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

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