Natalie Biggs Mentor: Dr. Joseph Beckman Copper Binding to Superoxide Dismutase Mutants in Amyotrophic Lateral Sclerosis (ALS) Natalie Biggs Mentor: Dr. Joseph Beckman

ALS (Lou Gehrig’s Disease) Fatal disease that causes death of motor neurons 30,000 Americans afflicted currently In 1993 mutations of Cu/Zn superoxide dismutase (SOD) were linked to the disease Dr. Beckman’s research focuses on understanding ALS in an attempt to find a cure Role of Cu/Zn SOD http://www.altonweb.com/cs/downsyndrome/index.htm?page=sod.html

Superoxide Dismutase (SOD) SOD protects the body from oxidative damage Superoxide (O2.-) is a radical that can harm the body SOD reduces (scavenges) superoxide into hydrogen peroxide, a less dangerous radical Michael Potter, UC San Diego

Determining the Cause of ALS Dr. Beckman’s hypothesis Zinc deficiency of SOD makes copper act toxically and causes oxidative stress Leads to motor neuron death

The Controversy Continues Another prevalent hypothesis Aggregation (clumping) of SOD creates toxicity in the motor neurons Cause of aggregation unknown Copper has no role in causing ALS

Copper’s Role Challenged Jiou Wang, et al. Fibrillar Inclusions and Motor Neuron Degeneration in Transgenic Mice Expressing Superoxide Dismutase 1 with a Disrupted Copper-Binding Site. Neurobiology of Disease. 2002 Mutation of two copper-binding histidines H46R/H48Q mutant SOD Conclusions Aggregation of SOD is responsible for disease Copper has no effect in ALS development

Project Hypothesis The H46R/H48Q mutant SOD still binds copper Poor evidence in paper that mutated SOD cannot bind copper Test hypothesis Determine H46R/H48Q mutant SOD’s affinity for binding copper arginine glutamine

Project Goals Recreate plasmid of H46R/H48Q SOD Transform and express mutated SOD in Escherichia coli cells Purify mutated SOD Test H46R/H48Q SOD for affinity to bind copper

Site-Directed Mutagenesis Use methylated double stranded plasmid Replace with unmethylated H46R/H48Q SOD plasmid Plasmid Preparation Temperature Cycling Digestion Transformation https://www.stratagene.com/manuals/200518.pdf

Plasmid Purification Select a few E.coli colonies to grow up Obtain bacterial pellet and add purification buffers Centrifuge again and apply supernatant to QIAGEN tip Precipitate DNA, wash, and resuspend in TE buffer

Transformation and Expression H46R/H48Q SOD plasmid is inserted into E.coli cells Colonies are selected to induce mutant SOD expression Large quantities of H46R/H48Q SOD produced

Purification Lyse (break open) cells to release contents Precipitate unwanted proteins using pH changes Anion exchange column

PAR (4-pyridylazoresorcinol) Assay PAR binds copper and zinc Measure absorbance at 500 nm Absorbance increases as PAR binds the metals Absorbance decreases if SOD can compete to bind copper and zinc Test H46R/H48Q SOD Determine affinity for copper

Results and Conclusions Created H46R/H48Q SOD plasmid DNA sequencing Screened a few colonies of E.coli None contained mutated SOD Started again at the transformation step Check purified SOD in mass spectrometer If correct mutant, begin testing with PAR assay

Acknowledgments Howard Hughes Medical Institute Linus Pauling Institute Dr. Joseph Beckman Dr. Kevin Ahern Keith Nylin Valerie Bomben