Transcript The Role of Copper in Amyotrophic Lateral Sclerosis

Copper Binding to Superoxide
Dismutase Mutants in
Amyotrophic Lateral Sclerosis
(ALS)
Natalie Biggs
Mentor: Dr. Joseph Beckman
ALS (Lou Gehrig’s Disease)
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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
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Role of Cu/Zn SOD
http://www.altonweb.com/cs/downsyndrome/index.htm?page=sod.html
Superoxide Dismutase (SOD)
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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
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Dr. Beckman’s hypothesis
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Zinc deficiency of SOD makes copper act
toxically and causes oxidative stress
Leads to motor neuron death
The Controversy Continues
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Another prevalent hypothesis
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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
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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
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Mutation of two copperbinding histidines
 H46R/H48Q mutant SOD
Conclusions
 Aggregation of SOD is
responsible for disease
 Copper has no effect in ALS
development
Project Hypothesis
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The H46R/H48Q mutant SOD still binds copper
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Poor evidence in paper that mutated SOD cannot bind copper
Test hypothesis
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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
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Use methylated double stranded plasmid
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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
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PAR binds copper and zinc
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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
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Determine affinity for copper
Results and Conclusions
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Created H46R/H48Q SOD plasmid
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Screened a few colonies of E.coli
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DNA sequencing
None contained mutated SOD
Started again at the transformation step
Check purified SOD in mass spectrometer
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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