Zinc-deficient SOD in ALS
Emily Clark
Dr. Joe Beckman
Department of Biochemistry/Biophysics
Amyotrophic Lateral Sclerosis
(ALS)
Louis Gehrig Disease




Fatal disease
Progressive motor
neuron death in the brain
and spinal cord
5,000 people diagnosed
per year
Mutations to superoxide
dismutase (SOD) occur
in only 2-3% of
individuals with ALS
Cu-Zn Superoxide Dismutase (SOD)



153 amino acid
protein
Exists as a
homodimer,
containing one
copper and one zinc
atom per monomer
SOD functions as a
superoxide
scavenger in cells
throughout the body
SOD Function
Superoxide (O2-)
O2
Zn2+
Superoxide (O2-)
Cu2+
Zn2+
Oxidized SOD
Hydrogen
Peroxide
Cu1+
Reduced SOD
Mutations in SOD

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Over 100 mutations causing ALS have been
discovered in the gene coding for SOD
It is not known exactly what about these mutations
causes toxicity in cells
The toxicity is not due to reduced superoxide
scavenging ability
Mutant SOD has reduced affinity for binding Zn,
leaving a more reactive Cu atom
Zn-deficient SOD Hypothesis
Peroxynitrite
(ONOO-)
2+
Cu
Oxidized
SOD
(1 e-)
Reduced by
Ascorbate or
Thiols
Oxygen
e-
Cu2+-Superoxide
(Cu2+ -O2.-)
(Cu1+... O2)
Cu1+
Reduced
SOD
. NO
e- 2+
Cu
Reoxidizing
SOD
The Disulfide Bond
• When SOD loses
the Zn atom, the
disulfide bond is
more easily reduced
+2e-
Reduced
When the
disulfide bond is
reduced, is Zn
more readily
separated from
SOD?
Native Gels
+
Experiment
1
2
3
4
5
6
7
8
1. Cu, Zn Wt SOD
2. SOD, TCEP
3. SOD, TCEP, EDTA
4. SOD, EDTA
5. Zn(-) Wt SOD
6. SOD, TCEP
7. SOD, TCEP,EDTA
8. SOD, EDTA
Experiment
1. Cu, Zn Wt SOD
2. SOD, TCEP
1
2
3
4
5
6
7
8
3. SOD, TCEP, EDTA
4. SOD, EDTA
5. Zn(-) Wt SOD
6. SOD, TCEP
7. SOD, TCEP,EDTA
8. SOD, EDTA
NFL
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Most abundant neuron filament protein in
neurons
Essential for neurofilament assembly
Binds Zn
Expression and Purification of
NFL

Human plasmids used to express NFL in
E. coli
Acknowledgments

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
The Beckman Lab
HHMI
Kevin Ahearn