Zinc-deficient SOD in ALS Emily Clark Dr. Joe Beckman Department of Biochemistry/Biophysics.
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Transcript of Zinc-deficient SOD in ALS Emily Clark Dr. Joe Beckman Department of Biochemistry/Biophysics.
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Zinc-deficient SOD in ALS
Emily Clark
Dr. Joe Beckman
Department of Biochemistry/Biophysics
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Amyotrophic Lateral Sclerosis (ALS)
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
Louis Gehrig Disease
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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
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SOD Function
Zn2+ Cu2+
Superoxide (O2-)
O2Superoxide (O2-) Hydrogen
Peroxide
Zn2+ Cu1+
Oxidized SOD Reduced SOD
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Mutations in SOD
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
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Zn-deficient SOD Hypothesis
aa
OxygenPeroxynitrite(ONOO-)(1 e-)Reduced byAscorbate orThiols
Cu2+OxidizedSODCu -Superoxide(Cu -O2.-)2+2+Cu1+ReducedSOD
(Cu O2)1+... Cu2+NO.e-ReoxidizingSODe-
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The Disulfide Bond
• When SOD loses the Zn atom, the disulfide bond is more easily reduced
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When the disulfide bond is reduced, is Zn more readily separated from SOD?
Reduced
+2e-
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Native Gels
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+
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Experiment
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
1 2 3 4 5 6 7 8
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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
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NFL
Most abundant neuron filament protein in neurons
Essential for neurofilament assembly Binds Zn
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Expression and Purification of NFL
Human plasmids used to express NFL in
E. coli
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Acknowledgments
The Beckman Lab HHMI Kevin Ahearn