Post on 28-Jan-2016
description
Zinc-deficient SOD in ALS
Emily Clark
Dr. Joe Beckman
Department of Biochemistry/Biophysics
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
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
Zn2+ Cu2+
Superoxide (O2-)
O2Superoxide (O2-) Hydrogen
Peroxide
Zn2+ Cu1+
Oxidized SOD Reduced SOD
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
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-
The Disulfide Bond
• When SOD loses the Zn atom, the disulfide bond is more easily reduced
When the disulfide bond is reduced, is Zn more readily separated from SOD?
Reduced
+2e-
Native Gels
-
+
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
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
NFL
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
The Beckman Lab HHMI Kevin Ahearn