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