comparing the toxicity of zinc deficient superoxide dismutase (sod) and the quad sod mutant:...

Comparing the Toxicity of Zinc Deficient Superoxide Dismutase (SOD) and the Quad SOD mutant: Implications for Amyotrophic

Lateral Sclerosis

Jesse Fitzpatrick

Dr. Joe Beckman’s Lab

The Linus Pauling Institute

Jesse Fitzpatrick (2008)Dr. Beckman’s LabThe Linus Pauling Institute

Amyotrophic Lateral Sclerosis (ALS)

• ALS is neurodegenerative disease

• Two Forms of ALS– Sporadic– Familial (fALS) (10-20%)

• fALS caused by mutant Superoxide Dismutase (SOD) (10-20% of fALS cases)

• Mutant SOD becomes toxic (gain of function)


Superoxide Dismutase (dimer)

http://pages.prodigy.net/macknife13/gehrig.jpg

Wild Type SOD


O2- O2

Cu2+ Cu1+

Cu, Zn SOD Cu, Zn SOD

ZnZn

Cu2+

Cu2+

e-

O2-

H2O2


Mutant SOD


ZnZn

Cu2+

Cu2+


Copper Toxicity: Mutant SOD


O2- O2

Cu2+ Cu1+

O2-

H2O2 ½ Rate

Cellular Reductases

Mutant Zn- SOD

Mutant Zn- SOD


Summary

Zn- Mutant SOD

O2-

Superoxide

NONitric Oxide

OONO-

Peroxynitrite(toxic!)

Cu

Mouse with ALS“Quad” Mutant

*Cu binding site mutated


Why do Quad SOD Mice get sick?

1. Cu toxicity may not contribute significantly to ALS symptoms

2. Quad SOD causes ALS because Cu binds to another part of the SOD protein



Research Aims

1. Determine whether Quad SOD binds Cu

2. If Quad SOD binds Cu, determine whether it still exhibits deleterious redox behavior


Quad SOD Binds Copper


• Metalation Variables– pH– Cu concentration– Folding Agents– Urea

• Successful Conditions– Combine Quad with

Urea (conc. 1.5M)– Dialyze against Tris

buffer pH 7.5 with 300uM CuCl2

– Dialyze against Tris buffer pH 7.5

0

2 104

4 104

6 104

8 104

1 105

1.2 105

15650 15700 15750 15800 15850 15900

Quad Cu Binding

Ion

co

un

ts

Mass

Apo Quad SOD

Cu Quad SOD

63.5 am


Quad SOD Produces Superoxide


Mutant Zn- SOD

O2-

NONitric Oxide

OONO-Cu

NOSensor

0

1000

2000

3000

4000

5000

0 200 400 600 800 1000

B

Time (sec)

Cu Quad Injection

NO

con

cent

ratio

n

NO injection

Mutant SOD injection

0

1000

2000

3000

4000

5000

0 200 400 600 800 1000

NO

con

cent

ratio

n

Time (sec)

NO injection

0

1000

2000

3000

4000

5000

0 200 400 600 800 1000

B

Time (sec)

NO

con

cent

ratio

n


NO injection

0

1000

2000

3000

4000

5000

0 200 400 600 800 1000

B

Time (sec)

Cu Quad Injection

NO

con

cent

ratio

n

NO injection


NO


Effects of Concentration on Toxicity


0

500

1000

1500

2000

2500

3000

0 200 400 600 800 1000 1200

NO

co

nc.

(nM

)

Time (sec)

Zn- SOD injection to 1.07uM Zn- SOD injection to .5uM

Rate = 7.18 nM/sec Rate = 3.38 nM/sec

0

500

1000

1500

2000

2500

3000

3500

4000

1840 1920 2000 2080 2160 2240

NO

co

nc.

(nM

)

Time (sec)

Quad injection to 3.5uMQuad injection to 1.75 uM

Quad injection to 3.5 uM

0

500

1000

1500

2000

2500

3000

3500

4000

0 500 1000 1500 2000

NO

co

nc.

(nM

)

Time (sec)

Quad Zn- SOD

Quad Injection

2nd NO injection2nd NO injection

Zn- SOD injection

Concentration and Zn- SOD

Concentration and Quad

Conclusions

- Quad SOD does not act catalytically and is consumed in the reaction


Effects of pH on Toxicity

0

50

100

150

200

250

6 6.5 7 7.5 8

pHN

O c

on

su

mp

tion

(n

m/

sec)


Effect of pH on Quad Activity

0

5

10

15

20

25

6 6.5 7 7.5 8

pH

NO

con

su

mp

tion

(n

m/

sec) Effect of pH on Zn- SOD Activity

Conclusions- Zn- SOD activity is pH dependent while Quad activity is not

- Supports that Cu is binding to the Zn site in the Quad mutant

The Big Picture


Quad SOD binds Cu (possibly at the Zn site)

Cu Quad exhibits toxic activity invitro

Could explain why transgenic Quad mice

develop ALS

Allays the dissonance between the Cu toxicity theory and the Quad transgenic mouse study

Future Directions

• Mutate both the copper and zinc binding site and test for Cu binding

• Investigate the redox differences between Zn- SOD and Cu Quad SOD

• Analyze invivo Quad SOD from Quad SOD transgenic mice

• Look for evidence of peroxynitrite damageJesse Fitzpatrick (2008)Dr. Beckman’s LabThe Linus Pauling Institute

Acknowledgments

Dr. Joe Beckmen

Keith Nylin

Nathan Lopez

Dr. Mark Levi

Dr. Keri Trumbull

Megan Hirko

Kevin Ahern

HHMI

The Linus Pauling Institute


Results



Cu2+

His 46

His 48

His 63

His 120

Mutate(Again)

Cu2+

Arg 46

Gln 48

Gly 63

Gly 120

Cu2+

Quad SOD Cu Binding Site

Mouse with ALS

What Function does Mutant SOD acquire?

Mutant SOD has lower affinity for metals (Zn and Cu)


Cu Toxicity Theory• Loss of metals cause SOD to

function backwards, creating super oxides which indirectly trigger apoptosis

What Function Does Mutant SOD Acquire?

Mutant SOD has lower affinity for metals (Zn and Cu)


SOD Aggregation Theory• Loss of metals leads to SOD

aggregation which triggers apoptosis

Cu Toxicity Theory• Loss of metals cause SOD to

function backwards, creating superoxides which indirectly trigger apoptosis

Consequences of Zn Deficiency


1. Copper becomes more exposed

2. Regeneration of Cu2+ SOD is decreased

3. Cu2+ SOD is reduced by cellular reductase

4. Cu2+ availability decreases while Cu1+ increases

5. Superoxide concentrations increase

6. Superoxides reacts with NO to produce peroxynitrate

7. Peroxynitrate nitrates tyrosine residues and triggers cell death

The Problem with Cu Toxicity Theory


Mouse with ALS

Quad Mutant

The Problem with Cu Toxicity Theory


His

His

His

His

Cu2+

Arg

Gly

Gln

Gly

Amyotrophic Lateral Sclerosis (ALS)

• Symptoms– Neuronal death– Muscle weakness– Paralysis

• Death occurs in 3-5 years from onset

• Two Forms of ALS– Sporadic– Familial (2-3% of cases)


Upper motor neuron

Lower motor neuron

Familial ALS: The Genetic Link

• Familial ALS patients have mutations in the Superoxide Dismutase (SOD) protein

• SOD– Structure

• Binds a Cu and a Zn cofactors• Exists as a homodimer

– Normal function• Scavenges superoxides such as O2

-

• Protects the cell from oxidative stress

• SOD Mutations do not cause ALS by hindering normal SOD activity

• SOD Mutations likely result in gain of harmful function


Superoxide Dismutase (dimer)

comparing the toxicity of zinc deficient superoxide dismutase (sod) and the quad sod mutant:...

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