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 Lab
The 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) (1020% of fALS cases)
• Mutant SOD becomes toxic (gain
of function)
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Superoxide Dismutase (dimer)
Wild Type SOD
e-O2
O2
Cu2+
Cu2+
Cu, Zn SOD
Zn
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Cu2+
Cu1+
Cu, Zn SOD
Zn
H2O2
O2-
Mutant SOD
Cu2+
Cu2+
Zn
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Zn
Copper Toxicity: Mutant SOD
O2
O2-
Mutant ZnSOD
Mutant ZnSOD
H2O2
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Cu1+
Cu2+
½ Rate
Cellular Reductases
O2-
Summary
Cu
O2
OONOPeroxynitrite
(toxic!)
Superoxide
Zn- Mutant SOD
*Cu binding
site mutated
“Quad” Mutant
NO
Nitric Oxide
Mouse with ALS
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
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
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
Quad Cu Binding
5
1.2 10
Apo Quad SOD
5
1 10
4
8 10
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
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Cu Quad SOD
Ion counts
•
4
6 10
4
4 10
63.5 am
4
2 10
0
15650
15700
15750
Mass
15800
15850
15900
Quad SOD Produces Superoxide
Cu
NO
Mutant Zn- SOD
NO
Nitric Oxide
5000
5000
5000
5000
NO
concentration
NO
NO
concentration
NOconcentration
concentration
NO
Sensor
OONO-
O2-
Cu Quad
Quad Injection
Injection
Cu
4000
4000
4000
4000
Mutant SOD
SOD injection
injection
Mutant
B
B
3000
3000
3000
3000
2000
2000
2000
2000
NO
injection
NO
injection
NO
injection
1000
1000
1000
1000
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
0000 00
00
200
200
200
400
600
400
600
400
600
400
600
Time (sec)
(sec)
Time
Time
(sec)
Time (sec)
800
800
800
800
1000
1000
1000
Effects of Concentration on Toxicity
Concentration and Zn- SOD
3000
Zn- SOD injection to 1.07uM
Zn- SOD injection to .5uM
NO conc. (nM)
2500
2000
1500
Zn- SOD
Quad
3500
NO conc. (nM)
Rate = 3.38 nM/sec
Rate = 7.18 nM/sec
4000
1000
3000
500
2000
Zn- SOD injection
Quad Injection
2500
0
0
200
400
600
Time (sec)
1500
800
1000
1000
Concentration and Quad
30
500
Quad500
injection to 3.5uM
0
NO conc. (nM)
2nd NO injection
2nd NO injection
500
4000
3000
1200
Quad injection to 1.75 uM
1000
T i m e (sec)
2500
1500
2000
Quad injection to 3.5 uM
2000
Conclusions
1500
1000
- Quad
SOD does not act catalytically and is consumed in the reaction
500
0
1840
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
1920
2000
2080
T i m e (se c)
2160
2240
Effects of pH on Toxicity
250
Effect of pH on Zn- SOD Activity
NO consumption (nm/sec)
NO consumption (nm/sec)
25
20
15
10
5
Effect of pH on Quad Activity
200
150
100
50
0
0
6
6.5
7
7.5
8
6
6.5
7
7.5
pH
pH
Conclusions
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
- Zn- SOD activity is pH dependent while Quad activity is not
- Supports that Cu is binding to the Zn site in the Quad mutant
8
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
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
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 damage
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The 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
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Results
His 46
His 48
Cu2+
Arg 46
Mutate
(Again)
His 63
Gln 48
2+
Cu
Quad SOD Cu Binding Site
Gly 63
Cu2+
His 120
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Gly 120
Mouse with ALS
What Function does Mutant SOD acquire?
Mutant SOD has lower affinity for metals (Zn and Cu)
Cu Toxicity Theory
•
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
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
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
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
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
The Problem with Cu Toxicity Theory
Quad Mutant
Mouse with ALS
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
The Problem with Cu Toxicity Theory
Gln
His
Cu2+
Gly
His
Arg
His
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
Gly
His
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)
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute
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
Superoxide Dismutase (dimer)
• SOD Mutations do not cause ALS by hindering normal SOD
activity
• SOD Mutations likely result in gain of harmful function
Jesse Fitzpatrick (2008)
Dr. Beckman’s Lab
The Linus Pauling Institute