Transcript Investigation of the Rates of Dimer Exchange and Electron Transport

An Investigation of the Interactions
Between Zinc-deficient and Copper,
Zinc Superoxide Dismutase
Katie Meyers
Dr. Joe Beckman
Department of Biochemistry/Biophysics
Amyotrophic Lateral Sclerosis
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Fatal neurodegenerative disease that is
characterized by selective motor neuron death
Majority of ALS cases are sporadic but
approximately 10% of all cases are familial
Of these familial cases, 20% of individuals inherit
dominant autosomal mutations in the SOD1 gene
SOD1 gene codes for copper, zinc superoxide
dismutase (SOD)
Superoxide Dismutase (SOD)
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Small: 153 amino acid
protein
Exists as a dimer of
identical subunits,
containing one copper
and one zinc atom per
monomer
SOD functions as a
superoxide scavenger in
cells throughout the
body
SOD Function
SOD Mutations
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Over 100 different ALS causing mutations
have been discovered dispersed throughout
the gene
The toxicity of these mutations is not due
to reduced superoxide scavenging ability
Something about these mutations causes
them to become toxic to cells
SOD Chemistry
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Mutant SODs have a reduced affinity for binding
zinc, leaving the copper more reactive
In Cu, Zn(-) SODs, copper can react with
ascorbate and facilitate the transfer of electrons
from ascorbate to oxygen, producing superoxide
Superoxide reacts with nitric oxide to form
peroxynitrite, which can nitrate tyrosine residues
and is harmful to motor neurons
Peroxynitrite
(ONOO-)
2+
Cu
Oxidized
SOD
(1 e-)
Reduced by
Ascorbate or
Thiols
Oxygen
e-
Cu2+-Superoxide
(Cu2+ -O2.-)
(Cu1+... O2)
Cu1+
Reduced
SOD
. NO
e- 2+
Cu
Reoxidizing
SOD
Zinc Deficiency
Rapid motor
neuron death
Wild-type
Cu, Zn
Protects motor
neurons
Mutant
Cu, Zn
Protects motor
neurons
Mutant Cu,
Zn(-)
Motor neuron
death
Objective

Why does Cu, Zn SOD make Cu, Zn(-) SOD more
toxic to motor neurons in vitro?
Can subunits exchange between dimers?
Cu, Zn
homodimer
31,808 Da
Cu, Zn(-)
homodimer
31,732 Da
Cu, Zn + Cu, Zn(-)
heterodimer
31,780 Da
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0
10
20
30
40
50
60
90
120
12hrs
Time, min
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SOD dimers do exchange their subunits and form
heterodimers
The exchange is rapid with a half life of 15-20
minutes
Why would heterodimers be toxic?
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Cu, Zn(-) SOD may be able to transfer an
electron to Cu, Zn SOD increasing the rate
of reduction and resulting in tyrosine
nitration
The presence of Cu, Zn SOD could slow
down the aggregation of Cu, Zn(-) SOD, a
protective mechanism, resulting in
increased cell damage
Does the Presence of Cu, Zn(-) SOD Increase
the Rate of Reduction of Cu, Zn SOD?
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Cu, Zn and Cu, Zn(-) SODs were
individually reduced by ascorbate and
analyzed using a stopped flow
spectrophotometer
An equimolar mixture of Cu, Zn and Cu,
Zn(-) SOD was reduced by ascorbate and
rates of reduction were measured as a
function of time
Cu, Zn
Cu, Zn(-)
0.014
0.04
0.012
m1
m2
0.0062662 8.1802e-05
0.01
m3
Chisq
0.042882
0.00018687
0.00112
NA
R
0.95168
NA
abs 680nm
y = m1 + m2 * e^(-m3*M0)
Value
Error
0.0044145 3.0205e-05
0.008
0.035
0.03
m1
m2
m3
Value
0.0065316
0.02868
1.5796
Error
3.6495e-05
0.00022097
0.01893
0.025
Chisq
0.00058678
NA
R
0.98646
NA
0.02
0.015
0.006
0.01
0.004
0.005
0.002
0
0
20
40
60
80
100
120
140
0
2
Cu, Zn + Cu, Zn(-)
0.028
y = m1 + m2 * e^(-m3*M0)
Value
Error
0.016099
0.01027
0.2333
4.0846e-05
0.00020494
0.0074866
Chisq 6.4622e-05
NA
0.026
m1
m2
m3
0.024
abs 680nm
abs 680nm
y = m1 + m2 * e^(-m3*M0)
R
0.022
0.97079
NA
0.02
0.018
0.016
0.014
0
10
20
30
40
50
4
6
8
10
Rates of Reduction
2
1.8
1.6
rate, abs/sec
1.4
1.2
Cu, Zn
1
Cu, Zn + Cu, Zn(-)
0.8
Cu, Zn(-)
0.6
0.4
0.2
0
time
Does Cu, Zn SOD Affect the
Aggregation of Cu, Zn(-) SOD?
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Cu, Zn(-) aggregates as a
mechanism to protect motor
neurons from apoptosis
Analytical ultracentrifugation
was used to measure aggregation
as a function of absorbance
When Cu, Zn(-) is mixed with
Cu, Zn SOD, aggregation is
severely reduced
Interactions increase the toxicity
of Cu, Zn SOD
Analytical Ultracentrifugation
% Aggregation
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40
35
30
25
20
15
10
5
0
40
8
Cu,Zn(-)
Cu,Zn(-) +
Cu,Zn
9
Cu,Zn
Conclusions
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Cu, Zn and Cu, Zn(-) SODs exchange monomers
with a half life of 15-20 minutes
The presence of Cu, Zn(-) SOD may cause the
Cu, Zn rate of reduction to increase, but further
data collection and analysis are necessary
Cu, Zn SOD interferes with the aggregation of
Cu, Zn(-) SOD, preventing this protective
mechanism and increasing the toxicity of Cu,
Zn(-) SOD
Acknowledgements
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Dr. Joe Beckman
Howard Hughes Medical Institute
Linus Pauling Institute
Keith Nylin, Blaine Roberts, Val Bomben,
Kristine Robinson
Kevin Ahern