An Investigation of the Interactions An Investigation of the Interactions Between Zinc-deficient and Copper, Zinc Between Zinc-deficient and Copper, Zinc Superoxide DismutaseSuperoxide Dismutase
Katie Meyers
Dr. Joe Beckman
Department of Biochemistry/Biophysics
Amyotrophic Lateral SclerosisAmyotrophic Lateral Sclerosis 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)Superoxide Dismutase (SOD)
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 FunctionSOD Function
SOD MutationsSOD Mutations 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 ChemistrySOD Chemistry 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
aa
Oxygen
Peroxynitrite(ONOO-)
(1 e-)Reduced by
Ascorbate orThiols
Cu2+
OxidizedSOD
Cu -Superoxide(Cu -O2
.-)2+
2+
Cu1+
ReducedSOD
(Cu O2)1+...
Cu2+
NO.
e-
ReoxidizingSOD
e-
Zinc DeficiencyZinc Deficiency
Wild-type Cu, Zn
Protects motor neurons
Motor neuron death
Mutant Cu, Zn
Mutant Cu, Zn(-)
Protects motor neurons
Rapid motor neuron death
ObjectiveObjective Why does Cu, Zn SOD make Cu, Zn(-) SOD more
toxic to motor neurons in vitro?
Cu, Zn homodimer31,808 Da
Cu, Zn(-) homodimer 31,732 Da
Cu, Zn + Cu, Zn(-) heterodimer 31,780 Da
Can subunits exchange between dimers?Can subunits exchange between dimers?
SOD dimers do exchange their subunits and form heterodimers
The exchange is rapid with a half life of 15-20 minutes
0 10 20 30 40 50 60 90 120 12hrs
Time, min
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Why would heterodimers be toxic?Why would heterodimers be toxic?
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 Does the Presence of Cu, Zn(-) SOD Increase the Rate of Reduction of Cu, Zn SOD?the Rate of Reduction of Cu, Zn SOD?
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
0.014
0.016
0.018
0.02
0.022
0.024
0.026
0.028
0 10 20 30 40 50
ab
s 6
80
nm
y = m1 + m2 * e^(-m3*M0)
ErrorValue
4.0846e-050.016099m1
0.000204940.01027m2
0.00748660.2333m3
NA6.4622e-05Chisq
NA0.97079R
Cu, Zn + Cu, Zn(-)
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0 2 4 6 8 10
ab
s 6
80
nm
y = m1 + m2 * e (̂-m3*M0)
ErrorValue
3.6495e-050.0065316m1
0.000220970.02868m2
0.018931.5796m3
NA0.00058678Chisq
NA0.98646R
Cu, Zn(-)
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0 20 40 60 80 100 120 140
abs
68
0nm
y = m1 + m2 * e (̂-m3*M0)
ErrorValue
3.0205e-050.0044145m1
8.1802e-050.0062662m2
0.001120.042882m3
NA0.00018687Chisq
NA0.95168R
Cu, Zn
Rates of ReductionRates of Reduction
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
time
rate
, abs
/sec
Cu, Zn
Cu, Zn + Cu, Zn(-)
Cu, Zn(-)
Does Cu, Zn SOD Affect the Does Cu, Zn SOD Affect the Aggregation of Cu, Zn(-) SOD?Aggregation of Cu, Zn(-) SOD?
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
40
8 9
05
10152025303540
% A
gg
reg
atio
n
Cu,Zn(-) Cu,Zn(-) +Cu,Zn
Cu,Zn
Analytical Ultracentrifugation
ConclusionsConclusions
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
AcknowledgementsAcknowledgements
Dr. Joe Beckman Howard Hughes Medical Institute Linus Pauling Institute Keith Nylin, Blaine Roberts, Val Bomben,
Kristine Robinson Kevin Ahern