an investigation of the interactions between zinc-deficient and copper, zinc superoxide dismutase
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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. Fatal neurodegenerative disease that is characterized by selective motor neuron death - PowerPoint PPT PresentationTRANSCRIPT
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 MeyersDr. Joe BeckmanDepartment 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 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
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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
abs
680n
m
y = m1 + m2 * e (̂-m3*M0)ErrorValue
4.0846e-050.016099m1 0.000204940.01027m2
0.00748660.2333m3 NA6.4622e-05ChisqNA0.97079R
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
abs
680n
m
y = m1 + m2 * e (̂-m3*M0)ErrorValue
3.6495e-050.0065316m1 0.000220970.02868m2
0.018931.5796m3 NA0.00058678ChisqNA0.98646R
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
680n
m
y = m1 + m2 * e^(-m3*M0)ErrorValue
3.0205e-050.0044145m1 8.1802e-050.0062662m2
0.001120.042882m3 NA0.00018687ChisqNA0.95168R
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, ZnCu, 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
ggre
gatio
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