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 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

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

AcknowledgementsAcknowledgements Dr. Joe Beckman Howard Hughes Medical Institute Linus Pauling Institute Keith Nylin, Blaine Roberts, Val Bomben,

Kristine Robinson Kevin Ahern