SOD1ALS link:

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Molecular Mechanisms of CuZnSOD-Linked ALS
Lecture 1 SOD1-ALS link Gain of
function mechanism Protein aggregation
vs. oxidative damage Chemistry of superoxide
and other ROS Lecture 2 ALS Clinical
aspects SODs and SOD mechanisms Lecture 3
Model studies in cell culture and ALS Tg mice
and rats WT and mutant SOD1
structures Lecture 4 Protein aggregation and
disease Oxidative stress in ALS? Lecture 5
Biophysical properties of WT and mutant SOD1s
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• Evidence for elevated oxidative stress in SALS
  and FALS
• Inclusions in FALS patients and G93A Tg mice
  consist of granule-coated fibrils were found to
  be modified by insoluble advanced glycation end
  products (AGE), and some of the CuZnSOD itself
  was AGE-modified.
• (S. Kato et al., Amyotroph. Lateral. Scler.
  Other Motor Neuron Disord. 1163184 2000.)
• SALS spinal cords (human) showed immunoreactivity
  to 4-hydroxy-2-nonenalhistidine (HNE-histidine)
  and (i) crotonaldehyde-lysine, indicative of
  lipid peroxidation (ii) carboxymethyl-lysine
  (CML), indicative of lipid peroxidation or
  protein glycoxidation and, (iii) pentosidine,
  indicative of protein glycoxidation. They did not
  detect pyrraline or imidazolone, markers of
  nonoxidative protein glycation.
• (N. Shibata et al., Brain Res. 91797104
  2001.)
• Proteomic analysis of G93 SOD1 mouse spinal cords
  showed elevated levels of HNE-modified proteins
  dihydropyrimidinase-related protein 2 (DRP-2),
  heat-shock protein 70 (Hsp70), and possibly
  ?-enolase.
• (Perluigi et al., FRBM 98960-968 (2005)
• But is not known if this oxidative stress is
• causative of the disease or a result of it!

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Figure 3 Introduction of carbonyl groups into
proteins and generation of protein-protein
cross-linkages by reaction with ,??-unsaturated
aldehydes.
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Figure 4 Generation of protein carbonyl
derivatives and protein-protein cross-linkages by
glycation and glycoxidation reactions.
Abbreviations P-Lys-NH2, ?-NH2 groups of lysine
residues of proteins Arg-P2, arginine residue of
another protein molecule.
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• Elevated oxidative stress has also been
  demonstrated in vivo in the Tg mice
• 1. Using the brain-permeable spin trap azulenyl
  nitrone, which reacts with free radicals to give
  azulenyl aldehyde, the G93A transgenic mice
  showed substantially enhanced free radical
  content, prior to motor neuron degeneration, in
  spinal cord but not brain, in G93A SOD1 mice
  relative to the WT transgenic control.
• (Liu, R. et al., Ann. Neurol. 44763770 1998.)
• In vivo microdialysis was used to measure the
  extent of oxidation of 4-hydroxybenzoic acid to
  3,4-dihydroxybenzoic acid (3,4-DHBA). Enhanced
  oxidation was found in the striatum of the G93A
  transgenic mice at baseline but not in mice
  overexpressing WT human SOD1.
• (Bogdanov, M. B. et al., J. Neurochem.
  7113211324 1998.)

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All mutants (tested) adopt detergent insoluble
structures to varying degrees
HEK 293 cells
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Are the insoluble SOD1-derived materials
oxidatively or otherwise covalently modified????
Accumulation of detergent-insoluble SOD1
monomers, fragments, and high molecular weight
species in spinal cords of affected mice and
human patients
(A) Mutant SOD1 from the spinal cords of affected
mice fractionated in the detergent-insoluble
pellet fraction (P3). insoluble species of SOD1
were abundant in symptomatic mutant mice. SOD1
monomers (open arrow), fragments (solid arrow),
and high molecular weight species (solid arrow
head) are indicated. Apparent dimers (asterisk)
and trimers (double asterisk) are noted.
(C) The spinal cord of a patient with the A4V
mutation contains SOD1 monomers, fragments and
oligomers that are insoluble in non-ionic
detergent (P2). A representative age-matched
non-ALS control case was examined in parallel.
Wang, et al., Human Molecular Genetics, (2003)
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Our Hypothesis
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Reaction with External Substrate
DMPO
DMPO-OH
H2O2
CuZnSOD
H
N
H
N
O
O
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EPR of Whole Yeast Treated with POBN and H2O2
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2-oxo-histidine
Hodgson Fridovich(1975) Biochemistry 14,
5294-5298
Uchida Kawakishi (1994) J. Biol. Chem. 269,
2405-2410
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• Specific residues that have been reported to be
  oxidized by hydrogen peroxide are His-46, His-48,
  Pro-62, His-63, and His-120.
• SOD inactivation is believed to be due to loss of
  copper ions.

Uchida, K. Kawakishi, S. (1994) J. Biol.
Chem. 269, 24052410. Kurahashi, T., Miyazaki,
A., Suwan, S.Isobe, M. (2001) J. Am. Chem. Soc.
123, 92689278.
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H2O2 oxidizes CuZnSOD at Metal Binding Sites

• Oxidative damage to metal-binding ligands in
  CuZnSOD destabilizes protein by causing loss of
  metal-binding ability

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Measuring Inactivation of CuZnSOD
SOD Activity
CuZnSOD Time 0 s 100 Active
Incubation with H2O2
100s
75 Active
200s 55 Active
300s 40 Active
400s 30 Active
600s 10
Active
Inactive CuZnSOD
Plot of enzyme activity as a function of time
yields rate of inactivation
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Inactivation of CuZnSOD as a Function of H2O2
Concentration
100
75
WT 3 mM H2O2 t1/2 545 s
50
25
WT 6.25 mM H2O2 t1/2 305 s
Superoxide Dismutase Activity
WT 25 mM H2O2 t1/2 120 s
10
WT 12.5 mM H2O2 t1/2 172 s
7.5
5
0
200
400
600
800
Time (s)
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Rates of inactivation of WT, L38V, and E133D by
hydrogen peroxide
Plot of SOD activity as a function of time after
addition of hydrogen peroxide. The L38V and the
E133D enzyme both have enhanced inactivation
rates relative to the wild-type enzyme
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Bicarbonate is Required for EPR Detection of
Peroxidative Activity
Phosphate inhibits this reaction Prediction
HCO3- should protect from inactivation by H2O2.
Zhang et. al., J Biol Chem 2000, 27514038-45
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Bicarbonate Enhances the Inactivation of CuZnSOD
by Hydrogen Peroxide!
WT t1/2 380 s 8 mM H2O2, no Bicarbonate, 0.5
mM Tris
WT t1/2 510 s no Bicarbonate, 100 mM Phosphate
1e9
1e9
kcalc (m-1s-1)
Dismutation of Superoxide
WT t1/2 480 s 10 mM Bicarbonate, 100 mM
Phosphate
1e8
1e8
WT t1/2 128 s 8 mM H2O2, 25 mM Bicarbonate,
0.5 mM Tris
1e7
1e7
0
200
400
600
800
0
200
400
600
800
Time (s)
Time (s)
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Formation of Peroxycarbonate

or
OCO HOO-
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Cu 2 ?OH CO3 2-   Cu 2 OH- ?
O-CO2 -   His oxidation and inactivation
Cu -O-O-CO2  
Protein aggregates?
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D125H with Sulfate Bound to Zinc in Copper Site
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D125H with Carbonate Bound to Zinc in Copper Site
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H2O2 oxidizes CuZnSOD at Metal Binding Sites

• Oxidative damage to metal-binding ligands in
  CuZnSOD destabilizes protein by causing loss of
  metal-binding ability

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Model for Toxicity
Aggregates
?
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Destabilized Metal-Free Proteins
Model for Toxicity
Damaged demetallated SOD
Stability
2 Cu 2 Zn
2 Cu 2 Zn
2 Cu 2 Zn
2 Cu 2 Zn
H2O2
Cu
Cu
Cu
Cu
Cu
Cu
Zn
Zn
Zn
Zn
Zn
Zn
Metal Binding Region Mutants
WT-like mutants
Wild Type CuZnSOD
Aggregates
Aggregates