Investigation of mutated Cu/Zn
Superoxide Dismutase
Sam Schuberg
Beckman Laboratory
Howard Hughes Medical Institute:
Summer 2007
Amyotrophic Lateral Sclerosis
• Neurodegenerative
disease caused by the
destruction of the motor
neurons along the spinal
cord
• Gradual loss of voluntary
muscular function
– Not only difficulty in
moving, but also in
speaking and swallowing
• Eventually affects
respiratory system
Types of ALS
• Two types of ALS
– Sporadic and Familial
– 98% of patients have
sporadic ALS
– 2% have genetically
inherited the disease
• Nature 1993
• Common link in a mutation
in chromosome 21
Beckman et al. Superoxide dismutase and the death of motor
nuerons in ALS. Trends Nueroscience, 2001.
– The gene with the
mutations is for SOD1
– Currently over 100
mutations identified
Wild Type Superoxide Dismutase
• Scavenges
superoxide produced
by normal metabolism
QuickTime™ and a
Sorenson Video 3 decompressor
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• The dimer interface is
stabilized by a
disulfide bond
• A cytosolic disulfides
• Active site contains
two metal ions
• Rate: 2 x 109 M-1s-1
Mutated SOD
•It is widely accepted
that these mutations
not only deactivate
SOD1’s scavenging
feature, but cause it to
gain a toxic function
•Debate is on what this
toxic function is
•One theory is
aggregation
The hypothesis of Zn deficient SOD
• Mutations destabilize the
enzyme and cause it to
lose its affinity for Zn
• Alters coordination of the
neighboring Cu through a
shared histidine ligand
• The exposed Cu is readily
reduced by antioxidents
• Reduced Cu donates an
electron to oxygen to produce
superoxide
• O2.- react with NO to form
peroxynitrite
– Peroxynitrite modifies
important biological molecules
leading to apoptosis
Beckman et al. Superoxide dismutase and the death of motor
nuerons in ALS. Trends Nueroscience, 2001.
• Dr. Wang and his
research group created
SOD-mutated mice
deficient in four histidines
• These coordinate and
hold Cu in its active site
– Their quad mice still get
ALS like symptoms
– Their in vitro data was
interpreted as consistent
with aggregation
– Their data suggests that
CuII is not important in
disease pathology
Wang et al. Journal of Neuroscience. Copper-binding-site-null SOD1
causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a
common feature. 2003.
Cu
My project
• To further investigate the aggregation of
mutant superoxide dismutases in spinal
cord punches from transgenic mice.
Spinal cord punches
Tissue Samples for MS
SOD Assay
Dorsal
Ventral
Do the Quad and G93A mutants
aggregate in animals?
• Western Blot Analysis
– Location of SOD1
• Supernatant versus pellet
• Mice
– Mutated SOD1
• Quad and G93A
• Punches from the spinal
cords
• ventral and dorsal side are
taken
ventral dorsal ventral dorsal
Pellet
Supernatant
Pellet
Supernatant
Pellet
15.78 ng SOD std
63.12 ng SOD std
31.56 ng SOD std
Heterozygous
G93A
Quad
80 days
190 days
Supernatant
Pellet
Supernatant
Pellet
Supernatant
Pellet
Pellet
Supernatant
Supernatant
Pellet
Supernatant
Heterozygous Homozygous
Quad
Quad
210 days
210 days
ventral dorsal ventral dorsal
15.78 ng SOD std
31.56 ng SOD std
63.12 ng SOD std
Mutated SOD is primarily located in the supernatant
Conclusions
• My results are not consistent with
aggregation
• Previous results could be artifacts of
sonication and grinding of the spinal cord
Acknowledgments
• Howard Hughes Medical Institute
• Dr. Joseph Beckman, Nathan Lopez and
the Beckman Lab
• Dr. Kevin Ahern
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