investigation of mutated cu/zn superoxide dismutase sam schuberg beckman laboratory howard hughes...
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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– The gene with the
mutations is for SOD1– Currently over 100
mutations identified Beckman et al. Superoxide dismutase and the death of motor nuerons in ALS. Trends Nueroscience, 2001.
Wild Type Superoxide Dismutase
• Scavenges superoxide produced by normal metabolism• 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
QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.
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
Cu
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.
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
Mutated SOD is primarily located in the supernatant
63.1
2 ng
SO
D s
td31
.56
ng S
OD
std
15.7
8 ng
SO
D s
td
G93A80 days
Heterozygous Quad 190 days
Sup
erna
tant
Sup
erna
tant
Sup
erna
tant
Sup
erna
tant
Pel
let
Pel
let
Pel
let
Pel
let
15.7
8 ng
SO
D s
td
31.5
6 ng
SO
D s
td
63.1
2 ng
SO
D s
td
Homozygous Quad 210 days
Heterozygous Quad 210 days
Sup
erna
tant
Sup
erna
tant
Sup
erna
tant
Sup
erna
tant
Pel
let
Pel
let
Pel
let
Pel
let
ventral ventralventralventral dorsal
dorsaldorsal
dorsal
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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