Copper Binding of Mutant Quad SOD1
And Its Relationship to Disease
Progression of ALS in
Transgenic Mice
Nick Classen
Dr. Joseph Beckman
Linus Pauling Institute
HHMI Summer 2011
Amyotrophic Lateral Sclerosis
•
Loss of motor functions (eventually
paralysis and loss of lung function)
•
More than 3,000 Americans are
diagnosed with ALS each year, and
3,000 deaths are attributed to ALS
every year
•
Average life span is 2-5 years after diagnosis
•
Familial (<10%) vs. sporadic
(>90%)
• Mutations in superoxide dismutase
(SOD1) gene were the first known
familial link for ALS
•
Transgenic mice with human mutant
sod1 genes develop disease
Superoxide Dismutase
(SOD1)
•
The major cytosolic antioxidant within cells
•
Active site characterized by a Cu ion, which participates in
redox reactions of superoxide radicals. The Zn ion adjacent to
the Cu stabilizes the structure
Cu-Toxicity Hypothesis
•
•
Dr. Beckman’s group has
shown Zn-deficient SOD1 to
activate death cascades in
motor neurons due to a gain of
function
Mutations weaken structure, and
decrease binding affinity for
metals, therefore it is possible
that they lose Zn more easily
than normal SOD1
Understanding Metal Binding
of SOD1
•
•
Cu and Zn ions are adjacent to each other in the periodic
table and have similar binding sites
Each binding site is primarily histidine residues, with the Znsite containing a negatively charged aspartic acid
•
Dr. Beckman’s group
hypothesizes that the gain of
function occurs due to Zn being
removed, thus exposing the Cu
ion, which is redox active and
initiates oxidative stress
•
The “Quad” Mutant
Mouse
This diseased transgenic
mouse was developed by Borchelt
to better understand the role of Cu in disease progression
•
•
All four Cu-binding histidine ligands were mutated to
non-Cu-binding residues
Seemed to dismiss the Cu-toxicity hypothesis when a study
showed that these mutant proteins lack bound Cu (Wang,
Journal of Biological Chemistry, Vol. 282, No. 1, pp345-352,
2007)
• However, to remove adventitious
metals from their protein
X
extraction, they used 5 mM
X
EDTA, which is strong enough to
X
remove Cu from Quad-SOD1
X
•
It may be possible to keep Cu in
Quad-SOD1 by not introducing
EDTA
Aims of Our Study
Goal
• To determine whether Cu can bind to Quad-SOD1 in
vitro
Hypothesis
• Copper binds to Quad for one of two reasons:
- Cu binds to Zn site
- A new site is formed on protein
Rationale
• It is known that Cu is capable of
binding to the Zn site
•
Other Cu binding sites have
been observed
Obtaining Cu-Bound
Quad SOD1 Proteins
•
Quad-SOD1 is extracted from auto-lysing E.
coli containing human Quad gene in a pET3d
plasmid
• Copper is loaded into the Quad mutants by
slightly unfolding the protein with urea in the
presence of Cu, and then refolding the
protein by dialyzing out the urea
•
Urea loosens the electrostatic loop,
but the -barrel is unaffected
•
Metalation status is analyzed using
mass spectrometry
•
•
Mass Spectrometry
Results
Initial results show small amounts
of Cu binding to Quad after in vitro
addition
This graph shows the isotopic distribution of all the charge states of
metalated Quad-SOD1 from a mass spectrometer assay
Relative
Intensity
Apo-Quad-SOD1
63.5
amu
63.5
amu
Mass
Conclusions
•
Initial results from mass spectrometry support the
hypothesis that Cu is able to bind to Quad-SOD1
Future Aims
•
Perfect methods of in vitro Cu-binding
analysis of the Quad-SOD1
•
Develop methods to explore in vivo binding
of Cu to Quad-SOD1
•
Develop methods to compare Cu and Zn
binding both in vitro and in vivo.
•
Explore the possibility that Copper Chaperone
for SOD1 (CCS) is stabilizing Cu in QuadSOD1
Acknowledgements
I would like to thank:
•
Joe Beckman
•
Jared Williams
•
Nathan Lopez
•
The HHMI Program
•
Kevin Ahern