Dissertation Abstract
Amyotrophic lateral
sclerosis (ALS) is a relentless neurodegenerative
disease characterized by the progressive degeneration of the
upper and
lower motor neurons leading to paralysis and death within ~2-5
years of
symptom onset. Over 90% of ALS cases occur sporadically (sporadic
ALS or
SALS) and ~10% are inherited (familial ALS or FALS). In
1993, a genetic
linkage was made between a subset of familial ALS cases and mutations
in
the gene encoding the antioxidant protein copper-zinc superoxide
dismutase (CuZnSOD or SOD1).
To date, over 100
dominantly inherited single site missense, truncation
and frameshift mutations have been identified in the CuZnSOD
gene.
Studies of transgenic mice with their SOD1 gene disrupted or
a human
FALS-associated gene inserted strongly indicate that SOD1mediated
FALS
occurs by a gain-of-function mechanism rather than by a loss
of CuZnSOD
superoxide disproportionation activity. The presence of mutant
SOD1
proteins in individuals heterozygous for any one of the CuZnSOD
mutations is sufficient to initiate ALS pathogenesis, therefore
these
proteins must possess some property that is cytotoxic to motor
neurons.
Although a significant amount of biochemical data had been accumulated
on the pathogenic SOD1 mutant proteins when the studies described
in
this dissertation were initiated, little structural information
on these
molecules was available. Similarly little was known about the
structure
and function of a protein found to specifically load copper into
CuZnSOD, the copper chaperone for SOD1 (CCS). Therefore, our
overall
objective was to understand at the molecular level how single
site amino
acid substitutions in CuZnSOD initiate the neuropathology observed
in
FALS.
Structures of three
pathogenic SOD1 molecules that cause FALS were
determined to high resolution by X-ray crystallographic methods.
The
aspartate 125 to histidine mutation (D125H) was the first structure
to
show zinc bound in the copper site of the enzyme. Combined with
data
showing hydrogen peroxide-mediated self-inactivation of CuZnSOD
is
significantly enhanced in the presence of bicarbonate anion through
the
enzyme's well-established peroxidase activity, the presence of
a sulfate
ion bound above the zinc in the copper site in the D125H structure
allowed us to propose a structure based mechanism for
bicarbonate-mediated peroxidation for SOD1 proceeding through
an
enzyme-associated peroxycarbonate intermediate. This peroxidase
activity
of CuZnSOD may be involved in the pathogenesis of ALS by directly
oxidizing critical neuronal components or indirectly by making
mutant
SOD1 proteins more susceptible to misfolding and aggregation.
Structures of the
metal free (apo) form of the histidine 46 to arginine
(H46R) SOD1 mutant and a partially metal-loaded form of the serine
134
to asparagine (S134N) SOD1 mutant showed these mutations have
a
surprisingly similar effect on the architecture of the protein.
A
significant amount of disorder in the electrostatic and zinc
loop
elements of these proteins leads to the formation of novel
intermolecular contacts that provide a model for how aggregation
of SOD1
molecules might occur. This model may have relevance to the
SOD1-containing protein inclusions observed in the neural tissue
of ALS
patients and transgenic animal models of the disease.
Since copper is essential
in catalysis and may play an important role
in the stability of CuZnSOD, we also performed functional studies
of the
human copper chaperone for SOD1 (hCCS) in yeast lacking the yeast
form
of this protein (yCCS). We found that the three distinct hCCS
domains
function similarly to those of yCCS and that expression level
can affect
their functional requirements, specifically in the CXC motif
of domain
III.
Overall, these studies
are leading to a greater understanding of the
molecular determinants of mutant SOD1 pathogenesis and stimulating
new
research that will hopefully lead to the design of new therapies
for
individuals with ALS.
Posted: 4/22/04