Rodriguez, J. A., J. S. Valentine, et al. (2002). "Familial amyotrophic lateral
sclerosis-associated mutations decrease the thermal stability of distinctly
metallated species of human copper/zinc superoxide dismutase." J Biol Chem277(18): 15932-7.
We report the thermal stability of wild type (WT) and 14 different variants of
human copper/zinc superoxide dismutase (SOD1) associated with familial
amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions
were observed by differential scanning calorimetry for partially metallated SOD1
enzymes isolated from a baculovirus system. We correlated the metal ion contents
of SOD1 variants with the occurrence of distinct melting transitions. Altered
thermal stability upon reduction of copper with dithionite identified
transitions resulting from the unfolding of copper-containing SOD1 species. We
demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants
(A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Delta) conferred a similar
degree of incremental stabilization as did metal ion binding to WT SOD1.
However, these mutants were all destabilized by approximately 1-6 degrees C
compared with the corresponding WT SOD1 species. Most of the "metal binding
region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions
that probably resulted from unfolding of metal-free species at approximately
4-12 degrees C below the observed melting of the least stable WT species. We
conclude that decreased conformational stability shared by all of these mutant
SOD1s may contribute to SOD1 toxicity in FALS.
Niwa, J. I., S. Ishigaki, et al. (2002). "Dorfin ubiquitylates mutant SOD1 and
prevents mutant SOD1-mediated neurotoxicity." J Biol Chem.
Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder
resulting from degeneration of motor neurons in the cerebral cortex, brainstem,
and spinal cord. The cytopathological hallmark in the remaining motor neurons of
ALS is the presence of ubiquitylated inclusions consisting of insoluble protein
aggregates. In this paper, we report that Dorfin, a RING-finger type E3
ubiquitin ligase, is predominantly localized in the inclusion bodies of familial
ALS with Cu/Zn-superoxide dismutase (SOD1) mutation as well as sporadic ALS.
Dorfin physically bound and ubiquitylated various SOD1 mutants derived from
familial ALS patients and enhanced their degradation, but it had no effect on
the stability of the wild-type SOD1. Overexpression of Dorfin protected against
the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions.
Our results indicate that Dorfin protects neurons by recognizing and then
ubiquitylating mutant SOD1 proteins, followed by targeting them for proteasomal
degradation.
Sampson, J. B. and J. S. Beckman (2001). "Hydrogen peroxide damages the
zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase." Arch Biochem
Biophys392(1): 8-13.
Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately
20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset
neurodegenerative disease affecting motor neurons. These mutations decrease
protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has
altered redox activities and is toxic to motor neurons in vitro. Using bovine
SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and
Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding
activity six times faster than superoxide dismutase activity, whereas
inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD
and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous
generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine.
Although urate, xanthine, and ascorbate can protect superoxide dismutase
activity of Cu,Zn SOD from inactivation, they were not effective at protecting
Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but
not the secondary structure of Cu,E SOD as detected by near and far UV circular
dichroism. Our results suggest that low levels of hydrogen peroxide could
potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS
by rendering zinc loss from SOD irreversible.
Oeda, T., S. Shimohama, et al. (2001). "Oxidative stress causes abnormal
accumulation of familial amyotrophic lateral sclerosis-related mutant SOD1 in
transgenic Caenorhabditis elegans." Hum Mol Genet10(19): 2013-23.
Mutations in the Cu/Zn superoxide dismutase (SOD1) genes are present in
approximately 20% of families suffering from familial amyotrophic lateral
sclerosis (FALS). Results from several transgenic studies in which FALS-related
SOD1 mutations have been expressed have suggested that mutant SOD1 proteins
induce cytotoxicity through a toxic gain of function, although the specific
mechanism of this has not been fully clarified. To investigate the mechanism of
toxicity induced by the mutant SOD1 associated with FALS, we generated
transgenic Caenorhabditis elegans strains that contain wild-type and mutant
human A4V, G37R and G93A SOD1 recombinant plasmids. The transgenic strains
expressing mutant human SOD1 showed greater vulnerability to oxidative stress
induced by 0.2 mM paraquat than a control that contained the wild-type human
SOD1. In the absence of oxidative stress, mutant human SOD1 proteins were
degraded more rapidly than the wild-type human SOD1 protein in C.elegans. In the
presence of oxidative stress, however, this rapid degradation was inhibited, and
the transgenic C.elegans co-expressing mutant human SOD1 and green fluorescent
proteins (GFPs) in muscle tissues demonstrated discrete aggregates in the adult
stage. These results suggest that oxidative damage inhibits the degradation of
FALS-related mutant human SOD1 proteins, resulting in an aberrant accumulation
of mutant proteins that might contribute to the cytotoxicity.
Fukada, K., S. Nagano, et al. (2001). "Stabilization of mutant Cu/Zn superoxide
dismutase (SOD1) protein by coexpressed wild SOD1 protein accelerates the
disease progression in familial amyotrophic lateral sclerosis mice." Eur J
Neurosci14(12): 2032-6.
Transgenic mice carrying familial amyotrophic lateral sclerosis (FALS)-linked
mutant Cu/Zn superoxide dismutase (SOD1) genes such as G93A (G93A-mice) and G85R
(G85R-mice) genes develop limb paresis. Introduction of human wild type SOD1
(hWT-SOD1) gene, which does not cause motor impairment by itself, into different
FALS mice resulted in different effects on their clinical courses, from no
effect in G85R-mice to acceleration of disease progression in G93A-mice.
However, the molecular mechanism which causes the observed difference, has not
been clarified. We hypothesized that the difference might be caused by the
stability of mutant SOD1 proteins. Using a combination of mass spectrometry and
enzyme-linked immunosorbent assay, we found that the concentration of G93A-SOD1
protein was markedly elevated in tissues of transgenic mice carrying both G93A-
and hWT-SOD1 genes (G93A/hWT-mice) compared to that in G93A-mice, and also found
that the concentration of G93A-SOD1 protein had a close relation to the disease
duration. The concentration of metallothionein-I/II in the spinal cord,
reflecting the degree of copper-mediated oxidative stress, was highest in G93A/hWT-mice,
second in G93A-mice, and normal in the mice carrying hWT-SOD1 gene. These
results indicated that the increase of G93A-SOD1 protein was responsible for the
increase of oxidative stress and disease acceleration in G93A/hWT-mice. We
speculate that coexpression of hWT-SOD1 protein is deleterious to transgenic
mice carrying a stable mutant such as G93A-SOD1, because this mutant protein is
stabilized by hWT-SOD1 protein, but not to transgenic mice carrying an unstable
mutant such as G85R-SOD1, because this mutant protein is not stabilized by
hWT-SOD1.
Johnston, J. A., M. J. Dalton, et al. (2000). "Formation of high molecular
weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for
familial amyotrophic lateral sclerosis." Proc Natl Acad Sci U S A97(23):
12571-6.
Deposition of aggregated protein into neurofilament-rich cytoplasmic inclusion
bodies is a common cytopathological feature of neurodegenerative disease. How-or
indeed whether-protein aggregation and inclusion body formation cause
neurotoxicity are presently unknown. Here, we show that the capacity of
superoxide dismutase (SOD) to aggregate into biochemically distinct, high
molecular weight, insoluble protein complexes (IPCs) is a gain of function
associated with mutations linked to autosomal dominant familial amyotrophic
lateral sclerosis. SOD IPCs are detectable in spinal cord extracts from
transgenic mice expressing mutant SOD several months before inclusion bodies and
motor neuron pathology are apparent. Sequestration of mutant SOD into
cytoplasmic inclusion bodies resembling aggresomes requires retrograde transport
on microtubules. These data indicate that aggregation and inclusion body
formation are mechanistically and temporally distinct processes.
Hall, J. R. and C. A. Strathdee (2000). "Disease-associated mutations in SOD1
are impervious to dominant positive or negative effects." Biochem Biophys Res
Commun276(3): 1056-61.
The familial form of amyotrophic lateral sclerosis is caused by mutations in the
SOD1 gene encoding the cytosolic antioxidant enzyme Cu,Zn superoxide dismutase.
Although there is no clear correlation between disease and dismutating catalytic
activity among the various disease-associated SOD1 alleles, all of the known
missense mutations significantly alter the half-life of the encoded
polypeptides. Using transient transfection studies in mammalian cells, it was
demonstrated that a frameshift mutation in SOD1 which results in a truncated
polypeptide is similarly destabilized. Using an epitope-tagging strategy to
discriminate between mutant and wild-type SOD1 polypeptides, no evidence for
dominant effects on polypeptide stability was detected, including that of a
positive effect of the wild-type on mutant SOD1 polypeptides or that of a
negative effect of mutant on wild-type SOD1 polypeptides. These experiments thus
favor a non-catalytic role of mutant forms of SOD1 in disease progression.
Liochev, S. I., L. L. Chen, et al. (1998). "The familial amyotrophic lateral
sclerosis-associated amino acid substitutions E100G, G93A, and G93R do not
influence the rate of inactivation of copper- and zinc-containing superoxide
dismutase by H2O2." Arch Biochem Biophys352(2): 237-9.
Inactivation of copper- and zinc-containing superoxide dismutase (Cu,ZnSOD) by
H2O2 is the consequence of several sequential reactions: reduction of the active
site Cu(II) to Cu(I) by H2O2; oxidation of the Cu(I) by a second H2O2, thus
generating a powerful oxidant, which may be Cu(I)O or Cu(II)OH or Cu(III); and
finally oxidation of one of the histidines in the ligand field, causing loss of
SOD activity. Three familial amyotrophic lateral sclerosis (FALS)-associated
mutant Cu,ZnSODs, i.e., E100G, G93A, and G93R, did not differ from the control
enzyme in susceptibility to inactivation by H2O2. It thus appears that an
increased peroxidase activity of the FALS-associated Cu,ZnSOD variants might not
be a factor in the development of this disease. This leaves the loss of Zn, and
the consequent increase in peroxidase activity, or in nitration activity, as a
viable explanation (J. P. Crow et al., 1997, J. Neurochem. 69, 1936-1944), among
other possibilities.
(1998). "Volume 241, Number 2 (1997), in Article No. RC977804, "Stability of
Mutant Superoxide Dismutase-1 Associated with Familial Amyotrophic Lateral
Sclerosis Determines the Manner of Copper Release and Induction of Thioredoxin
in Erythrocytes," by Yasuko Ogawa, Hiroaki Kosaka, Toyohumi Nakanishi, Akira
Shimizu, Nagato Ohoi, Hiroshi Shouji, Takehiko Yanagihara, and Saburo Sakoda,
pages 251-257." Biochem Biophys Res Commun244(2): 605.
Copyright
Orrell, R. W., S. L. Marklund, et al. (1997). "Familial ALS is associated with
mutations in all exons of SOD1: a novel mutation in exon 3 (Gly72Ser)." J
Neurol Sci153(1): 46-9.
Mutations of the SOD1 gene, which encodes the enzyme copper/zinc superoxide
dismutase, are associated with familial amyotrophic lateral sclerosis (ALS).
SOD1 consists of five exons, and over 50 different mutations have been described
involving exons 1,2,4 and 5. The absence of mutations in exon 3 has been
attributed to a critical function of this exon, its integrity being necessary
for the toxic effect of mutant SOD1, and it has been suggested that such
mutations may be lethal rather than leading to adult onset disease. We
identified the heterozygote mutation Gly72Ser (exon 3) in a family with two
individuals affected by ALS. SOD enzyme activity was reduced by 45% when
measured in erythrocytes indicating reduced enzyme activity, or reduced
stability of the mutant protein. These findings indicate that exon 3 is not a
privileged region from mutation; that all five exons should be investigated when
seeking SOD1 mutations in human disease; and may help in a better understanding
of the pathogenicity of these mutations in ALS.
Ogawa, Y., H. Kosaka, et al. (1997). "Stability of mutant superoxide dismutase-1
associated with familial amyotrophic lateral sclerosis determines the manner of
copper release and induction of thioredoxin in erythrocytes." Biochem Biophys
Res Commun241(2): 251-7.
We analyzed mutant superoxide dismutase-1 (SOD-1) in erythrocytes from patients
with familial amyotrophic lateral sclerosis (FALS) by using ion exchange
chromatography and HPLC/electrospray ionization mass spectrometry and were able
to divide mutant SOD-1 proteins into a stable form including G37R and H46R, and
an unstable form including I149T and a two base pair deletion mutant. Each
mutant sample showed abnormal copper peaks in different chromatographic
fractions without relation to SOD-1 activities. In addition, thioredoxin, known
as an antioxidant molecule, was markedly increased in the stable form but not in
the unstable form. These results suggest the presence of different pathways
leading to motor neuron death between stable and unstable mutants.
