From the Department of Neurology/Neurosurgery and Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada
Received for publication, November 29, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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Mutations in the Cu/Zn-superoxide
dismutase (SOD-1) gene are responsible for a familial form
of amyotrophic lateral sclerosis. In humans and experimental models,
death of motor neurons is preceded by formation of cytoplasmic
aggregates containing mutant SOD-1 protein. In our previous studies,
heat shock protein 70 (HSP70) prolonged viability of cultured motor
neurons expressing mutant human SOD-1 and reduced formation of
aggregates. In this paper, we report that mutant SOD-1 proteins have
altered solubility in cells relative to wild-type SOD-1 and can form a
direct association with HSP70 and other stress proteins. Whereas
wild-type human and endogenous mouse SOD-1 were detergent-soluble, a
portion of mutant SOD-1 was detergent-insoluble in protein extracts of
NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures
established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal
cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and Amyotrophic lateral sclerosis
(ALS)1 is an adult-onset
neurodegenerative disease characterized by death of motor neurons in the cerebral cortex, brain stem, and spinal cord. Approximately 10% of
ALS cases are familial, and about 20% of these familial cases
are caused by dominantly inherited mutations in the gene encoding the
enzyme Cu/Zn-superoxide dismutase (SOD-1) (1). Evidence indicates that
some "toxic gain of function" is responsible for motor neuron loss
rather than a decreased ability of mutant SOD-1 to carry out its
primary enzymatic function, dismutation of superoxide to hydrogen
peroxide. Most SOD-1 mutant proteins have significant enzymatic
activity, and any impairment relative to wild-type does not correlate
with clinical severity of the disease (2, 3). SOD-1 knockout mice do
not develop motor neuron disease (4), whereas mice expressing mutant
SOD-1 transgenes develop an ALS-like phenotype (5-7).
Over 60 different mutations have been identified and associated with
ALS (reviewed in Ref. 8). The distribution of such a large number of
disease-causing mutations throughout all exons of the SOD-1
gene suggests that altered protein conformation may contribute to the
toxic gain of function. Additional evidence comes from x-ray
crystallography showing that conserved interactions within the protein
that determine the conformation of the active channel are altered in
mutant SOD-1 proteins (9). How this contributes to toxicity is not
clear. Improperly folded SOD-1 enzyme could allow greater access of
abnormal substrates to the active site; could enhance other known
enzymatic functions of SOD-1, such as catalysis of protein nitration;
or could alter metal binding, all of which would ultimately lead to
increased oxidative damage (reviewed in Refs. 10-12). Alternatively,
proteins with altered conformation could form insoluble precipitates or participate in abnormal protein-protein interactions unless prevented from doing so by association with chaperone proteins. Thus, depletion of chaperones could result in formation of mutant SOD-1 aggregates, as
well as compromising cellular chaperoning function in general (13).
Mutant SOD-1 proteins appear to be degraded by the proteasome, a
pathway generally involved in proteolysis of denatured or misfolded
protein (14, 15). Moreover, several mutant SOD-1 proteins have a
tendency to precipitate in solution relative to wild-type (16).
Proteinaceous aggregates of SOD-1 have been found in the cytoplasm of
cultured primary motor neurons expressing several different SOD-1
mutants (17), in motor neurons and astrocytes of mice expressing mutant
SOD-1 transgenes (7, 18), in familial ALS patients at autopsy (19, 20),
and in transfected cell lines (15, 21). In cultured motor neurons,
coexpression of HSP70 and mutant SOD-1 expression vectors considerably
reduced formation of SOD-containing aggregates and prolonged viability (13). Mutant SOD-1-transfected NIH3T3 cell lines showed up-regulation of HSP70, HSP25, and In this study, we have demonstrated that mutant SOD-1
coimmunoprecipitates with HSP70, HSP40, and Antibodies and Plasmids--
The following primary antibodies
were used: Rabbit anti-SOD-1 antibodies (SOD-100 diluted 1:500 and
SOD-101 diluted 1:250, Stressgen Biotechnologies Corp., Victoria,
British Columbia, Canada) (SOD-100 reacts with both human and mouse
SOD-1 but favors human SOD-1; SOD-101 also reacts with both human and
mouse SOD-1 but favors mouse SOD-1); rabbit anti- NIH3T3 Cell Lines--
NIH3T3 cell lines stably expressing human
SOD-1 gene constructs (wild-type or with G93A or G41S
mutations) have been described previously (13). Cultures were
maintained under standard conditions in minimum essential medium
supplemented with 3.7 g of NaHCO3, 5 g of
dextrose, and 10% fetal bovine serum. Stable cell lines were
supplemented with G418 (Life Technologies, Inc.) (250 µg/ml for G41S and G93A lines and 700 µg/ml for the wild-type line). For
transient transfection, NIH3T3 cells were grown to 80-100% confluency, subcultured at a density of 2 × 105
cells/35-mm culture well, and transfected with the
G93A-SOD-1/pcDNA3 construct according to the standard protocol for
LipofectAMINETM reagent (Life Technologies, Inc.). Protein
extracts were prepared 43 h after transfection (see below).
Transgenic Mice--
Two lines of mice transgenic for G93A
mutant human SOD-1, B6SJL-TgN(SOD1-G93A)1Gur and
B6SJL-TgN(SOD1-G93A)1Gurdl, as well as one line of mice
transgenic for wild-type human SOD-1, B6SJL-TgN(SOD-1)2Gur, were
maintained in our animal facility (breeding pairs were purchased from
The Jackson Laboratory, Bar Harbor, ME). All experiments were approved
by the McGill University Animal Care Committee and followed the
guidelines of the Canadian Council on Animal Care.
B6SJL-TgN(SOD1-G93A)1Gur (fast line) mice develop paralysis in
the limbs and die at 4-5 months of age.
B6SJL-TgN(SOD1-G93A)1Gurdl (slow line) mice develop
paralysis in the limbs and die at 6-7 months of age.
Single Embryo Spinal Cord Cultures--
Cultures were prepared
from 13-day-old mouse embryos as previously described (22)
except that spinal cords from transgenic mouse embryos were dissociated
and plated individually at a density of 650,000/35-mm culture well.
Embryos were genotyped for the presence of human SOD-1 transgenes using
the polymerase chain reaction method provided by The Jackson Laboratory.
Preparation of Detergent-soluble and Detergent-insoluble Protein
Extracts--
NIH3T3 cell lines and spinal cord cultures were lysed on
ice in phosphate-buffered saline, pH 8.0, containing 0.5% Nonidet P-40, 0.2% digitonin, and 0.23 mM phenylmethylsulfonyl
fluoride (lysis buffer) and centrifuged to obtain detergent-soluble
supernatant and detergent-insoluble pellet fractions. Pellets were
washed three times in lysis buffer and then solubilized in 1× sample buffer (55 mM Tris, pH 6.8, 10% glycerol, 1% SDS, 5%
Western Blotting--
After electrophoresis, proteins were
transferred to nitrocellulose (Bio-Rad Trans-Blot® transfer medium).
Membranes were blocked overnight at 4 °C in TBST-milk (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20,
5% skim milk powder). Incubations in primary and secondary antibodies
were for 1 h at room temperature. Labeled proteins were detected
using the Renaissance® Western blot chemiluminescence reagent
(PerkinElmer Life Sciences).
Immunoprecipitation--
NIH3T3 cell lines were lysed in 700 µl of lysis buffer containing 9% Me2SO and then
incubated for 1 h on ice and for 10 min at room temperature.
Following the addition of 70 µl of 50 mM glycine, cells
were centrifuged at 15,000 × g, and supernatants were
collected. For total cell extracts, the centrifugation step was
eliminated. For immunoprecipitation, 3 µl of SOD-1 antibody (SOD-100), 12.5 µl of HSP70 antibody, 5 µl of HSP40
antibody, or 5 µl of Altered Solubility of Mutant Human SOD-1 in Transfected NIH3T3
Cells--
The tendency of mutant SOD-1 proteins to form aggregates in
cultured cells and spinal cord tissue suggested that they might distribute differently in subcellular compartments compared with wild-type enzyme. NIH3T3 cell lines stably expressing human wild-type, G41S mutant, or G93A mutant SOD-1, established for previous studies (13), were used to compare the solubility of wild-type and mutant SOD-1
in nonionic detergent. Detergent-soluble supernatant and detergent-insoluble pellet fractions were prepared from these cell
lines, as well as from untransfected NIH3T3 cells, and subjected to
SDS-polyacrylamide gel electrophoresis. The endogenous murine, wild-type human, and mutant human SOD-1 proteins were detected by
Western blotting. A blot probed with SOD-1 antibody (SOD-100, which has
a higher affinity for human SOD-1 relative to mouse) is shown in Fig.
1. Endogenous murine SOD-1 and wild-type
human SOD-1 were detected only in the supernatant, not in the pellet fraction. In contrast, G41S and G93A mutant human SOD-1 were present in
both fractions. Larger amounts of pellet fraction were loaded on the
gel relative to supernatant to demonstrate absence of wild-type and
presence of mutant SOD-1 in the pellet. Using NIH Image software to
compare relative band densities on Western blots and correcting for
differential loading of sample, it was estimated that about 1% of
total cellular mutant SOD-1 was detergent-insoluble in this experiment.
This estimate was consistent with the relative band intensities
observed when equal percentages of the total volume of supernatant and
pellet fractions were loaded on the gel (data not shown).
Because Ratovitski et al. (3) reported that several
different mutant SOD-1 proteins expressed in COS cells by transient transfection were essentially soluble in nonionic detergent, the experiment was repeated using NIH3T3 cells transiently transfected with
the G93A mutant SOD-1 construct. As shown in Fig.
2A, mutant human SOD-1, but
not murine SOD-1, was detected in the detergent-insoluble pellet by
Western blotting with antibody SOD100 and was estimated to correspond
to about 10% of total cellular mutant SOD-1. The absence of mouse
SOD-1 in the pellet fraction was confirmed using a SOD-1 antibody with
greater specificity for mouse SOD-1 (SOD101) (Fig. 2B).
Reprobing the blots shown in Fig. 1 with antibody SOD101 also failed to
detect murine SOD-1 in detergent-insoluble fractions of any cell line
(not shown).
Altered Solubility of Mutant Human SOD-1 in Spinal Cord Cultures
and Spinal Cord Tissue--
Whereas cell lines are convenient models
for biochemical analysis, the properties of mutant protein in these
cells do not necessarily reflect properties in primary tissue affected
by the disease. We examined the solubility of G93A mutant SOD-1 in
primary spinal cord cultures prepared from G93A and wild-type human
SOD-1 transgenic mouse embryos (Fig. 3)
and in lumbar spinal cord isolated from adult, presymptomatic
(214-day-old) transgenic mice (Fig. 4).
These experiments confirmed our findings in transfected NIH3T3 cells in
that both detergent-soluble and detergent-insoluble forms of G93A SOD-1
were detected. In extracts of primary cultures, but not lumbar spinal
cord, a small amount of transgenic wild-type SOD-1 was detected in the
detergent-insoluble pellet fraction, but this amount was considerably
less than the amount of mutant protein detected. Endogenous
mouse SOD-1 was not detected in the detergent-insoluble pellet.
Coimmunoprecipitation of Mutant SOD-1 and HSP70--
In response
to different types of stresses, cells up-regulate production of various
heat shock/stress proteins. When NIH3T3 cells were transfected with
G41S or G93A mutant human SOD-1, many of the cells died; however, those
cells that were able to survive and were selected as stable
transfectants had elevated levels of heat shock proteins as compared
with wild-type-transfected and untransfected cells (13). Furthermore,
coexpression of HSP70 and mutant SOD-1 expression
vectors protected primary cultured motor neurons from toxicity of
several different SOD-1 mutants, reducing formation of SOD-containing
aggregates and prolonging viability (13). To determine whether heat
shock proteins protect cells from mutant SOD-1 by direct
protein-protein interaction, coimmunoprecipitation experiments were
performed. Because NIH3T3 cell lines expressing mutant SOD-1 have
elevated endogenous levels of heat shock proteins (13), this model was
best suited for this purpose. When total cell extracts were
immunoprecipitated with antibody to SOD-1 (SOD100), HSP70 was
coimmunoprecipitated from both G93A and G41S expressing cell lines but
not from untransfected NIH3T3 or lines expressing wild-type human SOD-1
(Fig. 5A). Immunoprecipitation of total cell extracts with antibody to HSP70 (W27) pulled down G93A
and G41S mutant SOD-1 but not wild-type human or endogenous murine
SOD-1 (Fig. 5B). The same results were obtained when the cross-linking agent, dithiobis(succinimidyl propionate), was used to
stabilize HSP-SOD interactions (data not shown). HSP70·mutant SOD-1 complexes were distributed mainly in the detergent-insoluble fraction. Regardless of whether anti-SOD-1 or anti-HSP70 antibodies were used for immunoprecipitation, the amount of HSP70 or mutant SOD-1,
respectively, coprecipitated from detergent-soluble supernatants was
minimal compared with the amount coprecipitated from total cell
extracts. This was not due to absence of HSP70 in supernatants of cell
lines expressing mutant SOD-1; HSP70 was immunoprecipitated by W27
anti-HSP70 antibody from supernatant fractions of both mutant cell
lines (Fig. 5C) and was detected in both detergent-soluble supernatant and detergent-insoluble pellet fractions of
nonimmunoprecipitated samples (Fig. 5D). Similarly, G41S and
G93A mutant SOD-1 were localized to both supernatant and pellet
fractions (Fig. 1) and were immunoprecipitated from supernatants by the
SOD100 antibody (not shown).
The data shown in Fig. 5D also confirm our previous findings
that HSP70 is up-regulated in cell lines expressing mutant SOD-1. Although no association of HSP70 with wild-type human SOD-1 was detected (Fig. 5, A and B), this could have been
due to the relatively low level of HSP70 in this cell line. To test
this hypothesis, cells stably expressing wild-type human SOD-1
were transiently transfected with HSP70. Despite high level
expression of HSP70, verified by Western blotting of total cell lysates
with antibody to HSP70, neither wild-type human nor murine SOD-1
coimmunoprecipitated with HSP70 (data not shown).
Another important observation from Fig. 5D is that HSP70 was
not detected in pellet fractions of untransfected or wild-type human
SOD-1 expressing NIH3T3 cultures. Thus, HSP70 and wild-type SOD-1
proteins are largely detergent-soluble proteins; however, in G41S and
G93A-expressing cell lines, a proportion of both HSP70 and mutant SOD-1
are detergent-insoluble. How much of the detergent-insoluble forms
represents complexes of HSP70 and mutant SOD-1 cannot be determined
from these experiments.
Mutant SOD-1 Proteins Coimmunoprecipitate with HSP40 and
Similar results were obtained when immunoprecipitations were carried
out using antibody to In response to various physiological stresses, including heat
shock or exposure to toxic agents, cells up-regulate families of heat
shock/stress proteins. Their function is to prevent the accumulation of
improperly folded proteins by binding to and promoting proper refolding
of the damaged proteins or by facilitating their degradation by
proteasomes (23-25). Our studies have provided evidence that heat
shock proteins play a role in protecting cells from the toxicity of
mutant SOD-1 proteins associated with human familial ALS. Coexpression
of HSP70 prolonged viability of cultured motor neurons expressing
mutant SOD-1 proteins and reduced the formation of cytoplasmic
aggregates (13).2 Survival of
NIH3T3 cells expressing two different SOD-1 mutants, G41S and G93A,
correlated with up-regulation of heat shock proteins (13). Finally,
HSP70, HSP40, and SOD-1, being a cytoplasmic enzyme, is normally soluble in nonionic
detergent. However, a small percentage of mutant human SOD-1 protein
was consistently detected in the detergent-insoluble fraction of
protein extracts from NIH3T3 transfectants, spinal cord cultures
prepared from embryos of G93A mutant SOD-1 transgenic mice, and lumbar
spinal cord of adult G93A transgenic mice. Although mutant SOD-1
proteins can form homodimers with wild-type protein of different
species (26), the endogenous murine SOD-1 was not found in the
detergent-insoluble pellet fraction despite overloading of gels with
protein and blotting with antibody preferentially recognizing this species.
HSP70 and mutant SOD-1 were coimmunoprecipitated from total cell
extracts of the two mutant SOD-1 transfected NIH3T3 cell lines, whereas
only trace amounts were detected from the supernatant fractions,
indicating that HSP70·mutant SOD-1 complexes were
predominantly detergent-insoluble. Distribution of proteins, including
heat shock proteins, to the detergent-insoluble pellet occurs under a
variety of stressful conditions, including heat shock, oxidative stress, ATP depletion, calcium overload, and treatment with
sulfhydryl reagents (27-29). Whether all detergent-insoluble
mutant SOD-1 was complexed with HSP70 cannot be determined from the
present experiments. Future studies will address whether
posttranslational modification of mutant SOD-1 is required either to
alter solubility or to promote binding of HSPs. That a relatively small
fraction of total cellular mutant SOD-1 (estimated at 0.5-10%) was
found in the pellet fraction in which the HSP·SOD-1 complexes
were localized suggests the possibility that altered conformation
conferred by the mutation alone may not be sufficient. Regardless,
binding of heat shock proteins would be protective by preventing
inappropriate protein-protein interactions and/or facilitating degradation.
Although 3T3 cells may be physiologically different from spinal cord
cells affected in familial ALS, detergent-insoluble mutant SOD-1 also
was detected in spinal cord cultures prepared from embryos of G93A
mutant SOD-1 transgenic mice and from lumbar spinal cord of adult G93A
transgenic mice. Because significant expression of HSP70 was not
detected in spinal cord (13), additional studies are under way to
characterize expression of heat shock proteins and their interaction
with mutant SOD-1 in these models.
Although a proportion of mutant SOD-1 is detergent-insoluble in the
transfected NIH3T3 cell lines, no large aggregates are visible by
immunocytochemistry in these cells (13). However, in the study by
Johnston et al. (15), formation of large aggregates of
mutant SOD-1 was induced in transfected HEK cells by inhibiting proteasomal activity with lactacystin. The detergent insolubility of
mutant SOD-1 and association with heat shock proteins observed in our
study could represent an intermediate stage between soluble mutant
SOD-1 and protein aggregated into inclusions. Inclusions may form when
insufficient heat shock proteins are expressed to sequester mutant
protein and facilitate its degradation in proteasomes. This could
result from proteasomal inhibition, as in the experiment by Johnson
et al. (15), or from an inadequate stress response to
increase levels of heat shock proteins.
The propensity of mutant SOD-1 proteins to form aggregates differs
according to cell type (17). Motor neurons are particularly susceptible, perhaps in part because they have a high threshold for
activation of the stress response (30-33). For example, when spinal
cord cultures are heat shocked, glial cells up-regulate HSP70, but
motor neurons do not, even when the level of thermal stress is
lethal.3 In cultured motor
neurons expressing mutant SOD-1 proteins, formation of aggregates is
tightly linked to loss of viability (22). However, this does not
necessarily mean that aggregates cause toxicity. The presence of
aggregates may reflect the inability of the cell to elicit a sufficient
stress response to maintain general chaperoning function in the cell,
thus increasing its vulnerability to a variety of other physiological
and environmental stresses, which ultimately contribute to cellular
dysfunction and death.
Similar arguments apply to other motor neuron diseases involving
altered protein conformation. Colocalization of heat shock proteins and
mutant protein within proteinaceous aggregates occurs in many
neurodegenerative disorders, including those caused by expansion of CAG
(polyglutamine) repeat sequences (reviewed in Ref. 34). Spinal bulbar
muscular atrophy (Kennedy's disease) is a motor neuron disease
resulting from CAG repeat expansion in the androgen receptor gene (35).
Overexpression of heat shock proteins (HSP70, HSP40, and HDJ2/HSDJ) in
HeLa or Neuro2A cell culture models of spinal bulbar muscular atrophy
reduced both formation of aggregates and toxicity (36, 37). Aberrant
distribution and altered ratios of the 20 S proteasomal core and PA700
proteasomal cap proteins, the two major components of 26 S proteasomes,
were found in HeLa cells with aggregates of polyglutamine-expanded androgen receptor. Thus, both deficiency of chaperones and impaired proteolysis could contribute to pathogenesis (36).
The data presented here indicate that HSP70 may protect cells from
mutant SOD-1 proteins at least in part by direct interaction with the
mutant protein. This does not exclude other cytoprotective mechanisms
for heat shock proteins, such as chaperoning other proteins damaged as
a result of mutant SOD-1 expression or a general antiapoptotic effect
(38-40). Additional studies are required to assess the feasibility of
targeting stress protein pathways to treat motor neuron disease and
other neurodegenerative disorders characterized by accumulation of
aberrant proteins.
B-crystallin with mutant SOD-1 (G93A or G41S), but not
wild-type or endogenous mouse SOD-1, was demonstrated by
coimmunoprecipitation. Mutant SOD-1·HSP70 complexes were
predominantly in the detergent-insoluble fraction. However, only
a small percentage of total cellular mutant SOD-1 was
detergent-insoluble, suggesting that mutation-induced alteration of
protein conformation may not in itself be sufficient for direct
interaction with heat shock proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin compared with untransfected cells
or cells transfected with the wild-type SOD-1 construct (13). Heat
shock proteins could protect cells from mutant SOD-1 by direct
interaction, thus preventing aggregation and facilitating degradation,
or indirectly by protecting cells from downstream consequences of some
other toxic property.
B-crystallin.
Furthermore, a portion of mutant SOD-1 was detergent-insoluble in three
different experimental models of familial ALS: NIH3T3 cell lines stably or transiently transfected with mutant SOD-1 constructs, spinal cord
cultures derived from G93A transgenic embryonic spinal cords, and
lumbar spinal cord from adult G93A transgenic mice.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin
(SPA-223) (1:250) and rabbit anti-HSP40 (SPA-400) (1:1000) antibodies
from Stressgen Biotechnologies Corp.; mouse monoclonal HSP70 antibody
(W27 catalog number SC-24, Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) (1:250) (may cross-react with HSC70); mouse monoclonal actin
antibody, clone C4 (691001) (1:1000) and rabbit polyclonal tubulin
antibody (650951) (1:500) from ICN Biomedicals Inc. (Irvine, CA).
Secondary antibodies used were goat anti-rabbit horseradish
peroxidase (P0399, Dako Corp., Mississauga, ON) (1:3000) and
sheep anti-mouse horseradish peroxidase (515035062, Jackson
ImmunoResearch Laboratories, Willow Grove, PA) (1:10,000). cDNA
encoding human HSP70 was a kind gift from D. Mosser and was subcloned
into pcDNA3.
-mercaptoethanol, and 0.04% bromophenol blue). The lumbar region of
mouse spinal cord was homogenized in 500 µl of lysis buffer and
centrifuged at 15,000 × g for 15 min at 4 °C. After
collection of supernatants, pellets were washed five times with lysis
buffer and then resuspended in 100 µl of 1× sample buffer. Protein
concentrations of supernatants were determined by Bio-Rad DC
protein assay.
B-crystallin antibody was added to 400 µg
of protein and incubated at 4 °C overnight on a rotating platform.
Immune complexes were captured on 40 µl of protein G-Sepharose, 4 Fast Flow (Amersham Pharmacia Biotech) by incubation on a rotating platform at 4 °C for 3 h; the beads were washed with lysis
buffer, resuspended in 30 µl of 1× sample buffer, and boiled for 5 min. The beads were pelleted, half of the supernatant was
electrophoresed on a 12.5% SDS-polyacrylamide gel, and
immunoprecipitated proteins were detected by Western blotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
In stable transfectants of NIH3T3 cells, a
proportion of mutant human SOD-1, but not wild-type human or mouse
SOD-1, is insoluble in nonionic detergent. Cultures of
untransfected cells (NIH3T3) and lines expressing wild-type-human SOD-1
(WT-SOD-1), G41S mutant SOD-1, or G93A mutant SOD-1 were
lysed in buffer containing nonionic detergent. Shown are supernatant
(S) and pellet (P) fractions after
electrophoresis on a 12.5% SDS-polyacrylamide gel and Western blotting
with the anti-SOD-1 antibody SOD-100, which binds preferentially to
human SOD-1 (hSOD-1) but also labels the endogenous mouse
SOD-1 (mSOD-1). Both mutant human SOD-1 proteins were
detected in the pellet as well as the supernatant fraction.
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Fig. 2.
In transient transfectants of NIH3T3 cells, a
proportion of mutant SOD-1, but not endogenous mouse SOD-1, is
insoluble in nonionic detergent. Two days after transfection with
the G93A SOD-1 construct, cultures were lysed in buffer containing
nonionic detergent. Shown are supernatant (S) and pellet
(P) fractions of two separate transfectants (#1
and #2) after electrophoresis on a 12.5% SDS-polyacrylamide
gel and Western blotting with anti-SOD-1 antibody: SOD-100, which binds
preferentially to human SOD-1 (hSOD-1) but also binds to the
endogenous mouse SOD-1 (mSOD-1) (A) and SOD-101,
which binds preferentially to mouse SOD-1 (B). A fraction of
mutant human SOD-1, but not the endogenous enzyme, was detected in the
pellet fraction.
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Fig. 3.
A proportion of mutant human SOD-1 from
spinal cord cultures prepared from G93A SOD-1 transgenic mouse embryos
is detergent-insoluble. Spinal cord cultures were prepared
from G93A SOD-1 (fast line) or wild-type (WT) human SOD-1
transgenic mouse embryos. After 3-4 weeks in vitro,
cultures were lysed with buffer containing nonionic detergent.
Distribution of SOD-1 in the supernatant (S) and pellet
(P) after electrophoresis on 12.5% SDS-polyacrylamide gels
and Western blotting with SOD-100 antibody. G41S SOD-1/NIH3T3:
supernatant fractions from the NIH3T3 line stably expressing G41S
mutant human SOD-1 were run as a control to identify human
(h) and mouse (m) SOD-1. To compare protein
loading, blots were reprobed with antibody to tubulin, a protein that
distributes to both detergent-soluble and detergent-insoluble
fractions. Shown are samples from three different cultures for each
transgenic mouse line.
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Fig. 4.
A proportion of mutant human SOD-1 isolated
from lumbar spinal cord of G93A SOD-1 transgenic mice is
detergent-insoluble. Lumbar spinal cords from 190 day-old
wild-type (WT) and G93A SOD-1 (slow line) transgenic mice
were homogenized in lysis buffer containing nonionic detergent. Shown
is the distribution of human (h) and mouse (m)
SOD-1 in the supernatant (S) and pellet (P)
fractions after electrophoresis on 12.5% SDS-polyacrylamide gels and
Western blotting with SOD-100 antibody. To compare protein loading,
blots were reprobed with antibody to tubulin, a protein that
distributes to both detergent-soluble and detergent-insoluble
fractions.
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Fig. 5.
A-C, SOD-1 mutants, but not wild-type
human or mouse SOD-1, interact with the stress protein HSP70. Total
cell extracts or detergent-soluble extracts (supernatant) of NIH3T3
cell lines were immunoprecipitated (IP) with either SOD-1
antibody (SOD-100) (A) or HSP70 antibody (W27)
(B and C). Proteins were separated on 12.5%
SDS-polyacrylamide gels, and Western blots were probed for the presence
of HSP70 (A and C) and SOD-1 (B).
Ig indicates the immunoglobulin light chain of
immunoprecipitating antibody. Also shown is nonimmunoprecipitated
(not IP'd) G41S detergent-soluble extract to show the
location of human (h) and mouse (m) SOD-1 and
HSP70. D, HSP70 is up-regulated in NIH3T3 cell lines stably
expressing human SOD-1 and is partially detergent-insoluble in the
presence of mutant SOD-1. Cultures of untransfected cells (NIH3T3), a
cell line expressing wild-type human SOD-1 (WT) and lines
expressing G41S or G93A mutant human SOD-1 were lysed in buffer
containing nonionic detergent. Shown are supernatant (S) and
pellet (P) fractions after electrophoresis on a 12.5%
SDS-polyacrylamide gel and Western blotting with antibody to HSP70
(W27). Blots were reprobed with antibody to actin.
B-crystallin--
The interaction of mutant SOD-1 with stress
proteins was not limited to HSP70. Both SOD-1 mutants, but neither
wild-type human nor endogenous murine SOD-1, were pulled down when
total cell extracts of NIH3T3 cell lines were immunoprecipitated by
antibodies to HSP40 (Fig. 6A).
As in experiments with HSP70, failure to detect complexes of HSP40 and
wild-type human SOD-1 was not due to lack of HSP expression.
Considerable HSP40 was present in both supernatant and pellet fractions
of all cell lines (Fig. 6B).
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Fig. 6.
Mutant SOD-1, but not wild-type human or
mouse SOD-1, coimmunoprecipitates with the stress proteins HSP40
and B-crystallin. Total cell extracts of
NIH3T3 cell lines were immunoprecipitated (IP) with antibody
to HSP40 (A) or
B-crystallin (C). Proteins
were separated on 12.5% SDS-polyacrylamide gels, and Western blots
were probed for the presence of SOD-1 using antibody SOD-100.
Ig indicates the immunoglobulin light chain of the
immunoprecipitating antibody. Also included is nonimmunoprecipitated
G41S extract to show the location of human (h) and mouse
(m) SOD-1. B, although HSP40 is found in both
supernatant and pellet fractions of all cell lines, only mutant, not
wild-type SOD-1, coimmunoprecipitates with HSP40. Shown is a Western
blot of supernatant (S) and pellet (P) fractions
of untransfected NIH3T3 cells and lines expressing wild-type human
SOD-1 (WT) or G41S mutant SOD-1 probed with HSP40 antibody.
B-crystallin. Both G41S and G93A SOD-1
mutants, but not wild-type human or endogenous murine SOD-1, were
coprecipitated (Fig. 6C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin were coimmunoprecipitated with mutant
SOD-1 proteins from mutant SOD-1-transfected cell lines (this study).
These data support the hypothesis that heat shock proteins protect
cells, at least in part, by direct association with mutant protein.
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FOOTNOTES |
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* This research was supported by the Amyotrophic Lateral Sclerosis Association, the Amyotrophic Lateral Sclerosis Society of Canada, the Muscular Dystrophy Associations of Canada and the United States, and the Canadian Institutes for Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Killam Scholar. To whom correspondence should be addressed: Rm.
649, Montreal Neurological Institute, 3801 University St., Montreal,
Quebec H3A 2B4, Canada. Tel.: 514-398-8509; Fax: 514-398-1509; E-mail: mddm@musica.mcgill.ca.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010759200
2 J. Roy, S. Minotti, D. A. Figlewicz, and H. D. Durham, unpublished observations.
3 Z. Batulan, S. Minotti, and H. D. Durham, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: ALS, amyotrophic lateral sclerosis; HSP, heat shock protein; SOD-1, Cu/Zn-superoxide dismutase.
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