 |
INTRODUCTION |
Amyotrophic lateral sclerosis
(ALS)1 is an
age-dependent degenerative disorder of motor neurons in the
spinal cord, brain stem, and brain (1). Approximately 10% of ALS cases
are familial, and ~20% of these individuals inherit one of
>90 autosomal dominant mutations in the gene encoding
copper/zinc superoxide dismutase 1 (SOD1)
(2).2
SOD1 is a 32-kDa homodimeric enzyme expressed predominantly in the
cytosol that decreases the intracellular concentration of superoxide
radicals (O
) by catalyzing their dismutation to
O2 and H2O2. ALS-associated
mutations of conserved residues throughout the protein impart a toxic
property to the enzyme that appears unrelated to its normal dismutase
activity (reviewed in Ref. 3). Whereas transgenic mice that overexpress mutant SOD1s consistently develop lethal motor neuron degeneration (4-8), mice that overexpress the wild type (WT) enzyme exhibit only
subtle motor abnormalities (9). In addition, SOD1 knock-out mice are
not susceptible to motor neuron loss unless following axonal injury
(10).
Mutant SOD1 enzymes have been proposed to facilitate aberrant
copper-mediated chemistry, disrupt protein recycling or chaperone function, form toxic aggregates, or induce organelle dysfunction or
apoptosis (3, 11, 12), but the precise mechanism of specific motor
neuron toxicity has not been elucidated. The observation that some
mutant SOD1s exhibit accelerated turnover in vivo or increased proteolytic susceptibility compared with the WT enzyme (13-15) suggests that biologically significant perturbations of mutant
SOD1 conformation occur. The induction of chaperone proteins that can
protect cultured motor neurons from mutant SOD1 toxicity (16) and
appear to associate with SOD1 mutants (17) provides further evidence
that destabilization or unfolding of mutant SOD1s in vivo
may be related to their toxicity.
We previously purified 14 different biologically metallated ALS mutant
SOD1s and observed that one group of "WT-like" mutants (A4V,
L38V, G41S, G72S, D76Y, D90A, G93A, and E133
) bound copper in a
fully active coordination environment remarkably similar to that of
normal SOD1 despite causing a lethal phenotype (18). The other six
"metal-binding region" mutants (H46R, H48Q, G85R, D124V, D125H, and
S134N) were clearly distinguished from WT SOD1 according to decreased
metal ion contents, altered visible absorption spectra, or decreased
specific activities. In a further analysis by differential scanning
calorimetry, we found that metal-binding region mutants generally
exhibited a larger fraction of species that unfolded at low
Tm values compared with those of the WT-like mutants
and normal SOD1 (19). Although purified SOD1 mutants can lose in
vitro metal ion binding specificity following partial denaturation
at non-physiological pH (20), the retention of many native properties
in the "as-isolated" WT-like mutants suggests that other influences
may destabilize these SOD1 mutants in vivo.
Fully metallated bovine SOD1 is active in 4% SDS or 8 M
urea (21) and melts in solution at temperatures above 90 °C (22). SOD1 also retains its dimeric quaternary structure upon exposure to 1%
SDS in the absence of other denaturing stresses such as heat, urea,
reducing agents, or EDTA (23-25). Structural properties of SOD1 that
contribute to its extreme thermochemical stability include an
eight-stranded
-barrel motif, binding sites for copper and zinc
ions, hydrophobic interactions associated with dimerization, and an
unusual intrasubunit disulfide bond bridging a loop residue, Cys-57,
and Cys-146 of the
-barrel (24, 26). The loop that includes Cys-57
also strongly influences the conformation of Arg-143, which regulates
steering of superoxide anion and reactivity of the copper ion via a
local hydrogen bond network and the disulfide linkage to Cys-146
(27-29). Furthermore, portions of this loop contribute to the dimer
interface and form the zinc ion binding site (26). Conservation of
these structural features in all eukaryotic SOD1s suggests that
conformational stability of the enzyme, in general, and the disulfide
loop, in particular, is critical under physiological conditions.
The ratio of reduced (GSH) to oxidized glutathione ([GSH]/[oxidized
glutathione]) of ~30
100:1 in the cytosol (30) normally inhibits
the formation of disulfide linkages in cytosolic proteins (31), yet WT
SOD1 maintains a strong disulfide bond. We hypothesized that the
disulfide linkage in ALS-related SOD1 mutants could be vulnerable to
cleavage under cellular reducing conditions, which might further
destabilize even WT-like mutants. In this study, we correlated changes
in electrophoretic mobility, cysteine accessibility to modifying
reagents, and protease susceptibility upon incubation of purified SOD1
mutants with disulfide-reducing agents. We then compared sulfhydryl
accessibility of WT versus mutant SOD1 proteins in tissue
lysates from transgenic mice expressing WT, G85R, or G93A SOD1s under
reducing conditions. Our findings suggest that SOD1 destabilization
related to thiol-reducing influences in the spinal cord and brain may
contribute to the toxicity of mutant SOD1 enzymes in familial ALS.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All solutions were prepared using Milli-Q
ultrapurified water (Millipore). EDTA was from Invitrogen; sodium and
potassium phosphate (monobasic and dibasic), NaCl, and Tris base were
from J. T. Baker; ascorbate, reduced glutathione (GSH),
iodoacetamide, and human erythrocyte SOD1 were from Sigma;
Tris(2-carboxyethyl)phosphine (TCEP) was from Pierce; NaOH was from
Mallinckrodt; 1,4-dithiothreitol (DTT), SDS, HCl, methanol, and acetic
acid were from EM Science; and Coomassie Brilliant Blue R-250 and
bromphenol blue were from Bio-Rad. Formic acid and acetonitrile were
from Fisher. Maleimide coupled to polyethylene glycol (Mal-PEG,
molecular mass of 5 kDa) was from Shearwater Polymers
(Huntsville, AL). Transgenic mice overexpressing either WT SOD1 (line
B6SJL-TgN(SOD1)2Gur) or G93A SOD1 (line B6SJL-TgN(SOD1-G93A)1Gur) (4)
were obtained from the Jackson Laboratory (Bar Harbor, ME), whereas
those overexpressing G85R SOD1 (7) bred to homozygosity were provided
by Dr. Zuoshang Xu.
SOD1 Protein Preparation--
Human WT or ALS-related mutant
SOD1 enzymes (A4V, L38V, G41S, H46R, H48Q, G72S, D76Y, G85R, D90A,
G93A, D124V, D125H, E133
, and S134N) containing biologically
incorporated metal ions were isolated from a baculoviral expression
system (18). Protein concentrations were estimated using a dimeric
molar extinction coefficient at 280 nm of 10,800 M
1 cm
1 (32) or 13,800 M
1 cm
1 for D76Y SOD1 (33). The
purity, molecular mass, and metal ion contents for copper and zinc were
previously determined by mass spectrometry (18), and these same
preparations were used in the present study unless otherwise indicated.
The fully metallated enzyme is expected to contain 2 equivalents each
of copper and zinc/dimer. The D76Y and E133
mutants each eluted as
two distinct fractions during the final purification by ion exchange
chromatography. A previous metal content analysis (in equivalents per
dimer) revealed that fraction 1 for these mutants (D76Y-1:
0.58 Cu, 1.27 Zn; and E133
-1: 0.31 Cu, 1.26 Zn) contained more copper and zinc than did fraction 2 (D76Y-2: 0.36 Cu, 0.82 Zn; and
E133
-2: 0.16 Cu, 0.84 Zn) (18).
Tissue Lysates--
Frozen mouse tissues (spinal cord, brain,
brainstem, cerebellum, skeletal muscle, heart, kidney, or liver) were
pooled from at least three animals and homogenized in ice-cold lysis
buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, and
protease inhibitors ("CompleteTM EDTA-free," Roche
Molecular Biochemicals) using a motorized pestle. Approximately 70 mg
of tissue/ml lysis buffer was processed, and the lysate was centrifuged
at 14,000 × g for 10 min at 4 °C. 0.2% SDS was
added to the supernatant, and samples were stored at
80 °C. The
protein concentration of the mouse spinal cord lysates was 2.8 ± 0.3 mg/ml, and the G85R tissue lysates ranged from 1.8-3.5 mg/ml as
determined by the bicinchonic acid method (34).
Polyacrylamide Gel Electrophoresis--
Native PAGE of the
purified SOD1 enzymes was performed as described previously (18).
Partially denaturing PAGE was a modification of native PAGE in which 1)
SDS was added to the protein sample and running buffer to favor
migration based on the conformation-dependent degree of SDS
binding and 2) samples were incubated with variable amounts of reducing
agents or metal chelators prior to loading. The method differed from
standard denaturing SDS-PAGE in that the amount of SDS was lower
(0.1
0.4%), the amount of reducing agent during preincubation was
variable, and the sample was not boiled. Proteins were separated on
15% polyacrylamide Tris-HCl gels (Bio-Rad) run at 12 V/cm (80 V total)
for 2 h and stained with Coomassie Blue dye. The sample loading
buffer for partially denaturing PAGE contained 62 mM Tris
(pH 6.8), 10% glycerol, 0.05% bromphenol blue, and SDS, reducing
agents, or chelators as specified. The standard gel running buffer
contained 25 mM Tris (pH 8.3), 192 mM glycine,
and 0.1% SDS. All protein size markers and samples run under
denaturing conditions were boiled for 3 min in denaturing buffer (62 mM Tris (pH 6.8), 10% glycerol, 2% SDS, 5%
-mercaptoethanol, and 0.01% bromphenol blue) before loading. For
the partially denaturing gels (Figs. 1-3), the migration of dimeric WT
SOD1 in the absence of disulfide-reducing agent was indicated by an
arrow marked D. The migration of fully denatured
and reduced monomeric WT SOD1 was indicated by an arrow
marked M, which corresponded to the migration of a 21.5-kDa
denatured marker protein. Gels were photographed with a Kodak DC120
digital camera, and the images were analyzed using Adobe Photoshop and
Scion Image 4.0.2 software. Relative protein amounts were estimated by
subtracting the background from the total signal for specific protein bands.
Mass Spectrometry--
The number of accessible cysteine
residues in SOD1 variants was determined by the reaction of SOD1
proteins with iodoacetamide (35, 36) and detection of the number of
adducts formed per subunit by electrospray ionization mass spectrometry
(ESI-MS). WT or mutant SOD1 was incubated for 1-4 days in the absence
or presence of 10 mM TCEP at 37 °C. Proteins were
reacted with 5 mM iodoacetamide for 1 h before loading
on an liquid chromatography/MS column (C18, 5 µm, 300-Å Vydac
column, 1.0 mm, inner diameter × 15 mm) equilibrated with buffer
A (1% formic acid, 2% acetonitrile) and were eluted by a gradient to
buffer B (1% formic acid, 80% acetonitrile). Protein concentration
during incubation was 150 µg/ml, and 3 µl was loaded/injection.
ESI-MS was calibrated using porcine insulin (average mass = 5,734.60 Da).
Protease Susceptibility--
Digestion buffer contained 5 mM DTT, 150 mM NaCl, and 50 mM
sodium phosphate at pH 7.0. WT and mutant SOD1 proteins were divided into aliquots of 3 µg each and incubated at 37 °C in digestion buffer alone for 6 h, in buffer containing 50 µg/ml proteinase K
for 2 h, or in buffer containing 100 µg/ml trypsin for 6 h. The digestion was terminated by the addition of 10 mM
phenylmethylsulfonyl fluoride, and the proteins were boiled immediately
in denaturing buffer for 3 min before loading for standard SDS-PAGE.
Proteins that remained intact were detected by Coomassie Blue staining.
Western Blot Detection of Accessible SOD1 Cysteines in Tissue
Lysates--
Soluble tissue lysates containing 0.375 mg/ml total
protein and protease inhibitors (Complete, EDTA-free, catalog number
1873580, Roche Diagnostics) were incubated for 12 h in the
presence of 0, 0.3, 1.0, or 3.0 mM TCEP at 25 °C.
Mal-PEG (molecular mass of 5 kDa) was then added to a final
concentration of 3 mM for covalent modification of
accessible cysteines for 1 h at 25 °C (37). The addition of
Mal-PEG to accessible cysteines increases the subunit mass of SOD1 by
~5 kDa/modification. The reaction was competitively terminated upon
addition of loading buffer for denaturing SDS-PAGE that contained 5%
-mercaptoethanol (see above). For Western blot analysis, samples
were immediately boiled at 100 °C for 3 min, separated by denaturing
SDS-PAGE, and detected using a sheep polyclonal antibody to human SOD1
(catalog number 574597, Calbiochem) as described previously (18).
 |
RESULTS |
Both WT-like and Metal Binding Region ALS-related SOD1 Mutants
Exhibited Aberrantly Increased Electrophoretic Mobility during
Partially Denaturing SDS-PAGE--
Human and bovine WT SOD1s retain
their homodimeric subunit association, activity, and metal binding in
1% SDS (21, 24, 25, 38). To compare the electrophoretic behavior of
biologically metallated human WT and ALS mutant SOD1 variants (18)
under partially denaturing conditions, we incubated these enzymes for 30 min with 0.4% SDS and increasing concentrations of the
disulfide-reducing agent DTT without boiling prior to PAGE (Fig.
1).

View larger version (83K):
[in this window]
[in a new window]
|
Fig. 1.
ALS mutants were more susceptible than WT
SOD1 to aberrant migration during partially denaturing SDS-PAGE.
Human SOD1 proteins (3 µg/lane) isolated from erythrocytes (WT-C) or
from an insect cell expression system (all others) were preincubated
for 30 min at 37 °C in sample loading buffer consisting of 62 mM Tris (pH 6.8), 10% glycerol, 0.05% bromphenol blue,
and the indicated amounts of SDS and DTT. Samples were not boiled, and
the gel running buffer contained 0.1% SDS. Gels were stained with
Coomassie Blue. The migration of dimeric WT SOD1 under these conditions
is indicated by an arrow marked D, whereas the
migration of denatured and reduced WT SOD1 monomer is shown by an
arrow marked M. The metal contents of these
proteins were determined previously by inductively coupled plasma mass
spectrometry (18) and were (in equivalents per dimer) WT:
0.38 Cu, 1.37 Zn; A4V: 0.11 Cu, 1.36 Zn; L38V:
0.23 Cu, 1.32 Zn; G41S: 0.52 Cu, 1.35 Zn; H46R:
0.02 Cu, 0.07 Zn; H48Q: 0.63 Cu, 1.08 Zn; G72S:
0.74 Cu, 0.78 Zn; D76Y-1: 0.58 Cu, 1.27 Zn;
D76Y-2: 0.36 Cu, 0.82 Zn; G85R: 0.00 Cu, 0.01 Zn;
D90A: 0.34 Cu, 1.77 Zn; G93A: 0.45 Cu, 1.46 Zn;
D124V: 0.01 Cu, 0.03 Zn; D125H: 0.09 Cu, 0.36 Zn;
E133 -1: 0.31 Cu, 1.26 Zn;
E133 -2: 0.16 Cu, 0.84 Zn; and
S134N: 0.15 Cu, 0.33 Zn. The fully metallated enzyme is
expected to contain 2 equivalents each of copper and zinc/dimer.
|
|
In the absence of DTT (first two lanes of each
set), commercially available 32-kDa SOD1 holoenzyme from human
erythrocytes (WT-C) migrated as a single band. We showed previously
that our WT and WT-like as-isolated recombinant SOD1s typically
contained <30% full copper site occupancy, most probably as a
consequence of limited copper availability during overexpression in
insect cells (18). Partially metallated recombinant WT SOD1
(WT) migrated as three distinct species under these
conditions. The mobility of all WT species was considerably slowed
compared with that of denatured SOD1 near the 21.5-kDa marker during
standard SDS-PAGE (18), consistent with low SDS binding under these
conditions. Upon exposure to 2-10 mM DTT, a small fraction
of the WT SOD1 enzymes exhibited accelerated mobility near the mobility
of the denatured WT monomer (Fig. 1, arrow marked
M).
Four WT-like ALS mutants located at one pole of the
-barrel (L38V,
G41S, D90A, and G93A) contained amounts of copper and zinc ions
comparable with as-isolated WT SOD1 (see Fig. 1 legend) and had normal
specific activity (18). In contrast to WT SOD1, this group exhibited
reproducible "smearing" when exposed only to 0.1% SDS in the
gel-running buffer and in the absence of preincubation with SDS or DTT
(Fig. 1, first lane in each set). Moreover, these mutants were distinguished from the WT enzyme by an accelerated mobility upon preincubation with DTT. This result suggested that these
mutants were more susceptible than WT SOD1 to altered conformation or
net charge induced by SDS or DTT.
Mutants that were partially deficient in either zinc (H48Q and G72S) or
copper (A4V) compared with WT SOD1 (18) exhibited a component that
migrated similarly to denatured WT SOD1 (Fig. 1, arrow
marked M). In addition, two distinct fractions that differed only in metal occupancy were isolated by ion exchange chromatography for both the D76Y mutant and the single-residue deletion mutant E133
(18). The D76Y-2 and E133
-2 enzymes, which contained ~50-65%
less copper and ~65% less zinc than did D76Y-1 and E133
-1 (18),
were more susceptible to the effects of SDS and DTT on mobility.
Five ALS mutants with substitutions near the active site of SOD1 (H46R,
G85R, D124V, D125H, and S134N) were severely copper- and zinc-deficient
upon isolation from insect cells (18). These metal-binding region
mutants exhibited nearly maximal mobility upon exposure to SDS either
in the running buffer only (Fig. 1, first lane of each
set) or after preincubation in 0.4% SDS (second lane
of each set). This result suggested increased SDS binding to
these mutants, possibly as a consequence of unfolding in this gel
system, which was consistent with the established importance of metal
binding to SOD1 for both chemical and thermal stability (19, 21,
22).
Exposure to EDTA Alone Did Not Mimic the Accelerated Mobility of
SOD1 Mutants Caused by Disulfide-reducing Agents--
The results of
Fig. 1 suggested that disulfide bond reduction or decreased metal
occupancy or both contributed to faster migration of the mutant enzymes
compared with WT SOD1. DTT could have either reduced the single
intrasubunit disulfide bond (Cys-57 to Cys-146) (24) or chelated Zn(II)
ions (Kd = 10
6.9 M at pH
7.4) or Cu(I) ions (Kd = 10
11.1
M at pH 7.4) (39). To determine whether the SOD1 mutants
were sensitive to a strong metal ion chelator, we incubated the enzymes with EDTA in the absence or presence of DTT. Exposure to 20 mM EDTA alone did not mimic the gel mobility change induced
by 2 mM DTT (Fig.
2A). However, the combination
of 2 mM DTT and 20 mM EDTA altered the mobility
of even the WT enzyme. This finding suggested but did not directly
prove that reduction of the disulfide bond by DTT was important to
facilitate metal ion chelation from SOD1 by either DTT or EDTA.

View larger version (77K):
[in this window]
[in a new window]
|
Fig. 2.
Incubation of SOD1s with EDTA alone
did not mimic the effect of DTT. A, SDS-PAGE was performed
as in Fig. 1 with the exception that the sample buffer contained 0.4%
SDS and the indicated concentrations of EDTA and DTT. Proteins were
incubated for 24 h at 25 °C prior to loading (3 µg/lane).
SOD1 mutants were more susceptible than WT SOD1 to accelerated mobility
after incubation with TCEP. B, SDS-PAGE was performed as in
Fig. 1 with the exception that the sample buffer contained 0.1% SDS
and the indicated concentration of TCEP. Proteins were incubated for
24 h at 37 °C before loading (3 µg/lane). The metal contents
of these SOD1 preparations were (in equivalents per dimer)
WT: 0.67 Cu, 1.62 Zn; A4V: 0.33 Cu, 1.81 Zn;
L38V: 0.61 Cu, 1.91 Zn; and G93A: 0.63 Cu, 1.77 Zn.
|
|
To discern more clearly whether disulfide reduction was related to the
observed SOD1 mobility changes, we incubated the SOD1 enzymes with
TCEP, a highly stable reducing agent (40, 41). TCEP does not bind metal
ions but specifically reduces disulfide bonds between protein regions
(denoted by R and R') according to the following reaction.
Fig. 2B shows that 2 mM TCEP was
sufficient to fully shift the mobility of A4V SOD1 but had little
effect on WT SOD1. Exposure to 10 mM TCEP for 24 h
accelerated only ~25% WT, >60% G93A, and nearly all of A4V and
L38V SOD1s as judged by Coomassie Blue staining (Fig. 2B).
Similar results were obtained upon exposure of WT and mutant SOD1 to
-mercaptoethanol (data not shown).
ALS-related SOD1 Mutants Were Susceptible to Incubation
with GSH--
The cytosolic environment of the nervous system may
expose proteins to strong reducing influences. For example, GSH is an abundant intracellular thiol (present at ~1-10 mM in
neurons and glia) that can reduce disulfide bonds or be conjugated to
accessible cysteine residues (42). Also, among other reducing agents
that function as antioxidants by their free radical scavenging
activity, ascorbate accumulates in neurons to ~3 mM (43).
To determine whether the mobility of ALS mutants was sensitive to these
agents, we incubated the purified enzymes with either GSH or ascorbate for 24 h at 37 °C and then analyzed the samples by PAGE. Fig. 3 demonstrates that while WT SOD1 was
relatively resistant to 10-20 mM GSH, the mutant enzymes
exhibited faster migration suggestive of unfolding or monomerization.
In contrast, exposure to ascorbate (up to 100 mM) had no
effect on mutant SOD1 mobilities (data not shown). Overall, the
findings in Figs. 1-3 demonstrated strikingly distinct behavior during
partially denaturing PAGE of purified WT and all of the 14 ALS-related
mutants of SOD1 but did not precisely specify the origin of those
differences.

View larger version (97K):
[in this window]
[in a new window]
|
Fig. 3.
SOD1 mutants were susceptible to disulfide
reduction by GSH. SDS-PAGE was performed as in Fig. 1 with the
exception that samples contained 0.1% SDS and the indicated
concentration of GSH. Proteins were incubated for 24 h at 37 °C
before loading (3 µg/lane). Metal contents of these SOD1 preparations
were as shown in Fig. 2 for WT, A4V, L38V, and G93A, and they were as
shown in Fig. 1 for H48Q and D90A proteins.
|
|
TCEP Preferentially Reduced the Disulfide Bond of SOD1
Mutants and Contributed to Increased Accessibility of a Cysteine
Residue within the
-Barrel Core--
We next correlated
TCEP-induced changes in SOD1 mobility during native PAGE with altered
accessibility of cysteine residues as determined by mass spectrometry.
Fig. 4 shows the migration of WT and
mutant SOD1 enzymes after native PAGE in the absence or presence of
incubation with 10 mM TCEP for 1 day (left
panel) or 4 days (right panel). Migration of the WT
protein was not significantly altered after 1 day but was accelerated
after 4 days, suggestive of increased net negative charge resulting
from partial metal ion loss. In contrast, the mutant enzymes A4V, L38V,
G85R, D90A, and G93A exhibited species that migrated more slowly on
native PAGE after exposure to TCEP for 1-4 days. In the absence of
significant conformational change, metal ion loss alone would be
expected to accelerate rather than retard mobility of the mutants. For example, the as-isolated G85R mutant contained <5% metal occupancy (18) and migrated more rapidly than WT SOD1 before exposure to TCEP.
These native PAGE results suggested that a conformational change
related to either disulfide reduction by TCEP or metal ion loss impeded
the mobility of the mutant but not WT enzymes.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4.
TCEP slowed the electrophoretic mobility of
SOD1 mutants during native PAGE. Native PAGE of SOD1 proteins (5 µg/lane) following incubation for 1 day (left panel) or 4 days (right panel) at 37 °C in the absence or presence of
10 mM TCEP. C, carbonic anhydrase; L,
lactalbumin; A, albumin.
|
|
To determine cysteine accessibility before and after exposure
to TCEP, SOD1 proteins were incubated with 5 mM
iodoacetamide, which reacts with each accessible sulfhydryl group to
form an adduct that adds 57 Da to the subunit mass. Changes in the
masses of WT and mutant SOD1 proteins following reaction with
iodoacetamide were detected under denaturing conditions by ESI-MS. The
deconvoluted spectra in Fig. 5 indicate
the subunit masses of each resolved species, and the deduced number of
modified cysteine residues is shown as a circled number for
each peak. In the absence of incubation with TCEP, both the WT and
mutant SOD1 proteins exhibited only a single prominent modification,
which most probably corresponded to adduct formation at the
surface-accessible Cys-111 residue.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
ALS SOD1 mutants exposed to TCEP exhibited
greater accessibility of cysteine residues to alkylation by
iodoacetamide. WT and mutant SOD1 proteins were exposed to 5 mM iodoacetamide for 1 h before loading on an liquid
chromatography/MS column for ESI-MS under denaturing conditions. Shown
are deconvoluted spectra with the masses of each protein subunit
indicated. Circled numbers for each species indicate the
number of cysteine residues alkylated by iodoacetamide as inferred from
the measured masses (+57 Da change/modification). Protein concentration
was 150 ng/µl, and 3 µl was loaded/injection. Metal contents of
these preparations were as described in Fig. 1, and the masses of
unmodified subunits were reported previously (18).
|
|
All 14 mutants were evaluated for cysteine accessibility by mass
spectrometry, and all showed increased reactivity to iodoacetamide in
the presence of TCEP. Following exposure to TCEP for 1 day, there was
no change in cysteine accessibility for WT SOD1, but A4V, L38V, D76Y-1,
D76Y-2, G85R, D125H, E133
-1, E133
-2, and S134N mutants exhibited
species containing either three or four modified cysteines (Fig. 5). A
total of three modified cysteines would be expected if the disulfide
bond (Cys-57 to Cys-146) was cleaved, whereas all four cysteines of the
subunit could be modified only if disulfide cleavage was accompanied by
at least transient exposure of the side chain of Cys-6, which is
normally buried in the interior of the
-barrel. Similar results were
also obtained for the mutants G41S, H46R, H48Q, G72S, and D124V after a
1-day incubation with TCEP (data not shown). The results for D76Y and E133
(Fig. 5) also clearly indicated that fractions containing lower
metal occupancy were more susceptible to reaction with iodoacetamide. For the metal-depleted forms, the increased cysteine accessibility in
Fig. 5 correlated with the observed mobility shifts in Fig. 1 and the
protease susceptibility in Fig. 6.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 6.
Mutant SOD1s were more susceptible than WT
SOD1 to digestion by proteinase K or trypsin in the presence of
DTT. Digestion buffer contained 5 mM DTT, 150 mM NaCl, and 50 mM sodium phosphate at pH 7.0. SOD1 proteins (3 µg/lane) were incubated at 37 °C in buffer alone
for 6 h (lane 1 for each set), buffer with 50 µg/ml
proteinase K for 2 h (lane 2), or buffer with 100 µg/ml trypsin for 6 h (lane 3). The reactions were
terminated by adding 10 mM phenylmethylsulfonyl fluoride,
and samples were immediately boiled in loading buffer for denaturing
SDS-PAGE followed by Coomassie Blue staining. Metal contents for WT,
A4V, and L38V SOD1s were as shown in Fig. 2, and for the remaining
preparations they were as shown in Fig. 1.
|
|
After exposure to TCEP for 4 days (Fig. 5), even the WT enzyme
exhibited three modified cysteines indicative of disulfide cleavage by
TCEP, whereas A4V, L38V, D90A, and G93A not only appeared disulfide-reduced but also exhibited species in which all four cysteines were modified. These results provided strong evidence that
the mutant enzymes were more susceptible than WT to disulfide cleavage.
Moreover, the reduction of the mutants but not WT SOD1 contributed to
exposure of the buried Cys-6 residue and destabilization of the
-barrel itself.
Mutant SOD1s Were All Susceptible to Digestion by Proteinase K or
Trypsin in the Presence of DTT--
WT SOD1 and some ALS mutants
expressed in COS-1 cells are resistant to digestion by proteinase K
under non-reducing conditions (15). We hypothesized that a
disulfide-reducing environment may partially unfold mutant but not WT
SOD1 and thereby unmask a sensitivity to protease digestion. Therefore,
we compared the fraction of purified WT or ALS mutant SOD1s remaining
after exposure to either proteinase K or trypsin in the presence of
DTT.
Standard denaturing SDS-PAGE in Fig. 6 revealed that the metal-binding
region mutants (H46R, H48Q, G85R, D124V, D125H, and S134N) and the A4V
mutant were completely digested by proteinase K or trypsin when
incubated with 5 mM DTT at 37 °C. Other mutants at one
pole of the
-barrel (L38V, G41S, D90A, and G93A) or in loop regions
(G72S, D76Y, and E133
) exhibited partial digestion under these
conditions, whereas the WT protein remained intact. D90A SOD1, which
exhibited relative resistance to the effects of DTT (Fig. 1), TCEP
(Figs. 2 and 5), or GSH (Fig. 3), was also the mutant most resistant to
protease digestion (Fig. 6). However, as judged by Coomassie Blue
staining, less than half of the D90A and G93A SOD1s remained after a
6-h digestion with trypsin.
We hypothesized that increased stability afforded by metal ion binding
to SOD1 (19, 21, 22) could contribute to protease resistance despite
the presence of a destabilizing mutation. To test this possibility,
preparations of D76Y and E133
that differed only with respect to
metal ion occupancy (18) were exposed to these proteases under reducing
conditions (Fig. 6). The preparations with higher metal contents
(D76Y-1 and E133
-1) were more resistant to proteolysis than were
those with lower metal contents (D76Y-2 and E133
-2). The results of
Fig. 6 also demonstrated that SDS, which was absent from the reaction
buffer, was not required to unmask the protease sensitivity of the ALS
mutant SOD1s upon exposure to DTT.
TCEP Increased SOD1 Cysteine Accessibility in Neural Tissue Lysates
from ALS Mutant but Not WT SOD1 Transgenic Mice--
We next compared
SOD1 cysteine reactivity in soluble lysates from transgenic mouse
tissues expressing WT, G85R, or G93A SOD1s. Lysates were treated for
12 h with variable amounts of TCEP to simulate a reducing
environment, and proteins were then covalently tagged at accessible
cysteine residues with Mal-PEG. Reaction with Mal-PEG increased the
subunit mass by ~5 kDa/modification, which was resolved by Western
blot analysis (37). Fig. 7, left panel, shows that >90% SOD1 in spinal cord lysates from WT SOD1 transgenic mice was modified at a single cysteine residue, in agreement
with the results obtained by mass spectrometry for the purified
proteins in Fig. 5. Exposure of the WT lysate to TCEP for 12 h did
not affect labeling by 3 mM Mal-PEG except at 3 mM TCEP, which inhibited the labeling reaction. In
contrast, spinal cord lysates from G85R or G93A SOD1 mutant mice
contained SOD1 species that were modified at 1-4 cysteine residues,
and labeling at multiple residues was increased by exposure to TCEP
(Fig. 7, left panel).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
SOD1 cysteines in tissue lysates from ALS
transgenic mice exhibited increased accessibility to modification by
Mal-PEG. Soluble tissue lysates were obtained from spinal cord
(SC), brain (BN), cerebellum (CB),
brain stem (BS), skeletal muscle (SK), heart
(HT), kidney (K), and/or liver (L)
tissues from WT (age 15 weeks), G85R (age 27 weeks), or G93A (age 15 weeks) human SOD1-expressing transgenic mice. Lysates were incubated
with the indicated concentration of TCEP for 12 h at 25 °C,
exposed to 3 mM Mal-PEG (MP) for 1 h, and
subjected to SDS-PAGE (3 µg/lane) followed by Western blot detection
with an anti-SOD1 antibody.
|
|
The middle and right panels of Fig. 7 shows that
SOD1 in brain (BN), cerebellum (CB), brain stem
(BS), skeletal muscle (SK), and heart
(HT) tissues from G85R mice also exhibited susceptibility to
modification by Mal-PEG at 2, 3, or all 4 cysteines in the presence of
1 mM TCEP. In contrast, only a small fraction of the total
SOD1 in G85R mouse kidney (K) or liver (L)
tissues was labeled by Mal-PEG, even in the presence of TCEP. Because
even the surface Cys-111 was inefficiently labeled, this finding
suggests the presence of an inhibitor of the labeling reaction in
kidney and liver lysates. Overall, these results independently confirm
the mass spectrometry results with the purified SOD1s and indicate that
the presence of both an ALS mutation and a disulfide-reducing
environment may decrease SOD1 stability.
 |
DISCUSSION |
The conformation and stability of ALS-related SOD1 mutants in
affected tissues may be influenced directly by the mutant substitutions or indirectly as a consequence of disulfide bond reduction, decreased metal ion binding, monomerization, or other vulnerabilities under conditions in vivo. In this study, we correlated the effects
of disulfide-reducing agents upon electrophoretic mobility, cysteine accessibility, and protease sensitivity among purified WT and ALS-related SOD1 variants of known metal ion content (18). We further
demonstrated that G85R and G93A mutant SOD1s in transgenic mouse tissue
lysates were susceptible to modification at normally inaccessible
cysteine residues under reducing conditions.
Our initial observation that all 14 of the ALS mutants exhibited
accelerated migration during partially denaturing PAGE (Figs. 1-3)
indicated that ALS mutants share properties distinct from WT SOD1. To
clarify the nature of those properties, we showed that the mutant
proteins were more susceptible than WT SOD1 to disulfide reduction by
TCEP (Fig. 5) and proteolysis in the presence of DTT (Fig. 6).
Metal-binding region SOD1 mutants (H46R, G85R, D124V, D125H, and S134N)
that were severely deficient in copper and zinc ions (18) exhibited the
greatest increase in PAGE mobility (Fig. 1), increased reactivity to
iodoacetamide in the presence of TCEP (Fig. 5), and greater
susceptibility to proteolysis (Fig. 6). This behavior was consistent
with the known importance of metal ion binding to SOD1 stability.
However, even WT-like mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A,
and E133
), which had more normal metal ion coordination and specific
dismutase activity (18), could be distinguished from WT SOD1 under
disulfide-reducing conditions (Figs. 1 and 5).
The large loop containing residues 49-84, which is anchored by the
disulfide bond to the
-barrel, not only composes part of the dimer
interface but also forms the zinc binding site that links directly to
the copper ion via His-63 (44). In addition, the backbone oxygen atoms
of residues Cys-57 and Gly-61 in this loop form three hydrogen bonds
that correctly orient the side chain of Arg-143, an important
determinant of copper ion accessibility in SOD1 (27, 28, 45-48). These
structural features increase the likelihood that disulfide reduction at
Cys-57, and the disorder of this loop could facilitate partial
unfolding, monomerization, metal ion loss, or altered reactivity of
bound copper. Human SOD1 also contains two cysteine residues (Cys-6 and
Cys-111) that increase the irreversibility of SOD1 thermal unfolding
(49). The buried side chain of Cys-6, packed tightly within the
interior of the
-barrel, should remain inaccessible to solvent
unless the SOD1 core is severely disrupted. For example, sulfhydryl
reactivity of Cys-6 in bovine SOD1 occurs during exposure to 6 M guanidinium chloride but not 8 M urea (25).
In contrast, the side chain of Cys-111 is exposed on the protein
surface near the dimer interface and may be conjugated to GSH in
vivo (50). Our mass spectrometry results (Fig. 5) suggest that
increased susceptibility to disulfide reduction of SOD1 mutants also
contributed to partial unfolding of the
-barrel as indicated by
accessibility of all four cysteine residues to modification by
iodoacetamide. Consistent with these findings, Mal-PEG labeling of SOD1
at multiple cysteine residues was observed in tissue lysates from G85R
and G93A but not WT SOD1 transgenic mice (Fig. 7).
SOD1 is unusual in its ability to form a stable intrasubunit disulfide
bond in the reducing environment of the cytosol, and this property may
also be important for proper folding of the enzyme (51, 52). We
demonstrated that ALS-related SOD1 mutants were sensitive to
physiological concentrations of glutathione (Fig. 3), the main
cytosolic thiol-disulfide redox buffer. It is also possible that
thiol-disulfide oxidoreductases of the thioredoxin and glutaredoxin
pathways (53) could impair disulfide formation or stability
during folding of the mutant SOD1 enzymes. In this regard, thioredoxin
is up-regulated in erythrocytes of familial ALS patients (54), and its
transcript is also increased ~6-fold in ALS spinal cord tissue (55),
possibly as a defense against oxidative stress. The requirement to
maintain cellular redox homeostasis and prevent the formation of
non-native disulfide bonds thus might strongly influence the degree of
mutant SOD1 misfolding or unfolding in specific tissues.
How might the susceptibility of mutant SOD1 enzymes to disulfide
reduction relate to motor neuron toxicity in familial ALS? One
possibility is that destabilization of the zinc-binding loop upon
disulfide cleavage could promote selective zinc ion loss while
retaining reactive copper ions at the active site. Zinc-deficient, copper-containing SOD1 is neurotoxic in vitro, possibly by a
mechanism involving nitric oxide (56). Alternatively, disulfide
reduction could alter copper ion reactivity by perturbing the position
of Arg-143, which influences substrate selectivity or by creating a
novel copper binding site upon exposure of free cysteine residues. On
the other hand, disease progression in mutant SOD1 transgenic mice is
not affected by genetic ablation of copper
chaperone-dependent copper loading into SOD1 (57) or by
expression of SOD1 mutants that severely disrupt the copper binding
site (58). However, the toxicity of bound copper has not been
completely excluded because unshielded copper ions may be toxic at even
nanomolar concentrations (59).
Disulfide reduction of the ALS mutants may also contribute to toxicity
by mechanisms independent of abnormal copper reactivity. For example,
the exposure of hydrophobic residues or reactive cysteines could favor
abnormal interactions of SOD1 with itself or with other cellular
constituents. Mutant SOD1 can form insoluble protein complexes at early
stages in G93A SOD1 transgenic mouse tissues and following proteasome
inhibition in cultured cells (60). Similarly, a fraction of total
mutant SOD1 in brain and spinal cord tissues from SOD1 mice forms high
molecular weight complexes that accumulate with disease progression
(61) and mouse tissues can exhibit thioflavin-S-positive inclusions
(58). Oxidative stress may also be linked to the accumulation of mutant SOD1 either by decreased proteasome activity or by impaired degradation of oxidatively damaged SOD1 (62). An increased burden of partially unfolded SOD1 proteins or complexes could ultimately impair cellular chaperone capacity (16), perturb mitochondrial function (63), sequester
anti-apoptotic factors (12), disrupt protein recycling, or impose an
unsustainable metabolic cost to vulnerable tissues (reviewed in Ref.
3).
Our data suggest that cellular disulfide reducing influences at
physiological temperature and pH are sufficient to convert relatively
well folded WT-like SOD1 mutants (18) or less stable metal-binding
region mutants (19) into more severely destabilized species. These
non-native mutant forms might resemble the subset of highly unstable
SOD1 C-terminal truncation mutants (15, 64), which also lack the
disulfide bond consequent to deletion of Cys-146. Overall, these
results implicate susceptibility to conformational destabilization of
SOD1 by a cellular reducing environment as a shared property that may
be relevant to ALS mutant neurotoxicity.