From the Academic Unit of Neurology, Division of
Genomic Medicine, University of Sheffield, the
Academic Unit of
Pathology, Division of Genomic Medicine, University of Sheffield,
Sheffield S10 2RX, United Kingdom, ** Laboratory of
Neurogenetics, National Institute on Aging, National Institutes of
Health, Bethesda, Maryland 20892, and
Cancer Research UK, Clinical Cancer Centre,
St. James's University Hospital, Leeds LS9
7TF, United Kingdom
Received for publication, September 26, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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Injury to motor neurons associated
with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial
amyotrophic lateral sclerosis (FALS) results from a toxic
gain-of-function of the enzyme. The mechanisms by which alterations to
SOD1 elicit neuronal death remain uncertain despite intensive research
effort. Analysis of the cellular proteins that are differentially
expressed in the presence of mutant SOD1 represents a novel approach to
investigate further this toxic gain-of-function. By using the motor
neuron-like cell line NSC34 stably transfected with wild-type, G93A, or
G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches. Seven
up-regulated proteins were identified as argininosuccinate synthase,
argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding
motif protein 3, peroxiredoxin I, proteasome subunit Amyotrophic lateral sclerosis
(ALS),1 the most common form
of motor neuron disease, is a fatal, adult-onset neurodegenerative disorder, characterized by selective loss of lower and upper motor neurons from the spinal cord and brain. Approximately 10% of ALS cases
are inherited, and 20% of these familial ALS (FALS) cases result from
dominantly inherited missense mutations in the gene encoding
Cu,Zn-superoxide dismutase (SOD1) (1). As most FALS SOD1 mutants retain
dismutase activity close to that of the wild-type enzyme (2), the
injury to motor neurons associated with mutant SOD1 may result from a
toxic gain-of-function of the enzyme rather than loss of its ability to
catalyze the conversion of superoxide to hydrogen peroxide.
Furthermore, mice with targeted deletion of the sod1
gene do not develop an ALS phenotype (3) in contrast to transgenic mice
expressing mutant human SOD1 (4-6).
Several non-mutually exclusive hypotheses have been proposed to
describe the toxic gain-of-function of mutant SOD1 (7, 8). These
include altered free radical handling, altered copper/zinc binding, and
formation of high molecular weight protein aggregates. Evidence for
altered free radical handling has come from numerous observations; for
example, indices of free radical damage are increased in the transgenic
mutant SOD1 mice (9) and human ALS cases (10, 11). Cultured cells
expressing SOD1 mutants have been shown to exhibit increased oxygen
radical production and sensitivity to exogenously produced free
radicals (12, 13). Intracellular superoxide can react with nitric oxide
to produce the oxidant peroxynitrite (14). Several studies have
provided evidence for the role of peroxynitrite and nitric oxide in
SOD1-related ALS. Increased nitrosylation of proteins by peroxynitrite
has been suggested by elevated levels of 3-nitrotyrosine in transgenic mutant SOD1 mice (15, 16). Reduced zinc binding has been demonstrated for several SOD1 mutants (17). Interestingly, zinc-deficient wild-type
SOD1, as well as mutant SOD1, generates superoxide, which in turn
increases peroxynitrite production. Apoptosis of cultured primary
neurons induced by zinc-deficient SOD1 can be reduced by treatment
with inhibitors of nitric-oxide synthase (18).
Protein aggregation as a toxic gain-of-function was initially proposed
after the demonstration of anti-SOD1-reactive cytoplasmic inclusions in
motor neurons and surrounding astrocytes in mutant SOD1 transgenic
mouse models (19) and cell culture models (20) of human FALS.
Aggregation may lead to toxicity in a number of ways. SOD1 aggregates
may have altered free radical and metal ion chemistry as described
above. They may also challenge the protein folding and degradative
machinery of the cell, compromising housekeeping protein functions
essential for cell viability (8). Indeed only modest inhibition of the
proteasome complex is required to generate mutant SOD1 aggregates in
transfected cell lines (21). Whatever the relative contributions of
these potentially inclusive modes of SOD1 toxicity are, the molecular
pathways they trigger that ultimately lead to neuronal degeneration are
poorly understood. Studies using cell models (13) or transgenic mutant
SOD1 mice (22) have demonstrated biochemical markers suggesting an
apoptotic mode of programmed cell death for degenerating neurons
(reviewed in Ref. 23).
Recent developments in gene expression profiling technology have
provided new impetus in the identification of molecular pathways activated by mutant ALS SOD1. Two independent studies using microarray analysis of transgenic mice expressing the human FALS-associated G93A
SOD1 mutant (24, 25) and microarray analysis of human ALS spinal cord
(26) have detected some characteristic gene expression changes.
Differential regulation of apoptosis-, inflammation-, and
antioxidant-related genes were findings common to all of these studies.
The use of spinal cord tissue in these studies also underlined the
possible involvement of protein expression changes in non-neuronal cells including microglia and astrocytes in motor neuron degeneration. Attributing any of these changes specifically to the motor neurons that
account for a very small proportion of spinal cord tissue may not be
possible. Primary alterations within degenerating motor neurons in
response to mutant ALS SOD1 may go undetected among the protein changes
occurring within the complex mixture of more abundant cell types in the
central nervous system. We have addressed this issue by developing a
cell culture model of FALS based upon a murine motor neuron-like cell
line, NSC34, stably transfected with human FALS associated SOD1 mutants
G93A and G37R (27-29). The NSC34 cells are a hybrid motor neuron × neuroblastoma cell line that exhibits several features of motor
neurons including neurofilament expression, the ability to generate
action potentials, and induction of twitching in co-cultured muscle
cells (30). Alterations in gene expression in this model were analyzed
previously using a microarray approach. Expression of genes involved in
the regulation of axonal transport, vesicular trafficking, and
apoptosis were found to be altered by expression of ALS mutant hSOD1
(28). Here we extend these studies using proteomic techniques to
analyze the alterations in protein expression. We have demonstrated
that expression of both G37R and G93A hSOD1 results in the differential expression and altered function of proteins that regulate nitric oxide
metabolism, intracellular redox conditions, and protein degradation.
Reagents--
All two-dimensional gel electrophoresis reagents
were ultra-pure grade and purchased from Bio-Rad and Sigma. COMPLETE
EDTA-freeTM protease inhibitor mixture was purchased from
Roche Molecular Biochemicals. Cell culture media and reagents were
purchased from Invitrogen. Enhanced chemiluminescence (ECL) kits and
glutathione-Sepharose were purchased from Amersham Biosciences. Reduced
glutathione assay kit, reduced glutathione, 7-amino-4-methylcoumarin
(AMC), Suc-LLVY-AMC, Z-ARR-AMC, and Z-LLE-AMC were purchased from
Calbiochem. Owl silver stain and rabbit anti-rat glutathione
transferase-Mu antibody were purchased from Autogen Bioclear (Calne,
UK). Sheep anti-bovine SOD1 antibody was purchased from The Binding
Site (Birmingham, UK). Mouse anti-actin (AC-40), anti-mouse inducible nitric-oxide synthase monoclonal antibodies, and donkey anti-sheep IgG
horseradish peroxidase conjugate were purchased from Sigma. Rabbit
anti-rat neuronal nitric-oxide synthase antibody was purchased from
Zymed Laboratories Inc. Rabbit anti-human nucleotide
diphosphate kinase A antibody (nm23-H1 C-20) was purchased from Santa
Cruz Biotechnology, Inc. Rabbit anti-bovine endothelial nitric-oxide synthase antibody was purchased from Bioquote Ltd. (York, UK). Rabbit
anti-rat argininosuccinate synthase antibody was a gift from Masataka
Mori (University of Kumamoto, Japan). Rabbit anti-rat argininosuccinate
lyase antibody was a gift from Heinrich Wiesinger (University of
Tuebingen, Germany). Rabbit anti-mouse LMP7 antibody was a gift from
John Monaco (University of Cincinnati). Rabbit anti-human proteasome
subunit X antibody was a gift from Klavs Hendil (University of
Copenhagen, Denmark). Rabbit anti-mouse LMP2 antibody was purchased
from Affiniti Research Products Ltd. (Exter, UK). Rabbit anti-porcine
leukotriene B4 12-hydroxydehydrogenase antibody was a gift
from Takehiko Yokomizo and Takao Shimizu (University of Tokyo, Japan).
Swine anti-rabbit IgG horseradish peroxidase conjugate and goat
anti-mouse IgG horseradish peroxidase conjugate were purchased from
Dako (Ely, UK).
Cell Lines and Cytosol Preparation--
NSC34 single cell clones
stably expressing pCEP4 expression vector only, wild-type hSOD1, G93A
hSOD1, and G37R hSOD1 have been described previously (27). Cytosol was
prepared from NSC34 cells using a modified version of the method of
Yang and co-workers (31). NSC34 cells were seeded into T175 flasks at a
density of 9 × 105 cells per flask and maintained in
Dulbecco's modified Eagle medium containing 10% v/v fetal bovine
serum at 37 °C in a humidified atmosphere with 5% CO2
for 96 h. The medium was aspirated, and the cells were resuspended
in 10 ml of ice-cold PBS, pH 7.4. Cells from two flasks were pooled for
each cytosol preparation. The cells were centrifuged at 600 × g for 5 min. The pellets were washed twice with 30 ml of
ice-cold PBS and pelleted as above. The pellets were resuspended in 200 µl of extract buffer (20 mM HEPES/KOH, pH 7.4, containing
10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), 2 M sucrose, COMPLETE
EDTA-freeTM protease inhibitor mixture 1 tablet per 10 ml).
The cells were homogenized with 30 passes of a mini homogenizer. The
homogenates were centrifuged at 3000 rpm for 6 min at 4 °C; the
post-nuclear S1 supernatant was harvested; the pellet was resuspended
in 100 µl of extract buffer and homogenized as above; and the
centrifugation step was repeated. The S1 supernatants were pooled and
centrifuged at 13,000 × g for 10 min at 4 °C. The
protein concentration of the resulting S2 supernatant was determined
using a Coomassie G-250 assay (Pierce).
Two-dimensional Gel Electrophoresis--
For analytical gels,
cytosol containing 25 µg of protein was made up to 300 µl with 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM DTT, and 0.2% v/v ampholyte, pH 3-10 (Bio-Rad). For
preparative gels, 100-500 µg of cytosolic protein was used. Each
300-µl sample was applied to a 17-cm, pH 3-10, immobilized pH
gradient gel strip (IPG). The IPG strips were then rehydrated actively
at 50 V for 16 h in a Bio-Rad Protean isoelectric focusing (IEF)
cell. IEF consisted of 250 V for 15 min, linear ramping from 250 to
10,000 V over 3 h, followed by 10,000 for 60,000 V-h. The strips
were then incubated at room temperature for 10 min in SDS-PAGE
equilibration buffer (0.375 M Tris, pH 8.8, containing 6 M urea, 2% w/v SDS, 20% v/v glycerol) containing 2% w/v
DTT followed by 10 min in SDS-PAGE equilibration buffer containing
2.5% w/v iodoacetamide. The strips were loaded onto 20 × 18 cm
14% SDS-polyacrylamide gels, and overlaid with 0.5% low melting
temperature agarose (Bio-Rad). Electrophoresis was performed in a
Protean II xi electrophoresis cell (Bio-Rad). Analytical and
preparative two-dimensional gels were stained with Owl silver stain and
Biosafe Coomassie G-250 stain (Bio-Rad), respectively, according to the
manufacturer's instructions. Molecular weight and pI values of
proteins were estimated with two-dimensional standards (Bio-Rad).
Two-dimensional Gel Image Analysis--
Analytical
two-dimensional gels were scanned using a Powerlook III scanner (UMax,
Ascot, UK) and analyzed using Phoretix two-dimensional software
(Non-linear Dynamics, Newcastle, UK). A previously described pairwise
approach (32) was used to compare the intensities of protein spots
between cell clones. Here we compared single cell clones of NSC34
stable transfectants (27) expressing pCEP4 vector only with those
expressing wild-type hSOD1 and NSC34 stable transfectants expressing
pCEP4 vector only with those expressing G93A hSOD1. Comparisons of spot
intensities were made between a pair of gels electrophoresed and
stained at the same time using samples prepared within the same
experiment. Differences in spot intensities were tested with Wilcoxon
t test using no less than six pairs of gels.
Glutathione-Sepharose Precipitation--
NSC34 cells grown in
T175 flasks were resuspended in ice-cold PBS, pH 7.4. The cells were
centrifuged at 600 × g for 5 min. The pellets were
washed twice with ice-cold PBS and pelleted as above. The resulting
pellets were resuspended in 0.5 ml per flask of GST extract buffer (20 mM potassium phosphate buffer, pH 7.0, containing 0.1% v/v
Triton X-100 and protease inhibitors). The cells were homogenized with
30 passes of a mini homogenizer and then centrifuged at 13,000 × g for 10 min at 4 °C. The protein concentrations of the
resulting supernatants were determined using the above Coomassie G-250
assay. Appropriate volumes containing 2.5 mg of protein were adjusted
to 1 ml with GST extract buffer and incubated with 100 µl of 25% v/v
glutathione-Sepharose, equilibrated in 20 mM potassium
phosphate buffer, pH 7.0, for 60 min at 4 °C with end-over
rotation. The glutathione-Sepharose precipitates were pelleted by
centrifugation at 13,000 × g for 30 s and then washed 6 times with 1 ml of 20 mM potassium phosphate
buffer, pH 7.0, containing 50 mM NaCl followed by 3 washes
with 1 ml of 20 mM potassium phosphate buffer, pH 7.0. The
pellets were thoroughly aspirated and resuspended each in 300 µl of 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM DTT, and 0.2% v/v ampholyte, pH 3-10. The resuspended pellets were incubated at room temperature for 10 min to denature and
dissociate the glutathione-protein complexes and then
centrifuged at 13,000 × g for 30 s. The resulting
supernatants were subjected to two-dimensional electrophoresis as
described above. The relative expression levels of
glutathione-Sepharose binding proteins between cell lines were analyzed
using silver staining and Wilcoxon t test as described
above. All glutathione-Sepharose binding proteins detectable by
Coomassie Blue staining were selected for identification by
MALDI-TOF-MS.
Western Blotting of One- and Two-dimensional
SDS-PAGE--
Samples were Western-blotted as described previously
(27) using antibodies diluted as indicated in the figure legends. The proteins were visualized using an ECL kit according to the
manufacturer's instructions. To determine the positions of spots
corresponding to endogenous mouse SOD1 (mSOD1) and G93A hSOD1,
two-dimensional gels were performed in duplicate using 25 µg of
cytosolic protein. Both gels were transferred onto polyvinylidene
difluoride membrane. One of the membranes was probed with 1:1000
primary sheep anti-SOD1 polyclonal followed by 1:2000 secondary donkey
anti-sheep IgG horseradish peroxidase conjugate. Antibody binding was
visualized with 3,3'-diaminobenzidine (DAB). Both membranes were
stained with Coomassie R-250. Spot positions of endogenous mouse SOD1 and G93A human SOD1 were located by comparing the DAB and
Coomassie-stained spot pattern.
Peptide Mass Fingerprinting by MALDI-TOF-MS and Data Base
Searching--
Protein identification of Coomassie Blue-stained spots
with tryptic peptide mass fingerprinting by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
and data base searching was performed at the Aberdeen Proteome Facility
(University of Aberdeen, UK). In-gel trypsin digestion of proteins of
interest was performed using an Investigator ProGest work station
(Genomic Solutions, Huntingdon, UK). MALDI-TOF-MS was performed using a Voyager-DE STR instrument (Perspective Biosystems). Data base searching
was performed using the MS-Fit (University of California, San
Francisco) and Mascot Software (Matrix Science, London, UK) to search
the Swiss Prot/TrEMBL (www.ca.expasy.org) and NCBI
(ncbi.nlm.nih.gov) data bases.
Enzyme and Metabolite Assays--
Glutathione
S-transferase assay reaction mixtures (33) consisted of 100 mM potassium phosphate, pH 6.5, containing 1 mM EDTA, 1 mM 1-chloro-2,4-dinitrobenzene, and 2 mM reduced glutathione. Reactions were initiated by adding
100 µg of NSC34 post-nuclear S1 protein per ml assay reaction. The
increase in absorbance at 340 nm was measured at 0-5 min at room temperature.
Glutathione reductase assay reaction mixtures (34) consisted of 50 mM HEPES/KOH, pH 8.0, containing 0.1 mM EDTA,
0.1 mM oxidized glutathione (Sigma), and 0.1 mM
NADPH. Reactions were initiated by adding 100 µg of NSC34
post-nuclear S1 protein per ml of assay reaction. The decrease in
absorbance at 340 nm was measured at 0-5 min at room temperature.
Proteasome assays were performed with modification to the method of
Beyette and co-workers (35). NSC34 cells grown in T175 flasks were
resuspended ice-cold PBS, pH 7.4. The cells were centrifuged at
600 × g for 5 min. The pellets were washed twice with
ice-cold PBS and pelleted as above. The resulting pellets were
resuspended in 0.3 ml of proteasome extract buffer (20 mM
Tris/HCl, pH 7.4, containing 0.1 mM EDTA, 1 mM
2-mercaptoethanol, 5 mM ATP, 20% v/v glycerol, and 0.04%
v/v Nonidet P-40). The resuspended cells were homogenized by 25 passes
through a 21-gauge needle. The resulting homogenates were centrifuged
at 13,000 × g for 15 min at 4 °C. The protein
concentrations of the supernatants were determined using the Coomassie
G-250 assay. Proteasome assay reaction mixtures consisted of 50 mM HEPES/KOH, pH 8.0, containing 5 mM EGTA, 100 µg of NSC34 extract protein per ml of assay reaction. The reactions were initiated by adding the appropriate proteasome substrate and
incubating the reactions at 37 °C for 45 min. Suc-LLVY-AMC was used
at 50 µM. Z-ARR-AMC and Z-LLE-AMC were used at 100 µM. Hydrolysis of the peptides was measured at 340 nm
excitation and 460 nm emission for AMC using a Denley
spectrofluorimeter. Standard curves were constructed using 0-50
µM AMC to convert fluorescence units to AMC concentration.
Reduced glutathione concentration in NSC34 cells was determined using a
commercial assay kit (Calbiochem) according to the manufacturer's instructions.
RT-PCR Analysis of Gene Expression in NSC34 Cells and Human Motor
Neurons--
NSC34 cells expressing pCEP4 vector or G93A hSOD1 were
harvested, washed in Hanks' buffered saline solution, and resuspended in TRIzol (Invitrogen). RNA was extracted according to the
manufacturer's protocol. Following treatment with DNase I
(Invitrogen), the sample was divided in two, and cDNA
synthesis was performed both with and without the addition of
Superscript II reverse transcriptase (Invitrogen), according
to the manufacturer's instructions.
The Arcturus Pixcell II Laser Capture Microdissector was used to
isolate motor neurons from 10 µm toluidine blue-stained sections of
human lumbar spinal cord. The material used was collected at autopsy
from two neuropathologically normal control subjects (1 male and 1 female, mean age 50 ± 3 years, post-mortem delay 16.5 ± 2.5 h), and two subjects with FALS resulting from an I113T
mutation in SOD1 (1 male and 1 female, mean age 62 ± 2 years,
post-mortem delay 23.5 ± 3.5 h). RNA was extracted using
TRIzol according to the manufacturer's instructions, except that 0.1 µg/ml glycogen was added to assist in the recovery of RNA. As above,
following DNase I treatment, samples were divided in two, and cDNA
synthesis was carried out.
PCR was performed using 1 µl of cDNA, 10 pmol of each primer (see
Table I), 200 µM total
dNTPs, 0.5 units of Taq polymerase, and 1× reaction buffer
(75 mM Tris/HCl, pH 8.8, 20 mM
(NH4)2SO4, 1.5 mM
MgCl2, 0.01% (v/v) Tween 20). NSC34 samples were denatured at 95 °C for 5 min, followed by 30 cycles of 95 °C for 15 s
and 60 °C for 1 min. Isolated human motor neurons required 35 cycles of amplification due to limited amounts of starting material. PCR
products were electrophoresed on 3% agarose, and relative band
intensities were determined by densitometry. The relative level of
expression of each of the genes of interest was calculated relative to
that of actin in the same sample.
Immunohistochemical Analysis of Motor Neurons--
Serial
sections were cut from paraffin-embedded lumbar spinal cords from
normal neurological controls and sporadic ALS cases. Following
de-waxing, immunohistochemistry was performed using standard
techniques. Antigen retrieval (microwave for 10 min in 0.01 M citrate buffer, pH 6.0) was used prior to primary
antibody incubation. Primary rabbit polyclonal antibodies to GST Mu,
LMP7, and ASS were used at a dilution of 1:200 and that to
LTB4 12HD was used at 1:50. Incubations were performed
overnight at 4 °C. Staining was performed using the Vector ABC
technique with DAB, except for LTB4 12HD staining, which
was performed using the Envision kit (Dako). Sections treated
identically, except for omission of the primary antibody, were used as
negative controls. The pattern of staining with each antibody was
recorded using light microscopic examination.
Two-dimensional Gel Analysis and Identification of Differentially
Expressed Proteins in NSC34 Cytosol--
Cytosol from NSC34 cells
stably transfected with pCEP4 vector only, wild-type hSOD1, and G93A
hSOD1 was separated by two-dimensional gel electrophoresis.
Representative silver-stained two-dimensional gels from cells stably
transfected with vector only and cells expressing G93A hSOD1 are shown
in Fig. 1. Typically, up to 700 individual protein spots were detected in NCS34 cytosol within the
10-100-kDa size range using pH 3-10 IPG strips. The positions of
endogenous mouse SOD1 and G93A hSOD1 determined as described are shown
with horizontal arrows (Fig. 1, A and
B). Several landmark spots were identified as follows.
Spots 1 and 2 (Fig. 1A) were identified as nucleotide diphosphate kinase A by Western blotting (results not shown). Spots 3-6 (Fig. 1B) were
all identified as cyclophilin A (cyph A) (Swiss-Prot accession number
P17742) with 75, 68, 57, and 74% sequence coverage, respectively, by
MALDI-TOF-MS analysis. Spot 7 (Fig. 1B) was
identified as glyceraldehyde-3-phosphate dehydrogenase (Swiss-Prot
accession number P16858) with 54% sequence coverage by MALDI-TOF-MS
analysis.
To detect changes in cytosolic protein expression due to G93A hSOD1
expression, two-dimensional gels of cytosol from NSC34 cells
transfected with pCEP4 vector only were compared with those of cytosol
from NSC34 cells expressing G93A hSOD1 in a pairwise fashion. To
control for changes resulting from hSOD1 expression and not the G93A
amino acid change, two-dimensional gels of cytosol from NSC34 cells
transfected with pCEP4 vector only were compared with those of cytosol
from NSC34 cells expressing wild-type hSOD1. We detected 4 spots
reduced (spots D1, D2, D3, and
D4, Fig. 1A, Fig.
2, and Table
II) and 4 spots increased (spots
U1, U2, U3, and U4, Fig.
1B, Fig. 2, and Table II) in intensity in the G93A hSOD1
gels compared with pCEP4 vector only gels. Only one of these 8 spot
changes (Fig. 2, spot D1) was found when pCEP4 vector only gels were compared with wild-type hSOD1 gels indicating that the other
7 spot changes were specific to G93A hSOD1 expression. This was the
only change detected comparing pCEP4 vector only gels with wild-type
hSOD1 gels; therefore, no spot changes were detected due to wild-type
hSOD1 expression that were not seen due to G93A hSOD1 expression.
For identification of the differentially displayed spots by
MALDI-TOF-MS, Coomassie Blue-stained two-dimensional preparative gels
loaded with 100-500 µg of cytosolic protein were used. Only 6 of the
8 differentially displayed spots detected by silver staining were
detected by Coomassie Blue staining even at the highest protein loads
(spots D2, D3, D4, U2, U3, and U4). This prevented the detection and
subsequent identification of the remaining 2 spots (spots D1 and U1) by
MALDI-TOF-MS. The 6 Coomassie Blue-stained differentially displayed
spots were identified by MALDI-TOF-MS analysis (Table III). Spot D2 that was reduced 2.3-fold
in intensity in G93A hSOD1 cells (p < 0.02) (Fig. 2
and Table II) was positively identified as glutathione
S-transferase Mu 1 (GST Mu 1) (Swiss-Prot accession number
P10649). Spots D3 and D4 that were undetectable in G93A hSOD1 cells
(p < 0.02) (Fig. 2 and Table II) were positively identified as hypothetical protein 251000C21Rik protein (Swiss-Prot accession number Q9CPS1) and 20 S proteasome
Spots U2 and U4 that were increased 1.9- (p < 0.02)
and 1.8-fold (p < 0.05) in intensity in G93A hSOD1
cells (Fig. 2 and Table II) were positively identified as
argininosuccinate synthase (ASS) (Swiss-Prot accession number P16460)
and peroxiredoxin I (Prx I) (Swiss-Prot accession number P35700),
respectively (Table III). The observed pI of Prx I (6.9) was more
acidic than the theoretical value (8.26) (Table III). To investigate
whether Prx I occupied an additional spot position, MALDI-TOF-MS
analysis was performed on spot 8 (Fig. 1B) that had an
apparent molecular mass of 25.5 kDa like spot U4, but with an apparent
pI of 8.2. The resulting tryptic peptides exhibited 79% sequence
coverage for Prx I, confirming that the protein also occupied a more
basic position that matched the theoretical pI. MALDI-TOF-MS analysis of spot U3, prepared from cells expressing G93A hSOD1, detected tryptic
peptides that exhibited 44 (9 matched peptides) and 42% (7 matched
peptides) sequence coverage for cyph A (Swiss-Prot accession number
P17742) and RNA-binding motif protein 3 (Rbm3) (Swiss-Prot accession
number O89086), respectively (Table III and Fig.
3B). MALDI-TOF-MS analysis of
spot U3 prepared from cells expressing pCEP4 vector alone detected
tryptic peptides that exhibited 50 (9 matched peptides) and 22% (3 matched peptides) sequence coverage for cyph A and Rbm3, respectively
(Fig. 3A). As none of the matched peptide masses were shared
between the two proteins, it was concluded that spot U3 contained both
cyph A and Rbm3. The number and intensities of peptides matched to Rbm3
were significantly increased in spot U3 from cells expressing G93A
hSOD1 (Fig. 3B) compared with those expressing pCEP4 vector only (Fig. 3A). In contrast the number and intensities of
peptides matched to cyph A were similar between both cell lines (Fig.
3, A and B). It was concluded that the increased
intensity of spot U3 in cells expressing G93A hSOD1 was due to
alteration of Rbm3 levels in the cytosol. We have also demonstrated
that as well as spot U3, cyph A occupies spot positions 3-6 with pI
values of 6.5, 6.7, 6.9, and 7.4 respectively (Fig. 1B and
Table III).
Western Blotting of NSC34 Cytosol with Antisera to Identified
Proteins--
To investigate whether the cytosolic steady-state levels
of these proteins were altered in the presence of G37R hSOD1 as well as
G93A hSOD1, Western blotting analysis was performed on NSC34 cytosol
with antisera to identified proteins where available. Faint 46-kDa
bands corresponding to ASS (Fig.
4A) were detected in cytosol
from cells expressing pCEP4 vector only and wild-type hSOD1
(lanes 1 and 3), whereas strong bands
corresponding to ASS (Fig. 4A) were detected in cytosol from
cells expressing both G93A and G37R hSOD1 (lanes 2 and
4). The antiserum to GST Mu (Fig. 4B) detected a
strong 25-kDa band corresponding to GST Mu in cytosol from cells
expressing pCEP4 vector only and wild-type hSOD1 (lanes 1 and 3), whereas this band was reduced in intensity in
cytosol from cells expressing both G93A and G37R hSOD1 (lanes
2 and 4). The antiserum to LTB4 12HD (Fig.
4C) detected a strong 35-kDa band corresponding to
LTB4 12HD in cytosol from cells expressing pCEP4 vector
only and wild-type hSOD1 (lanes 1 and 3). This
band was undetectable in cytosol from cells expressing both G93A and G37R hSOD1 (lanes 2 and 4). This provided further
evidence that the 251000C21Rik protein is the murine homologue of
LTB4 12HD. The antiserum to LMP7 (Fig. 4D)
detected a strong 23-kDa band corresponding to LMP7 in cytosol from
cells expressing pCEP4 vector only (lane 1). This band was
slightly reduced in intensity in the presence of wild-type hSOD1
(lane 3). In marked contrast this band was weakly detected
in cytosol from cells expressing both G93A and G37R (lanes 2 and 4). These results demonstrate that the cytosolic
steady-state levels of ASS, LMP7, GST Mu, and LTB4 12HD are
altered in G37R hSOD1-expressing cells as well as G93A hSOD1 when
compared with control cells expressing wild-type hSOD1 or pCEP4 vector
only. Although antisera raised to Rbm3 and Prx I were not available to
us, we have observed similar increases in intensities, to those seen in
G93A hSOD1 cells, of the corresponding spots (U3 and U4, respectively)
in cytosol from G37R hSOD1 cells on silver-stained two-dimensional gels
when compared with control cytosol (results not shown).
Argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL)
function to regenerate a pool of intracellular arginine that is
dedicated to the synthesis of nitric oxide via the action of
nitric-oxide synthase (NOS) (37, 38). As steady-state levels of ASS
were increased in cytosol from cells expressing G93A and G37R hSOD1,
the effects of mutant SOD1 expression on the levels of other enzymes
involved in arginine and nitric oxide cycling were determined. Western
blots of NSC34 cytosol were probed with antisera to ASL, neuronal NOS
(nNOS), endothelial NOS, and inducible NOS. There were significant
increases in the intensities of both ASL bands (Fig. 4E) and
nNOS bands (Fig. 4F) in the cytosol from cells expressing
G93A and G37R hSOD1 (lanes 2 and 4) compared with
those in the cytosol from cells expressing pCEP4 vector only and
wild-type hSOD1 (lanes 1 and 3). Neither
endothelial NOS nor inducible NOS were detected in any of the cytosol
samples by Western blotting (results not shown). As well as an increase
in the intensity of ASL bands in the cytosol from cells expressing G93A
and G37R hSOD1 compared with control cells (Fig. 4E), we
also observed an alteration in the pattern of ASL bands. An ASL doublet
was present in cytosol from cells expressing pCEP4 vector only or wild-type hSOD1 (lanes 1 and 3), whereas an ASL
triplet was present in cytosol from cells expressing G93A or G37R hSOD1
(lanes 2 and 4). This result indicated that
mutant SOD1 expression resulted in either differential
post-transcriptional or post-translational processing of the ASL
mRNA or protein, respectively. The nature of this mutant
hSOD1-dependent alteration to ASL expression is currently
under investigation.
Alterations to Glutathione S-transferase Family Members--
As
GST Mu 1 expression was reduced due to mutant hSOD1 expression, we
further investigated the expression and function of other GST family
members expressed in NSC34 cells. The broad range GST substrate
1-chloro-2,4-dinitrobenzene was used to compare the overall GST
activity of the NSC34 cell lines. The GST activity within NSC34 cell
extracts was significantly reduced to ~60% of normal
(p < 0.05) in cells expressing G93A and G37R hSOD1 but not in those expressing the wild-type enzyme (Fig.
5A). In contrast, glutathione
reductase activity was not significantly different in any of the cell
lines (Fig. 5B). The levels of reduced glutathione in the
NSC34 cells were also measured and found to be significantly reduced to
~75% of normal (p < 0.05) in cells expressing G93A and G37R hSOD1 but not in those expressing the wild-type enzyme (Fig.
5C).
It was reasoned to be unlikely that reduced GST Mu 1 expression would
be solely responsible for the dramatic reduction of glutathione
conjugating capacity of the mutant hSOD1 expressing cell lines toward
1-chloro 2,4-dinitrobenzene. A more global loss of GST family members
may have accounted for this markedly decreased activity. To investigate
this further, detergent extracts of NSC34 cells expressing either pCEP4
vector only or G93A hSOD1 were prepared. These extracts were
precipitated with glutathione-Sepharose, and the resulting precipitates
were subjected to analytical two-dimensional gel electrophoresis to
compare the relative expression levels of various GST sub-types between
the two cell lines. Out of 11 spots detectable by Coomassie Blue
staining (Fig. 5, D and E), 10 were positively
identified by MALDI-TOF-MS (Table III). Spot E that was significantly
reduced (p < 0.05) in cells expressing G93A SOD1 was
identified as hypothetical Rik061005A07 protein (Swiss-Prot accession
number Q9DD25) (Fig. 5D and Table
IV). The amino acid sequence of
hypothetical Rik061005A07 protein, derived from the mouse RIKEN
cDNA clone deposited in the FANTOM data base (36), was BLAST
searched. The five highest scoring protein sequences were rat GST Mu 3, human GST Mu 1, human GST Mu 2, rat GST Mu 2, and mouse GST Mu 2 with
95, 83, 81, 80, and 80% sequence identity, respectively, suggesting
that hypothetical Rik061005A07 protein was a Mu class GST. In addition
to hypothetical Rik061005A07 protein, we demonstrated significantly
reduced expression (p < 0.05) of three other Mu class
GST enzymes, namely GST Mu 1 (spot B) (Swiss-Prot accession number
P10649), GST Mu 2 (spot D) (Swiss-Prot accession number P15626),
and GST Mu 5 (spot C) (Swiss-Prot accession number P48774) in cells
expressing G93A hSOD1 (Fig. 5 and Table IV). The most abundant GST
precipitable by glutathione-Sepharose from NSC34 cells was GST Pi B
that occupied 5 individual spot locations (spots F-J). Four of the GST
Pi B spots were significantly less abundant (p < 0.05)
in cells expressing G93A hSOD1 (Table IV and Fig. 5, D and
E). In contrast to GST Mu and GST Pi, GST Alpha 2 (spot A)
(Swiss-Prot accession number P10648) was found to be significantly
increased (p < 0.05) in cells expressing G93A hSOD1
(Table IV and Fig. 5, D and E).
Alterations to Proteasome Activity and Proteasome Subunit
Expression--
Up-regulation of the LMP7 proteasome subunit is known
to promote cleavage after hydrophobic (chymotrypsin-like activity) and basic (trypsin-like activity) residues and suppress cleavage after acidic residues (postglutamyl cleavages) (39). As LMP7 expression was
significantly reduced due to G93A and G37R hSOD1 expression, the
effects of these mutant forms of SOD1 on proteasomal chymotrypsin-like activity, trypsin-like activity, and the postglutamyl hydrolase activity were investigated in the NSC34 cell lines using the model fluorogenic peptides Suc-LLVY-AMC, Z-ARR-AMC, and Z-LLE-AMC,
respectively (Fig. 6, A-C).
All three activities were significantly reduced (p < 0.02) in the presence of G93A and G37R hSOD1 expression, with
chymotrypsin-like activity showing the most marked reduction to ~70%
of normal. There was no significant change in chymotrypsin-like activity (Fig. 6A), but there were small reductions in
trypsin-like (Fig. 6B) and postglutamyl hydrolase activity
(Fig. 6C) in the presence of wild-type hSOD1 expression.
As there was significant reduction in the chymotrypsin-like activity
(Fig. 6A) and the cytosolic levels of the inducible
immunoproteasome subunit LMP7 ( Alterations to Expression of Antioxidant Enzymes, Proteasome, and
Nitric Oxide-related Enzymes of Motor Neurons from ALS Cases--
To
confirm whether the protein changes observed for GST Mu 1, LMP7, nNOS,
ASS, and LTB4 12-HD were accompanied by alterations in the
expression levels of the corresponding mRNAs, RT-PCR analysis was
performed on NSC34 cells expressing pCEP4 vector or G93A hSOD1 (Fig.
7A). Expression levels of ASS,
nNOS, LMP7, and LTB4 12-HD, but not GST Mu 1, were changed
significantly (p < 0.05) due to G93A hSOD1 in NSC34
cells. To investigate whether the expression levels of these
differentially regulated proteins were affected in motor neurons from
human FALS cases, we performed RT-PCR analysis of gene expression in
laser capture microdissected motor neurons from normal individuals or
individuals with FALS attributed to the I113T hSOD1 mutation (Fig.
7A). The expression levels of GST Mu 1, LMP7, and
LTB4 12-HD were significantly reduced (p < 0.05) (Fig. 7A) in FALS cases compared with normal human
controls. In contrast, no significant changes were detected for ASS and
nNOS expression (Fig. 7A).
We set out to investigate the cellular distribution of GST Mu, LMP7,
ASS, and LTB4 12-HD protein expression in normal human spinal cord. Immunohistochemical staining was performed on sections cut
from paraffin-embedded lumbar spinal cords from normal individuals along with tissue from SALS cases (Fig. 7B) as a comparison.
GST Mu, LMP7, and ASS were diffusely expressed in white and gray matter of the spinal cord. These enzymes were strongly expressed in the cytoplasm and in neurites of motor neurons. The intensity of neuronal cytoplasmic staining tended to be less for all three proteins in the
SALS cases (Fig. 7B). Neuronal NOS has already been shown to
be up-regulated at the protein level in human motor neurons from ALS
cases (40). LTB4 12-HD staining was only weakly detected in
the neuronal cytoplasm in both SALS cases and controls. Interestingly, LTB4 12-HD staining was observed in the nuclei of motor
neurons and glia in SALS cases and controls.
Several recent studies (24-26) employing genomic profiling
technologies to identify gene expression changes in ALS have
concentrated on whole spinal cord tissue from human ALS cases and
transgenic mouse models of FALS. However, due to the low proportion of
motor neurons compared with other cell types in spinal cord, the
primary responses of motor neurons to mutant SOD1 toxicity that trigger cell death pathways may go undetected among the overall tissue transcriptional changes responding to motor neuron degeneration. To
this end we have exploited a well characterized cell culture model of
mutant SOD1-related FALS (27-29) to enable us to analyze the proteome
changes that occur as a direct result of SOD1 toxicity in cells with a
motor neuron phenotype.
By using a combination of two-dimensional electrophoresis, mass
spectrometry, and Western blotting, we identified seven up-regulated proteins as ASS, ASL, nNOS, Rbm 3, Prx I, subunit X, and GST Alpha 2. Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu homologue, GST Pi B, LTB4
12HD, and LMP7. We also demonstrated that the mRNA expression levels of GST Mu 1, LTB4 12HD, and LMP7 were similarly
changed in motor neurons isolated from FALS cases. In the case of GST Mu 1 in the cell culture model, and nNOS in the model and isolated motor neurons, the protein changes were not reflected by the mRNA levels as determined by RT-PCR. This was probably due to the
semi-quantitative nature of the RT-PCR technique, as recent studies in
our laboratory employing the Affymetrix Murine Genome oligonucleotide
array have demonstrated 2.3-fold up-regulation and 7.6-fold
down-regulation of nNOS and GST Mu 1 mRNA
respectively,2 due to
expression of G93A hSOD1 in NSC34 cells. Surprisingly, only one protein
change (spot D1) was detected due to normal hSOD1 expression rather
than the G93A mutant SOD1 protein. Further changes may have been
detected using narrower pH ranges for the first dimension IEF along
with a staining technique with a wider range of linearity than silver.
However, it is of interest that our results using the Affymetrix
oligonucleotide array also reveal only minor effects of wild-type hSOD1
on the mRNA expression profile of NSC34 cells compared with those
of G93A hSOD1.2
The differentially regulated proteins fall into four categories: (i)
proteins involved in regulation of mRNA processing (Rbm3); (ii)
proteins involved in NO metabolism (ASS, ASL, and nNOS); (iii) proteins
involved in anti-oxidant defense (Alpha, Mu, and Pi class GSTs,
LTB4 12HD, and Prx I); and (iv) proteins involved in
protein degradation (LMP7, subunit X). Our results provide further
evidence for mutant SOD1-mediated alterations in the intracellular redox state and protein degradation machinery, which in turn supports the hypothesis that both altered free radical handling and abnormal protein aggregation are likely to be mechanisms contributing to motor
neuron injury.
Rbm3 was shown to have elevated cytosolic levels in the presence of
G93A hSOD1 expression. To date the exact function of this protein is
unclear. It is a heterogeneous nuclear ribonucleoprotein that contains
an N-terminal consensus sequence RNA binding domain and a C-terminal
glycine-rich domain. Proteins with such domains have been shown to
regulate mRNA stability and translation, mRNA splicing, and
export of mRNA from the nucleus to the cytoplasm (41). The raised
levels of this heterogeneous nuclear ribonucleoprotein in the cytosol
in the presence of G93A hSOD1 expression may indicate alterations to
protein biogenesis at the level of post-transcriptional mRNA
processing and/or mRNA translation.
ASS acts in conjunction with ASL to regenerate arginine from citrulline
for the purpose of nitric oxide production by NOS (37, 38). As with
ASS, we found that both ASL and nNOS were up-regulated by G93A and G37R
hSOD1. Additionally, ASL was shown to undergo mutant hSOD1-specific
alterations to either its post-transcriptional or post-translational
processing. The human ASL gene product has been shown previously to
undergo highly variable splicing (42). The nature of the alteration
here to the mouse ASL gene product is currently under investigation.
By using microelectrode biosensor measurements, we have determined
previously that NSC34 cells expressing wild-type hSOD1 exhibit enhanced
NO release, whereas those expressing ALS mutant hSOD1 exhibit reduced
NO release following cell stress induced by serum withdrawal (29). Both
groups of cells expressing wild-type or mutant hSOD1 show decreased
superoxide release in the same experimental paradigm. In addition, the
mutant SOD1-expressing NSC34 cells are more sensitive to apoptosis
stimulated by NO-releasing compounds (29). The mechanism by which NO
release is reduced in the mutant SOD1-expressing NSC34 cells has yet to
be determined. The reduction may result in a compensatory response by
the cells in the form of up-regulation of the arginine/NO recycling
pathway. The role of NO in ALS pathogenesis remains controversial due
to its dual role as a neuroprotective and neurotoxic agent. At low concentrations NO can protect cells against oxidative stress, presumably by induction of adaptive responses in the form of
up-regulation of antioxidant proteins (43, 44). In contrast, inhibition of nNOS has been shown to protect rat motor neurons from cell death
induced by oxidative stress (45). The findings that inhibition of nNOS
in a cell culture model of FALS did not confer resistance to mutant
SOD1 toxicity (46) and that blockade of NOS either by chemical means
(47) or targeted deletion of the nNOS gene (48) had little effect on
disease progression in a transgenic mouse model of human ALS have
raised uncertainties regarding the importance of NO production in ALS
pathogenesis. However, the relevance of our findings here to ALS is
reinforced by studies reporting nNOS up-regulation in human ALS cases
(40, 49) and inducible NOS up-regulation in transgenic mice expressing
ALS hSOD1 mutants (50). Furthermore, increased levels of nitrotyrosine have been shown in human cerebrospinal fluid in sporadic ALS (SALS) (51) as well the spinal cord of a transgenic mouse model (15). We
anticipate that exploitation of recently developed "nitroproteomic" techniques (52, 53) will eventually clarify the contribution of altered
nitric oxide metabolism to ALS.
Expression of mutant hSOD1 resulted in a complete loss of the
LTB4 12HD protein from NSC34 cells. LTB4 12HD
may have a wider role in antioxidant defense as well as in lipid
messenger metabolism. A recent study (54) demonstrated that
LTB4 12HD was effective at reducing a wide variety of
cytotoxic Other antioxidant enzymes that were down-regulated were GST Mu 1, GST
Mu 2, GST Mu 5, GST Pi B, and a hypothetical Mu class GST Rik061005A07.
The GST family catalyzes the conjugation of reduced glutathione to
toxic compounds to allow their elimination from cells by glutathione
conjugate transporters (58). Here we demonstrated this function of GST
to be significantly impaired in G93A and G37R hSOD1-expressing NSC34
cells. Previously, human neuroblastoma cells overexpressing mouse GST
Mu showed increased resistance to cell death induced by hydrogen
peroxide, peroxynitrite, and HNE (59). Human GST Mu when added to rat
neuronal cultures protected against HNE cytotoxicity (60). Additional
to their detoxification role, GST family members have also been shown
to regulate apoptotic pathways. Mouse GST Mu has been shown recently to
interact physically with and inhibit apoptosis signal-regulating kinase
1 (ASK1) that is known to activate the stress-activated protein
kinase/c-Jun N-terminal kinase and p38 pathways (61).
Prx I was shown to occupy at least two spot positions on our
two-dimensional gels. The most abundant of the two forms, which was
unchanged on our gels, had an apparent pI (8.2) in close agreement with
the theoretical value (8.26) for Prx I. The less abundant form, which
was up-regulated in the presence of mutant SOD1 expression, had a more
acidic pI of 6.9. Prx I, II, and III (62, 63) have all been shown to
undergo acidic shifts when cultured cells are subjected to
hydroperoxide-mediated oxidative stress. A recent study (64) has
demonstrated that the acidic shift of Prx II produced under conditions
of oxidative stress was due to conversion of an essential active site
cysteine to cysteic acid. The possibility that Prx I undergoes
over-oxidation of its cysteine residues due to mutant SOD1 expression
therefore warrants further investigation.
All of the antioxidant proteins shown here to be down-regulated (GST
family and LTB4 12HD) or post-translationally modified (Prx
I) have been shown previously to either contain the
antioxidant-response element in their genes, undergo nuclear factor E2
p45-related factor (Nrf2)- dependent transcriptional regulation
via antioxidant-response element binding, and/or undergo up-regulation
by chemical agents known to activate Nrf2 (65, 66). It is
tempting to speculate that this down-regulation is due to alterations
in Nrf2 status. The down-regulation of genes encoding
antioxidant proteins is likely to contribute significantly to the
increased sensitivity of the mutant SOD1-expressing NSC34 cells to
oxidative stress (27). Identification of the primary alteration to SOD1
biochemistry that drives this transcriptional down-regulation of
antioxidant proteins may prove to be insightful in determining how the
mutant enzyme triggers cell death in motor neurons.
The expression of both G93A and G37R hSOD1 resulted in a significant
reduction in amount of the LMP7 proteasome subunit ( In conclusion we have used proteomic approaches to identify protein
alterations in a cell culture model of SOD1-related FALS. The novel
approach in this study is that we have applied proteomic technology to
motor neuronal cells expressing mutant SOD1 at levels approximating
those seen in human FALS. Several of the protein changes were
corroborated at the transcriptional level in motor neurons isolated
from human FALS cases. The proteome alterations we have identified are
accompanied by functional consequences and may contribute to or
represent responses to cellular changes that ultimately trigger cell
death pathways resulting in neurodegeneration. These protein changes
provide further evidence for the altered free radical handling and
protein aggregation hypotheses to explain the toxic gain-of-function of
FALS-associated mutant SOD1.
5 (X), and
glutathione S-transferase (GST) Alpha 2. Seven
down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu
5, a hypothetical GST Mu, GST Pi B, leukotriene B4
12-hydroxydehydrogenase, and proteasome subunit
5i (LMP7). GST
assays demonstrated a significant reduction in the total GST activity
of cells expressing mutant human SOD1. Proteasome assays demonstrated
significant reductions in chymotrypsin-like, trypsin-like, and
post-glutamylhydrolase proteasome activities. Laser capture
microdissection of spinal cord motor neurons from human FALS cases, in
conjunction with reverse transcriptase-PCR, demonstrated decreased
levels of mRNA encoding GST Mu 1, leukotriene B4
12-hydroxydehydrogenase, and LMP7. These combined approaches provide
further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the
pathophysiology of FALS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primer pairs used for RT-PCR analysis of gene expression in human motor
neurones and mouse NSC34 cells
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Two-dimensional electrophoresis of NSC34
cytosol. Cytosol containing 25 µg of protein from NSC34 cells
stably transfected with pCEP4 vector only (A) or expressing
G93A hSOD1 (B) was separated by IEF using 17-cm, pH 3-10,
IPG strips. Second dimension SDS-PAGE was performed on 14%
polyacrylamide gels, and the proteins were visualized by silver
staining. Horizontal arrows indicate the positions of spots
corresponding to endogenous mouse SOD1 and recombinant G93A hSOD1.
Arrowheads indicate the positions of spots corresponding to
nucleotide diphosphate kinase A (A, spots 1 and
2), cyclophilin A (B, spots 3-6),
glyceraldehyde-3-phosphate dehydrogenase (B, spot
7), and peroxiredoxin I (B, spot 8).
Vertical arrows indicate spots decreased (A,
spots D1-4) and increased (B, spots U1-4) in
the presence of G93A hSOD1 expression. Molecular weight and pI
calibration were performed using two-dimensional molecular weight and
pI standards.
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Fig. 2.
Protein spots changed in intensity due to
G93A hSOD1 expression. Cytosol containing 25 µg of protein from
NSC34 cells stably transfected with pCEP4 vector only, expressing
wild-type hSOD1 or expressing G93A hSOD1, were separated by IEF using
17-cm, pH 3-10, IPG strips. Second dimension SDS-PAGE was performed on
14% polyacrylamide gels, and the proteins were visualized by silver
staining. Specific regions of the gels containing spots of interest are
shown in each panel. Diagonal arrows
indicate the positions of spots changed in the presence of G93A hSOD1
expression. Spots decreasing or increasing in intensity are prefixed
with D or U, respectively.
Pairwise analyses of protein spot intensities in vector only gels
compared with wild-type hSOD1 gels and vector only gels compared
with G93A hSOD1 gels
5i subunit (LMP7) (Swiss-Prot accession number P28063), respectively (Table III). The
hypothetical 251000C21Rik protein sequence derived from the mouse RIKEN
cDNA clone 251000C21RIK deposited in the Functional Annotation of
Mouse (FANTOM) data base (36), at
www.gsc.riken.go.jp/e/FANTOM, was BLAST searched. The three
highest scoring protein sequences were porcine, human, and rabbit
NADP-dependent leukotriene B4 12-hydroxydehydrogenase (LTB4 12HD), with 82.4, 79.7, and
76.9% sequence identity, respectively. This suggested that
251000C21Rik protein represented a mouse homologue of LTB4
12HD.
Identities of differentially displayed protein spots determined by
MALDI-TOF-MS analysis
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Fig. 3.
Relative intensities of cyph A and Rbm3
tryptic peptides from spot U3 detected by MALDI-TOF-MS. Tryptic
digests of protein spot U3 from NSC34 cells stably transfected with
pCEP4 vector only (A) or expressing G93A h SOD1
(B) were analyzed by MALDI-TOF-MS. Peptide peaks that match
to cyph A (open diamonds) and Rbm3 (filled
squares) are shown. Intensities of the peptide peaks are expressed
as percentages of the highest peak height of the matched peptides and
plotted against their masses.
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Fig. 4.
Western blotting of NSC34 cytosol with
antisera to differentially expressed proteins. Cytosol containing
20 (A-D) or 10 µg of protein (E-G) from NSC34
cells stably transfected with pCEP4 vector only (lane 1),
expressing G93A hSOD1 (lane 2), expressing wild-type hSOD1
(lane 3), or expressing G37R hSOD1 (lane 4) were
Western-blotted using antisera to ASS (A) diluted 1:500, GST
Mu (B) diluted 1:1000, LTB4 12HD (C)
diluted 1:1000, LMP7 (D) diluted 1:2000, ASL (E)
diluted 1:500, nNOS (F) diluted 1:250, and actin
(G) diluted 1:1000. Proteins were visualized using enhanced
chemiluminescence.
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Fig. 5.
Alterations to glutathione handling due to
ALS mutant SOD1 in NSC34 cells. A-C, assays. Lysates
prepared from NSC34 cells stably transfected with pCEP4 vector only
(white bars), expressing wild-type hSOD1 (black
bars), expressing G37R hSOD1 (light gray bars), or
expressing G93A hSOD1 (dark gray bars) were assayed for
glutathione S-transferase activity (A), glutathione
reductase activity (B), and reduced glutathione
concentration (C). For the enzyme activities (A
and B), assays were performed in triplicate on lysates
extracted on 4 separate occasions. Specific activities are expressed as
percentage of pCEP4 vector control. For reduced glutathione
determinations (C), measurements were performed in
triplicate on lysates extracted on 3 separate occasions. Reduced
glutathione concentrations are expressed as nanomoles of GSH/1 × 106 cells. Two-dimensional gel analysis (D and
E), lysates containing 2.5 mg of protein from NSC34 cells
stably transfected with pCEP4 vector only (D) or expressing
G93A hSOD1 (E) were precipitated with glutathione-Sepharose.
The resulting precipitates were subjected to IEF on pH 3-10 IPG strips
followed by SDS-PAGE on 14% polyacrylamide gels. Gels stained with
Coomassie Blue are shown.
Relative expression levels and MALDI-TOF-MS analysis of
glutathione-Sepharose binding proteins in vector only and G93A
hSOD1-expressing NSC34 cells
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Fig. 6.
Alterations to proteasome activity and
subunit expression due to ALS mutant SOD1 in NSC34 cells.
A-C, assays. Lysates prepared from NSC34 cells stably
transfected with pCEP4 vector only (white bars), expressing
wild-type hSOD1 (black bars), expressing G37R hSOD1
(light gray bars), or expressing G93A hSOD1 (dark gray
bars) were assayed for chymotrypsin-like activity using 50 µM Suc-LLVY-AMC (A), trypsin-like activity
using 100 µM Z-ARR-AMC (B), and post-glutamyl
hydrolase activity using 100 µM Z-LLE-AMC (C).
Six fluorescence measurements were taken from lysates extracted on 4 separate occasions. Specific activities are expressed as percentage of
pCEP4 vector control. D-F, Western blots and
densitometry. Lysates prepared from NSC34 cells stably transfected with
pCEP4 vector only (lane 1 of blot, white bars on
histogram), expressing wild-type hSOD1 (lane 2, black
bars), expressing G37R hSOD1 (lane 3, light gray
bars), or expressing G93A hSOD1 (lane 4, dark
gray bars) were Western-blotted using antisera to LMP7
(D), subunit X (E), and LMP2 (F) all
diluted 1:1000. Proteins were visualized using enhanced
chemiluminescence. The relevant bands, from lysates extracted on 4 separate occasions, were quantified by densitometry, and their
intensities are expressed as percentages of pCEP4 vector control.
5i) due to mutant hSOD1 expression
(Fig. 4), we investigated whether there were alterations in the level
of expression of the constitutive
5 proteasome subunit, subunit X,
and the other inducible immunoproteasome subunit LMP2 (
1i). Western
blotting of the proteasome assay extracts with anti-LMP7 serum (Fig.
6D) confirmed the dramatic reduction of LMP7 expression
(Fig. 4) in cytosol of cells expressing G93A and G37R hSOD1 (24 and
14%, respectively) and a less dramatic reduction in cells expressing
wild-type hSOD1 (66%). In the case of the constitutive subunit (Fig.
6E), Western blotting detected a significant increase
(p < 0.05) in expression of subunit X (
5) in cells
expressing G93A and G37R hSOD1 (142 and 158%, respectively) that
mirrored the decrease in LMP7. Paradoxically, this coincided with
decreased postglutamyl hydrolase activity (Fig. 6C). We
detected no significant change in the level of LMP2 expression (Fig.
6F) in cells expressing wild-type or G37R hSOD1, but we
observed a small reduction (p < 0.05) in LMP2
expression in the cells expressing G93A hSOD1 (Fig. 6F). As
there were substantial amounts of LMP2 remaining in the presence of
hSOD1 expression (Fig. 6F), this probably accounts for the
remaining chymotrypsin-like activity toward Suc-LLVY-AMC (Fig.
6A).
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Fig. 7.
RT-PCR and immunohistochemical
analysis of human motor neurons. RT-PCR (A), relative
expression levels of ASS, nNOS, GST Mu 1, LMP7, and LTB4
12-HD in NSC34 cells expressing pCEP4 vector (white bars) or
G93A hSOD1 (black bars), and in laser captured human motor
neurons from normal controls (white bars) or I113T SOD1 FALS
cases (black bars) were measured by densitometry of RT-PCR
products generated on at least 5 separate occasions. The levels are
expressed relative to that of actin. B,
immunohistochemistry. Human lumbar spinal cord sections from normal
neurological controls and sporadic ALS cases were stained using
antisera raised to ASS, GST Mu, LMP7, and LTB4 12-HD.
Cytoplasmic staining in anterior horn motor neurons is indicated with
an asterisk. Focal granular brown staining in the cytoplasm
of neurons from SALS cases represents lipofuscin, indicated with a
chevron. Nuclear staining of LTB4 12-HD, in
anterior horn motor neurons and glia, is indicated with
large and small arrows, respectively. Black
bar indicates 100 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated aldehydes and ketones including products
of lipid peroxidation such as 4-hydroxy-2-nonenal (HNE). HNE has been
shown to be elevated in the cerebrospinal fluid of SALS cases (55) and
SALS spinal cord (56, 57). A potential chemoprotective role for the
enzyme was suggested by its ability to confer resistance of
LTB4 12HD-transfected cell lines to HNE-induced apoptosis
(54). The association of a significant amount of LTB4 12HD
with membranes (54) and the nuclear associated staining of motor
neurons in this study supports the notion that the enzyme may help to
protect membranes from oxidative damage.
5i) that
coincided with a reduction in chymotrypsin-like activity and an
increase in the amount of subunit X (
5). The 20 S proteasome can
contain three interferon-
-inducible
subunits LMP7, LMP2, and
MECL that are homologous and interchangeable with three constitutive
subunits X, Y, and Z, respectively (39, 67-69). It is likely that
interferon-
-inducible
subunits favor the production of peptides
with basic or hydrophobic C termini that are most suited to transport
into the endoplasmic reticulum via the transporter associated with
antigen presentation prior to major histocompatibility complex class I
presentation. Several studies have reported proteasome activity and
subunit content alteration in response to disease-associated protein
aggregation and/or oxidative stress (70-73). SOD1-containing aggregates have been observed in both transgenic mouse (19) and cell
culture models of FALS (20, 21). Similarly ubiquitinated protein
aggregates have been observed in human SALS (74). The down-regulation
of LMP7 and up-regulation of its displacing partner, subunit X, may
suggest an adaptive response to a challenge to the proteasome resulting
from ALS mutant SOD1 expression. The observed reduction in proteasome
activity may contribute over time to the abnormal aggregation of
intracellular proteins observed in motor neurons, in cellular and
animal models as well as human disease.
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ACKNOWLEDGEMENTS |
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We thank Liz Stewart for performing the MALDI-TOF-MS analysis and data base searching; Gillian Forster, Lynne Baxter, and Catherine Gelsthorpe for performing the immunohistochemical analysis of spinal cord; Neil Cashman for providing the parent NSC34 cell line; Denise Figlewicz for providing the hSOD1 expression constructs; Michael Blackburn and Alan Spivey (Department of Chemistry, University of Sheffield) for use of the fluorimeter; and all of our colleagues who provided antibodies.
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FOOTNOTES |
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* 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.
§ Supported by the Wellcome Trust.
¶ Supported by the Motor Neurone Disease Association.
§§ To whom correspondence should be addressed: Academic Unit of Neurology, Division of Genomic Medicine, Beech Hill Rd., University of Sheffield, Sheffield, S10 2RX, UK. Tel.: 114-2712473; Fax: 114- 2261201; E-mail: simon.allen@sheffield.ac.uk.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M209915200
2 J. Kirby, P. R. Heath, and P. J. Shaw, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu,Zn-superoxide dismutase; GST, glutathione S-transferase; hSOD, human SOD; mSOD, mouse SOD1; FALS, familial amyotrophic lateral sclerosis; RT, reverse transcriptase; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ASS, argininosuccinate synthase; NOS, nitric-oxide synthase; nNOS, neuronal NOS; AMC, 7-amino-4-methylcoumarin; Z, benzyloxycarbonyl; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IPG, immobilized pH gradient gel; IEF, isoelectric focusing; DAB, 3,3'-diaminobenzidine; LTB4 12HD, leukotriene B4 12-hydroxydehydrogenase; cyph A, cyclophilin A; Prx I, peroxiredoxin I; ASL, argininosuccinate lyase; SALS, sporadic ALS; HNE, 4-hydroxy-2-nonenal; CSF, cerebrospinal fluid.
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