From the Departments of a Neuroscience and f Neurology, b Section of Old Age Psychiatry, Institute of Psychiatry, Denmark Hill, London SE5 8AF, United Kingdom and h Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, November 3, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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The neuronal adaptor protein X11 The X11s, also known as mints (munc18
interacting proteins 1-3), are a family of adaptor
proteins with three members ( X11 The full complement of ligands that bind to the X11 proteins is not yet
known. In this study, we demonstrate that, via its second PDZ domain
(PDZ2), X11 Yeast Two-hybrid System--
Interactive cloning experiments,
including relevant controls, were performed as described previously
(2). In brief, the sequence encoding the two human X11 Mammalian Expression Plasmids and Transfection--
Chinese
hamster ovary (CHO) cells and rat primary cortical neurones were
cultured as described previously (8, 30). CHO cells were transfected
using LipofectAMINE (Life Technologies, Inc.) as per the
manufacturer's instructions. Full-length C-terminal Myc-tagged human
X11 Antibodies--
X11 Coimmunoprecipitation Studies--
CHO cells were doubly or
singly transfected as indicated with Myc-tagged X11 GST Fusion Protein Binding Assays--
Glutathione
S-transferase (GST) fusion proteins with X11 Indirect Immunofluorescence--
Transfected CHO cells and rat
neuronal cultures were fixed and prepared for immunofluorescence as
described (8, 22, 34). X11 SOD1 Activity Assays--
CHO cells were transfected with 2.5 µg of SOD1, X11 X11
Human CCS is comprised of three functional domains (31, 33). The
N-terminal domain I (amino acids 1-86) is homologous to the secretory
pathway copper chaperone HAH1 and its yeast ortholog Atx1p (37); the
central domain II is ~50% identical to SOD1 (24); and the C-terminal
domain III is unique to CCS (Fig. 1B). As all of domain II
of CCS was encoded in the CCS cDNA isolated in the yeast two-hybrid
screen, this raised the possibility that X11 X11
In a complementary series of experiments, we used GST fusion proteins
containing different domains of CCS in pull-down experiments from
X11 Cellular Distribution of X11 X11 In the present study we have demonstrated that human X11 The anti-oxidant enzyme SOD1 catalyzes the disproportionation of
superoxide anions through redox cycling of the bound copper ion in each
monomer (40). Copper is delivered to SOD1 via CCS, and CCS is thus
required for SOD1 activity (24, 25, 27, 28, 43). CCS comprises three
domains as follows: an N-terminal domain I that is homologous to the
Atx1p metallochaperone and that is involved in the recruitment of
copper; a central domain II that exhibits homology to SOD1 and that
facilitates the interaction of CCS with SOD1; and a C-terminal domain
III that also binds copper but that additionally is involved in the
interaction with SOD1 (26, 31, 44, 45). We mapped the X11 Overexpression of X11 Altered copper homeostasis may be at least part of the pathogenic
process in several neurodegenerative diseases (for reviews see Refs. 46
and 47). In particular, mutations in SOD1 are the causative genetic
defect in some familial forms of amyotrophic lateral sclerosis (ALS)
(48, 49). ALS mutant SOD1 proteins can bind CCS, and it has been
suggested that mutant SOD1 may exert a toxic effect via altered
cellular copper chemistry (25). In addition to binding CCS, X11 participates
in the formation of multiprotein complexes and intracellular
trafficking. It contains a series of discrete protein-protein
interaction domains including two contiguous C-terminal PDZ domains. We
used the yeast two-hybrid system to screen for proteins that interact
with the PDZ domains of human X11
, and we isolated a clone encoding
domains II and III of the copper chaperone for Cu,Zn-superoxide
dismutase-1 (CCS). The X11
/CCS interaction was confirmed in
coimmunoprecipitation studies plus glutathione
S-transferase fusion protein pull-down assays and was shown
to be mediated via PDZ2 of X11
and a sequence within the carboxyl
terminus of domain III of CCS. CCS delivers the copper cofactor to the
antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for
its activity. Overexpression of X11
inhibited SOD1 activity in
transfected Chinese hamster ovary cells which suggests that X11
binding to CCS is inhibitory to SOD1 activation. X11
also interacts
with another copper-binding protein found in neurons, the Alzheimer's
disease amyloid precursor protein. Thus, X11
may participate in
copper homeostasis within neurons.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
) encoded by separate
genes on, respectively, human chromosomes 9, 15, and 19 (1-8).
Expression of X11
and X11
is restricted to neurons, whereas
X11
is ubiquitously expressed (1, 4, 6-9). The X11s diverge
substantially in their N-terminal regions, but they all contain a
centrally located phosphotyrosine binding domain (10), through which
all three X11s bind to the cytoplasmic domain of the Alzheimer's
disease amyloid precursor protein
(APP)1 (2, 6, 7, 11, 12), and
two contiguous C-terminal PDZ (PSD-95,
Drosophila disks-large, ZO-1)
domains (13). These, and a variety of other less well characterized
protein-protein interaction regions (Fig. 1A), mediate the
binding of the X11s to a number of proteins.
binds to the pre-synaptic adaptor protein CASK via a sequence
preceding the phosphotyrosine binding domain (9, 14, 15). CASK also
binds both Veli1 and members of the neurexin family of pre-synaptic
membrane-spanning proteins (14, 16). The X11
-CASK-Veli
pre-synaptic complex is highly evolutionarily conserved and is found in
orthologous form in Caenorhabditis elegans as the
LIN-10-LIN-2-LIN-7 complex (17) that regulates basolateral sorting of
the epidermal growth factor receptor LET-23 and maintains cell polarity
(18, 19). X11
also binds to the synaptic vesicle docking protein
Munc-18 through an N-terminal region (3, 4, 14). Via its first
PDZ domain (PDZ1), X11
has been reported to bind both the kinesin
superfamily motor protein KIF17 (20) and the C terminus of the N-type
Ca2+ channel pore-forming
1B-subunit, which
in turn can bind the SH3 region of CASK (21). Finally, we have shown
that X11
binds, via both of its PDZ domains, to presenilin-1 and
mediates interactions between presenilin-1 and APP (22). Thus, similar
to several other PDZ-bearing proteins (for reviews see Refs. 13 and
23), X11
appears to provide a framework, or scaffolding, for the
assembly of multimolecular complexes and functions in the trafficking
and sorting of proteins to different neuronal compartments.
interacts with the copper chaperone for copper/zinc
superoxide dismutase-1 (CCS). CCS delivers and inserts the copper
cofactor into the antioxidant enzyme copper/zinc superoxide dismutase-1
(SOD1) and is required for its activation (24-28). We also demonstrate
that overexpression of X11
inhibits SOD1 activity in transfected
cells, consistent with a role for X11
in regulating CCS function and
SOD1 activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PDZ domains
(PDZ1 and -2; X11
amino acid sequence 649-837) was amplified by
polymerase chain reaction (22), subcloned into the EcoRI
site of the yeast "bait" vector pY3 (29), and used to screen a
human brain cDNA library (CLONTECH). Yeast
colonies were grown using selective media lacking tryptophan, leucine,
and histidine. Vigorously growing colonies were subjected to
freeze-fracture
-galactosidase assays, and candidate library
plasmids were rescued from positive colonies by transformation into
Escherichia coli HB101. Brain library cDNA inserts were
sequenced using a Cyclist Exo
Pfu DNA
sequencing kit (Stratagene).
was as described previously (22). The cDNA encoding human
CCS (24) was subcloned into pCDNA3.1 (Invitrogen), and human SOD1
was expressed in pCIneo (Promega).
was detected using rabbit polyclonal
antibodies raised against X11
-(161-421) (22). The same immunogen
was also used to generate mouse polyclonal antibodies. Antibody
specificity was confirmed by competing out signals with immunogen (data
not shown). Rabbit polyclonal antibodies raised against CCS residues 1-85 were used to detect CCS (26). SOD1 sheep polyclonal antibody was
purchased from Calbiochem, and the 9E10 anti-Myc monoclonal antibody
was purchased from Sigma.
, CCS, and SOD1.
Transfected cells were harvested in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 5 µg/ml pepstatin, and 0.25 mM phenylmethylsulfonyl
fluoride) and incubated on ice for 20 min. In binding studies with
transfected SOD1, the cell lysates also underwent one freeze-thaw cycle
(26). Lysates were then centrifuged at 14,000 rpm for 10 min at
4 °C. For immunoprecipitation studies the supernatant was precleared
with protein A-Sepharose beads (Sigma). Myc-tagged X11
was
immunoprecipitated from 500 µg of total protein lysate using antibody
9E10. The antibody was captured using protein A-Sepharose beads that
were then washed four times with ice-cold lysis buffer.
Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting as
described (8).
PDZ1-(649-746), PDZ2-(742-837), and PDZ1 + 2-(649-837) were as
described (22). The partial CCS clone (CCS-(48-274)) isolated in the
library screen encoded the C-terminal 227 amino acids of CCS,
incorporating the C-terminal 39 residues of domain I and all of domains
II and III of CCS (Fig. 1B). Digestion of this clone with
BglII released cDNAs encoding CCS-(48-241) and
CCS-(243-274) that were subcloned into the BamHI site of
pGEX-5X-1 (Amersham Pharmacia Biotech). CCS-(48-241) encompasses all
of domain II of CCS (amino acids 87-234), whereas CCS-(243-274)
encodes the C-terminal 32 amino acids of CCS including the
CXC copper-binding motif (31-33). Digestion of the library
clone with EcoRI released the CCS-(48-274) insert, which
was subcloned into pGEX-5X-1. Fusion proteins containing full-length
CCS or the cytoplasmic domain of APP were as described previously (26,
34). Expression and purification of GST fusion proteins, plus their use
as ligands in pull-down binding assays, were essentially as described
(22). The immunoprecipitation and GST fusion assays to confirm the
X11
and CCS interaction were performed three times with similar results.
was detected in transfected CHO cells
using the 9E10 antibody to the Myc tag; endogenous X11
in primary
rat cortical neurons was detected with the mouse X11
antiserum. The
rabbit antiserum was used to detect CCS in both transfected CHO cells
and in neurons. Antibodies were visualized by goat anti-mouse Igs
coupled to Texas Red and goat anti-rabbit Igs coupled to Oregon Green
(Molecular Probes).
, and CCS DNA as indicated; transfections receiving
only one or two plasmids were balanced with empty vector such that all
received the same total amount of DNA. Transfected cells were washed
twice with PBS, harvested by scraping into cold PBS, and then
centrifuged at 10,000 × g for 5 min at 4 °C. The
pelleted cells were resuspended and lysed in water by a freeze-thaw
cycle as described (35), centrifuged at 10,000 × g for
5 min, and the supernatant collected. A sample of each supernatant was
removed for SDS-PAGE and immunoblot analyses; the remainder was
adjusted to 0.125 M Tris chloride, pH 6.8, 20% (v/v)
glycerol, 0.025% bromphenol blue, and 0.1% Nonidet P-40 (35). 30 µg
of protein from each sample was run on 10% nondenaturing
polyacrylamide gels, and SOD1 activities were determined by nitro blue
tetrazolium in-gel staining assays as described (36). 1 µg of protein
from each supernatant sample was analyzed by immunoblotting to
determine the expression of SOD1, X11
, and CCS. Protein quantities
were assayed using Bradford reagent (Sigma) as per the manufacturer's
instructions. SOD1 activity assay gels and immunoblots were further
analyzed by pixel densitometry using a Bio-Rad GS710 scanner and
Quantity 1 software to obtain the ratio of SOD1 activity to expressed
protein level for each sample (35). Within each set of transfections,
this ratio was set at 100% for the SOD1-only transfected cells, and
the remaining activities were expressed relative to this. Data from
three separate sets of transfections were analyzed together using the
Kruskal-Wallis one-way analysis of variance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Binds to CCS--
To identify binding partners for the
X11
PDZ1 + 2 region, we used the yeast two-hybrid system to screen a
human brain cDNA library. A cDNA encoding the C-terminal 227 amino acids of CCS (CCS-(48-274)) was isolated (Fig.
1B). To investigate if the
full-length proteins could interact in mammalian cells,
immunoprecipitation experiments were performed from X11
, CCS, and
X11
/CCS cotransfected CHO cells. X11
was immunoprecipitated using
the 9E10 antibody to the Myc tag. CCS was present only in
immunoprecipitates obtained from X11
/CCS-cotransfected cells but not
X11
or CCS alone transfected cells (Fig.
2A). Thus CCS
coimmunoprecipitates with X11
from transfected CHO cells.
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Fig. 1.
Schematic representation of human
X11 and CCS. A, structure of
human X11
. MI, Munc-18 interacting region;
CI, CASK interacting region; PTB, phosphotyrosine
binding domain; PDZ, PSD-95,
Drosophila disks-large, ZO-1 domain.
The sequences used in this study encompassing PDZ1, PDZ2, and PDZ1 + 2 are indicated. B, structure of CCS depicting the location of
domains I-III and the copper binding regions of CCS. The sequences
identified and used in this study are indicated as CCS-(48-241),
CCS-(243-274), and CCS-(48-274) as described in the text.
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Fig. 2.
X11 interacts with
CCS. A, immunoblot analysis of lysates and
immunoprecipitates (IPs) from CHO cells transfected with
Myc-tagged X11
, CCS, or X11
+ CCS. X11
was immunoprecipitated
with 9E10 monoclonal antibody to the Myc tag, and the samples were
analyzed by SDS-PAGE and immunoblotting with X11
or CCS antisera.
Immunoprecipitations were performed with (+) and without (
) 9E10
primary antibody. The lower level of CCS protein seen in cells
cotransfected with X11
in this particular experiment was not a
consistent feature. B, immunoprecipitation studies of CHO
cells transfected either individually or in combination with Myc-tagged
X11
and wild-type SOD1. X11
was immunoprecipitated with the 9E10
antibody, and the samples were probed with antibodies to X11
or
SOD1.
might also interact
with SOD1. However, under similar experimental conditions as above,
SOD1 did not coimmunoprecipitate with X11
(Fig. 2B).
Binding to CCS Is Mediated by PDZ2 and Sequences within CCS
Domain III--
To map further the regions required for binding of
X11
with CCS, we used GST fusion protein binding assays. GST fusion
proteins containing X11
PDZ1, PDZ2, and PDZ1 + 2 were used as baits
in pull-down assays from CCS-transfected CHO cells. CCS strongly interacted with PDZ2 and PDZ1 + 2, whereas no interaction could be
detected between CCS and PDZ1 or GST alone (Fig.
3A). Since domain II of CCS is
homologous to SOD1, we also tested whether SOD1 could bind to the
X11
PDZ domain fusion proteins. No interaction between any of the
X11
PDZ domains and SOD1 was detected (Fig. 3A), which is
consistent with the immunoprecipitation experiments (Fig.
2B).
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Fig. 3.
X11 PDZ2 interacts
with sequences within domain III of CCS. A, GST and
GST-X11
PDZ domain fusion proteins were used as baits to probe for
interactions with CCS and SOD1 from transfected CHO cell lysates.
X11
PDZ1 + 2 and PDZ2, but not PDZ1 or GST only, bound to CCS
(upper panel). None of the baits interacted with SOD1
(lower panel). B, GST and GST-CCS fusion proteins
were used as baits to probe for interactions with X11
or SOD1
(lower panel). Full-length CCS (CCS-(1-274)) and CCS baits
CCS-(243-274) and CCS-(48-274), but not CCS-(48-241) or GST alone,
bound X11
(upper panel). The C terminus of APP
(APPc) also bound X11
(upper panel). CCS baits
CCS-(48-241), CCS-(48-274), and CCS-(1-274), but not CCS-(243-274)
or GST alone, bound SOD1 (lower panel).
-transfected CHO cells. X11
bound to full-length CCS, CCS-(48-274), which includes both domains II and III and replicates the results of the yeast two-hybrid screen, and also to CCS-(243-274) (i.e. the C-terminal 32 amino acids of domain III) but not
to CCS-(48-241). GST alone did not bind X11
(Fig. 3B).
Thus sequences within domain III of CCS mediate the interaction with
X11
. The strength of the signals obtained using these CCS
baits was generally weaker than those with the X11
baits.
However, it is notable that in these experiments the strength of
signals obtained with the CCS baits was similar to that obtained with a
GST bait containing the C-terminal 47-amino acid cytoplasmic domain of
APP (Fig. 3B), which is known to bind to all X11 proteins
(2, 6, 7, 11, 12). We also tested whether the CCS baits would interact with SOD1 in these pull-down assays. Only sequences that included domain II bound SOD1, a finding consistent with previous reports (26)
(Fig. 3B). Taken together, these binding studies indicate that the interaction between human X11
and CCS is mediated by the
second PDZ domain of X11
and sequences within domain III of CCS.
and CCS in Cotransfected CHO Cells
and in Rat Primary Cortical Neurons--
Immunocytochemical labeling
of X11
/CCS cotransfected CHO cells revealed that both proteins had
overlapping distribution patterns, particularly in the perinuclear
region (Fig. 4A). X11
and
CCS also displayed an overlapping distribution pattern in rat primary cortical neurons where staining for both proteins was particularly pronounced in cell bodies with weaker labeling of neurites (Fig. 4B). These observations are consistent with previous reports
that X11
is present in cell bodies, possibly including the Golgi
apparatus and to a lesser extent in axons, dendrites, and synapses (9, 38), and that CCS is enriched in neuronal cell bodies (39).
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Fig. 4.
Subcellular distribution of
X11 and CCS. A, double
immunofluorescence in CHO cells cotransfected with Myc-tagged X11
and CCS. X11
was detected using the 9E10 antibody to the Myc tag,
and CCS was detected using CCS rabbit antiserum. B, double
immunofluorescence of rat primary cortical neurons with murine X11
polyclonal antiserum and CCS rabbit antiserum. In the overlaid images
(X11
/CCS) yellow indicates regions of overlapping
expression. Scale bar, 10 µm.
Inhibits SOD1 Enzymatic Activity--
CCS delivers the
metal ion copper cofactor to SOD1 (24-28), and copper is required for
SOD1 activity (40). We therefore tested whether expression of X11
could influence SOD1 activity. SOD1 activity assays were performed on
mock-transfected CHO cells, and CHO cells transfected with SOD1, SOD1 + X11
, SOD + CCS, and SOD1 + CCS + X11
(Fig.
5A). Immunoblot analyses
demonstrated that similar levels of transfected SOD1 were expressed in
the different transfections (Fig. 5B). However, SOD1
activity was significantly reduced in CHO cells expressing X11
(Kruskal-Wallis
2 = 8.74, D.F. = 3, p = 0.03; Fig. 5, A and C). This
included cells that were not cotransfected with CCS (Fig. 5A,
track 3) which demonstrates inhibition of the action of endogenous
CCS by X11
.
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Fig. 5.
X11 down-regulates
SOD1 activity. A, in situ nondenaturing gel
assay of SOD1 activity in lysates from CHO cells, either
mock-transfected (lane 1) or transfected with SOD1
(lane 2), SOD1 + X11
(lane 3), SOD1 + CCS
(lane 4) or SOD1 + X11
+ CCS (lane 5); human
SOD1 homodimer (hSOD1), is indicated by an arrow.
B, immunoblot analyses with antisera to detect SOD1, X11
,
and CCS in equivalent amounts of lysate from the same transfected cells
as depicted in A. Extended exposure of the blots revealed
the presence of endogenous CHO cell SOD1 but not CCS in the samples. We
attribute this absence of signal for CCS to a failure of our antibody
to cross-react with the CHO cell species. Indeed, transfected SOD1
displays significant activity in CHO cells in the absence of
transfected CCS (A, lane 2) which demonstrates activation by
endogenous CCS. The slight variation in the levels of X11
and CCS
seen in SOD1 + X11
+ CCS-transfected cells compared with cells
transfected with SOD1 + X11
and SOD1 + CCS was not a consistent
feature of these experiments. C, relative activities of SOD1
in the presence of X11
and/or CCS were calculated following
quantification of SOD activities and expression levels. The data shown
are the mean (S.D.) of three sets of experiments. S, SOD1;
S + X, SOD + X11
; S + C, SOD + CCS; S + C + X, SOD + X11
+ CCS. The activities are presented as a
percentage of activity found in SOD1 only (S) transfected
cell lysates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
interacts with CCS in a variety of biochemical assays and that X11
and CCS display overlapping subcellular distribution patterns in both
neurons and transfected CHO cells. The interaction between X11
and
CCS is mediated via the second PDZ domain of X11
and sequences
within the C-terminal 32 amino acids of CCS. CCS is the first reported
ligand specific for PDZ2 of X11
. The prototypical PDZ domain binding
sequence is the C-terminal motif X(S/T)X(V/I/L). It is now apparent, however, that PDZ domains bind to a much broader range of C-terminal sequences (for example see Ref. 41) and can also
bind to internal sequences as well as to other PDZ domains (13, 23,
42). It is thus possible that PDZ2 of X11
binds to the C terminus of
CCS (i.e. PAHL) or alternatively to an internal sequence
within the C-terminal 32 amino acids of domain III.
-binding
site to the C-terminal 32 amino acids of CCS, i.e. within
domain III.
led to a down-regulation of SOD1 activity in
transfected CHO cells, and this was not due to an effect of X11
on
expression of either SOD1 or CCS (Fig. 5B). These
observations suggest that X11
binding to CCS is inhibitory to CCS
function. Recent studies have highlighted the crucial importance of
domain III in the delivery of copper from CCS to SOD1 (31, 44, 45). One
suggestion is that domain III extends into the active site of SOD1 to
facilitate insertion of copper into the enzyme (31, 44, 45). Thus,
X11
may inhibit SOD1 activity by binding to CCS domain III and
disrupting this process in some way.
interacts with another copper-binding protein, APP, mutations in which
can cause Alzheimer's disease (see for review Ref. 50). APP has a
conserved copper binding region in its ectodomain (51) and may play a
role in the transport of copper to different cellular compartments
(46). APP has been shown to reduce Cu(II) to Cu(I) upon binding Cu(II)
and may thereby mediate copper-induced toxicity and oxidative stress in
neurons (52-54). Thus, through its interactions with both CCS and APP, X11
may play a general role in copper homeostasis within neurons. Defective copper metabolism, perhaps involving X11
, may be
mechanistic in both ALS and Alzheimer's disease.
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FOOTNOTES |
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* This work was supported in part by grants from the Wellcome Trust, Research into Aging, the Croucher Foundation (Hong Kong), the UK Motor Neurone Disease Association (MNDA), National Institutes of Health Grant DK44464, and the European Commission (Vth Framework Program).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.
c Wellcome Advanced Fellow.
d Research into Aging Prize Student.
e Croucher Foundation Research Fellow.
g Recipient of the UK Motor Neurone Disease Association Jim Tew Memorial studentship.
i Recipient of a Burroughs Wellcome Scholar award in Experimental Therapeutics.
j To whom correspondence should be addressed: Dept. of Neuroscience, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. Tel.: 44 020 78480393; Fax: 44 020 77080017; E-mail: chris.miller@iop.kcl.ac.uk.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M010023200
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ABBREVIATIONS |
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The abbreviations used are: APP, amyloid precursor protein; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; SOD1, superoxide dismutase-1; ALS, amyotrophic lateral sclerosis.
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