From Wyeth Neuroscience, Wyeth-Ayerst Research, CN 8000, Princeton, New Jersey 08543-8000
Received for publication, December 12, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Degeneration of neurons in Alzheimer's disease
is mediated by Genetic and biochemical data have coalesced to establish that
Yeast Two-hybrid Systems--
Yeast 2-hybrid (Y2H) expression
plasmids were constructed in the vectors pAS2 and pACT2 (19). Strain
CY770 (20) served as host for Y2H assays. Sequences encoding
A RACE and Genomic Cloning--
A modified rapid amplification of
cDNA ends (RACE) protocol was utilized to obtain 5'-BBP sequences
absent in the original Y2H clone. First strand DNA synthesis was
performed using the rTth thermal-stable polymerase system
(Perkin Elmer Life Sciences) with human hippocampus mRNA as
template. The MarathonTM cDNA synthesis kit
(CLONTECH) was used for subsequent second strand
cDNA generation and amplification. In addition, a human genomic
lambda library (Stratagene) was screened with a randomly primed BBP
protein coding region probe. Positive clones were purified and
subjected to further analysis by hybridization to a 45-nucleotide probe
directed to the most 5' sequences known from the original cDNA
clone. The nucleotide sequence of the upstream BBP region from a
positive Antibody Generation and Immunoblots--
Predicted BBP
ectodomain sequences were synthesized as five nonoverlapping peptides.
The peptides were pooled and conjugated to activated KLH carrier
protein per vendor's instructions (Pierce). Chickens were injected
intramuscularly with 0.1 mg of peptides/KLH each week for 4 weeks. Eggs
were collected and tested for IgY titer to each BBP peptide by ELISA.
IgY was partially purified from egg yolk by dilution and ammonium
sulfate precipitation (22). This sample was further purified by solid
phase affinity binding to BBP peptide composed of residues 42-81 (Fig.
1A). Expression of recombinant BBP protein was evaluated in
Chinese Hamster Ovary cell lysates. Cells were transfected with pBBP by
LipofectAMINE-PLUS per manufacturer's (Life Technologies, Inc.)
instructions. Cells were suspended in hypotonic buffer (50 mM Tris, pH 7.2; 1 mM EDTA) plus proteinase
inhibitors and were maintained on ice. Cells were disrupted using a
polytron and debris was removed by centrifugation at 2,000 rpm in a
microcentrifuge. Soluble and membrane fractions were separated by
centrifugation at ~200,000 × g using a 45Ti rotor in
a TL100 centrifuge (Beckman Instruments). The membrane pellet was
resolubilized in phosphate-buffered saline (PBS) with 1% Triton X-100
plus proteinase inhibitors. Laemmli's buffer with detergent and
2-mercaptoethanol were added to aliquots containing 50 µg of protein,
and samples were boiled for 5 min prior to electrophoresis in a 4-10%
Tris-glycine NuPage gel (NOVEX). Samples were transferred to a
polyvinylidene difluoride membrane by the semi-dry method (BIORAD).
Blots were probed with the chicken anti-BBP antibody described above,
using rabbit anti-IgY conjugated to horseradish peroxide (Promega) as a
secondary detection reagent. Proteins were visualized by development
with the ECL-Plus reagent and exposure to Hyperfilm (Amersham Pharmacia
Biotech). Deglycosylation of proteins was achieved using the enzymes
PNGase-F, NANase II, and O-glycosidase DS per
manufacturer's instructions (Bio-Rad).
Northern and RT-PCR Analyses--
Human multiple tissue mRNA
Northern blots were obtained from CLONTECH. BBP
sequences extending from the original Y2H fusion junction to the
poly(A) region were used to generate a radiolabeled probe. Similar BLP1
and BLP2 cDNAs were used to generate probes. Control In Situ Hybridization--
DNA templates for riboprobe synthesis
were prepared by PCR using each human BBP or BLP cDNA. Subsequent
riboprobes were generated to 3'-untranslated regions using the
Riboprobe GeminiTM System (Promega). In situ
hybridization histochemistry using sections of cynomolgus monkey
(Macaca fascicularis) forebrain was performed as described
previously (23). Emulsion autoradiograms were developed and fixed
according to the manufacturer's (Kodak) instructions, and the
underlying tissue sections were stained with hematoxylin. To assess
nonspecific labeling, a control probe was generated from a template
provided in the Riboprobe GeminiTM System kit. No specific
hybridization was observed in control sections.
In Vitro Binding Assays--
The tagged BBP segment used in
in vitro binding assays was produced by amplifying cDNA
encoding the BBP protein from the clone 14 junction to 1/2tm,
incorporating DNA encoding the c-Myc epitope EQKLISEEDL in the 5'-PCR
primer. This DNA was cloned into the pET28a vector and transformed into
E. coli strain BL21/DE3 (Novagen). Recombinant BBP protein
was isolated in inclusion bodies extracted by B-PER reagent (Pierce).
Samples were resuspended in 20 ml of 1× binding buffer (Novagen)
containing 6 M urea and purified using His-BindTM
chromatography (Novagen) in the presence of 6 M urea. Following elution, urea was removed by dialysis against binding buffer.
To prepare Sepharose-antibody complex, 1.5 mg of swelled protein
A-Sepharose (Sigma) was mixed with 3.3 ml of rabbit anti-mouse IgG
(Jackson Immunoresearch Laboratories, Inc.) in cold immunoprecipitation buffer (IPB; Ref. 24) plus 0.02% NaN3 with rotation
overnight at 4 °C. After washes, the 6E10 anti-A Cellular Assays--
Human Ntera-2 (Nt2) stem cells (26) were
maintained in Dulbecco's Modified Eagle's medium supplemented with
10% fetal bovine serum. Human SH-SY5Y cells were maintained in 1:1
Dulbecco's Modified Eagle's medium/F-12 Ham nutrients plus 15% fetal
bovine serum. BBP cDNAs were modified by PCR for expression from
the vector pcDNA3.1 (Invitrogen). Mutation of the arginine codon to
glutamate within the DRF motif of BBP cDNA was performed using the
QuickChangeTM system (Stratagene). Cell viability determinations were
performed with a CellTiter kit (Promega) measuring conversion of MTS
tetrazolium to formazan. For nuclear morphology assays, expression
constructs were introduced with pEGFP-N1 (CLONTECH)
into cells by electroporation. DNA amounts were 7.5 µg of subject DNA
plus 2.5 µg of pEGFP-N1. Approximately 24 h after transfection,
growth medium was replaced with medium containing 10 µM
A Identification of a Novel BBP Protein Expression--
The BBP cDNA contained in clone 14 lacked a potential native translation initiation site. BBP 5'-DNA
sequences were identified from both cDNA and genomic DNA sources.
The additional cDNA sequence contained three possible start codons
preceded by an inframe stop codon. The deduced sequence of the
translation product shown in Fig. 1A begins at the first
potential start site possessing suitable context for efficient
translation initiation (27), and as suggested experimentally by
recombinantly expressed BBP (data not shown). Peptides contained in the
predicted ectodomains of BBP were used to immunize chickens. The
chicken IgY was purified and utilized to probe partitioned soluble and
membrane fractions produced from cells transfected with a BBP cDNA
expression plasmid. A broad specific band of 36-42 kDa was observed in
the membrane-enriched fraction (Fig. 1B); no protein was
detected in the soluble fraction. Enzymatic removal of carbohydrates
resulted in a shift to 19 kDa, demonstrating that the BBP protein is
extensively glycosylated. These data suggest that mature BBP is an
integral membrane glycoprotein likely derived from a precursor
containing a cleavable secretory signal leader. To investigate this
prediction, recombinant human BBP protein lacking putative
transmembrane (tm) domains was produced as a fusion to immunoglobulin
Fc domain to facilitate protein stability and detection. This fusion
protein was found to be secreted from cultured cells. Amino terminal
sequencing of the protein revealed a single product processed to the
position indicated in Fig. 1A. The site of cleavage is
canonical to the signal peptidase site consensus (28). The cleaved
protein contains 170 amino acids, consistent with the migration
of deglycosylated protein at ~19 kDa (Fig. 1B).
BBP Relationship to the G Protein-coupled Receptor
Superfamily--
The BBP protein and translations of available ESTs
were assembled, aligned, searched for conserved segments, and evaluated by the MoST protein motif search algorithm (29). First, these analyses
revealed three distinct sets of ESTs in both the human and mouse
datasets, indicating that BBP is one member of a structurally related
protein family. Subsequently, orthologous sequences to mammalian BBP
and the BBP-like proteins were also identified in the Drosophila
melanogaster genome. No additional subtypes were detected in
available databases. Human BLP1 and BLP2, and mouse and fly BBP
cDNAs were isolated by RT-PCR methodologies using EST and genomic
DNA information to guide primer design. The cDNA sequences encoding
the mouse and fly BLP1 and BLP2 proteins were derived from EST and
genomic DNA consensus determinations. Alignment of the three
translation products from human, mouse, and fly revealed several key
features (Fig. 2A). Most
striking is a segment exhibiting a significant relationship to the G
protein-coupled receptor (GPCR) superfamily. Specifically, these
proteins contain two potential tm domains with a high degree of
similarity to the third and fourth tm domains of GPCRs (Fig.
2A and B). The similarity includes the intervening hydrophilic loop, which contains the well characterized three amino acid motif, aspartate (D), arginine (R), and an aromatic residue (Y or F) (commonly referred to as the DRY sequence), that is
conserved in most members of this receptor superfamily and has been
shown to serve as the molecular trigger for G protein activation (30).
In addition to a general similarity, >25% identity to the tm3 through
tm4 segment of some GPCR members, other very highly conserved amino
acids include a cysteine immediately preceding tm3 (BBP tm1) and a
lysine marking the beginning of tm4 (BBP tm2). A tryptophan found in
tm4 of ~95% of GPCRs is present at the equivalent position in the
BLP1 and BLP2 subtypes. Preceding the tm domains, there is little
homology between BBP/BLP subtypes, a common feature of receptor
families sharing a conserved signal coupling domain, with unique
activities determined by less conserved ectodomains. Each protein
possesses a region of strong hydrophobicity near the amino terminus,
indicative of an amino-terminal secretory signal. With the demonstrated
functionality of the amino-terminal signal sequence in BBP, and in
conjunction with the homologies to GPCR topology, it is predicted that
the proteins transverse cellular membranes twice, with both termini
luminal or extracellular as depicted in Fig. 2B. As with
prototypic 7-tm domain G protein-coupled receptors, the BBP/BLP
proteins contain the important DRF motif appropriately positioned
between two tm domains, juxtaposed to the first tm domain. This
suggests that the proteins could modulate a heterotrimeric G protein
regulatory pathway.
BBP Gene Expression--
Tissue expression of BBP and BLP
mRNAs was evaluated, revealing major transcripts of 1.25 kb, 1.35 kb, or 1.40 kb for BBP, BLP1, and BLP2, respectively, in all samples
(Fig. 3A). Analysis of
in situ hybridization autoradiograms of non-human primate
forebrain obtained using riboprobes directed to each BBP mRNA
demonstrated that all three genes are prominently expressed in medium
to large cells in a pattern indicative of elevated expression in
neurons as opposed to glial cells. BBP/BLP transcripts were observed in largely overlapping regional patterns, with greatest expression in the
hippocampus and neocortex (Fig. 3B). The BBP riboprobe was
also used to evaluate a human hippocampus/entorhinal cortex sample,
revealing expression in virtually all neurons (data not shown). In
summary, BBP mRNAs were observed in all tissues examined, and
in situ analyses of brain samples revealed extensive
expression in neurons of the hippocampus/entorhinal cortex and
neocortical regions. This pattern is similar to the
regionally-restricted neurodegeneration observed in Alzheimer's
disease (31, 32). More rigorous histopathological studies will be
necessary to fully assess correlations between BBP expression and
disease pathology.
BBP Interactions with A Binding of BBP to A
A solid phase assay was developed to initiate pharmacological analyses
of BBP/A BBP Confers Cellular Sensitivity to A
It is only aged (i.e. aggregated) preparations of human A Evaluation of Endogenous BBP Activity--
The BBP-R
Nt2 stem cells can be differentiated into cells possessing the
morphological, genetic, and physiological properties of neurons by
treatment with retinoic acid (26). BBP mRNA levels were evaluated in Nt2 stem cells and neurons, and a >20-fold increase in
BBP gene expression was observed in the differentiated cells
(Table I). Stem cells and neurons were
transfected with pEGFP plus vector, pBBP, or pBBP-R BBP, BLP1, and BLP2 constitute a novel family of proteins
containing a module related to the G protein-coupled receptor
superfamily. The normal physiological activities of these proteins
remain to be elucidated, but the demonstration that the BBP subtype can modulate the apoptotic response to A The search for molecular mechanisms underlying Alzheimer's disease
became more sharply focused with the discovery that the biochemical
basis of inherited, aggressive forms of the disease was a phenotypic
change in A-amyloid peptide by diverse mechanisms, which include
a putative apoptotic component stimulated by unidentified signaling
events. This report describes a novel
-amyloid peptide-binding
protein (denoted BBP) containing a G protein-coupling module.
BBP is one member of a family of three proteins containing this
conserved structure. The BBP subtype bound human
-amyloid peptide
in vitro with high affinity and specificity. Expression of
BBP in cell culture induced caspase-dependent vulnerability
to
-amyloid peptide toxicity. Expression of a signaling-deficient
dominant negative BBP mutant suppressed sensitivity of human Ntera-2
neurons to
-amyloid peptide mediated toxicity. These findings
suggest that BBP is a target of neurotoxic
-amyloid peptide and
provide new insight into the molecular pathophysiology of Alzheimer's disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid peptide (A
)1
is a causative factor in neuron death and the consequent dimunition of
cognitive abilities observed in Alzheimer's disease (1, 2). Plasma
lipoproteins and their cell surface receptors influence sequestration
and clearance of soluble A
, contributing to the etiology of the
disease (3-6). Inflammatory responses and oxidative damage also appear
to contribute to the loss of neurons in Alzheimer's disease (7-10).
Although the earliest cellular perturbations remain unclear, recent
findings indicate that A
may act as an initiating factor in the
death of neurons by inducing signaling pathways leading to apoptosis
(11-17). However, the specific molecular target(s) transducing these
A
effects has not been identified. The intracellular protein ERAB
can bind A
in vitro, and neuroblastoma cells expressing recombinant ERAB undergo apoptosis when treated with exogenously added
A
(18), but the mechanism by which ERAB may affect apoptotic signaling remains obscure. We identified a novel human
-amyloid peptide binding protein (BBP) utilizing yeast
2-hybrid technology. Analysis of the BBP amino acid sequence revealed
the presence of a structural module related to that of the 7 transmembrane domain G protein-coupled receptor superfamily and known
to be important in heterotrimeric G protein activation. Data suggest that BBP mediates cellular vulnerability to A
toxicity through a G
protein-regulated program of cell death. Two related proteins (BLP1,
BLP2; BBP-like proteins) were
identified by sequence and structural similarities to BBP, but only the
BBP subtype regulates a response to A
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
42 were amplified by PCR using primers incorporating
restriction sites for subsequent ligation into pAS2, using a human APP
(amyloid precursor protein) cDNA clone as template. A Y2H plasmid
library consisting of cDNA fragments isolated from human fetal
brain cloned into the yeast 2-hybrid expression vector pACT2
(CLONTECH Laboratories) was screened with
pAS2-A
. Interactors were identified on synthetic complete yeast
growth medium lacking histidine and containing 3-amino-triazole at a
concentration of 25 mM. Y2H expression plasmids for BBP
deletion analyses were generated by standard methods. Expression of all hybrid proteins was confirmed by immunoblot using antibodies
(CLONTECH) directed to the Gal4 sequences.
-galactosidase assays to measure Y2H interactions were conducted in
strain CY770 containing the indicated Y2H expression plasmids plus the
GALUAS-lacZ reporter plasmid pRY131 (21).
clone was identical to cDNA sequences identified by
RACE. Mouse BBP cDNA and human BLP1 and BLP2 cDNA sequences
were initially assembled from expressed sequence tag (EST) data
extracted from GenbankTM/EBI. These segments, which did not
include potential translation start sequences but extended 3' to
poly(A) sequences, were amplified by RT-PCR from mouse or human brain
mRNA. Subsequently, RACE was used to isolate 5' sequences for human
BLP1 and BLP2. RACE failed for mouse BBP, but 5' sequences were
obtained to a potential initiating ATG from genomic cloning (data not
described). The three human cDNAs were assigned accession numbers
AF353990, AF353991, and AF353992; mouse BBP AF353993.
-actin DNA
was provided by the manufacturer. Hybridizations were performed per
manufacturer's (CLONTECH) instructions.
Quantitative RT-PCR was performed utilizing a PE Applied Biosystems
7700 instrument per manufacturer's instructions, probing with
sequences contained within the BBP protein coding region. Detection
reagents for glyceraldehyde phosphate dehydrogenase mRNA were used
to qualitatively assess RNA samples and to standardize reactions.
antibody
(Senetek PLC) was added, and the mixture was rotated overnight at
4 °C. Purified BBP was combined with 500 ng of A
40 at
a 1:1 molar ratio in 200 µl of 50 mM Tris pH 7.6, 150 mM NaCl, 1% Nonidet P-40. For A
40-1 competition
experiments, 25 µg (50× molar excess) of A
40-1 was combined with
BBP and A
1-40. Samples were agitated at room temperature for 1 h, washed three times with 200 µl of IPB, resuspended in 20 µl of
2× Laemmli sample buffer containing
-mercaptoethanol, boiled for 5 min, and loaded on a 10-20% Tris-HCl polyacrylamide gel. Standard
Western blot procedures utilizing Ni2+-horseradish
peroxidase were used to detect BBP. ELISA plates for solid phase
binding measurements were coated with A
40 peptide and
blocked with 5% BSA as described elsewhere (25). Forty-residue human
or rodent
-amyloid peptide and reverse human peptide were obtained
from AnaSpec, Inc. Peptides were dissolved and stored in
hexafluoro-isopropyl alcohol at 1 mg/ml. Samples were lyophilized by
pervasion with nitrogen and then resuspended in PBS and immediately frozen as disaggregated peptide. To achieve consistent aggregation, the
peptides were resuspended in cell growth medium and divided into
0.13-ml aliquots in a 96-well plate. The plate was shaken at 500 rpm
for 5 h. Samples were then combined and normalized to a final A
concentration of 50 µM and immediately frozen. The human
A
peptide was evaluated for circular dichroism and thioflavin-S fluorescence and shown to be predominantly aggregated, but not fibrillar. Purified tagged BBP protein was prepared in PBS plus 5%
BSA. Protein was applied to A
-coated plates for 1 h at room temperature, washed 5× with PBS plus 0.1% Tween 20. Bound BBP was
detected by application of mouse anti-Myc antibody (CalBiochem) followed by five washes, with subsequent application of donkey anti-mouse IgG conjugated to alkaline phosphatase (Jackson
Immunoresearch Laboratories, Inc.). Following five washes and
development with an alkaline phosphatase substrate kit (BIORAD),
A405 values were obtained. Values obtained from
wells lacking A
peptide were subtracted from total bound BBP1 to
calculate specific binding. Competition assays were performed by adding
peptides to BBP (50 nM) in PBS, 5% BSA and incubating at
4o overnight prior to application to A
-coated plates.
Approximate binding coefficients were calculated by nonlinear
regression analysis using PRISM software (Graphpad, Inc.).
peptide. Forty-eight hours after A
addition, the
chromatin-specific dye Hoechst 33342 (Molecular Probes, Inc.) was added
to a concentration of 10 ng/ml. Medium was removed after 10 min, and
cells were washed with PBS. Cells were then fixed by immersion in PBS
containing 4% paraformaldehyde. A minimum of 200 transfected
(EGFP+) cells per sample were scored manually by
fluorescence microscopy. Statistical comparisons of apoptotic nuclei
counts were conducted using Yates G-test of probability for categorical
data. All experiments were repeated greater than three times with the
same results. Human Nt2 neurons were derived by treatment of stem cells
with retinoic acid (26). Neurons were transfected by the
ProfectionTM system (Promega) using 4.5 µg of test
plasmid plus 1.5 µg of pEGFP-N1 per sample. Similar to the other
cellular assays, growth medium was replaced with medium containing 10 µM aggregated A
24 h after transfection.
Following incubation for 48 h, cells were fixed and nuclear
morphologies of transfected (i.e. EGFP+) cells
were scored as described above. Transfection efficiencies, determined
as the fraction of green fluorescent cells, for Nt2 stem cells and
neurons were routinely 5-6% and 1-2%, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Amyloid Peptide-binding
Protein--
A Y2H genetic screen was developed to identify proteins
that interact with human A
. The Y2H assay strain CY770, expressing A
42 fused to the yeast Gal4 DNA-binding domain, was
transformed with a human fetal brain cDNA Y2H library. A single
clone, denoted no. 14, was selected for further characterization as it
produced consistent reporter gene activation and contained a
substantial open reading frame continuous with that of the Gal4
transcriptional activation domain. The cDNA insert of clone 14 comprised 984 base pairs, terminating in a poly(A) tract. This sequence
encoded 201 amino acids with two regions of sufficient hydrophobicity
and length to transverse a cellular membrane, indicated in Fig.
1A as tm1 and
tm2. Examination of available databases revealed numerous ESTs, but no entry contained a complete open reading frame or attributed a potential function to the protein, which we designate BBP.
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Fig. 1.
BBP is a membrane-associated
glycoprotein. A, BBP protein sequence and features. The
overline indicates a region of hydrophobicity predicted to
direct the nascent protein to the secretory pathway. The
arrow indicates the site of signal peptidase cleavage.
Predicted transmembrane domains (tm1 and tm2) are
boxed. The DRF motif is circled and possible
sites of N-linked glycosylation are underlined.
The diamond indicates the site of fusion in the yeast
2-hybrid clone 14. B, anti-BBP immunoblot. Membrane
fractions were isolated from CHO cells transfected with vector or a BBP
expression plasmid and evaluated by Western blot analysis using
anti-BBP IgY antibody. Each sample was also treated by deglycosylation
enzymes, indicated by +. A bracket marks mature BBP protein.
The arrow marks BBP minus carbohydrates. The slightly higher
band was observed inconsistently, and likely results from incomplete
deglycosylation. The positions of molecular mass markers (in kDa) are
indicated.
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Fig. 2.
BBP/BLP proteins are related to the G
protein-coupled receptor superfamily. A,
sequence comparison of BBP/BLP translation products. The deduced amino
acid sequences of human, mouse, and D. melanogaster (fly)
BBP, BLP1, and BLP2 proteins were aligned using the ClustalW algorithm
(46). The fly BLP2 protein has been tentatively identified as almondex
(amx; accession AF217797). Gaps, indicated by dashes, were
introduced to optimize the alignment. Amino acids common within a
subtype are shaded. Amino acids invariant for all proteins are
indicated by arrows. Predicted transmembrane domains
(tm1 and tm2) are indicated. Asterisks
indicate translation stops. B, cartoon depiction comparing
the predicted topology of the BBP proteins with a 7-tm domain G
protein-coupled receptor. The two tm domains of BBPs correspond to tm
domains 3 and 4 of GPCRs.
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Fig. 3.
The BBP genes are widely
expressed, with prominent expression in neurons of the
hippocampus/entorhinal cortex and neocortex. A, nylon
membranes blotted with 2-µg size fractionated poly(A) RNA isolated
from the indicated tissues were sequentially hybridized with the
indicated radiolabeled cDNA probe. A single predominant band
corresponding to the indicated length was observed in all
lanes for each probe. Blots were also probed with -actin
as a loading and RNA integrity control. B, autoradiograms of
coronal sections of cynomolgus monkey forebrain, taken at mid to caudal
levels, processed to visualize the distribution of BBP, BLP1, or BLP2
mRNA by in situ hybridization histochemistry.
Darker areas correspond to areas of higher expression of
mRNA. The images are not normalized relative to each other.
--
Deletion variants of BBP were
examined in Y2H assays to delineate the A
-binding domain. Results
are shown in Fig. 4A. The original clone 14 produced only a weak response with the A
fusion protein. Protein transmembrane domains have been observed to
substantially attenuate Y2H responses,2 as
the reconstitution of transcription activator function must occur in
the cell nucleus. Deletion of the second tm domain and carboxyl-terminal sequences of BBP resulted in much more robust Y2H
activity, measured by activation of either HIS3 or
lacZ reporter genes (clone14-
tm2). Moving in from the
amino end, protein deleted for approximately half of the region between
the clone 14 junction and tm1 maintained a positive interaction with
A
(
60-
tm2). The deletion of 26 additional amino acids resulted
in the loss of a Y2H response with A
(
86-
tm2). From the
carboxyl end, one-half of the tm1 domain could be deleted with no
effect on Y2H interaction (
60-1/2tm). However, complete
removal of the first tm domain (
60-
tm1) resulted in loss of
activity. These results indicate that crucial A
binding determinants
within BBP are contained within the segment delineated by
60 and
1/2tm. The elimination of the A
association upon further
deletion from either terminus could result from structural disruption
of a single binding site by a distant deletion, or more likely,
suggests that two important contacts with A
exist (between
60 and
86, and
tm1 and 1/2tm), each necessary but not sufficient
for binding alone.
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Fig. 4.
BBP binds A in
vitro. A, determination of the A
-binding
domain of BBP by Y2H analysis. A series of Y2H BBP fusion protein
expression plasmids was generated as described in the text and as
drawn. Transmembrane domains are indicated by rectangles.
Clone 14 is fused at residue 5 as indicated by the arrow.
The extent of amino-terminal deletions is indicated by residue number.
Each protein was tested with the A
fusion protein for Y2H responses,
measured as both histidine prototrophy on solid growth medium and by
activation of a lacZ reporter. Y2H responses are indicated
as positive (+ or ++) or negative (
). The minimal A
-binding domain
begins at the
60 location and extends to the 1/2tm site.
B, Western blot image of samples immunoprecipitated with the
anti-A
antibody 6E10 and probed with Ni2+-HRP to detect
His6-BBP. Lane 1, molecular mass standards;
lane 2, sample lacking A
; lane 3,
His6-BBP plus A
; lane 4, same as lane
3 plus 50-fold excess reverse A
40-1. Slower
migrating bands in lanes 2-4 are immunoglobulin heavy and
light chains. C, saturation binding of BBP segment to
immobilized A
; Kd
150 nM.
Nonspecific binding was determined by measuring BBP bound to wells
lacking A
, and those values were subtracted from total binding to
obtain specific binding. D, comparison of BBP binding to
aggregated or disaggregated A
in vitro. A
peptide was
disaggregated by suspension in hexafluoro-isopropyl alcohol. A portion
of disaggregated peptide was subsequently aggregated as described.
These peptide samples were immobilized and incubated with vehicle (PBS + 5% BSA) or vehicle plus purified BBP. Values represent the mean
absorbance of triplicate wells, with standard deviation.
E, demonstration of specificity of binding. Human
A
1-40, reverse A
40-1, or rodent A
were added to Myc-tagged BBP (150 nM) over a range of
competitor concentrations. Nonspecific binding was subtracted. The
IC50 value for free A
1-40 was ~300
nM.
in Vitro--
The amino-terminal
A
-binding segment of BBP (between the clone 14 and 1/2tm
sites; see Fig. 4A) was expressed in bacteria with amino-terminal His6 and c-Myc tag sequences. This protein
was purified and utilized in in vitro binding assessments
with synthetic A
peptide. In pull-down experiments, BBP was mixed
with disaggregated A
, and proteins were immunoprecipitated with the
anti-A
antibody 6E10. Denatured protein complexes were subsequently
evaluated using a Ni2+-peroxidase conjugate to detect the
His6 tag of the BBP protein. As controls, A
was omitted
or 50-fold excess reverse A
40-1 was added as a
potential competitor of binding. Associated BBP protein could be
readily observed in the A
immunocomplexes, and the signal was not
affected by the addition of reverse peptide (Fig. 4B). These
results demonstrate that BBP binds non-fusion A
peptide,
complementing the Y2H results.
binding. A
was immobilized in 96-well plates, and bound
BBP was measured with an anti-Myc antibody. Specific binding of BBP to
A
was determined by subtracting nonspecific binding in wells lacking
A
peptide. The binding of BBP protein to A
exhibited saturable,
high affinity characteristics (Kd = 150 nM, Fig. 4C). For the binding experiments, A
peptide was disaggregated to provide a homogeneous preparation.
However, disaggregated A
has much reduced neurotoxicity in cell
culture. Only A
aggregates possess substantial toxic properties
(33). BBP binding to wells coated with disaggregated or aggregated A
was compared to determine whether BBP could bind neurotoxic-aggregated
A
. BBP bound to A
in vitro regardless of the peptide
state prior to application (Fig. 4D). BBP binding to A
in
solution (Fig. 4C) was also independent of peptide
aggregation state (data not shown). Binding could be competed by free
A
with an IC50 value of ~300 nM (Fig.
4E). BBP binding to A
was not affected by the addition of
peptide of reverse sequence. Rodent A
peptide, differing from the
human sequence by three amino acid substitutions (positions 5, 10, and 13), also failed to substantially inhibit BBP binding to
human A
(Fig. 4E). Interestingly, the rodent peptide
demonstrates reduced neurotoxicity and an absence of binding to human
brain homogenates (33).
--
Potential
involvement of BBP in A
toxicity was investigated in cultured human
SH-SY5Y cells transfected with vector or pBBP. The efficiency of
transfections was 40-50% (determined by independent transfection of
pEGFP). Samples were treated with aggregated A
peptide for 48 h
and evaluated for viability. Under these experimental conditions, A
treatment had no significant toxic effect in control samples (Fig.
5A). However, transfection
with pBBP resulted in a significant increase in sensitivity to A
,
with an average loss of 22% of total cells (Fig. 5A),
indicating that expression of BBP stimulated sensitivity to A
.
Neurons exposed to toxic aggregated A
exhibit characteristics of
apoptosis before dying (11-13, 15-17, 34). To determine whether
BBP-specific A
toxicity includes apoptotic events, nuclear
morphology assays were conducted. SH-SY5Y cells were doubly transfected
with pEGFP plus test plasmids, treated with toxic A
, and nuclear
morphologies of transfected cells were evaluated by fluorescence
microscopy following staining with a Hoechst chromatin dye. Included in
these experiments was a BBP expression plasmid mutated to substitute
glutamate for the arginine in the DRF motif. The corresponding R
E
substitution has been shown to eliminate the activity of 7-tm domain
GPCRs (35, 36). Transfection with pBBP resulted in a substantial and
significant increase in pyknotic nuclei, and this response was
prevented by the R
E substitution (Fig. 5B). An anti-BBP
immunoblot of cell lysates is shown to demonstrate that the R
E
substitution does not alter protein expression (Fig. 5C).
The absence of a response in the pBBP-R
E sample suggested that BBP
modulates A
toxicity by coupling to heterotrimeric G proteins. To
further investigate this possibility, samples were treated with the
G
i/o inhibitor pertussis toxin. This treatment
eliminated cellular sensitivity to A
via BBP (Fig. 5B).
The same results were observed in transfected Nt2 stem cells.
Furthermore, Nt2 stem cells transfected with pBBP were treated with the
non-selective caspase inhibitor BOC-Asp(Ome)-fluoromethylketone (BAF)
to evaluate the involvement of caspases. Treatment with BAF abrogated
the induction of nuclear condensation mediated by A
in
BBP-transfected cells (Fig. 5D). These data were replicated in SH-SY5Y cells. These findings demonstrate that BBP mediates A
-induced apoptosis by a G protein-regulated
caspase-dependent signaling pathway in neurotypic
cells.
View larger version (14K):
[in a new window]
Fig. 5.
Recombinant expression of BBP induces
cellular vulnerability to A toxicity.
A, transfection of cells with pBBP results in increased cell
loss upon treatment with A
. SH-SY5Y cells were transfected with
vector or pBBP. Samples were treated with 10 µM aged A
for 48 h, then evaluated for cell viability compared with
untreated control samples. Values represent the mean ± S.D. of
three independent experiments. The asterisk indicates
p < 0.01 (Student's t test). B,
A
-induced apoptosis in cells transfected with pBBP is transduced
through G proteins. SH-SY5Y cells were transfected with pEGFP plus pBBP
or pBBP-R
E expression plasmids. Samples were treated with 10 µM A
, and nuclear morphologies were evaluated in
transfected (EGFP+) cells as described in the text. One
pBBP sample was simultaneously treated with pertussis toxin
(PTX) at 100 ng/ml to obtain the value labeled
pBBP+PTX. Values are the means of duplicate samples of >100
EGFP+ cells, with S.D. The asterisk indicates
significant (p < 0.01; Yates G-test) effect of pBBP
versus vector. C, expression of BBP and BBP-R
E
protein. The image shows an anti-BBP immunoblot of membrane fractions
isolated from cells transiently transfected with vector, pBBP or
pBBP-R
E. D, the BBP-mediated response to A
is
caspase-dependent. Nt2 stem cells were transfected with
pEGFP plus vector or pBBP and treated with 10 µM A
.
Duplicate pBBP samples were also treated with 25 µM
BOC-Asp(Ome)-fluoromethylketone (BAF), a nonspecific caspase
inhibitor. Samples were scored for apoptotic nuclei and significance
determined as described in the legend to B. E, BBP-specific
apoptotic response to A
is selective for aged (i.e.
aggregated) human peptide. Nt2 stem cells were transfected with pEGFP
plus vector or pBBP. Samples were treated for 48 h with the
indicated peptide at 10 µM and examined for nuclear
morphology as described for B. F, transient
transfection assays demonstrate that the BBP-R
E variant acts in a
dominant negative manner to suppress the activities of wild-type
protein. Nt2 stem cells were transfected with the indicated mixtures of
DNAs, maintaining total DNA concentrations constant (1.65 µg).
Duplicate samples were treated with 10 µM A
and scored
for apoptotic cells as described in B. Transfection with
pBBP in the absence of pBBP-R
E resulted in a significant
(p < 0.01) induction of apoptosis versus
vector control. In dually transfected samples, there was a consistent
(n = 5) and significant (p < 0.01)
dominant negative effect of pBBP-R
E versus pBBP alone.
The intermediate value of the pBBP plus pBBP-R
E dual transfection
versus pBBP-R
E alone was not statistically significant
(p > 0.05; Yates G-test).
that elicit substantial toxicity on primary neurons; disaggregated human peptide or aggregated rodent peptide confer greatly reduced toxicity (33, 37, 38). Cells transfected with pBBP exhibited the same
selectivity for A
preparations, failing to show effects with
disaggregated A
, aged reverse peptide, or aged A
composed of the
rodent sequence (Fig. 5E). The absence of a response to A
composed of the rodent sequence correlates with the inability of human
BBP to interact with this peptide in binding assays (Fig. 4E). These data demonstrate that selectivity for peptide
state and type leading to BBP/A
toxicity in cell culture matches
that required for A
toxicity in neurons. Of further note, A
toxicity is specific for only the BBP subtype, as no change in
apoptotic response to A
was observed in cells transfected with BLP1
or BLP2 expression plasmids (data not shown).
E variant
is unable to mediate an apoptotic response to A
. Transient
transfection assays were utilized to determine whether BBP-R
E could
act as a dominant negative protein, which, if so, would then allow for
the possibility of assessing endogenous BBP activities in human
neurons. Nt2 stem cells were transfected with pEGFP plus equal
quantities of mixed DNAs consisting of either vector, vector plus pBBP,
vector plus pBBP-R
E, or both pBBP plus pBBP-R
E. These samples
were challenged with A
, and transfectants were scored for
nuclear morphology. As shown previously, BBP stimulated A
-mediated
apoptosis, and protein containing the R
E substitution was inactive.
Cells transfected with pBBP plus pBBP-R
E exhibited the negative
phenotype (Fig. 5F), demonstrating that the BBP-R
E inactive variant is phenotypically dominant over wild-type protein.
E, and examined
for A
-induced apoptosis. Results are shown in Table I. Nt2 stem
cells became sensitive to A
either by differentiation into neurons
or by transfection with pBBP. Transfection of neurons with pBBP did not
have an additive effect. Transfection of neurons with the pBBP-R
E
dominant negative variant substantially reduced the induction of
apoptosis by A
exposure, presumably by inhibiting the activity of
the endogenous BBP protein. These data indicate that the BBP protein
plays a central role in A
-induced apoptosis in human neurons.
BBP gene induction in differentiated Nt2 cells and apoptotic
responses to A
for 48 hrs, and nuclear
morphologies of transfected cells were determined as described in the
legend to Fig. 5. Values indicate the average of three independent
experiments with S.E. Statistical significance of pBBP or pBBP-R
E
transfection samples were determined by testing against the vector
control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
through a G protein and caspase-dependent mechanism suggests important regulatory
roles for BBP/BLPs in cell death or proliferation signal cascades. The involvement of heterotrimeric G proteins in the regulation of apoptotic
pathways has been reported (39-42) but remains only partially characterized. It is possible that BBP/BLPs act as key components of
integral membrane protein complexes, providing the molecular trigger
for G protein activation in the context of other proteins serving
structural roles. We have preliminary evidence that BBP can associate
with the amyloid precursor protein APP, which has been shown to
modulate apoptosis through binding to heterotrimeric Go protein
(41-43). It has not yet been determined whether the A
and APP
association domains overlap within BBP. A simple
model would place a BBP/APP heteroduplex as the source of a G protein regulated signal transduction cascade modulating apoptosis with the two
proteins each providing functional components more commonly contained
within a single 7-tm domain
protein. The biophysical mechanism by which the binding of aggregated A
to such a BBP/APP complex might induce apoptosis remains to be determined, but the numerous correlations between this description of BBP as a regulator of
pertussis-sensitive G protein-dependent apoptosis and the
findings of Nishimoto and co-workers (41-42) suggest a common pathway.
Recently, it was shown that a cell surface protein complex containing
APP binds fibrillar A
and may contribute to its toxicity (44). It
will be important to determine whether BBP is also a component of this complex.
peptide, manifested as increased generation of total
peptide or elevated production of the more amyloidogenic 42-residue
form relative to shorter species (1). However, the subsequent
mechanisms of neurotoxicity mediated by A
are reported to include
many different biochemical perturbations, making the elucidation of the
critical initiating events challenging. The cumulative data pertaining
to BBP are consistent with a possible activity for this protein in
these early events. The gene is prominently expressed in neurons in a
regional pattern consistent with the known pathophysiology of
Alzheimer's disease. BBP binds A
peptide with high affinity and
selectivity in vitro. In cell culture, the BBP protein
selectively responds to aggregated human A
, failing to respond to
disaggregated or rodent peptides. These findings correlate with
descriptions of sharply reduced A
-mediated toxicity on primary
neuronal preparations if the peptide is not multimeric or is composed
of the rodent amino acid sequence (33, 37, 38). It is important to note
that both aggregated and disaggregated A
bound BBP in
vitro, yet only A
aggregates were able to induce BBP-specific
cell toxicity. Demonstration of a physical association between BBP and
A
in vivo is lacking in this report, leaving open the
possibility that BBP expression potentiates A
toxicity by a
mechanism that does not include direct binding between the molecules.
However, the apparent discrepancy between the in vitro binding of disaggregated A
, and the absence of toxicity in cell culture invokes a potential molecular model for the requisite aggregation of A
to achieve toxicity. Ligand-mediated receptor clustering is a common mechanism of signal activation. As one example,
parallel dimers of the nerve growth factor protein activate TrkA by
bridging the extracellular domains of two receptor proteins, thereby
stimulating intrinsic tyrosine kinase activity (45). Similarly, we
speculate that A
aggregates may promote oligomerization, and
consequent activation, of BBP by a bridging mechanism unachievable by
A
monomers. Central to implicating BBP as a molecular target of A
was the finding that a signaling-deficient variant of BBP could block
the activity of native BBP in human Nt2 neurons, inhibiting the
induction of apoptosis by A
. These data strongly suggest that the
BBP protein regulates neuronal apoptosis initiated by A
. The
discovery of BBP introduces an important new molecule to be considered
in the complex pathophysiology of Alzheimer's disease and presents a
promising new target in the intensive search for novel therapeutic approaches.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the important contributions of
G. Krishnamurthy, D. Fairman, and R. Tasse for A peptide
preparation, J. Wang and R. Gassaway for protein sequencing, X.-Y. Tan,
A. Knight, and D. Ohara for antibody preparations, and K. Sidik for
statistical analyses. Special thanks are extended to J. Barrett, M. Cockett, E. Eppler, D. Howland, D. Kirsch, J. Moyer, P. Spence, and K. Young for advice and assistance.
![]() |
FOOTNOTES |
---|
* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF353990, AF353991, Af353992, AF353993.
These authors contributed equally to this work.
§ Present addresses: Biology Dept., Whitman College, Walla Walla, WA 99362, and Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA 99352.
¶ To whom correspondence should be addressed: Wyeth Neuroscience, Wyeth-Ayerst Research, CN 8000, 865 Ridge Rd., Monmouth Junction, NJ 08852. Tel.: 732-274-4437; Fax: 732-274-4755; E-mail: ozenbeb@war.wyeth.com.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M011161200
2 B. A. Ozenberger, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid-
peptide;
BBP,
-amyloid-binding protein;
Y2H, yeast 2-hybrid;
APP, amyloid precursor protein;
RT-PCR, reverse transcription-polymerase
chain reaction;
RACE, rapid amplification of cDNA ends;
ELISA, enzyme-linked immunosorbent assay;
Nt2, Ntera-2;
PBS, phosphate-buffered saline;
tm, transmembrane;
EST, expressed sequence
tag;
GPCR, G protein-coupled receptor;
kb, kilobase;
BSA, bovine serum
albumin.
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