From the Laboratory of Molecular Neurobiology,
Division of Neurobiology, Department of Psychiatry,
Department of Neuroscience, and ** The Program in Cellular and
Molecular Medicine, The Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205-2196
Received for publication, September 5, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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Huntington's disease is caused by an expanded
CAG trinucleotide repeat coding for a polyglutamine stretch within the
huntingtin protein. Currently, the function of normal huntingtin and
the mechanism by which expanded huntingtin causes selective
neurotoxicity remain unknown. Clues may come from the identification of
huntingtin-associated proteins (HAPs). Here, we show that huntingtin
copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is
encoded by the open reading frame factor VIII-associated gene A (F8A)
located within intron 22 of the factor VIII gene. In transfected
cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is
cytoplasmic in the presence of huntingtin but is actively targeted to
the nucleus in the absence of huntingtin. These data indicate that
HAP40 is likely to contribute to the function of normal huntingtin and
is a candidate for involvement in the aberrant nuclear localization of
mutant huntingtin found in degenerating neurons in Huntington's disease.
Huntington's disease
(HD)1 is an autosomal
dominant neurodegenerative disorder characterized by motor and
cognitive impairments that begin in mid-life and progress over 15-20
years to death. The genetic defect underlying HD is an expanded CAG
trinucleotide repeat encoding a polyglutamine stretch within the
huntingtin protein (1). Normally, huntingtin's CAG repeat codes for
6-35 consecutive glutamines, whereas in HD, it is expanded beyond 35. At least seven other progressive neurodegenerative diseases result from
polyglutamine repeat expansions within otherwise unrelated host
proteins (2). In each disorder, polyglutamine expansion causes a toxic
gain of function, with longer repeats resulting in increased severity.
Accumulating data are consistent with the hypothesis that expanded
polyglutamine repeats adopt a conformation that facilitates toxic
protein-protein interactions and aggregation (3, 4).
Despite sharing similar pathogenic mechanisms, polyglutamine repeat
disorders display distinctive neuropathologies. In each disease, a
different subset of central nervous system neurons is targeted that
cannot be attributed to the expression pattern of the expanded protein.
For example, HD most severely affects medium spiny neurons in the
striatum (5, 6), whereas spinocerebellar ataxia 1 targets cerebellar
Purkinje cells, although both cell types express the huntingtin and
ataxin-1 proteins (for further review, see Ref. 7). Thus, the selective
vulnerability to polyglutamine toxicity is influenced by properties of
the host protein. Protein characteristics known to alter polyglutamine
toxicity in animal and cellular models include nuclear localization,
high local protein concentration, protein-protein interactions, and
protein size or truncation. Each of these features is likely to be
determined by the protein's normal function. Thus, it may be necessary
to identify huntingtin's normal cellular roles to understand how polyglutamine expansion in huntingtin results in the selective toxicity
characteristic of HD.
Huntingtin is a 348-kDa protein that lacks close similarity to known
proteins and has no known function (1). It is expressed in all tissues,
with the highest levels found in brain and testis (6). Within the
brain, huntingtin is expressed in the cytoplasm of neurons (8).
Targeted mutagenesis that significantly reduces huntingtin expression
in mice causes aberrant neurogenesis and perinatal death (9). Complete
disruption of the huntingtin gene in mice results in embryonic
lethality at gastrulation with increased neuronal apoptosis (10-12).
Recent data from cultured neurons support an antiapoptotic role for
normal huntingtin, although the mechanism remains unclear (13).
Yeast two-hybrid screens have identified a number of proteins that
interact with huntingtin, known as huntingtin-interacting proteins
(HIPs), huntingtin-yeast partners (HYPs), and huntingtin-associated proteins (HAPs). These associated proteins implicate huntingtin in a
number of cellular processes (reviewed in Refs. 7 and 14). Among the
interactors, HAP1, HYP-J ( The known huntingtin-associated proteins are likely to represent only a
partial list of proteins that complex with huntingtin in
vivo. All 18 of the huntingtin-interacting proteins identified in
yeast bind to the N-terminal 18% of huntingtin. The absence of
proteins binding the central and C-terminal regions may reflect technical limitations of expressing large proteins in yeast. The central 1500 amino acids of huntingtin comprise 10 HEAT repeats that
are likely to form a single extended structure and mediate protein-protein interactions. No domain information is available to
guide the design of small yeast baits from within the C-terminal 1500 amino acids of huntingtin. Thus, to identify proteins that associate
with full-length huntingtin in vivo, we purified huntingtin complexes from rat brain extracts and identified the protein components.
Fractionation--
Rat brain extracts were prepared in a
glass/Teflon homogenizer in 10 volumes of PBS containing Complete
protease inhibitors (Roche Molecular Biochemicals) and 1 mM
phenylmethylsulfonyl fluoride. Homogenates were successively
centrifuged at 1,000 × g for 10 min, 10,000 × g for 20 min, and 100,000 × g for 1 h.
Pellets were washed once and resuspended in PBS. Protein concentration
was determined by BCA assay (Pierce).
Antibody Preparation--
Antibodies were prepared according to
standard procedures (Cocalico Biological). A goat polyclonal antibody
was generated against a fusion, Hnt Immunoaffinity Purification--
Protein complexes were purified
from rat brain (Pel-Freez, Rogers, AZ). Brains were homogenized in 10 volumes of PBS, 1% Triton X-100. Complete protease inhibitors
(Roche Molecular Biochemicals), and 1 mM
phenylmethylsulfonyl fluoride. This homogenization buffer was modified
with either 1 mM EDTA or 2 µM
Ca2+, with or without phosphatase inhibitors, and with or
without detergent added after 100,000 × g
centrifugation. In all experiments, extracts were cleared by
centrifugation at 100,000 × g for 1 h. For
immunoaffinity purification, 20 ml of cleared brain extract was
combined with antibody resins prepared by coupling 5 mg of affinity-purified antibody to 2 ml of Affi-Gel-10. Beads and brain extract were mixed in batch for 2 h and collected in a column. Beads were successively washed with 50 ml of PBS/1% Triton X-100, 10 ml of PBS/0.1% Triton X-100, and 5 ml of PBS. Proteins were eluted with 5 ml of 0.2 M glycine, pH 2.5. Samples were
concentrated by lyophilization or trichloroacetic acid
precipitation. For immunoprecipitation, 2 µl of antibody was combined
with 1 ml of extract according to the standard procedures (34).
Protein Identification--
Samples were resolved by
SDS-polyacrylamide gel electrophoresis on 4-15% gradient gels
(Bio-Rad) and stained with Coomassie Blue. Proteins were
excised, and sequence analysis was performed at the Harvard
Microchemistry Facility, Cambridge, MA by microcapillary reverse-phase high pressure liquid chromatography nano-electrospray tandem mass spectrometry. For two-dimensional SDS-polyacrylamide gel
electrophoresis, isoelectric focusing was performed in tube
gels (Kenderick Laboratories, Madison, WI) or on pH 3-10 linear
IPGphor strips. Duplicate two-dimensional gels were run for all
samples. Immunoblotting was performed as described previously (33).
HAP40 Cloning and Expression--
A mouse EST known to contain
at least the 5' 763 nucleotides of the F8A/HAP40 open reading frame was
obtained from the Japanese National Institute of Infectious Disease
(Tokyo, Japan) (EST MNCb-6402, GenBank accession number
AU035947). The 1589-base insert was subcloned into the
EcoRI/XbaI sites in pcDNA3.1 and sequenced on
both strands. An epitope-tagged construct was prepared by cloning oligonucleotides encoding the C-terminal 21 amino acids of HAP40 and a
FLAG tag into the BlpI and XbaI sites. The
amino acids KKR in the putative nuclear localization signal were
changed to STS. HEK293 cells were transfected with LipofectAMINE Plus
(Life Technologies, Inc.) and immunolabeled as described previously
(35). Fluorescence images are 0.8-µm optical sections captured on a
Zeiss LSM-410 confocal microscope.
HAP40 in HD Mouse Models and HD Patient Brain--
Brain
sections from mice expressing an N-terminal 171-82Q (36) or littermate
nontransgenic controls were immunostained using the Vectastain ABC
labeling system (Vector Laboratories). Biochemical enrichment of
huntingtin aggregates from post-mortem human brains was performed by
homogenizing aggregates in PBS and centrifuging them at 1,000 × g for 5 min. Pellets were collected, boiled in 2%
SDS, and resolved by SDS-polyacrylamide gel electrophoresis. Immunoblotting with anti-peptide antibodies against huntingtin but not
HAP40 clearly detected proteins in the stacking gel from HD patients
(data not shown).
To prepare quantities of antibody suitable for immunoaffinity
purification, we immunized goats with a fusion protein corresponding to
huntingtin exon 1 with an in-frame deletion of part of the polyglutamine and polyproline regions. We chose this immunogen because
N-terminal fusion proteins with deletions have been shown to generate
rabbit antibodies that strongly label native huntingtin, especially
with an expanded polyglutamine
(37).2
Consistent with previous observations in rodents (38), we noted that
rat huntingtin was found predominately in the soluble fraction (Fig.
1A). Lower levels of
huntingtin were consistently detected in particulate fractions,
suggesting specific association with membranes, the nucleus, or other
cellular structures. To extract the maximum amount of soluble
huntingtin for purification, we used rat brain homogenates treated with
Triton X-100.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-adaptin-C), SH3GL3 (endophilin III), and
HIP3 are believed to be involved in vesicular recycling or trafficking
(15-21). Several interactors, including nuclear corepressor, HYP-A,
and HYP-I (symplekin), have been linked to nuclear functions (20, 22).
Other interactors include: (a) HIP1, which is related to
yeast Sla2p, a transmembrane protein with a talin-like domain (23, 24);
(b) HIP2, which is an E2 ubiquitin-conjugating enzyme (25);
(c) HYP-F, which is part of the 26S proteosome (20); and
(d) cystathionine
-synthase, which is a key enzyme in the
generation of cysteine (26). Six additional HYPs have been identified
but have no known cellular roles (20). Other data suggest that
cAMP-response element-binding protein-binding protein,
TATA-binding protein, epidermal growth factor receptor, mixed-lineage
kinase 2, and calmodulin can associate with huntingtin under some
conditions (27-32).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ex1, corresponding to amino acids
1-23 and 69-92 of human huntingtin. For immunization, a
Hnt
ex1-glutathione S-transferase fusion protein
was expressed, purified on glutathione-agarose, resolved by
SDS-polyacrylamide gel electrophoresis, and injected as a
Coomassie Blue-stained gel slice. A purified
Hnt
ex1-His-tagged fusion protein was used for affinity purification
of huntingtin antibodies from serum and anti-glutathione
S-transferase antibodies. Rabbit polyclonal antibodies
(rAbs) were prepared against peptides coupled to keyhole limpet
hemocyanin through a terminal cysteine. Huntingtin rAbs were
generated against the peptides MATLEKLMKAFESLKSFQC and CITEQPRSQHTLQ
corresponding to amino acids 1-18 and 497-508, respectively. HAP40
rAbs were prepared against peptides CRY- RQVSNKLKKRFLRKPN, CPQPPSGPQPPLSGPQPRP, and CDGHGQDTSGQLPEE corresponding to amino acids
25-42, 226-243, and 314-326, respectively. With the exception of the
crude antisera used for the immunoprecipitation in Fig. 5A,
all antibodies were affinity-purified using antigen coupled to Affi-Gel
(Bio-Rad) as described previously (33). MAP2 and PYK2 antibodies were
purchased from Transduction Laboratories. The huntingtin monoclonal
antibody (mAb) 2166 was purchased from Chemicon. The M2
anti-FLAG antibody was obtained from Sigma. The HAP1 antibody
was obtained from Alan Sharp, the Johns Hopkins University, Baltimore,
MD. The HIP2 antibody was a gift of Cecile Pickart, the Johns Hopkins
University, Baltimore, MD.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunoaffinity purification of
huntingtin. A, rat brain extracts were successively
centrifuged at 1,000 × g, 10,000 × g,
and 100,000 × g. 40 µg of protein from the resulting
pellets (P1, P10, and P100, respectively) and the
final supernatant (S100) were immunoblotted for huntingtin
(mAb 2166). B, control and huntingtin samples were
immunoaffinity-purified from Triton X-100 solubilized rat brain
extracts using goat IgG and goat polyclonal antibody Hnt ex1 columns.
Samples (0.5 µl) were blotted for huntingtin (mAb 2166).
C, goat IgG and goat polyclonal antibody Hnt
ex1
immunoaffinity-enriched samples (5 µl) were resolved on 4-15% gels
and stained with Coomassie Blue. Four protein bands of ~350,
115, 70, and 40 kDa were coenriched in huntingtin samples compared with
IgG samples. The 40-kDa protein migrates below a doublet of more
intensely stained nonspecific proteins at ~41 and 45 kDa. Protein
sequencing identified these four proteins as huntingtin, PYK2, MAP2,
and a novel protein termed HAP40, respectively. PYK2, MAP2(b), and
MAP2(c) have predicted molecular masses of 118, 199, and 70 kDa,
respectively.
Immunoaffinity purification with the anti-Hntex1 antibody
dramatically enriched for huntingtin (Fig. 1B). Purification
was specific because immunoblotting did not detect huntingtin in
samples eluted from goat IgG columns (Fig. 1B). As expected
from a one-step preparation, Coomassie Blue-stained gels
revealed that huntingtin samples were highly enriched in a
350-kDa protein but were not pure (Fig. 1C).
Compared with the proteins present in control samples, four major
proteins of
40, 70, 115, and 350 kDa were consistently
enriched in the anti-huntingtin samples (Fig. 1C; data not
shown). Several variations in the extraction conditions (see
"Materials and Methods") did not significantly alter the proteins
that specifically coenriched in huntingtin samples (data not shown).
The four protein bands were identified by mass spectroscopy/mass spectroscopy sequencing of tryptic peptides. The 350-kDa band yielded 71 nonoverlapping peptide sequences corresponding to huntingtin and 5 peptides corresponding to MAP2(a/b). The 115-kDa band was comprised of PYK2. The 70-kDa band contained MAP2(c). The sequence from
the 40-kDa protein band corresponded to open reading frame F8A located
on chromosome X within intron 22 of the factor VIII gene (39). We refer
to this 40-kDa huntingtin-associated protein as HAP40.
Immunoblot analysis with commercial antibodies to PYK2 and MAP2 and
newly prepared HAP40 antibodies (described below) confirmed that each
protein was highly enriched in samples eluted from anti-Hntex1 columns compared with those eluted from goat IgG columns (data not
shown). However, sequence comparisons revealed a common proline-rich sequence, PPPXP, in the Hnt
ex1 fusion protein, PYK2, and
MAP2(a/b/c). This raises the possibility that both PYK2 and MAP2 were
enriched by binding to antibodies against huntingtin's proline-rich
region rather than by associating with the huntingtin protein. To
distinguish between these possibilities, we prepared new antibodies
against huntingtin peptides 1-18 and 497-508, which lack consecutive
prolines. Immunoaffinity purification with each of these antibodies
greatly enriched for huntingtin and HAP40 but not for PYK2 or MAP2.
With long exposures, PYK2 and MAP2 could be detected nonspecifically in
both anti-huntingtin and rabbit IgG samples (Fig.
2A). In contrast, HAP40 was
highly enriched in both rAb Hnt1-18 and rAb
Hnt497-508 samples in comparison with the IgG control
(Fig. 2A). Thus, we conclude that HAP40 is tightly
associated with huntingtin after Triton X-100 solubilization.
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To identify additional proteins copurifying with huntingtin in our
assay, we immunoblotted for the previously identified
huntingtin-interacting proteins. Immunoblots for HAP1 and HIP2 clearly
detected proteins migrating at the predicted size in crude soluble
fractions. However, neither protein was found to copurify with
huntingtin despite increasing the amount of sample loaded by 20-fold
compared with that needed to saturate the immunoblot signal for
huntingtin (Fig. 2B). Similar immunoblots probed for other
huntingtin-binding proteins including HIP1, SH3GL3, symplekin (HYP-I),
-adaptin (HYP-J), calmodulin, epidermal growth factor receptor, and
the nuclear corepressor failed to reveal any enrichment in huntingtin
samples (data not shown). We conclude that none of these proteins form Triton X-100-resistant complexes with soluble huntingtin (see "Discussion").
To search for additional interacting proteins that may be present at
levels too low to be detected by Coomassie Blue staining or
obscured by comigrating contaminate proteins, we analyzed
immunoaffinity-purified huntingtin samples by two-dimensional gel
electrophoresis and silver staining. Samples eluted from rAb
Hnt1-18, rAb Hnt497-508, goat polyclonal
antibody Hntex1, rabbit IgG, and goat IgG columns were compared
(Fig. 3; data not shown). The pattern of
protein spots was highly similar in all samples and reproducible in
several experiments. Each of the five samples contained some proteins that were unique to that antibody column (Fig. 3, compare double arrows). Two 40-kDa protein spots were detected in all three
huntingtin samples but were not detected in either IgG control (Fig. 3,
single arrows). No other potential HAPs were identified in
more than one huntingtin sample and absent in IgG samples.
Immunoblotting similar gels with HAP40 antibodies revealed a pair of
40-kDa spots in a pattern indistinguishable from the two silver-stained
spots (Fig. 3D). The reason for two HAP40 spots is unknown.
We conclude that HAP40 is a major component of the huntingtin protein
complex.
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For expression analysis, we obtained mouse EST MNCb-6402, which was
previously reported to code for at least the N-terminal 254 amino acids
of HAP40/F8A. Complete sequencing of this EST revealed a Kozak start
site followed by an open reading frame encoding 381 amino acids with a
calculated molecular mass of 40,472.78 Da. This EST sequence
differed from the previously reported mouse F8A sequence (39) by one
amino acid insertion and two substitutions (Fig.
4). These changes are unlikely to result
from errors in the mouse EST sequence because each of these three
residues in the mouse EST was identical to human ESTs. None of the
differing residues were confirmed by mass spectroscopy/mass
spectroscopy peptide sequences (Fig. 4).
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To further test the huntingtin-HAP40 association, we prepared
anti-HAP40 peptide antibodies. Two HAP40 antisera coimmunoprecipitated huntingtin from mouse brain extracts (Fig.
5A). This was judged to be
specific because preimmune serum failed to coimmunoprecipitate huntingtin. When considered together, the coimmunoprecipitation of
huntingtin with two anti-HAP40 antibodies and the copurification of
HAP40 with three anti-huntingtin antibodies strongly indicate that
huntingtin and HAP40 form a stable complex.
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To determine the region of huntingtin required for association with HAP40, HEK293 cells were cotransfected with HAP40 and huntingtin constructs of various lengths. Full-length huntingtin specifically coimmunoprecipitated with HAP40 (Fig. 5B). Despite being expressed at levels higher than full-length huntingtin, a construct corresponding to the N-terminal 513 amino acids failed to coimmunoprecipitate with HAP40 (Fig. 5B). HAP40 is the first protein known to require either the central or C-terminal regions of huntingtin for association. Although additional experiments will be needed to prove that the HAP40-huntingtin interaction is direct, it seems unlikely that other proteins mediate this interaction because none were detected in the purified huntingtin complexes (Fig. 3).
To compare the subcellular localization of HAP40 and huntingtin, we
immunolabeled sections of mouse brain and transfected cells. None of
the anti-peptide antibodies against HAP40 labeled normal brain
sections. Because each antibody recognized native protein in
transfected cells, these data are consistent with low expression levels
of HAP40 proteins in normal brain. On sections of HD-N171-82Q
transgenic mouse brain, the anti-huntingtin antibodies but not the
anti-HAP40 antibodies labeled inclusions (data not shown). In HEK293
cells transiently transfected with full-length huntingtin and HAP40,
immunostaining for both proteins showed a similar cytoplasmic
distribution (Fig. 6, top
panels). Cotransfection of either normal (23Q) or expanded (82Q)
full-length huntingtin with HAP40 resulted in similar colocalization in
the cytoplasm (data not shown). Surprisingly, in cells that lack
detectable endogenous huntingtin, HAP40 transfected alone was
concentrated in the nucleus, both diffusely and in punctate nuclear
structures (Fig. 6, bottom panels). The HAP40 nuclear dots
did not overlap with staining for a splicing factor (mAb SC35) or
promyelocytic leukemia protein oncogenic domains detected with
anti-PML antibodies (data not shown). Although the nature of
HAP40 nuclear structures remains unclear, they are unlikely to result
from nonspecific aggregation of overexpressed protein because they were
observed in individual cells expressing lower levels of protein (Fig.
6).
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HAP40 is smaller than the 45-kDa threshold for passive
diffusion through nuclear pores. However, passive diffusion alone would
not be expected to result in the relative concentration of HAP40 in the
nucleus. To test the possibility that HAP40 is actively targeted to the
nucleus, we examined the localization of a 68-kDa HAP40-GFP fusion
protein. The HAP40-GFP fusion was concentrated in the nucleus (Fig. 7,
upper panels). Mutation of a
lysine/arginine-rich region resembling a nuclear localization signal (NLS) resulted in a significant redistribution of HAP40-GFP to
the cytoplasm (Fig. 7, lower panels). Low levels of
HAP40-GFP mutant NLS were detectable in the nucleus, suggesting that
HAP40 may be actively targeted to the nucleus by multiple mechanisms (Fig. 7).
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DISCUSSION |
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We immunoaffinity-purified huntingtin and characterized the associated proteins. We found that most rat huntingtin is soluble and that soluble huntingtin associates with a novel protein, HAP40. None of the previously identified huntingtin-associated proteins were found to copurify with huntingtin in our assay. This result may be due to several reasons, the most notable of which is that our purification system requires soluble huntingtin and the presence of detergent to reduce nonspecific protein associations. Thus, we would not expect to purify huntingtin-associated protein complexes that were either detergent-sensitive or detergent-insoluble. Interestingly, HAP1 and SH3GL3 have been shown to form detergent-resistant complexes with huntingtin from transfected cells (15, 19), yet neither was found to copurify with huntingtin from brain, raising the possibility that a number of proteins may associate with huntingtin in a regulated fashion. The stable association of HAP40 with huntingtin suggests that it is likely to hold important clues to the cellular role of huntingtin.
Peptide sequencing of HAP40 revealed that it is encoded by an open reading frame previously termed F8A. F8A was identified as a putative gene located within intron 22 of the factor VIII gene but transcribed in the direction opposite factor VIII. In humans, a portion of factor VIII intron 22 including F8A is duplicated with two additional copies located nearer to the Xq telomere. Intrachromosomal recombination between intron 22 and either of these two copies interrupts the factor VIII gene and underlies almost half of all severe hemophilia A cases (40). Our data prove that the single F8A sequence in mouse is expressed. However, because the three intron 22 sequences in human are 99% identical, it remains unclear which of the three human F8A sequences are expressed.
F8A/HAP40 mRNA is expressed in a wide range of mouse tissues, similar to huntingtin (39). When recombinant HAP40 and huntingtin are coexpressed in cells, they colocalize in the cytoplasm. In the absence of huntingtin, HAP40 is concentrated in the nucleus, where it is both diffuse and in punctate nuclear structures. We found that a HAP40-GFP fusion protein too large to diffuse through nuclear pores was concentrated in the nucleus. Mutation of a NLS largely redistributed HAP40-GFP to the cytoplasm, indicating that HAP40 is actively targeted to the nucleus. Future experiments will be needed to determine the function of nuclear HAP40.
The mechanism by which huntingtin redistributes HAP40 to the cytoplasm
in transfected cells is unclear. Huntingtin is normally concentrated in
the cytoplasm and may simply anchor HAP40. Alternatively, several lines
of evidence are consistent with regulated active nuclear transport of
huntingtin. Huntingtin contains several consensus NLSs (41) and >10
sequences matching leucine-rich nuclear export signals.3 Huntingtin is
comprised of 10 HEAT repeats (42) plus an additional flanking sequence
highly similar to -importin, a key component of the nuclear
transport machinery.3 Low levels of full-length huntingtin
have been detected in purified nuclei from brain (43) and in the the
nucleus of cultured cells (44). Thus, under various conditions,
huntingtin may import and export HAP40 through nuclear pores.
HAP40 is unlikely to be directly involved in the toxic gain of function
caused by huntingtin's expanded polyglutamine. HAP40 does not
associate with the N-terminal region of huntingtin that contains the
repeat and is not easily detected in huntingtin aggregates of either
mice transgenic for an expanded N-terminal fragment or HD post-mortem
brain. However, events that regulate nuclear localization of the
huntingtin-HAP40 protein complex are candidates for contributing to the
selective neuronal vulnerability in HD. In mouse models of HD generated
by inserting an expanded polyglutamine stretch into mouse huntingtin,
full-length (43) and N-terminal fragments of expanded huntingtin (45)
are found in the nucleus. The nuclear redistribution is selective for
the striatal neurons most vulnerable in HD. Nuclear targeting
exacerbates the toxicity of both expanded huntingtin and ataxin-1 (35,
46, 47). A key role for the nuclear localization is consistent with
recent findings that expanded polyglutamine repeats cause toxicity via changes in cAMP-response element-binding protein-binding
protein-mediated transcription (30, 48, 49). Investigating the
mechanisms that regulate the localization of the normal
huntingtin-HAP40 complex may be important to understanding the aberrant
nuclear localization and toxicity of expanded huntingtin.
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ACKNOWLEDGEMENTS |
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We thank Jon Wood for the
Hntex1-glutathione S-transferase plasmid, Jon Kushi for
affinity-purifying antibodies, Cecile Pickart for providing
the HIP2 antibody, Erich Wanker for the SH3GL3 and HIP1 antibodies,
Bill Lane for helpful discussions regarding protein sequencing, and
Fred Nucifora and Dave Borchelt for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Huntington's Disease Society of America "Coalition for the Cure" and National Institutes of Health Grants NS16375, NS34172, and NS38144 (to C. A. R.).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) AF299331.
§ Supported by a Huntington's Disease Society of America postdoctoral fellowship.
¶ To whom correspondence may be addressed: Laboratory of Molecular Neurobiology, Departments of Psychiatry and Neuroscience, Johns Hopkins University School of Medicine, Ross 618, 720 Rutland Ave., Baltimore, MD 21205-2196. Tel.: 410-614-0011; Fax: 410-614-0013; E-mail: mfpeters@jhmi.edu (M. F. P.) or caross{at}jhu.edu (C. A. R.).
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008099200
2 J. D. Wood and C. A. Ross, unpublished data.
3 M. F. Peters and C. A. Ross, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: HD, Huntington's disease; HAP, huntingtin-associated protein; NLS, nuclear localization signal; rAb, rabbit polyclonal antibody; mAb, monoclonal antibody; HIP, huntingtin-interacting protein; HYP, huntingtin-yeast partner; PBS, phosphate-buffered saline; MAP, mitogen-activated protein; PYK, proline-rich tyrosine kinase; EST, expressed sequence tag; GFP, green fluorescent protein; F8A, factor VIII-associated gene A.
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REFERENCES |
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