Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2007.00352.x |
Sequestration of
Glyceraldehyde-3-phosphate Dehydrogenase to Aggregates Formed by Mutant
Huntingtin
Junchao WU, Fang LIN, and
Zhenghong QIN*
Department
of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow
University School of Medicine, Suzhou 215123, China
Received: April 18,
2007
Accepted: June 20,
2007
This work was partially
supported by the grants from the National Natural Science Foundation of China
(No. 30370506) and the Specialized Research Fund for the Doctoral Program of
Higher Education, China (No. 20050285017)
*Corresponding author:
Tel, 86-512-65880406; Fax, 86-512-65880406; E-mail, [email protected]
Abstract Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) has been reported to interact with proteins containing the
polyglutamine (polyQ) domain. The present study was undertaken to evaluate the
potential contributions of the polyQ and polyproline (polyP) domains to the
co-localization of mutant huntingtin (htt) and GAPDH. Overexpression of
N-terminal htt (1–969 amino acids) with 100Q and
46Q (htt1-969-100Q and httl-969-46Q, mutant htt) in human mammary gland
carcinoma MCF-7 cells formed more htt aggregates than that of htt1-969-18Q
(wild-type htt). The co-localization of GAPDH with htt aggregates was found in
the cells expressing mutant but not wild-type htt. Deletion of the polyP region
in the N-terminal htt had no effect on the co-localization of GAPDH and mutant
htt aggregates. These results suggest that the polyQ domain, but not the polyP
domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt.
This effect might contribute to the dysfunction of neurons caused by mutant htt
in Huntington’s disease.
Keywords huntingtin; GAPDH; polyglutamine; polyproline
Huntington抯 disease (HD) is an adult-onset, autosomal-dominant neurodegenerative
disorder. Patients with HD are characterized by hyperkinetic involuntary
movement, cognitive impairment, and depression [1]. The gene responsible for HD
was identified as IT15. This gene contains a CAG repeat that encodes the
polyglutamine (polyQ) tract in the N-terminus of huntingtin (htt). This
trinucleotide repeat is highly polymorphic. The normal range of the repeat is
from 10 to 35Q. The disease-causing mutation was identified as an expansion of
this repeat to more than 36 triplets [2]. In a variety of HD animal models,
cellular accumulation of mutant htt (mhtt) is a critical step to HD pathology
[3–6].
The polyproline (polyP) domain is a proline-rich region located immediately
after polyQ. It is an important domain for many protein-protein interactions.
It has been reported that polyP interacts with proteins containing WW domains
including huntingtin-protein interactors, htt yeast partners (HYPs; HYPA, HYPB
and HYPC), that appear to have a higher affinity for expanded polyQ htt [7].
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) has
been well studied as a classical glycolytic protein in energy production. In
fact, GAPDH is a multifunctional protein and plays roles in a variety of
activities, including endocytosis and membrane fusion, microtubule bundling and
translational regulation, nuclear transfer RNA export, as well as DNA
replication and DNA repair [8]. In vivo, GAPDH has three forms: tetramer
of identical 37 kDa subunits; dimer; and monomer. These forms are related to
its diverse functions. Its glycolytic activity is restricted to the tetramer,
whereas the dimeric and monomeric structures are required for its other
functions.
In the last 10 years, the contribution of GAPDH to neurodegenerative
diseases has been reported [9–11]. Some in vitro studies have shown an interaction
between GAPDH and the mutated forms of several CAG trinucleotide
repeat-containing proteins. It was thought to be the causative factor in
certain neurodegenerative diseases [12,13]. The interaction between htt and
GAPDH was proposed as one possible cause of cell death in HD [8]. As polyQ and
polyP are important domains in htt and are involved in many protein
interactions [14], this study was designed to investigate whether the polyQ and
polyP domains took part in the interactions between GAPDH and htt.
Materials and Methods
Materials
Huntingtin 1-969 expression plasmids, including pcDNA3-htt1-969-18Q,
pcDNA3-htt1-969-46Q, pcDNA3-htt1-969-46Q with proline deletion, and pcDNA3-htt1-969-100Q,
were kindly provided by Dr. Marian DiFIGLIA (Massachusetts General Hospital and
Harvard Medical School, Boston, USA). These constructs encode an N-terminal htt
fragment (1–969 amino acids) with 18, 46, or 100Q and a Flag tag. To study the
role of polyP in the interaction between htt and GAPDH, a polyP rich region was
deleted from htt1-969-46Q. Human mammary gland carcinoma cell line MCF-7 was
purchased from the Institute of Biochemistry and Cell Biology, Shanghai
Institutes of Biological Sciences (Shanghai, China). Fetal bovine serum was
purchased from Hangzhou Sijiqing Biological Company (Hangzhou, China). The
transfection reagent SuperFect was purchased from Qiagen (Valencia, USA).
Cell culture and transient
transfection
MCF-7 cells are epithelial-type cells derived from human mammary
gland carcinoma. Cells were cultured in 25 cm2 flasks
in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 mg/L
streptomycin, and 100 U/ml penicillin, and incubated in a humidified atmosphere
of 5% CO2 at 37 篊. SuperFect was used for transfection. At 80% confluence, cells were
incubated in 2 ml RPMI 1640 containing 5–10 mg htt cDNA and 36 ml SuperFect.
After incubation for 3 h, cells were given 3 ml fresh RPMI 1640 medium, and
culturing was continued to the desired time.
Protein preparation and
Western blot analysis
For preparation of whole cell lysates, cells were scraped off the
flasks and centrifuged at 500 g for 5 min, 24 h after transfection. The
cell pellets were rinsed with ice-cold phosphate buffer solution (PBS) twice.
Five volumes of Western blot lysing buffer (containing 20 mM Tris-HCl, pH 7.0,
20 mM EDTA, 5% Triton X-100 supplemented with a cocktail of protease inhibitors
containing 1 mM pefablock, 100 U/ml aprotinin, and 10 mg/ml leupeptin) for each
volume of cell pellets was added, then the mixture was incubated on ice for 10
min and sonicated on ice (1 s/ml per sonicate, an interval of 30 s, a total of
five times). The lysate was microcentrifuged at 10,600 g at 4 篊 for 10 min and supernatant
was preserved at –70 篊 for later use. Protein concentration was determined with a BCA kit
(Pierce, Rockford, USA). Samples were mixed with loading buffer and boiled for
5 min. An aliquot of 30 mg protein from each sample was separated on 10% sodium dodecyl
sulfate-polyacrylamide gel, and protein was subsequently transferred to
nitrocellulose membranes. Membranes were blocked with 5% skimmed milk in 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20, and immunoblotted with
anti-GAPDH monoclonal antibody (1:1000 dilution; Advanced Immunochemical, Long
Beach, USA) at 4 篊 overnight. Excess primary antibody was removed by three washes with
0.1% Tween-20. The reaction of primary antibodies was detected using
horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Jackson
ImmunoResearch Laboratories Inc., West Grove, USA) with a dilution of 1:5000 in
blocking solution for 1 h at room temperature. Immunoreactivity was detected by
enhanced chemiluminescence (ECL kit; Amersham Pharmacia Biotech, USA) and
visualized by autoradiography. Actin was used as a loading control and detected
with a mouse monoclonal antibody (Sigma, St. Louis, USA).
Immunofluorescence
MCF-7 cells were cultured on poly-L-lysine-coated microslips and
transfected with different cDNAs encoding htt proteins as described above.
Cells were washed with PBS and fixed with 4% paraformaldehyde (Fluka, Buchs,
Schweiz) in PBS for 15 min at room temperature. Cells were subsequently washed
with PBS and incubated in PBS containing 0.1% Triton X-100 for 10 min. After
being washed again with PBS, the cells were then incubated for 1 h in PBS
containing 2% non-fat milk (Bright Dairy and Food, Shanghai, China) at room
temperature. Cells were then incubated overnight at 4 篊 in a blocking solution
containing rabbit anti-htt polyclonal antibody Ab1 (gift from Dr. Marian
DiFIGLIA) with a dilution of 1:1500 and mouse anti-GAPDH monoclonal antibody
(Immunochemical) with a dilution of 1:1000. Cells were then incubated with
fluorescein-isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G
antibody with a dilution of 1:400 and Cy3-conjugated donkey anti-mouse
immunoglobulin G antibody (Jackson ImmunoResearch Laboratories) with a dilution
of 1:600 for 2 h at room temperature [15]. Immunostained cells were examined
with a confocal microscope (Radiance 2001; Bio-Rad, Hercules, USA) using a 100
oil immersion lens. Confocal images were captured with Laser Sharp 2000
software and merged in Adobe Photoshop (Version 8.0). To quantify the
co-localization of GAPDH and htt aggregates in the cells expressing htt, 100
htt aggregate-positive cells per microslip were randomly scanned with a
confocal microscope, and six microslips were used for each group. The examiner
was unaware of the experimental conditions.
Statistics analysis
Statistical analysis was carried out with one-way anova.
P<0.05 was considered to be significant.
Results
Expression of GAPDH in MCF-7 cells
after transfection of htt
The level of GAPDH in MCF-7 cells after expression of htt was
determined by Western blot analysis. GAPDH was detected as a major protein
band, using immunoblotting, with the expected molecular weight. The level of GAPDH
was not significantly altered by expressed htt (Fig. 1; P>0.05).
Co-localization of mhtt and
GAPDH
Twenty-four hours after transfection of htt1-969-18Q, htt1-969-100Q
and htt1-969-46Q, double immunofluorescence of htt and GAPDH was used for identification
of the expression of the two proteins in MCF-7 cells. As shown in the confocal
images, htt aggregates were observed in the cytoplasm in the cells expressing
htt1-969-18Q, htt1-969-100Q or htt1-969-46Q. GAPDH in control cells [Fig.
2(A), top panel] and cells expressing htt1-969-18Q [Fig. 2(A)]
appeared dispersed in the cytoplasm. No co-localization of high levels of GAPDH
in htt aggregates was formed by htt1-969-18Q. In contrast, immunofluorescence
patches of GAPDH were found in the cells expressing htt1-969-100Q and
htt1-969-46Q. These GAPDH patches were co-localized with htt aggregates [Fig.
2(A)]. Quantitative analysis of htt-positive cells bearing mhtt aggregates
revealed that sequestration of GAPDH was found in approximately 60% of these cells.
There was no sequestration of GAPDH by htt aggregates formed by wild-type htt [Fig.
2(B)].
Influence of polyP domain on
interaction between mhtt and GAPDH
Double immunofluorescence was used to identify the expression of
GAPDH and mhtt proteins in MCF-7 cells transfected with htt1-969-46Q and
htt1-969-46Q with polyP deletion. The results showed that GAPDH localized to
htt aggregates in the cells transfected with either htt1-969-46Q or
htt1-969-46Q with polyP deletion [Fig. 3(A)]. Quantitative analysis of
htt-positive cells bearing mhtt aggregates revealed that sequestration of GAPDH
was not altered in the cells expressing htt1-969-46Q with or without polyP
deletion [Fig. 3(B); P>0.05].
Discussion
Many studies have found that mhtt obtained abnormal interactions
with other cytoplasmic and nuclear proteins [16–19]. Recruitment or
sequestration of cellular proteins might is possible causes of cell death in HD
[20]. Htt interacts with a number of proteins including GAPDH, a key glycolytic
enzyme in the process of glycolysis, and such binding was relevant to the
nuclear targeting and cytotoxicity of mhtt [21].
The present study confirmed the domains of htt involved in its
interaction with GAPDH. Four constructs of htt cDNA were used, one was
wild-type htt1-969-18Q and the other three were mutant types, htt1-969-100Q,
htt1-969-46Q, and htt1-969-46Q with polyP deletion. Using Western blot
analysis, we found that expression of either wild-type (wt) or mutant htt did
not change the overall levels of GAPDH. However, GAPDH formed patches and
localized to htt aggregates in the cells expressing mhtt. In contrast,
expression of wt htt had no effect on cellular distribution of GAPDH. This
suggests that GAPDH might be sequestered by mhtt into htt aggregates. Our
previous research revealed that htt was cleaved by several proteases, such as
caspases and calpains [22]. The cleavage of htt by these proteases produces
N-terminal htt fragments. N-terminal htt is prone to aggregate in cells and
might be the cause of neuronal death in HD. It is critical for cytotoxicity
induced by HD. In our previous studies, we found that htt bodies formed either
by wt htt or mhtt sequestered many cytoplasmic proteins in a polyP-dependent manner
[14]. The present study revealed that aggregates formed by mhtt, but not wt
htt, could sequestrate GAPDH, suggesting that this kind of sequestration is
dependent on expanded CAG repeats. We also examined the relationship of GAPDH
and the polyP domain in htt. We found that GAPDH still co-localized with mhtt
aggregates in the absence of the polyP domain. This suggests that sequestration
of GAPDH by mhtt is dependent on an expanded polyQ, but not polyP domain. The
sequestration of GAPDH by mhtt might affect cell viability as GAPDH has
important cellular functions.
In fact, many proteins with CAG expansions bind to GAPDH. mhtt-GAPDH
interactions through this expanded polyQ domain could not only affect energy
production but also result in pleiotropic effects involving various biochemical
pathways in HD cells. Other in vitro studies have also shown that GAPDH
glycolytic activity was specifically reduced in Alzheimer抯 disease and HD [23].
Furthermore, mitochondria dysfunction and impairment of energy metabolism have
long been implicated in HD pathogenesis [24]. There are several reports
suggesting that interactions between mhtt and GAPDH might play important roles
in HD pathogenesis. This notion is supported by data from HD patients showing
reduced ATP production and increased lactate production [25], and supported by
animal studies showing that compromising mitochondria function produced
striatal lesions mimicking human HD pathology [26]. In HD fibroblasts, reduced
GAPDH activity has been reported in subcellular fractions [10]. Additionally,
the ability of GAPDH to translocate to the nucleus might also be related to
apoptosis of cells [27], causing neuronal cell death in HD. GAPDH might act as
a chaperon through its selective binding to the polyQ region of htt. Senatorov
et al. also reported that GAPDH was directly or indirectly involved in the
initiation of apoptotic cascades in a transgenic mouse model of HD [28].
Mazzola and Sirover identified an abnormal nuclear GAPDH structure in HD, a
high molecular weight species that alters GAPDH function [10]. Nuclear GAPDH
will interact with DNA to affect nuclear transfer RNA transport, DNA
replication, and DNA repair [29], and to modulate calcium release [30]. All of
these lines of evidence suggest that interactions between mhtt and GAPDH could
play a role in cell death in HD. The present results showed that GAPDH could be
sequestered to htt aggregates formed by mhtt, providing additional evidence in
support of the notion that mhtt might have an effect on physiological functions
of GAPDH. However, the exact function of GAPDH in the pathogenesis of HD
remains unclear. Further studies are warranted to examine the sequestration of
GAPDH in HD patients and the functional impact of GAPDH sequestration in the
cells expressing mhtt.
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