|
|
|
Original Paper
|
|
|||
|
Acta Biochim Biophys
Sin 2007, 39: 885�890 |
||||
|
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.
References
1��� Haddad MS, Cummings JL. Huntington�s disease. Psychiatr Clin North Am 1997, 20: 791-807
2��� The Huntington�s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington�s disease chromosomes. Cell 1993, 72: 971-983
3��� Hodgson JG, Agopyan N, Gutekunst CA, Leavitt BR, LePiane F, Singaraja R, Smith DJ et al. A YAC mouse model for Huntington�s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 1999, 23: 181-192
4��� Wheeler VC, White JK, Gutekunst CA, Vrbanac V, Weaver M, Li XJ, Li SH et al. Long glutamine tracts cause nuclear localization of a novel form of huntingtin in medium spiny striatal neurons in HdhQ92 and HdhQ111 knock-in mice. Hum Mol Genet 2000, 9: 503-513
5��� Sawa A. Mechanisms for neuronal cell death and dysfunction in Huntington�s disease: Pathological cross-talk between the nucleus and the mitochondria? J Mol Med 2001, 79: 375-381
6��� Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA, Scherzinger E et al. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 1997, 90: 537-548
7��� Faber PW, Barnes GT, Srinidhi J, Chen J, Gusella JF, MacDonald ME. Huntingtin interacts with a family of WW domain proteins. Hum Mol Genet 1998, 7: 1463-1474
8��� Mazzola JL, Sirover MA. Alteration of intracellular structure and function of glyceraldehyde-3-phosphate dehydrogenase: A common phenotype of neurodegenerative disorders? Neurotoxicology 2002, 23: 603-609
9��� Sirover MA. New insights into an old protein: The functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta 1999, 1432: 159-184
10�� Mazzola JL, Sirover MA. Alteration of nuclear glyceraldehyde-3-phosphate dehydrogenase structure in Huntington�s disease fibroblasts. Brain Res Mol Brain Res 2002, 100: 95-101
11�� Mazzola JL, Sirover MA. Subcellular alteration of glyceraldehyde-3-phosphate dehydrogenase in Alzheimer�s disease fibroblasts. J Neurosci Res 2003, 71: 279-285
12�� Burke JR, Enghild JJ, Martin ME, Jou YS, Myers RM, Roses AD, Vance JM et al. Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. Nat Med 1996, 2: 347-350
13�� Koshy B, Matilla T, Burright EN, Merry DE, Fischbeck KH, Orr HT, Zoghbi HY. Spinocerebellar ataxia type-1 and spinobulbar muscular atrophy gene products interact with glyceraldehyde-3-phosphate dehydrogenase. Hum Mol Genet 1996, 5: 1311-1318
14�� Qin ZH, Wang Y, Sapp E, Cuiffo B, Kegel KB, Aronin N, DiFiglia M. Huntingtin bodies sequester vesicle-associated proteins by a polyproline-dependent interaction. J Neurosci 2004, 24: 269-281
15�� Qin ZH, Wang Y, Chen RW, Wang X, Ren M, Chuang DM, Chase TN. Prostaglandin A(1) protects striatal neurons against excitotoxic injury in rat striatum. J Pharmacol Exp Ther 2001, 297: 78-87
16�� Li XJ, Li SH, Sharp AH, Nucifora FC Jr, Schilling G, Lanahan A, Worley P et al. A huntingtin-associated protein enriched in brain with implications for pathology. Nature 1995, 378: 398-402
17�� Kalchman MA, Koide HB, McCutcheon K, Graham RK, Nichol K, Nishiyama K, Kazemi-Esfarjani P et al. HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nature Genet 1997, 16: 44-53
18�� Wanker EE, Rovira C, Scherzinger E, Hasenbank R, W�lter S, Tait D, Colicelli J et al. HIP-I: A huntingtin interacting protein isolated by the yeast two-hybrid system. Hum Mol Genet 1997, 6: 487-495
19�� Kalchman MA, Graham RK, Xia G, Koide HB, Hodgson JG, Graham KC, Goldberg YP et al. Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme. J Biol Chem. 1996, 271: 1938519394
20�� Di Prospero NA, Tagle DA. Normal and mutant huntingtin: Partners in crime. Nat Med 2000, 6: 1208-1209
21�� Bae BI, Hara MR, Cascio MB, Wellington CL, Hayden MR, Ross CA, Ha HC et al. Mutant huntingtin: Nuclear translocation and cytotoxicity mediated by GAPDH. Proc Natl Acad Sci USA 2006, 103: 3405-3409
22�� Qin ZH, Gu ZL, Lin F. Research progress in molecular pathology of Huntington�s disease. Chin Pharmacol Bull 2004, 20: 378-382
23�� Mazzola JL, Sirover MA. Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimer�s disease and in Huntington�s disease fibroblasts. J Neurochem 2001, 76: 442-449
24�� Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 2006, 443: 787-795
25�� Lodi R, Schapira AH, Manners D, Styles P, Wood NW, Taylor DJ, Warner TT. Abnormal in vivo skeletal muscle energy metabolism in Huntington�s disease and dentatorubropallidoluysian atrophy. Ann Neurol 2000, 48: 72-76
26�� Trushina E, Dyer RB, Badger JD, Ure D, Eide L, Tran DD, Vreize BT et al. Mutant huntingtin impairs axonal trafficking in mammalian neurons in vivo and in vitro. Mol Cell Biol 2004, 24: 8195-8209
27�� Dastoor Z, Dreyer JL. Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress. J Cell Sci 2001, 114: 1643-1653
28�� Senatorov VV, Charles V, Reddy PH, Tagle DA, Chuang DM. Overexpression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase in a transgenic mouse model of Huntington�s disease. Mol Cell Neurosci 2003, 22: 285-297
29�� Sundararaj KP, Wood RE, Ponnusamy S, Salas AM, Szulc Z, Bielawska A, Obeid LM et al. Rapid shortening of telomere length in response to ceramide involves the inhibition of telomere binding activity of nuclear glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem 2004, 279: 6152-6162
30�� Patterson, RL, van Rossum, DB, Kaplin, AI, Barrow, RK, and Snyder, SH. Inositol 1,4,5-trisphosphate receptor/GAPDH complex augments Ca2+ release via locally derived NADH. Proc Natl Acad Sci USA 2005, 102: 1357-1359