Altered Expression of HSPA5, HSPA8 and PARK7 in Spinocerebellar Ataxia Type 17 Identiﬁed by 2-Dimensional ﬂuorescence Difference in Gel Electrophoresis

Clinica Chimica Acta 400 (2009) 56–62

Contents lists available at ScienceDirect

Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Altered expression of HSPA5, HSPA8 and PARK7 in spinocerebellar ataxia type 17 identiﬁed by 2-dimensional ﬂuorescence difference in gel electrophoresis

Li-Ching Lee a,1, Chiung-Mei Chen b,1, Fen-Lin Chen a, Pei-Ying Lin a, Ya-Chin Hsiao a, Pin-Rong Wang a, Ming-Tsan Su a, Hsiu-Mei Hsieh-Li a, Ji-Chuu Hwang a, Chung-Hsin Wu a, Guan-Chiun Lee a, Sher Singh a, Yenshou Lin a, Sen-Yung Hsieh d, Guey-Jen Lee-Chen a,⁎, Jung-Yaw Lin a,c,⁎ a Department of Life Science, National Taiwan Normal University, Taipei, Taiwan b Department of Neurology, Chang Gung Memorial Hospital and Chang-Gung University College of Medicine, Taipei, Taiwan c Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan d Clinical Proteomics Center, Chang Gung Memorial Hospital, Taipei, Taiwan article info abstract

Article history: Background: Expansion of the CAG repeat of the TATA-box binding protein (TBP) gene has been identified as the Received 3 March 2008 causative mutations in spinocerebellar ataxia 17 (SCA17). TBP is ubiquitously expressed in both central nervous Received in revised form 11 September 2008 system and peripheral tissues. The underlying molecular changes of SCA17 are rarely explored. Accepted 8 October 2008 Methods: To study the molecular mechanisms underlying SCA17, we generated stably induced isogenic 293 Available online 30 October 2008 cells expressing normal TBP-Q36 and expanded TBP-Q61 and analyzed the expressed proteins using two- dimensional difference in gel electrophoresis (2D-DIGE), followed by mass spectrometry and immunoblotting. Keywords: fi Spinocerebellar ataxia type 17 Results: Upon induction with doxycycline, the expanded TBP-Q61 formed aggregates with signi cant increase in TBP expansion the cell population at subG1 phase and cleaved caspase-3. Proteomics study identified a total of 16 proteins 2D-DIGE with expression changes greater than 1.5 fold. Among the 16 proteins, PARK7, GLRX3, HNRNPA1, GINS1, ENO1, HSPA5 HNRPK and NPM1 are increased, and SERPINA5, HSPA5, VCL, KHSRP, HSPA8, HNRPH1, IMMT, VCP and HNRNPL HSPA8 are decreased in cells expressing TBP-Q61 compared with those expressing TBP-Q36. The altered expression of PARK7 HSPA5, HSPA8 and PARK7 were further validated by 2D and Western immunoblot analyses. Conclusions: The results illustrate the utility of proteomics to identify alterations of proteins which underlie pathogenesis of SCA17, and may serve as potential therapeutic targets. © 2008 Elsevier B.V. All rights reserved.

1. Introduction seizures [4–10]. In addition to marked cerebellar atrophy and Purkinje cell loss in human with SCA17, less pronounced neurodegeneration Expansion of the CAG repeat of the TATA-box binding protein (TBP) occurs in other brain regions [11,12]. gene has been identified as the causative mutations in spinocerebellar In addition to SCA17, inherited neurodegenerative diseases ataxia 17 (SCA17) [1,2]. TBP is a transcription initiation factor including Huntington's disease, dentatorubral–pallidoluysian atrophy, containing a polyglutamine (polyQ) tract in the N-terminal encoded spinal and bulbar muscular atrophy, and SCA1, 2, 3, 6, 7 are also caused by a mixed CAG/CAA repeat [3]. Wild type TBP in humans contains a by expansion of unstable CAG trinucleotide expansion mutation long polyQ stretch ranging in size from 29 to 42. Expanded alleles encoding polyQ tracts in the responsible genes [13]. As these polyQ ranging from 43 to 66 repeats were shown to be associated with disease proteins are widely expressed and some are critical for cellular SCA17 characterized by ataxia, dystonia, parkinsonism, dementia and function, the selective neurodegeneration is paradoxical. How the polyQ domain contributes to the normal function of proteins and the expanded polyQ induces selective neuropathology remain unclear. A mechanism for aberrant polyQ gain of function, increased Cre- Abbreviations: 2D-DIGE, two-dimensional difference in gel electrophoresis; HSPA5, dependent transcriptional activity, in SCA17 has been suggested heat shock 70 kDa protein 5; HSPA8, heat shock 70 kDa protein 8; IPG, immobilized pH gradient; PARK7, Parkinson disease 7; PMF, peptide mass fingerprinting; polyQ, [14]. In SCA17 transgenic mice, the small heat shock protein HSPB1 polyglutamine; SCA17, spinocerebellar ataxia type 17; TBP, TATA-box binding protein. was downregulated and TFIIB occupancy of the Hspb1 promoter was ⁎ Corresponding authors. Department of Life Science, National Taiwan Normal decreased, implicating the role of transcriptional regulation played by University, Taipei, Taiwan. 88 Ting-Chou Road, Section 4, Taipei 116, Taiwan. Tel.: +886 2 polyQ domain of TBP [15]. 29336875; fax: +886 2 29312904. Difference in gel electrophoresis (DIGE) is an improved two- E-mail addresses: [email protected] (G.-J. Lee-Chen), [email protected] (J.-Y. Lin). dimensional (2D) electrophoresis for comparative proteomics research, 1 The first two authors contributed equally to this work. enabling accurate analysis of differences in protein abundance between

Table 1 at room temperature in Cy5 or Alexa Fluor-conjugated secondary antibody (Zymed) Experimental design of 2D-DIGE gels and ﬂuorophore labeling scheme diluted to 1:500 in PBS containing 1% BSA, and washed in PBS. Nuclei were detected using 4¢-6-diamidino-2-phenylindole (DAPI). The stained cells were examined for dual Gel no. Cy2 Cy3 Cy5 ﬂuorescent imaging using a Leica TCS confocal laser scanning microscope.

A Pool Vector only TBP-Q36

B Pool TBP-Q36 TBP-Q61 2.4. Real-time PCR

C Pool TBP-Q61 Vector only Total RNA was extracted from isogenic TBP and vector only cells using the Trizol Each gel was loaded with 50 µg of Cy2-labeled protein pool from all 3 samples (equal (Invitrogen). The RNA was DNase treated, quantified, and reverse-transcribed to amounts of each) as an internal standard, 50 µg of Cy3-labeled and 50 µg of Cy-5 labeled cDNA using Superscript III as described. The transcribed cDNA (12.5 ng) were used for as indicated. The images of the gels A, B and C correspond to Fig. 2A–C. real-time PCR with TaqMan fluorogenic probes Hs00920494_m1 for TBP and 4326321E for HPRT1 (endogenous control) (Applied Biosystems). Fold change was ΔCt calculated using the formula 2 , ΔCT = CT (control)− CT (target), in which CT indicates cycle threshold. or among samples [16,17]. As most SCA genes including SCA17 TBP are ubiquitously distributed and cell culture systems are generally a single 2.5. Western blot cell type, presenting fewer confounding factors that might influence the Total proteins from isogenic TBP and vector only cells were prepared using lysis experimental results, we established SCA17 isogenic 293 cell lines buffer containing 5% glycerol, 0.5% Triton X-100, 1 mM dithiothreitol (DTT), and carrying 36 and 61 combined CAG/CAA repeats. These cell lines were protease inhibitor cocktail (Sigma). Proteins (25 µg) were separated on 10% SDS- originated from human embryonic kidney (HEK) 293 cells which polyacrylamide gel electrophoresis (SDS-PAGE) and blotted unto nitrocellulose express the neurofilament proteins as well as many other proteins membranes by reverse electrophoresis. After blocking, the membrane was stained with antibody to TBP or HA tag (1:5000 dilution). The immune complexes were typically found in neurons [18]. Given that neuronal phenotypes detected using horseradish peroxidase-conjugated goat anti-mouse (Jackson Immu- associated with 293 cells [19] and a large amount of studies on other noResearch) or goat anti-rabbit (Rochland) IgG antibody (1:10000 dilution) and repeat expansion diseases with similar neuronal pathology used this cell chemiluminescent substrate. line [20], we applied the established SCA17 isogenic 293 lines to investigate the molecular pathogenesis underlying the SCA17 cell 2.6. DNA cell cycle and caspase-3 activity dysfunction by DIGE technology. The isogenic TBP and vector only cells were trypsinized, washed twice with cold PBS, and resuspended in 70% ethanol for 15 min at 4 °C. After washing with cold PBS, the 2. Materials and methods cells were suspended in propidium iodide (PI)-staining solution (0.05% Triton X-100 and 100 µg/ml RNase in PBS) and incubated at 37 °C for 30 min. The cells were then 2.1. SCA17 and HSPA5 cDNA constructs stained with PI (50 µg/ml) for 15 min and analyzed in a FACStar flow cytometry (Becton- Dickinson). Caspase-3 activity in cell extracts was analyzed by Western blotting using Human cerebellum polyadenylated RNA (200 ng) (Clontech) was reverse-transcribed antibody to caspase-3 (1:1000 dilution, Cell Signaling Technology). using the SuperScript™ III reverse transcriptase (Invitrogen). Sense and antisense primers used for amplification of TBP cDNA were 5'-CTGGTTTGCCAAGAAGAAAGTG-3' and 5'- 2.7. 2D-DIGE AGGCAAGGGTACATGAGAGCCA-3', respectively. The 1.1-kb full-length, (CAG)3(CAA)3 7 (CAG)9CAACAGCAA(CAG)16CAACAG (total 36 combined repeats) containing cDNA was Isogenic TBP and vector only cells (10 ) were washed (10 mM Tris, 250 mM sucrose, cloned into pGEM-T Easy vector (Promega) and sequenced. The cloned TBP cDNA pH 7.0), collected and lysed in lysis buffer (30 mM Tris, 8 M urea, 4% CHAPS, pH 9.0). containing 54 CAG/CAA combined repeats was first made by replacing a 138 bp EarI–PstI After ultrasonic homogenizing and high-speed centrifugation (12,000 rpm) at 15 °C for fragment with a 192 bp fragment from the PCR clone of a SCA17 patient [(CAG)2(CAA)3 30 min, the extracted protein solution was purified by a 2-D Clean-Up Kit (Amersham (CAG)8CAACAGCAA(CAG)36CAACAG]. The repeat number was then further expanded Biosciences) and quantified using a 2-D Quant Kit (Amersham Biosciences). Soluble to 61 [(CAG)2(CAA)3(CAG)16CAACAGCAA(CAG)35CAACAG] or 109 [(CAG)2(CAA)3 proteins from isogenic TBP and vector only cells were minimally labeled with CyDye (CAG)16CAACAGCAA(CAG)45CAACAGCAA(CAG)35CAACAG] by ligating Fnu4HI par- DIGE fluors (8 nmol/l dye/mg protein) (GE Healthcare) according to the manufacturer's tially digested fragments. A 27-amino-acid HA tag was fused in-frame to the C-terminal instructions. Each gel was loaded with 50 µg of Cy3-labeled and 50 µg of Cy-5 labeled

TBP sequence and cloned into the NotI and SmaI sites of pEF-IRES/hrGFP vector [21].The proteins from TBP-Q36,-Q61 or vector only cells (Table 1). Also, 50 µg of an internal constructs were verified by DNA sequencing and used for transient expression study in standard comprising equal amounts of all three samples labeled with Cy2 was added to HEK-293 cells. Then the TBP cDNA was cloned into pcDNA5/FRT/TO vector (Invitrogen) for each gel. The mixture of labeled proteins and rehydration buffer (8 mol/l urea, 2% establishing isogenic 293 cell lines. CHAPS, 0.5% IPG buffer, and 18 mmol/l DTT) was rehydrated onto 13 cm, pH 3–10 The sense and antisense primers used for HSPA5 cDNA amplification was 5'- (linear), immobilized pH gradient (IPG) strips using IPGphor II (GE Healthcare) at 30 V CACAGACAGATTGACCTATTGGGGTGTTTC-3' and 5'-ACAGCACTAGCAGATCA- for 12 h. Isoelectric focusing was then performed as recommended by the supplier. After GTGTCTACAACTC-3', respectively. After cloning and sequencing, the full-length 2.2-kb reduction and alkylation of disulfide bonds with 1% DTT and 2.5% iodoacetamide, HSPA5 cDNA was excised with NotI and subcloned into pcDNA3 (Invitrogen). respectively, the second dimensional separation was performed by 12.5% SDS-PAGE.

2.2. Cell culture, transfection and isogenic 293 cell lines 2.8. Image acquisition and protein identiﬁcation

HEK-293 and HEK-293-derived Flp-In™-293 (Invitrogen) cells were cultivated in The 2D gels were scanned on a Typhoon 9400 imager (GE Healthcare). Excitation Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a 37 °C and emission wavelengths were chosen specifically for each of the dyes according to the fi humidi ed incubator with a 5% CO2 atmosphere. For transient overexpression, HEK-293 manufacturer's recommendations. The 2D maps were compared using ImageMaster 2D 5 cells were plated into 12-well dishes (2×10 /well), grown for 20 h, and transfected by Platinum 6.0 software DIGE gels analysis (GE Healthcare). Differences in expression the lipofection method (GibcoBRL) with the pEF-IRES/hrGFP vector or TBP cDNA greater than 1.5-fold (Pb0.05) were considered significant. The protein spots interested plasmid (1 µg). For co-transfection, equal amounts of TBP cDNA plasmid and HSPA5 were punched out and subjected to reduction and alkylation by DTT/iodoacetamide, cDNA plasmid (1 µg each) were employed. The cloned pcDNA5/FRT/TO-TBP plasmids followed by in-gel digestion with freshly prepared Trypsin Gold (20 ng in 5 µl, Promega) and vector were used to generate the isogenic TBP and vector only cell lines by targeting at 37 °C for overnight. The obtained peptides were extracted with 50% acetonitrile insertion into Flp-In™-293 cells, according to the supplier's instructions. The repeats in containing 1% trifluoroacetic acid and peptide mass fingerprinting (PMF) were these TBP cell lines were examined by PCR. These cell lines were grown in medium generated by matrix-assisted laser desorption/ionization time-of-flight mass spectro- containing 5 µg/ml blasticidin and 100 µg/ml hygromycin. Doxycycline (dox, 1 µg/ml) metry (MALDI-TOF MS) at Clinical Proteomics Center, Chang-Gung Memorial Hospital. was added to induce HA-tagged TBP expression. PMFs were matched to the theoretical tryptic digests of proteins in database NCBInr by MASCOT software (www.matrixscience.com). 2.3. Immunocytochemical staining For confirmation of differential expression, total soluble proteins from isogenic TBP and vector only cells were prepared as described (2D-DIGE). For Western blotting, Transiently transfected HEK-293 (with or without HSPA5 overexpression) or isogenic proteins (25 µg) were separated on 10% SDS-PAGE and blotted. For 2D-immunodetec- TBP cells on coverslips were washed with phosphate buffered saline (PBS) and fixed in 4% tion, proteins (100 µg) were first separated using IPG DryStrip (7 cm, pH 3–10) and paraformaldehyde in PBS for 10 min, followed by 20 min incubation with 0.1% Triton X- further separated by a 12% SDS-PAGE. The membranes were stained with antibody to 100 in PBS to permeabilize cells, overnight incubation with 0.5% bovine serum albumin heat shock 70 kDa protein 5 (HSPA5), heat shock 70 kDa protein 8 (HSPA8) (1:500 (BSA) in PBS to block non-specific binding. The primary antibodies TBP (Calbiochem), 1C2 dilution, Santa Cruz Biotechnology), Parkinson disease 7 (PARK7) (1:10000 dilution, (Chemicon) and HA tag (Abcam), diluted 1:500 in 1% BSA in phosphate buffered saline Chemicon) or C4 (actin) (1:10000 dilution, Chemicon). The immune complexes were (PBS), were used to stain cells at 4 °C overnight. After washing, cells were incubated for 2 h detected using horseradish peroxidase-conjugated donkey anti-goat (1:10000 dilution, 58 L.-C. Lee et al. / Clinica Chimica Acta 400 (2009) 56–62 L.-C. Lee et al. / Clinica Chimica Acta 400 (2009) 56–62 59

Fig. 2. 2D image of soluble proteins from isogenic 293 cells. Proteins were extracted as described and separated in pH 3 to 10 IPG strips for the first dimension and in 12.5% SDS-PAGE for the second dimension. Image was acquired on a Typhoon 9400 scanner. Gel images in (A), (B) and (C) correspond to the gel numbers shown in Table 1. The number of each spot in a CYPRO Ruby-stained image (D) indicates proteins identified by MALDI/TOF MS, and the details of identified proteins are shown in Table 2. White numbers indicate proteins that are decreased and red numbers indicate proteins that are increased in cells expressing TBP-Q61 when compared with those expressing vector only or TBP-Q36.

Santa Cruz Biotechnology), goat anti-mouse or goat anti-rabbit IgG antibody as for the number of CAG/CAA combined repeats. TBP-RNA levels were ﬁrst described. examined by real-time PCR using TBP-speciﬁc probe and primers. As shown in Fig. 1B, in the presence of dox, the two TBP lines make 18–20 3. Results times more TBP RNA than in the absence of dox. Expression of HA-tagged TBP proteins were then examined by Western blotting using TBP and HA 3.1. Cloning and expression of TBP constructs antibodies (Fig.1C). In addition to the 43 kDa endogenous TBP protein, the

TBP antibody detected HA-tagged 47 kDa TBP-Q36 and 50 kDa TBP-Q61 The TBP cDNA containing normal 36 and expanded 54 CAG repeats proteins in dox induced TBP cells. Although HA antibody detects a non- were cloned. The TBP with 54 CAG repeats was used to generate the specific band comparable in size with the HA-tagged TBP-Q36,thesame clones with 61 and 109 CAG repeats. The HA-tagged TBP cDNAs with 47 and 50 kDa proteins can also be detected using anti-HA tag antibody. various CAG repeats were then cloned into pEF-IRES/hrGFP vector which The induced levels of TBP-Q36 and TBP-Q61 protein were relatively equal. permits the humanized green fluorescent protein to be translated from While overexpressed TBP-Q36 was seen as diffuse cytoplasmic and the same RNA containing the TBP. The normal (TBP-Q36) and expanded nuclear staining, positive nuclei with punctate inclusions were visible (TBP-Q61 and TBP-Q109) TBP constructs were transiently expressed in within cells expressing TBP-Q61 (Fig. 1D). Cell cycle analysis revealed a HEK-293 cells for immunocytochemical staining and fluorescence significant increase in the cell population at subG1 phase for TBP-Q61 cells microscopy examination. As shown in Fig. 1A, the expressed TBP-Q61 (8.0%) when compared to TBP-Q36 cells (0.3%) or vector only cells (0.5%) and TBP-Q109 protein formed aggregates and the expanded polyQ (Fig. 1E, Pb0.001). Cleaved caspase-3 (17 kDa) was significantly increased stretches in TBP-Q61 and TBP-Q109 were 1C2 antibody-staining positive. in these dox induced TBP-Q61 cells (Fig. 1F, P=0.009).

3.2. Isogenic TBP cell lines 3.3. 2D-DIGE analysis of the isogenic TBP cell lines

The pcDNA5/FRT/TO-TBP constructs containing Q36,Q61 and Q109 Total proteins were extracted from isogenic TBP and vector only were used to generate TBP lines. While TBP-Q109 line cannot be lines. Approximately 2500 CyDye labeled proteins were resolved using established, the derived TBP-Q36 and TBP-Q61 lines are isogenic except 2D-DIGE technology (Fig. 2). Differentially expressed proteins (N1.5-

Fig. 1. Expression of TBP cDNA constructs in transiently transfected HEK-293 (A) and stably induced isogenic 293 (B–F) cells. (A) Immunofluorescent microscopy examination directly (green) or after immunostaining using antibody specific for TBP or expanded polyQ (red). Nuclei were counterstained with DAPI (blue). (The scale bar=10 µm) (B) Real time PCR quantification of TBP mRNA level relatively to HPRT mRNA after 2 (gray bar), 4 (charcoal gray bar) or 6 (black bar) days induction. (C) Western blot analysis using TBP and HA antibodies. (D) Fluorescence microscopy examination after immunostaining using 1C2 antibody (green). Nuclei were counterstained with DAPI (blue). (The scale bar=25 µm) (E)

Representative ﬂow cytometric image of cellular DNA content for TBP-Q61 expressing cells (left). Data in the right panel of graph represent the mean±SEM (n=3) for vector only, TBP-

Q36 and TBP-Q61 cells. ⁎ indicates Pb0.05, TBP-Q61 versus TBP-Q36 at subG1. (F) Representative Western blot images (left) using caspase-3 and β-actin antibodies. Levels of caspase-3 were normalized with an internal control (β-actin) (right). Data (mean±SEM; n=3) in the right panel of graph represent the relative immunoreactivity for the cleaved caspase-3. ⁎Indicates Pb0.05. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) 60 L.-C. Lee et al. / Clinica Chimica Acta 400 (2009) 56–62

Table 2 Summary of the proteins identiﬁed by using 2D-DIGE and MALDI/TOF MS

b Spot Protein name Accession no. Score Theoretical Ratio Q61/ t Functional classiﬁcation a c d e No (NCBI) (coverage %) MW/pI Q36 value 1 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), gi|21361195 45 (23%) 45673/9.30 −3.40 3.16 Regulatory molecule member 5 (SERPINA5) 2 Parkinson disease (autosomal recessive, early onset) 7 (DJ-1 protein) gi|31543380 84 (60%) 19878/6.33 +2.85 4.39 Regulatory molecule/ (PARK7) chaperone 3 heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) gi|6470150 85 (30%) 70888/5.23 −2.60 8.57 Chaperone (BiP protein) (HSPA5) 4 glutaredoxin 3 (GLRX3) gi|48257132 50 (33%) 32595/5.36 +2.46 3.87 Redox regulation 5 vinculin (VCL) gi|4507877 94 (18%) 116649/5.83 −2.38 18.61 Cytoskeleton/structural 6 KH-type splicing regulatory protein (FUSE binding protein 2) (KHSRP) gi|154355000 120 (28%) 73070/6.85 −2.37 8.70 Transcription/ translation 7 heat shock 70 kDa protein 8 (HSPA8) gi|5729877 96 (33%) 70854/5.37 −2.20 3.74 Chaperone 8 heterogeneous nuclear ribonucleoprotein H1 (H) (HNRPH1) gi|5031753 40 (22%) 49198/5.89 −2.08 6.61 Transcription/ translation 9 heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) gi|119617171 58 (48%) 33876/9.27 +1.98 28.00 Transcription/ translation 10 Inner membrane protein, mitochondrial (mitoﬁlin) (IMMT) gi|154354966 67 (17%) 82574/6.15 −1.94 5.25 Cytoskeleton/structural 11 GINS complex subunit 1 (Psf1 homolog) (GINS1) PSF1_HUMAN 46 (50%) 22974/6.96 +1.92 3.00 Regulatory molecule 12 enolase 1, (alpha) (ENO1) gi|13279239 52 (43%) 29864/5.63 +1.79 2.96 Metabolism 13 valosin-containing protein (VCP) gi|6005942 56 (30%) 89266/5.14 −1.74 15.84 Chaperone 14 heterogeneous nuclear ribonucleoprotein K (HNRPK) gi|55958544 81 (39%) 47528/5.46 +1.60 4.37 Transcription/ translation 15 heterogeneous nuclear ribonucleoprotein L (HNRNPL) gi|119577230 96 (27%) 60066/6.81 −1.58 4.98 Transcription/ translation 16 nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1) gi|40353734 70 (30%) 29446/4.47 +1.51 4.55 Transcription/ translation

a Spot number corresponding to the annotation shown in Fig. 2D. b,c,d Accession numbers, scores and theoretical MW/pI were obtained by Mascot PMF searching following MALDI/TOF MS. e Pb0.05 (tN2.92, df=2).

fold increase/decrease, P-valueb0.05) were selected for PMF by 3.4. 2D and Western immunoblot confirmation of HSPA5, HSPA8 and PARK7 MALDI-TOF mass spectrometry. A total of 16 spots representing 16 different proteins were identified with expression changes varying Among the identified protein spots, we focused on the down- between −3.40 and 2.85-fold (Table 2). Identified spots corresponding regulated HSPA5 and HSPA8 chaperones and up-regulated PARK7. To to 7 up-regulated and 9 down-regulated proteins are involved mainly confirm the identified proteins and expression changes, proteins from in six functional groups: regulatory molecules (3), chaperones (3), TBP and vector only lines were subjected to 2D and Western blotting redox regulation (1), cytoskeleton/structural (2), transcription/trans- with appropriate antibodies. Consistent with the 2D-DIGE results, lation (6) and metabolism (1) (Table 2). significantly decreased HSPA5 and HSPA8 and increased PARK7 in 293

Fig. 3. 2D (A) and Western (B) immunoblot analyses of the vector only and isogenic TBP lines using HSPA5, HSPA8, PARK7 and β-actin antibodies. The left panel of (B) shows representative Western blot images. Levels of HSPA5, HSPA8 and PARK7 were normalized with an internal control (β-actin). The relative immunoreactivity of HSPA5, HSPA8 and

PARK7 of cells expressing vector only (open bar), TBP-Q36 (gray bar) and TBP-Q61 (black bar) are shown in the right panel of (B). Data are expressed as the mean±SEM values from three independent experiments. ⁎Indicates Pb0.05. L.-C. Lee et al. / Clinica Chimica Acta 400 (2009) 56–62 61

Fig. 4. Overexpression of HSPA5 in SCA17 transient cell models. HEK-293 cells were co-transfected with plasmids encoding vector (Vec), TBP-Q36, TBP-Q61 or TBP-Q109, and pcDNA3 plasmid with (+HSPA5) or without (−HSPA5) HSPA5 cDNA. Images of ﬂuorescence microscopy examination directly (green) and after immunostaining using 1C2 antibody (red) were merged. Nuclei were counterstained with DAPI (blue). (The scale bar=40 µm). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

cells expressing TBP-Q61 were observed (Fig. 3, P=0.014 for HSPA5, Sixteen differentially expressed proteins were identiﬁed through 0.028 for HSPA8 and 0.031 for PARK7). proteomics study (Table 2). Among them, only HSPA5 and HSPA8 contain conserved A-T-rich TATA box. Nevertheless, TBP is involved in 3.5. Overexpression of HSPA5 to reduce the aggregate formation caused basal transcription from both TATA and TATA-less promoter sites. The by expanded TBP mechanisms of altered expression of these proteins are not clear and may be postulated as the followings. (1) The N terminus of TBP can To determine whether HSPA5 could suppress aggregation of mutant activate transcription when fused to a heterologous DNA binding

TBP, we co-expressed HSPA5 with vector, TBP-Q36,TBP-Q61 or TBP-Q109 domain [25]. Expanded polyQ tract may influence this activation. (2) A in HEK-293 cells. As shown in Fig. 4, the overexpressed HSPA5 effectively long polyQ tract in soluble TBP can enhance the interaction of the C suppressed aggregate formation in TBP-Q61 as well as in TBP-Q109 cells. terminus of TBP with TFIIB [15] and the TBP-TFIIB interaction is required for proper function of certain activator proteins [26]. Thus 4. Discussion the expanded polyQ tract may influence TBP function by modulating this TBP-TFIIB interaction. (3) The increased interaction with TFIIB may SCA17 is one of polyQ expansion diseases which share a common also affect transcription by reducing the availability of the TFIIB at pathological feature: inclusions of mutant protein, often in the nuclei of other certain promoters. (4) PolyQ inclusions recruit various tran- affected nerve cells [22]. That the formation of aggregates may mediate scription factors to cause transcriptional dysregulation [27]. (5) The the cellular vulnerability to neuronal dysfunction and death has been expanded polyQ tract in TBP interacts with other polyQ-containing suggested, but remains debatable. To examine whether SCA17 CAG proteins, such as CREB binding protein, to alter gene expression [14]. expansion results in intracellular aggregates and impair cell viability and (6) High mobility group protein 1 (HMGB1) interacts with the N to explore the effects of expanded TBP accumulation, we cloned and terminal domain of TBP to interferes with TFIIB within the pre- transfected normal (TBP-Q36) and expanded (TBP-Q61 and TBP-Q109)TBP initiation complex [28]. The length of polyQ may influence TBP constructs in 293 cells. Aggregates only formed in the cells expressing function by modulating this TBP–HMGB1 interaction. expanded TBP-Q61 or TBP-Q109 protein but not in those expressing TBP- It has been reported that down-regulation of Hsp27 chaperone and Q36 protein (Fig. 1A and D), which is compatible with the observation the protection conferred by its overexpression in polyQ disease that TBP-positive inclusions were detected in the cerebellum and basal models [15,29–31]. Probably due to the induced TBP overexpression ganglia of SCA17 patients, with an apparent CAG length-dependent of that might reduce the availability of the TFIIB at HSPB1 promoter, the age of onset [4]. PolyQ-expanded cell death in SCA3 and SCA7 is expression of Hsp27 chaperone was significantly repressed in both our preceded by activation of caspases 3 and 9 [23,24], whereas SCA17 TBP-Q36 and -Q61 lines, and the difference in Hsp27 was not detected associated with apoptosis has not yet been reported. The increased by our 2D-DIGE analysis (data not shown). Nevertheless we identified subG1 population and caspase-3 activity in our SCA17 TBP-Q61 and validated another two down-regulated chaperones that function expressing cells (Fig. 1E and F) provide evidence of pathogenic potential in protein refolding and are ubiquitously expressed in brain and other of polyQ expansion in the context of this critical transcription protein. tissues (http://bioinfo.weizmann.ac.il/cards/index.shtml). The greater 62 L.-C. Lee et al. / Clinica Chimica Acta 400 (2009) 56–62 sensitivity of 2D-DIGE than Western blot may explain the observed [10] Lasek K, Lencer R, Gaser C, et al. Morphological basis for the spectrum of clinical fi – − de cits in spinocerebellar ataxia 17 (SCA17). Brain 2006;129:2341 52. HSPA5 ratio difference between 2D-DIGE ( 2.60=38%) and Western [11] Bauer P, Laccone F, Rolfs A, et al. Trinucleotide repeat expansion in SCA17/TBP inwhite blot (65%). HSPA5, also known as immunoglobulin heavy chain- patients with Huntington's disease-like phenotype. J Med Genet 2004;41:230–2. binding protein (BiP) or 78 kDa glucose-regulated protein (GRP78), is [12] Lin IS, Wu RM, Lee-Chen GJ, Shan DE, Gwinn-Hardy K. The SCA17 phenotype can include features of MSA-C, PSP and cognitive impairment. Parkinsonism Relat involved in the folding and assembly of proteins in the endoplasmic Disord 2007;13:246–9. reticulum (ER). Abnormal protein folding due to decreased rate of [13] Cummings CJ, Zoghbi HY. Fourteen and counting: unraveling trinucleotide repeat HSPA5 hydrolysis and disturbed SIL1–HSPA5 interaction is the cause diseases. Hum Mol Genet 2000;9:909–16. of Marinesco–Sjogren syndrome that is characterized by ataxia, [14] Reid SJ, Rees MI, van Roon-Mom WM, et al. Molecular investigation of TBP allele length: a SCA17 cellular model and population study. Neurobiol Dis 2003;13:37–45. progressive myopathy and cataract [32]. The product encoded by the [15] Friedman MJ, Shah AG, Fang ZH, et al. Polyglutamine domain modulates the TBP– HSPA8 gene is a constitutively expressed heat-shock cognate protein TFIIB interaction: implications for its normal function and neurodegeneration. Nat – (HSC70) binding to nascent polypeptides to facilitate correct folding. Neurosci 2007;10:1519 28. [16] Swatton JE, Prabakaran S, Karp NA, Lilley KS, Bahn S. Protein profiling of human HSC70 is associated with ER/Golgi SNAREs in vivo, which is consistent postmortem brain using 2-dimensional fluorescence difference gel electrophor- with a modulatory role for HSC70 in transport [33]. The down- esis (2-D DIGE). Mol Psychiatry 2004;9:128–43. regulation of molecules functioning in protein refolding suggests the [17] Damodaran S, Dlugos CA, Wood TD, Rabin RA. Effects of chronic ethanol administration on brain protein levels: a proteomic investigation using 2-D DIGE role of chaperones in SCA17 pathogenesis. The enrichment for system. Eur J Pharmacol 2006;547:75–82. proteins involved in processes of protein quality control and recycling [18] Shaw G, Morse S, Ararat M, Graham FL. Preferential transformation of human in ataxia interactome network [34] further supports the role of neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J 2002;16:869–71. unfolded proteins in the course of SCA. [19] Dautzenberg FM, Higelin J, Teichert U. Functional characterization of corticotropin- In addition to HSPA5 and HSPA8 chaperones, we also validated the releasing factor type 1 receptor endogenously expressed in human embryonic up-regulated PARK7 (+2.85 by 2D-DIGE and 181% by Western blot). kidney 293 cells. Eur J Pharmacol 2000;390:51–9. [20] Handa V, Goldwater D, Stiles D, et al. Long CGG-repeat tracts are toxic to human cells: PARK7, also known as DJ-1, was the causative gene of autosomal implications for carriers of Fragile X premutation alleles. FEBS Lett 2005;579:2702–8. recessive early-onset Parkinson disease 7. In addition to involving in [21] Chung S, Andersson T, Sonntag KC, Bjorklund L, Isacson O, Kim KS. Analysis of transcriptional regulation [35,36], it may function as a redox-sensitive different promoter systems for efficient transgene expression in mouse embryonic – chaperone and a sensor for oxidative stress to protect neurons against stem cell lines. Stem Cells 2002;20:139 45. [22] DiFiglia M, Sapp E, Chase KO, et al. Aggregation of huntingtin in neuronal oxidative stress and cell death [37–39]. The increased expression of intranuclear inclusions and dystrophic neurites in brain. Science 1997;277:1990–3. PARK7 in TBP-Q61 cells may be a compensatory response to the [23] Chou AH, Yeh TH, Kuo YL, et al. Polyglutamine-expanded ataxin-3 activates oxidative stress induced by polyQ mutation [40]. mitochondrial apoptotic pathway by upregulating Bax and downregulating Bcl-xL. Neurobiol Dis 2006;21:333–45. In conclusion, our study results suggest that expanded polyglutamine [24] Wang HL, Yeh TH, Chou AH, et al. Polyglutamine-expanded ataxin-7 activates length leads to aggregation and toxicity in cultured cells with the similar mitochondrial apoptotic pathway of cerebellar neurons by upregulating Bax and threshold as for clinical disease, indicating a causative connection. The downregulating Bcl-x(L). Cell Signal 2006;18:541–52. [25] Seipel K, Georgiev O, Gerber HP, Schaffner W. Basal components of the results also revealed that impaired protein folding may contribute the cell transcription apparatus (RNA polymerase II, TATA-binding protein) contain dysfunction of SCA17. Since the pathogenesis of the polyQ diseases is not activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase completely clear and treatment is not available, our cell models are II a “portable enhancer domain”? Mol Reprod Dev 1994;39:215–25. [26] Tansey WP, Herr W. Selective use of TBP and TFIIB revealed by a TATA–TBP–TFIIB valuable for identifying differentially expressed proteins which underlie array with altered specificity. Science 1997;275:829–31. pathogenesis of SCA17, and may serve as potential therapeutic targets. [27] Riley BE, Orr HT. Polyglutamine neurodegenerative diseases and regulation of transcription: assembling the puzzle. Genes Dev 2006;20:2183–92. [28] Sutrias-Grau M, Bianchi ME, Bernués J. High mobility group protein 1 interacts Acknowledgements specifically with the core domain of human TATA box-binding protein and interferes with transcription factor IIB within the pre-initiation complex. J Biol We thank the Clinical Proteomic Center of Chang Gung Memorial Chem 1999;274:1628–34. Hospital for the technical support. This work was supported by grants [29] Wyttenbach A, Sauvageot O, Carmichael J, Diaz-Latoud C, Arrigo AP, Rubinsztein DC. Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses NSC-96-3112-B-003-006 and NSC 96-2311-B-182A-001 from the the increase of reactive oxygen species caused by huntingtin. Hum Mol Genet National Science Council, Executive Yuan, 96TOP001 from National 2002;11:1137–51. Taiwan Normal University, and CMRGP 33108 from Chang Gung [30] Wen FC, Li YH, Tsai HF, et al. Down-regulation of heat shock protein 27 in neuronal cells and non-neuronal cells expressing mutant ataxin-3. FEBS Letters 2003;546:307–14. Memorial Hospital, Taipei, Taiwan. [31] Tsai HF, Lin SJ, Li C, Hsieh M. Decreased expression of Hsp27 and Hsp70 in transformed lymphoblastoid cells from patients with spinocerebellar ataxia type 7. References Biochem Biophys Res Commun 2005;334:1279–86. [32] Anttonen AK, Mahjneh I, Hamalainen RH, et al. The gene disrupted in Marinesco– Sjogren syndrome encodes SIL1, an HSPA5 cochaperone. Nat Genet 2005;37:1309–11. [1] Koide R, Kobayashi S, Shimohata T, et al. A neurological disease caused by an [33] Joglekar AP, Hay JC. Evidence for regulation of ER/Golgi SNARE complex formation expanded CAG trinucleotide repeat in the TATA-binding protein gene: a new by hsc70 chaperones. Eur J Cell Biol 2005;84:529–42. polyglutamine disease? Hum Mol Genet 1999;8:2047–53. [34] Lim J, Hao T, Shaw C, et al. A protein–protein interaction network for human inherited [2] Nakamura K, Jeong SY, Uchihara T, et al. SCA17, a novel autosomal dominant ataxias and disorders of Purkinje cell degeneration. Cell 2006;125:801–14. cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein. [35] Takahashi K, Taira T, Niki T, Seino C, Iguchi-Ariga SM, Ariga H. DJ-1 positively Hum Mol Genet 2001;10:1441–8. regulates the androgen receptor by impairing the binding of PIASx alpha to the [3] Imbert G, Trottier Y, Beckmann J, Mandel JL. The gene for the TATA binding protein receptor. J Biol Chem 2001;276:37556–63. (TBP) that contains a highly polymorphic protein coding CAG repeat maps to 6q27. [36] Shinbo Y, Taira T, Niki T, Iguchi-Ariga SM, Ariga H. DJ-1 restores p53 transcription Genomics 1994;21:667–8. activity inhibited by Topors/p53BP3. Int J Oncol 2005;26:641–8. [4] Fujigasaki H, Martin JJ, De Deyn PP, et al. CAG repeat expansion in the TATA box-binding [37] Mitsumoto A, Nakagawa Y, Takeuchi A, Okawa K, Iwamatsu A, Takanezawa Y. protein gene causes autosomal dominant cerebellar ataxia. Brain 2001;124:1939–47. Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in [5] Silveira I, Miranda C, Guimaraes L, et al. Trinucleotide repeats in 202 families with ataxia: response to sublethal levels of paraquat. Free Radic Res 2001;35:301–10. asmallexpanded(CAG)n allele at the SCA17 locus. Arch Neurol 2002;59:623–9. [38] Taira T, Saito Y, Niki T, Iguchi-Ariga SM, Takahashi K, Ariga H. DJ-1 has a role in [6] Maltecca F, Filla A, Castaldo I, et al. Intergenerational instability and marked antioxidative stress to prevent cell death. EMBO Rep 2004;5:213–8. anticipation in SCA-17. Neurology 2003;61:1441–3. [39] Yanagisawa D, Kitamura Y, Inden M, et al. DJ-1 protects against neurodegeneration [7] Rolfs A, Koeppen AH, Bauer I, et al. Clinical features and neuropathology of caused by focal cerebral ischemia and reperfusion in rats. J Cereb Blood Flow autosomal dominant spinocerebellar ataxia (SCA17). Ann Neurol 2003;54:367–75. Metab 2008;28:563–78. [8] Bruni AC, Takahashi-Fujigasaki J, Maltecca F, et al. Behavioral disorder, dementia, [40] Wyttenbach A, Sauvageot O, Carmichael J, Diaz-Latoud C, Arrigo AP, Rubinsztein ataxia, and rigidity in a large family with TATA box-binding protein mutation. Arch DC. Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses Neurol 2004;61:1314–20. the increase of reactive oxygen species caused by huntingtin. Hum Mol Genet [9] Toyoshima Y, Yamada M, Onodera O, et al. SCA17 homozygote showing 2002;11:1137–51. Huntington's disease-like phenotype. Ann Neurol 2004;55:281–6.