Differential expression of neuroblastoma cellular proteome due to AICD overexpression

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Journal of Alzheimer’s Disease xx (20xx) x–xx DOI 10.3233/JAD-130695 IOS Press

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Differential Expression of Neuroblastoma Cellular Proteome due to AICD Overexpression

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Arunabha Chakrabarti1 , Kasturi Roy1 and Debashis Mukhopadhyay∗

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Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, WB, India

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Handling Associate Editor: Inga Zerr

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Accepted 10 August 2013

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Keywords: Alzheimer’s disease, A␤PP intracellular domain, 2D-DIGE, MALDI-MS, transfection

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INTRODUCTION

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Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative disorder causing dementia [1]. The disorder is pathologically characterized by accumulation of amyloid plaques in the brain parenchyma and the main neurotoxic component responsible for the formation of these plaques, which in turn leads to neurodegeneration, is amyloid-␤ (A␤) peptides [2]. A␤ is one of the proteolytic fragments cleaved from the amyloid-␤ protein precursor (A␤PP) by two proteases, ␤- and ␥-secretase [3]. Although extensive studies have

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Abstract. Amyloid-␤ protein precursor intracellular domain (AICD), which exerts intracellular effects by interacting with proteins involved in a plethora of biological processes, is a key player behind the pathophysiology of Alzheimer’s disease (AD). Keeping in mind that overwhelming presence of AICD would mimic AD-like conditions in neuroblastoma cell lines, we hypothesized alteration in the proteomic expression pattern in these cells in the presence of AICD compared to their normal proteome. The rationale behind the study was to distinguish between symptomatic pathophysiological effects as opposed to any artifactual consequence due to protein overload in the cell lines. Using 2D-DIGE analysis and MALDI-MS identifications in neuro2A (mouse) and SHSY5Y (human) cell lines, we have identified several proteins belonging to different functional classes and involved in several biological pathways including protein folding, cytoskeletal dynamics, metabolism, and stress. Many of these were being upregulated or downregulated due to AICD effects and could be correlated directly with AD phenotypes.

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authors contributed equally to this manuscript. ∗ Correspondence to: Debashis Mukhopadhyay, Associate Professor, Structural Genomics Division, Saha Institute of Nuclear Physics. Postal address: 1/AF, Bidhannagar, Kolkata-700064, WB, India. Tel.: +91 33 2337 5345; Fax: +91 33 2337 4637; E-mail: [email protected].

been done with A␤ fragment, understanding the complexity of the disease is far behind expectation and search for new players has become a necessity [4]. The A␤PP intracellular domain (AICD), one of the C-terminal fragments of A␤PP, is one such potential candidate that plays an important role in determining cell fate and neurodegeneration [5]. AICD is produced in same amount like A␤ in the amyloidogenic pathway resulting from the same secretase cleavage, and the amino acid sequence of AICD is more conserved through evolution than that of A␤ [6], indicating the possibility of functional involvement of this fragment of A␤PP in the disease pathway. AICD expression is prevalent in AD. Earlier studies exploited AICD-overexpression to mimic AD-like conditions in both cell lines and transgenic mouse models [7, 8], and there have always been concerns about artifactual effects due to overexpression. To resolve

ISSN 1387-2877/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved

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MATERIALS AND METHODS Neuro2A and SHSY5Y cell culture, transfection of AICD, and protein extraction

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considered as the AICD-overexpressed proteome of these two cell lines. The corresponding control set of proteome was prepared following the same protein extraction protocol from the control N2A and SHSY5Y cells where transfections were carried out only with empty GFP-C1 vector, i.e., without the expression of AICD. This scramble was used to rule out modifications due to manipulation rather than resulting from AICD overexpression. To show that overexpression of AICD really took place in AICD-GFP transfected cells compared to only GFP transfected cells, western blot with anti-AICD antibody was done (Supplementary Fig. 4) for these two samples and the blots showed that the transfection efficiency and corresponding AICD overexpression was very high in AICD-GFP transfected cells compared to GFP-only transfected cells. Protein quantification was done with the dissolved protein samples by Bradford protein assay using BSA as standard. CyDye labeling

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The nucleotide sequence of the AICD protein (47 amino acids) was previously cloned in our lab into the mammalian fluorescent expression vector pEGFP-C1 (purchased from Clontech, USA), and this construct (AICD-GFP) was used for transfection into the mammalian neuroblastoma cells (both N2A and SHSY5Y). In the pEGFP-C1 vector, the multiple cloning site (MCS) is followed by GFP coding sequence under the same starting ATG. So genes cloned into the MCS have been expressed as fusion to C terminus of GFP protein. The vector backbone also contains an SV40 origin for replication in mammalian cells and an SV40 early promoter under which the synthesis of mRNA and further expression of the fusion protein occurs. Mouse neuroblastoma Neuro2A (N2A) and human neuroblastoma SHSY5Y cells were grown at 37◦ C in a humidified 5% CO2 incubator in DMEM culture media (Himedia) supplemented with 10% FBS (Biowest). AICD-GFP (4 ␮g in each 90 mm culture plate) was transfected in both the cell lines when it was 75% confluent. 24 h post-transfection, the cell pellets were collected and washed with PBS. The cell pellets were resuspended in lysis buffer (50 mM Tris/Cl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 2 mM PMSF, 0.5% NP-40, and 1X protease inhibitor cocktail, Pierce) and rapid freezing and thawing was carried out to rupture the cell walls. After centrifugation at 13,000 RPM for 15 min at 4◦ C, the supernatant containing the whole cell proteins was collected. This was

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this, we used AICD-overexpressed N2A and SHSY5Y cells to mimic the AD-like condition and identify the differentially expressed proteins due to AICD transfection into those cells. All the proteins we obtained in this study may not directly relate to or influence AD pathogenesis, but others are directly related functionally with the disease. We have shown previously the involvement of AICD in physical interaction with proteins with several different classes of molecular functions using in vitro pull-down experiments with N2A cells [9]. Involvement of these affected proteins in the disease condition is supposed to be mediated by their interactions with AICD, either directly or indirectly. This study is the first approach to understand and compare the effects of AICD on the neuroblastoma proteome from two different organisms.

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Both AICD-transfected and control samples were salt depleted using acetone precipitation method. The protein extracts were mixed with 1 : 4 (v/v) acetone, incubated for 3 h at −20◦ C with vortexing in between, and centrifuged at 13,000 rpm for 15 min. Protein precipitates were dissolved in 20 ␮l DIGE labeling buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris pH 8.8, Roche pI cocktail 1 : 100 dilution). In each of the experiments, 50 ␮g of transfected protein and 50 ␮g of control were used. An internal standard was also prepared for each DIGE experiment taking 25 ␮g protein each of the transfected and control proteins, dissolved in 10 ␮l of DIGE buffer, and then pooled. N-hydroxysuccinimide cyanine dyes (CyDyes, Amersham Biosciences, Uppsala, Sweden) were reconstituted in ice, with DMF and added to the protein samples such that there was 200 pM of CyDye per 50 ␮g protein sample with Cy5, Cy3, and Cy2 being used to label the transfected, control and internal standard samples respectively and incubated in ice for 30 min with gentle mixing every 10 min. The reaction was stopped with 10 mM lysine solution after 30 min of labeling. All reactions with the CyDyes were carried out in dark. Post reaction, all labeled samples were mixed up and the total volume was made up to 300 ␮l with rehydration buffer (8 M urea, 4% CHAPS, 2% DTT, 2% ampholyte pH 3–10, and 30 mM Tris/HCl pH 8.5). 17 cm IPG strip (pH 4–7) rehydration and subsequent steps for 2D-gel run were carried out in the dark to avoid quenching of CyDyes.

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Western blot

For validation of differential expression of eight of the proteins, western blot experiments were performed to compare the expression of peroxiredoxin 2, ␣-Tubulin, vimentin, HSPA8, 14-3-3, Pin1, PDI, and stathmin in the proteome of AICD-overexpressed cells and control cells with GFP-only transfection. Cells were transfected separately with AICD-GFP and GFP-only and 24 h post-transfection, proteins were extracted from both the transfected cells and compared. Amount of proteins from each sample run in each western blot experiment was 30 ␮g and for validation of each protein, the experiment was performed at least twice. Antibodies used for this study are as follows: mouse monoclonal anti-peroxiredoxin-2 (ab16748), mouse monoclonal anti-␣-tubulin (ab7291), mouse monoclonal anti-vimentin (V6389), rabbit monoclonal anti-HSPA8 (ab51052), rabbit polyclonal anti-14-33 (ab9063), mouse monoclonal anti-Pin1 (sc46660), rabbit monoclonal anti-PDI (3501S), and rabbit monoclonal anti-stathmin (ab52630). GAPDH was used as loading control and rabbit polyclonal anti-GAPDH antibody (ab9485) was used for this purpose. Densitometry analyses were performed using Versa doc (BioRad) and t-test p-values were calculated using Origin software.

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ative resolution of 50 (full width at half maximum) and data from 3000–5000 laser shots were collected. Spectral data was analyzed with GPS ExplorerTM version 3.6 (Applied Biosystems). Peptide identification was based on MASCOT database scoring algorithm from Swiss-Prot and NCBI protein databases using search settings of: single missed tryptic cut, fixed carbamidomethylation, variable methionine oxidation and N-terminal acetylation and 150 ppm mass accuracy. Autolytic tryptic peaks were excluded in the Mascot search parameter and p < 0.05 was taken to be significant during identification. Since we had taken a gel-based proteomics approach, we could identify from the protein list given by mascot server, the specific protein in a particular spot using the hint of molecular weight and pI.

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MALDI-MS identification

The excised spots from the DIGE gels were destained using destaining buffer suitable for different staining procedures and then tryptic digestion was performed overnight with the spots using In-Gel tryptic Digestion Kit (Pierce, USA). The digestion mixture was lyophilized in Heto vacuum centrifuge (Thermo) and resuspended in 50% Acetonitrile (ACN) and 0.1% Trifluoroacetic acid (TFA) solution. The dissolved samples were mixed 1 : 1 (v/v) with ␣-Cyano-4hydroxycinnamic acid (CHCA) matrix and spotted on the MALDI plate and air dried. MALDI analyses were performed with 4700 Proteomic Analyzer MALDI-TOF/TOF (Applied Biosystems) for identification of the protein spots. Peptide mass fingerprinting data was acquired in positive MS reflector mode using fixed laser intensity of 5500 with 2000–3000 laser spots in the range 800–4000 Da, signal to noise ratio 10 and mass exclusion tolerance of 150 ppm. Internal calibration was done in the instrument with minimum signal to noise of 20 and mass tolerance of ±300 ppm taking monoisotopic peaks only. The isolation of peptides of interest was done at a rel-

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After rehydration, isoelectric focusing (IEF) was performed according to the following protocol: 250 V for 35 min, 1000 V for 1 h, 10000 V for 8 h, and 10000 V up to 65000 Vh. After the first dimension (IEF), IPG strips were equilibrated for 10 min in reducing equilibration buffer (50 mM Tris/HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 50 mM DTT) followed by 15 min in alkylating equilibration buffer containing 2.5% (w/v) iodoacetamide instead of DTT. Equilibrated IPG strips were run in 12% SDS-PAGE. The gels from each 2D-DIGE experiment was scanned with a Typhoon imager (version 3.0) at excitation/emission values of 488/520 nm for Cy2 dye, 532/580 nm for Cy3 dye, 633/670 nm for Cy5 dye. Gel images were analyzed with DeCyder software (version 6.5, Amersham Biosciences) using the automated spot detection algorithm of Differential In-gel Analysis (DIA) module. A spot was considered as differentially expressed whenever the fold change in DIA was larger than +1.5 or smaller than −1.5 and if t-test p-value were less than 0.05. For each DIGE experiment, post-staining of the gel was done with sypro ruby and the differentially expressed spots as determined by DeCyder software were excised using an automatic spot-cutter (Bio-Rad) for MALDI-MS analysis.

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2D-DIGE analysis

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RESULTS Differentially expressed proteins in N2A and SHSY5Y cells Figure 1 represents differentially expressed N2A proteome upon AICD transfection. Overall, 41

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sifications of these altered proteins according to PANTHER are represented as a pie chart in the Supplementary Materials. Transfection of SHSY5Y cells with AICD and subsequent 2D-DIGE analyses, as represented in Fig. 2, revealed that several proteins showed altered expression pattern. 25 proteins were upregulated and 20 downregulated among the 45 differentially expressed proteins upon AICD transfection. The spot numbers in this figure is marked according to the spot numbers given in Table 2. Again, PANTHER classification of these altered proteins revealed them to be involved in a variety of molecular functions and biological pathways (Supplementary Materials). It was clear that the altered proteins from both the cell lines represented similar patterns of molecular functional classes involved in similar type of biological pathways.

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different proteins from N2A cells were found to be differentially expressed due to AICD overexpression. Among them, 21 proteins were upregulated and 20 were downregulated in AICD-GFP transfected cells compared to GFP-transfected control cells. The spot numbers in the figure, as marked match with the spot numbers given in Table 1. PANTHER classification of these proteins revealed that the proteins belonged to several protein classes involved in biological processes having a large spectra of molecular functions. The majority of the proteins possess catalytic activity, binding, and structural roles. The most prevalent biological processes involved cell communications, cell cycle, and cellular component organization. Additionally, some differentially expressed proteins were also involved in immune response as expected in a stressed condition. Clas-

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Fig. 1. Representative 2D-DIGE image showing differential proteomic expression of N2A cells due to AICD-GFP transfection. The whole cell proteome of AICD-GFP transfected cells were labeled with Cy5 and that of the GFP-only transfected cells were labeled with Cy3. The internal standard containing equal amount of both the proteome was labeled with Cy2. In the merged image, after DeCyder analyses, the spots appearing redder are upregulated and the spots that are greener are downregulated in AICD-transfected condition. The yellow spots were with similar expression patterns. The identified proteins showing differential expressions (both upand downregulation) were marked according to the numbers given in Table 1.

Fig. 2. Representative 2D-DIGE image showing differential proteomic expression of SHSY5Y cells due to AICD-GFP transfection. The whole cell proteome of AICD-GFP transfected cells were labeled with Cy5 and that of the GFP-only transfected cells were labeled with Cy3. The internal standard containing equal amount of both the proteome was labeled with Cy2. In the merged image, after DeCyder analyses, the spots appearing redder are upregulated and the spots that are greener are downregulated in AICD-transfected condition. The yellow spots were with similar expression patterns. The identified proteins showing differential expressions (both upand downregulation) were marked according to the numbers given in Table 2.

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Table 1 Differentially expressed N2A proteins upon AICD transfection

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

pI

seq cov

Peptides matched

Score

Status

83535.1 72493.5

4.95 5.07

40% 58%

50 54

163 590

up down

P11142 P27797 P07437 P68361 Q9C0J8 O75944 Q99798 Q9NVU0 Q9NX65 P06576

71086.2 48285.8 49370.5 50815.7 146235.3 65944.5 86126 79847.4 54893.7 56524.6

5.37 4.29 4.71 4.94 9.24 7.96 7.62 6.05 9.03 5.26

56% 35% 39% 47% 33% 41% 39% 23% 26% 42%

48 27 36 28 65 49 52 24 17 25

241 167 261 83 58 190 181 53 65 99

down down down down up up up down up down

P49368 P06733 Q6M063 P06733 P60709 P04075 P00558 P40925

60364.3 47487.4 59829.6 47515.4 41709.7 39264.3 44455.1 36272

6.1 7.01 9.16 7.01 5.29 8.39 8.3 6.89

31% 53% 38% 56% 50% 36% 38% 35%

27 44 38 49 20 16 17 19

114 68 127 108 284 63 90 262

up up up down down up up up

Q9NYQ7 P11766 Q04917 P32119 P62937 P18669 P63244 P06748 P63241

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MW

P08238 P11021

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13 14 15 16 17 18 19 20

Un

3 4 5 6 7 8 9 10 11 12

Accession No.

363022 41574.3 28172 21843.3 17999.9 28654.8 35088.5 32554.8 16690.4

6.22 7.83 4.76 6.84 7.68 6.75 7.6 4.64 5.08

15% 36% 63% 47% 76% 63% 67% 29% 84%

61 33 41 15 29 33 47 7 17

68 63 80 81 289 101 138 66 226

down down up up up up down down up

P16949 P25398 P15531 P28074

17160.9 14385.4 17137.7 22882.3

5.77 6.36 5.83 8.66

29% 62% 59% 33%

4 11 16 13

61 78 153 61

down down up down

O14904 Q9UF56 P38646

40294 41961.2 73634.8

9.08 7.81 5.87

31% 37% 41%

19 23 34

52 60 69

down down up

P10809 Q13087 P31948 P08670 P35232

61016.4 57080.7 62599.4 53619.1 29785.9

5.7 4.76 6.4 5.06 5.57

48% 51% 56% 51% 50%

41 41 56 26 20

53 72 76 157 313

up up up down down

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HSP90 ␤ dnaK type molecular chaperone HSPA5/GRP78 HSPA8 Calreticulin Precursor Tubulin ␤ Tubulin ␣ WD-repeat containing protein Aconitase Aconitase hydratase DNA-directed RNA polymerase III Zinc finger protein 434 ATP synthase ␤ chain, mitochondrial precursor T-complex protein 1, ␥ subunit Phosphopyruvate hydratase H+-transporting two-sector ATPase ␣-Enolase Actin-␤ Fructose bisphosphate aldolase A Phosphoglycerate kinase 1 Malate dehydrogenase, mitochondrial precursor Protocadherin Alcohol dehydrogenase 5 14-3-3η Peroxiredoxin 2 Peptidyl-prolyl cis-trans isomerase Phosphoglycerate mutase Guanine nucleotide binding protein B23 Nucleophosmin Eukaryotic translation initiation factor 5A Stathmin 40 S ribosomal protein S12 Nucleoside diphosphate kinase A Proteasome subunit ␤, type 5 precursor Wnt-9a protein precursor F-box/LRR repeat protein 17 Stress 70 protein/GRP75, mitochondrial precursor HSP60 Protein disulfide isomerase Stress-induced phosphoprotein 1 Vimentin Prohibitin

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Protein name

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Spot No.

Note: Status is represented as expression in AICD-overexpressed condition compared to that in control. ‘up’ means upregulation of at least 1.5 fold and ‘down’ means at least 1.5 fold downregulation in AICD-transfected condition.

274 275

276 277 278 279 280

Comparison of the altered proteomic expression in N2A and SHSY5Y Comparison of the proteins that were found to be altered in N2A and SHSY5Y due to AICD overexpression revealed some similarity of alteration in both the paradigms. 21 proteins were common in showing differential expression in both the cell lines. These

proteins are listed in Table 3 with short indication of their function along with their alteration pattern (upregulation or downregulation) as a result of AICD transfection. The most prevalent protein class to which the altered proteins from both cell lines belonged to is molecular chaperone. Six proteins out of 21 (∼28.6%), namely, HSP90 ␤, HSPA8, GRP78, HSP60, 14-3-3 protein, and

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A. Chakrabarti et al. / AICD-Dependent Altered Neuroblastoma Proteome Table 2 Differentially expressed SHSY5Y proteins upon AICD transfection

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

pI

seq cov

Peptides matched

Score

Status

41765.8 53651 61016 72071.2

5.3 5.19 5.7 5.03

46% 51% 57% 51%

26 26 49 46

410 157 261 322

down down up up

Q5CAQ5 Q2VPJ6 P55072 P14314 Q6IAW5 P08865 P06748 P09493 P06753 Q07021

92282 68328.8 89134.7 59258.8 36995.6 32702 32554.8 32688.7 29026.8 31342.6

4.7 5.11 5.14 4.34 4.5 4.79 4.64 4.69 4.73 4.74

51% 50% 52% 27% 28% 61% 40% 49% 71% 29%

56 39 48 23 21 27 24 22 32 10

310 243 138 146 71 148 209 138 237 199

down up down down up up up down down down

P61981 P11142 Q13087 O95672 P02992

28153.9 70854.2 56746.8 87761.5 49510.2

4.8 5.37 5.98 6.56 7.26

49% 56% 48% 12% 41%

24 40 24 10 16

202 288 173 57 109

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38448.8 26521.7 249154 38169 53881.2

7.56 6.51 6.49 6.2 5.7

49% 68% 11% 32% 26%

34 44 43 13 12

81 111 51 76 62

down down down down down

P06733 P04179

47139.3 22190.2

7.01 6.86

40% 47%

12 9

71 70

down up

A4D1P6

77165.5

6.28

20%

15

62

up

P32119 Q06830 Q5JC45 CAC88315 P09382 P62937 P36969

21843.3 18963.7 48654.9 19217.5 14592.1 17959.8 22113

6.84 6.41 5.26 7.82 5.33 7.74 8.64

47% 52% 34% 64% 61% 74% 56%

15 14 23 23 16 26 18

70 109 67 284 148 115 43

up up up down up up up

P40926 P18669 P00558 Q15293 P04075 P14625 P08238 P16949 Q9UNM1 P15531 O75944

35508.8 28654.8 44455.1 38866.2 39264.3 92411.3 83081.1 17160.9 10288.6 17137.7 65945

8.92 6.75 8.3 4.86 8.39 4.76 4.97 5.77 8.98 5.83 7.96

46% 27% 38% 33% 22% 51% 39% 35% 47% 55% 40%

20 8 17 17 9 56 42 6 13 17 41

239 62 80 58 64 310 243 70 187 134 180

up up up up up up up down down down down

P07355 P60174 Q96BY6 Q8TE01 P05091

or P

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MW

P63261 P08670 P10809 P11021

rre

20 21 22 23 24

co

15 16 17 18 19

Un

5 6 7 8 9 10 11 12 13 14

Accession No.

uth

Actin ␥ Vimentin HSP60 dnaK type molecular chaperone HSPA5/GRP78 Tumor rejection antigen HSP90AA1 TER ATPase Glucosidase 2, ␤-subunit precursor Calumenin 40 S ribosomal protein SA B23 Nucleophosmin Tropomyosin 1 alpha chain Tropomyosin 3 isoform 2 Complement component 1, Q-binding protein 14-3-3␥ HSPA8 Protein disulfide isomerase Endothelin converting enzyme-like1 Elongation factor Tu, mitochondrial precursor Annexin A2 Triose phosphate isomerase Dedicator of cytokinesis protein-10 DERP12 Mitochondrial aldehyde dehydrogenase ␣-Enolase Superoxide dismutase [Mn], mitochondrial WD repeat containing protein 91/ HSPC049 Peroxiredoxin2 Peroxiredoxin1 Transformation related protein2 Seq. 9 from patent WOO164904 Galactoside protein/Galectin 1 Peptidyl-prolyl cis-trans isomerase Glutathione peroxidase, mitochondrial precursor Mitochondrial Malate dehydrogenase Phosphoglycerate mutase Phosphoglycerate kinase1 Reticulocalbin1 precursor Fructose bisphosphate aldolase A Endoplasmin precursor HSP90 ␤ Stathmin Chaperonin 10-related protein Nucleoside diphosphate kinase A Aconitase

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Protein name

1 2 3 4

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Spot No.

Note: Status is represented as expression in AICD-overexpressed condition compared to that in control. ‘up’ means upregulation of at least 1.5 fold and ‘down’ means at least 1.5 fold downregulation in AICD-transfected condition.

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B23 nucleophosmin, represented this class. The proteins vimentin and actin were from the cytoskeletal protein class. Vimentin was also represented in structural protein class. Several proteins were found to have

catalytic activity and they represented different protein classes like isomerases (phosphoglycerate mutase, protein disulfide isomerase, and peptidyl-prolyl cistrans isomerase), kinases (phosphoglycerate kinase

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Table 3 List of proteins with altered expression in both the paradigms along with their molecular functions Molecular chaperone Molecular chaperone inside ER Molecular chaperone; acts as repressor of transcriptional activation Pre-mRNA 3’ end processing Citric acid cycle enzyme Glycolysis enzyme; takes part in various processes like growth control, hypoxia tolerance and allergic response Structural constituent of cytoskeleton; involved in various types of cell motility Key role in glycolysis and gluconeogenesis; may also function as scaffolding protein Glycolysis enzyme; also acts as primer recognition protein for polymerase Oxidoreductase function in TCA cycle Adaptor for various signalling pathway proteins; have chaperone activity Involved in redox regulation of cells; have oxidoreductase and peroxidase activity Has isomerase activity; also accelerate folding of proteins Intramolecular transferase activity Protein chaperone involved in rRNA metabolism, ribosome biosynthesis etc Involved in regulation of microtubule filament system by destabilizing microtubules- prevents assembly and promotes disassembly Required for assembly/stability of 40S ribosomal subunit; nucleic acid binding Nucleotide kinase; involved in cell proliferation, differentiation and development; also required for neural development, patterning and cell fate determination Molecular chaperone, implicated in mitochondrial protein import and macromolecular assembly Involved in protein modification process with disulfide isomerase activity Structural constituent of cytoskeleton

Actin Fructose bisphosphate aldolase A Phosphoglycerate kinase1 Malate dehydrogenase 14-3-3 Peroxiredoxin2 Peptidyl-prolyl cis-trans isomerase

Stathmin

40S ribosomal protein

HSP60 Protein disulfide isomerase Vimentin

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Nucleoside diphosphate kinase A

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Phosphoglycerate mutase B23 Nucleophosmin

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WD-repeat containing protein Aconitase ␣-Enolase

Status in N2A

Status in SHSY5Y

up down down

up up down

up up down

up down down

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Molecular function

HSP90 ␤ HSPA5/GRP78 HSPA8

or P

Protein name

down

down

up

up

up

up

up up

up up

up

up

up

up

up down

up up

down

down

down

up

up

down

up

up

up

up

down

down

298 299 300 301 302 303 304 305 306 307 308 309 310 311 312

and nucleoside diphosphate kinase), and lyases (fructose bisphosphate aldolase). Phosphoglycerate kinase and nucleoside diphosphate kinase also possessed transferase activity. These proteins have their major roles in different metabolic pathways. The proteins 40S ribosomal protein and WD-repeat containing protein have nucleic acid binding property. Peroxiredoxin 2 and malate dehydrogenase belonged to the class oxidoreductase. Although the pattern of alterations (upregulation or downregulation) for most of the proteins from both cell lines was similar, some of the proteins showed a reverse pattern in their change in expression upon AICD overexpression. For example, the proteins GRP78, WD-repeat containing protein, B23 nucleophosmin, and 40S ribosomal protein showed downregulation in

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Note: “up” and “down” represents upregulations and downregulations in AICD-transfected condition.

N2A but upregulation in SHSY5Y upon AICD transfection. On the other hand, the proteins aconitase and nucleoside diphosphate kinase showed upregulation and downregulation in N2A and SHSY5Y, respectively. 3D expression patterns for 12 proteins found to show similar patterns of alteration in both cell lines upon AICD transfection are represented in Supplementary Fig. 3. Validation of a few differential expression results Eight of the proteins showing altered expression in DIGE experiments were further validated by western blot. All the western blot validations were carried out in SHSY5Y cells. Peroxiredoxin 2 (PRDX2), found to be upregulated by 3.23 fold in

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western blot which was similar to the observations in DIGE experiments and ␣-tubulin and vimentin, both downregulated in DIGE experiments, were also shown to have downregulations in western blots (by 4.19-fold and 2.17-fold, respectively). Although the protein ␣-tubulin was identified to be differentially expressed only in N2A by DIGE experiments, validation of its downregulation in SHSY5Y represents the limitation of the high-throughput proteomic identification. Other validated proteins found to be upregulated were protein disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase (Pin1), and 14-3-3 (upregulated by 3.37-, 1.7-, and 1.68-fold, respectively). Stathmin and HSPA8 were also validated which showed downregulation (1.64- and 1.27-fold, respectively) due to AICD overexpression. Figure 3 represents the western blots for validation of these eight proteins showing altered expression due to AICD transfection.

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Fig. 3. Western blots for validation of eight of the proteins altered upon AICD transfection. Peroxiredoxin 2, PDI, Pin1, and 14-3-3 were found to be upregulated, and ␣-tubulin, vimentin, stathmin, and HSPA8 were downregulated, which was similar as obtained in DIGE experiments. All eight proteins showed significant alterations. P-values for PRDX2, PDI, Pin1, 14-3-3, ␣-tubulin, vimentin, stathmin, and HSPA8 were found to be 0.07, 0.05, 0.007, 0.03, 0.01, 0.05, 0.01, and 0.002, respectively, indicated with ‘*’ marks in the bars showing the fold change in protein expression with AICD-GFP transfected condition with respect to that for the GFP-only transfected condition.

DISCUSSIONS Our study is an attempt to decipher the effect of AICD-overexpression in two different neuroblastoma cell lines at the proteome level. The proteins which

show altered expression levels belong to several protein classes participating in different molecular functions. One major class of proteins which showed differential expression upon AICD transfection was cytoskeletal or structural proteins. Among them, actin and vimentin showed altered expression in both cell lines. Disruption of actin dynamics is already reported due to induction of AICD expression in neuronal cells [10]. Some isoforms of tubulin were found to be differentially expressed in N2A cells, and tropomyosin ␣ showed altered expression in SHSY5Y cells. We also obtained 40S ribosomal subunits, though different subunits in these two cell lines, showing altered expression in our study. Loss of structural integrity is a wellestablished event in neurodegenerative diseases like AD [11]. Altered expression of the structural proteins due to AICD overload, therefore, is definitely an important observation. We previously showed vimentin as one of the several interactors of AICD [9]. Alteration of vimentin found in our study further strengthens the role of AICD in cytoskeletal restructuring. Stathmin, downregulated in both cell lines in our study, functions as a regulatory protein of microtubule dynamics and was found to be associated with neurofibrillary tangles in brains of AD patients [12]. This protein is also reported to be downregulated by AICD-Fe65-Tip60 complex in

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level was also found to be decreased in adult brain with Down syndrome and AD [26]. It is generally perceived that there might be a link between Down syndrome and overproduction of AICD that leads to an AD-like condition [27]. Once again, decreased expression of stathmin due to AICD overexpression might have some role to link these two diseases. Other proteins which were reported to be altered during aging and also showed up- or downregulation in our study include triosephosphate isomerase, superoxide dismutase, annexin, enolase, and protein disulfide isomerase, among others [28]. Several heat shock proteins (HSPs), e.g., HSP90␤, GRP78, GRP75, HSPA8, HSP60, and T-complex protein1, were found to show altered level of expression in our experiments conforming with a previous study with AD brain [29]. AD-like neurodegenerative features are the result of gain of toxic functions of misfolded proteins/peptides (A␤ and tau), and aberrant chaperone activities are thought to be responsible for the shift of balance between folded to misfolded proteins [30]. Several studies have been conducted to explain the role of chaperones, mainly the HSPs, in AD [31, 32]. Our results, therefore, are in accordance with the hypotheses regarding the involvement of chaperones as well as AICD in AD pathophysiology. Although there were some inconsistencies in the direction of change of the HSPs in our study and studies by others, this may be explained by differential response of individual HSPs to a stressed condition [33]. Quite a few metabolic proteins involved in a variety of biochemical pathways including glycolysis, Krebs cycle, oxidative phosphorylation, mitochondrial protein synthesis, etc., showed altered expression in this study hinting that active metabolic changes had occurred due to AICD transfection. In a protein misfolding and aggregation disorder like AD, it is expected that metabolic protein expression should get altered due to changes in the energy need [34]. There is strong evidence in favor of mitochondrial dysfunction contributing to AD phenotype [35–37]. A number of mitochondrial proteins in this study were found to have differential expression in the AICDtransfected condition. An imbalance in mitochondrial bioenergetics was also previously reported due to the presence of AICD [10]. Among these, mitochondrial malate dehydrogenase, superoxide dismutase [Mn], fructose bisphosphate aldolase A, etc., were found to be upregulated in an AICD-overexpressed condition. These proteins were also reported to be upregulated in an earlier study of mitochondrial proteome with mouse model of AD [38]. However, the proteins fructose

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HEK293T cells [13]. Our result suggests the possibility of AICD-mediated role of stathmin in microtubule disorganization during AD progression. Oxidative stress is another feature for the pathogenesis of AD [14–16]. In the present study, we found several antioxidant proteins to be upregulated due to AICD-overexpression. The proteins peroxiredoxin 1, peroxiredoxin 2, and mitochondrial superoxide dismutase, which were observed to be altered (increased) in our study, were also previously reported to show increased expression in different regions of the brain of AD patients [17, 18]. Peroxiredoxin 1 and peroxiredoxin 2 play important antioxidant roles for protecting neurons from peroxide-induced damage. Elevated expression of these proteins (both PRDX1 and PRDX2 in SHSY5Y and PRDX2 in N2A) indicates the involvement of reactive oxygen species in AD pathogenesis [18]. Two other antioxidant proteins, glutathione peroxidase in SHSY5Y and alcohol dehydrogenase in N2A were also showed to have increased and decreased expressions, respectively. Although we did not find any protein involved in autophagy or vesicle formation to show altered expression, several signaling proteins were found to be altered in this study. The protein Pin1 was found to be upregulated in both the cell lines. Of late it has been reported that Pin1 is involved in several signaling pathways as well as in regulation of cell cycle and cell division [19]. Due to its involvement in aging process and age-related disorders, it plays a pivotal role in cancer as well as in AD and can act as an important link between the two [19, 20]. Wnt signaling is increased during normal aging [21] and this pathway is also involved with Pin1 and p53 linking aging, cancer, and AD [22]. We found Wnt-9a protein precursor, involved in developmental processes, having shown decreased expression in N2A cells upon AICD overexpression. This result, along with increased expression of Pin1, strengthen the hypothesis that normal aging and AD are coupled processes and the developmental defects linked with them, as well as cancer, do have some level of commonality. Additionally, AICD-mediated transcriptional activity is promoted by Wnt/␤-catenin signaling [23], further implying the influence of AICD on these pathways. Stathmin was earlier reported to have altered expressions in the neonatal mice brain as well as in old postmortem human brain in comparison to normal adults [24, 25]. This protein was found to be downregulated in this study with overexpression of AICD, and hence, its involvement in the age-dependent decline in neurogenesis as well as in the disease process can be thought to be mediated by AICD. Stathmin

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ACKNOWLEDGMENTS

The work was partially funded by the IBOP project, Department of Atomic Energy, Government of India. AC acknowledges Council of Scientific and Industrial Research, Government of India for his fellowship. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=1909). SUPPLEMENTARY MATERIAL

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bisphosphate aldolase A and superoxide dismutase [Mn] were also reported to be altered in human AD brain in several studies by different groups [39–43]. A recent review from the systems biology aspects of AD discussed the diverse molecular changes that might result in the memory impairment [44] and according to their compilation, a majority of the altered proteins from animal models of AD were involved in cytoskeleton organization, energy metabolism, response to oxidative stress, and chaperone activity, which is highly congruent to our data. Another differential proteomics study with A␤1-42 treatment in differentiated SHSY5Y cells (mimicking the AD condition) revealed similar kinds of molecular functional classes the altered proteins were involved in [45]. It is obvious that several pathways are engaged in AD pathogenesis and the ensemble of proteins obtained in our study as well as in studies by other groups strongly suggests that there must be a complex protein interaction network leading to AD with the short intracellular fragment of A␤PP, the AICD, situated in a central node of this complex network. It is likely that a complex pathway interaction network, rather than a protein interaction network, can explain the molecular mechanism behind AD.

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