Cytosolic RNA Recognition Pathway Activates 14 ... - ACS Publications

Cytosolic RNA Recognition Pathway Activates 14-3-3 Protein Mediated Signaling and Caspase-Dependent Disruption of Cytokeratin Network in Human Keratinocytes ¨ hman,†,‡ Niina Lietzeń,†,‡ Elina Va¨lima¨ki,§ Jesper Melchjorsen,| Sampsa Matikainen,§,⊥ Tiina O and Tuula A. Nyman*,†,⊥ Protein Chemistry Research Group, Institute of Biotechnology, University of Helsinki, Finland, Unit of Excellence in Immunotoxicology, Finnish Institute of Occupational Health, Finland, and Department of Infectious Diseases, Aarhus University Hospital, Skejby, Denmark Received November 13, 2009

The skin is the primary boundary between the body and the environment. In addition to its properties as a physical barrier, skin keratinocytes actively participate in many defense mechanisms. Viral doublestranded RNA (dsRNA) is the most important viral structure involved in activation of immune response. Intracellular detection of dsRNA by cytoplasmic receptors activates well-characterized antiviral response, as well as pro-inflammatory response and apoptosis of virus-infected cells. Here, we have used quantitative subcellular proteomics to characterize the signaling pathways activated by cytosolic dsRNA recognition pathway in human keratinocytes. Cytoplasmic and mitochondrial proteomes were analyzed using 2-DE in combination with MS, immunoblotting and confocal microscopy. We have identified 239 reproducibly differentially expressed proteins upon dsRNA stimulation. The identified proteins include several key proteins involved in cytoskeletal dynamics, cell signaling, cell death, and stress response. Our analysis provides novel information how the cytokeratin network is disrupted in a caspasedependent manner upon dsRNA stimulation as well as Encephalomyocarditis virus or Vesicular stomatitis virus infection. We show that this caspase-dependent disruption of cytokeratin is activated by cytoplasmic RNA recognition pathway. In addition, we show that viral infection activates 14-3-3 protein mediated signaling pathways in human keratinocytes which suggest an important role of 143-3 proteins in antiviral innate immune response. Keywords: subcellular proteomics • virus infection • innate immune response • apoptosis • cytokeratin • 14-3-3 proteins

Introduction Keratinocytes represent the major cell population of human epidermis which provides a first line defense barrier for the host. In addition, a number of studies have demonstrated that keratinocytes actively participate in immune response.1 During infection, keratinocytes express a lot of cytokines, chemokines, and accessory molecules, which can transmit both positive and negative signals to cells of innate and adaptive immunity. Dysregulation and abnormal expression of inflammatory mediators or their receptors in keratinocytes are relevant to the pathogenesis of chronic inflammatory skin diseases such as psoriasis, atopic dermatitis, and allergic contact dermatitis. * To whom correspondence should be addressed. Tuula A. Nyman. E-mail: [email protected]. Protein Chemistry Research Group, Institute of Biotechnology, University of Helsinki, P.O. Box 65, Viikinkaari 1, FI-00014, Finland. Phone: +358-9-191-59411. Fax: +358-9-191-59930. † University of Helsinki. ‡ These authors contributed equally to this work. § Finnish Institute of Occupational Health. | Aarhus University Hospital. ⊥ These authors contributed equally to this work. 10.1021/pr901040u

 2010 American Chemical Society

However, the role of keratinocytes in innate immunity is less well understood. Viral genomic RNA and viral replication intermediates including double-stranded RNA (dsRNA) are the most important viral pathogen associated molecular patterns (PAMPs). These PAMPs are sensed by the pattern recognition receptors resulting in activation of innate immune response. Viral RNA is able to trigger distinct signaling pathways by engaging either the endosomal membrane-bound Toll-like receptor 3 (TLR3) or soluble cytoplasmic receptors including retinoic acidinducible gene I (RIG-I) and melanoma differentiation-associated gene-5 (MDA-5). TLR3 detects extracellular viral dsRNA internalized into the endosomes whereas RIG-I and MDA-5 are focused on detecting viral genomic RNA or replication intermediates in the cytoplasm (reviewed in refs 2 and 3). RIG-I is implicated to recognize also viral ssRNA bearing 5′ phosphates found in the genome of many ssRNA viruses, such as influenza A virus.4,5 In contrast, MDA-5 is essential for interferon (IFN) production in response to cytosolic dsRNA analogue polyinosicpolycytidylic acid (polyI:C) and picornavirus infection.6,7 In addition to antiviral response, cytosolic RNA recognition pathway is involved in the activation of pro-inflammatory Journal of Proteome Research 2010, 9, 1549–1564 1549 Published on Web 01/13/2010

research articles response and programmed cell death, apoptosis of virusinfected cells.8 Mitochondria are highly dynamic organelles that contribute to many cellular events, including energy conversion, reactive oxygen species production, cell signaling, and cell death.9 Moreover, mitochondria have a unique function in viral infection and innate immunity, since the downstream adaptor molecule MAVS (mitochondrial antiviral signaling protein), which is critical for RIG-I and MDA-5 mediated signaling, is localized to the outer membrane of mitochondria.10 In addition, it was recently reported that two other mitochondrial proteins, NLRX1 and MITA, associate with MAVS and act as regulators for antiviral response.11,12 In addition, our recent report shows that several components of RIG-I/MAVS signaling pathway, including RIG-I, TRADD, TRIM25, and IKK, translocate onto mitochondria in response to influenza A virus infection in human macrophages.13 These indicate that multiprotein signaling complexes are formed onto mitochondrial membrane during viral infection. Thus, mitochondria play a key role in antiviral response by acting as a unique signaling platform. Proteomics has emerged as an important tool to extract the details of cellular signaling mechanisms. In the present study, we have used 2-DE based quantitative subcellular proteomics to characterize the signaling pathways activated by cytosolic dsRNA recognition pathway in human keratinocytes. We show that dsRNA stimulation of HaCaT keratinocytes results in caspase-dependent disruption of cytoskeletal network. In addition, we provide evidence that 14-3-3 adapter proteins are involved in the activation of signaling cascades during viral infection.

Experimental Procedures Cell Stimulation and Fractionation. Human keratinocyte HaCaT cells (American Type Culture Collection) were cultured in DMEM supplemented with 10% FCS, L-glutamate and antibiotics. Cells were transfected with a mimetic of dsRNA, polyinosic-polycytidylic acid (polyI:C) (Sigma-Aldrich) using Lipofectamine2000 transfection reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. For caspase inhibition experiments general caspase inhibitor zVAD-fmk was used (R&D Systems). Encephalomyocarditis virus (EMCV) and Vesicular stomatitis virus (VSV) were propagated in confluent L929 cells grown in DMEM with 2% FCS and antibiotics. When complete lysis was reached the preparations were twice frozen and thawn, followed by centrifugation for 60 min at 5000× g (4 °C) for removal of cellular debris. Cell supernatants containing virus were immediately frozen and kept at -80 °C until performing the experiments. Virus titers were determined in Vero cells using end point-dilutions and subsequent Reed-Mu ¨ nch quantification. Titers were determined to be 2.7 × 107 plaque forming unit (PFU)/ml and 1 × 107 PFU/ml for EMCV and VSV, respectively. Cells were infected with multiplicity of infection (MOI) of 2 for both viruses used. RIG-I, ∆RIG-I, and MAVS plasmids have been previously described.14,15 The mitochondrial and cytoplasmic cell fractions were isolated by Qproteome Mitochondria Isolation Kit (Qiagen). Cytoplasmic fractions were further purified with 2-D CleanUp Kit (GE Healthcare). For each cell fractionation, 10 million HaCaT cells were used. 2-DE and Quantitative Analysis. Protein separation by 2-DE was performed with 11 cm pI 4-7 IPG-strips (Bio-Rad) and the second dimension with Criterion Tris-HCl 8-16% precast gels (Bio-Rad). Isolated mitochondrial and cytoplasmic frac1550

Journal of Proteome Research • Vol. 9, No. 3, 2010

¨ hman et al. O tions from polyI:C-transfected and untreated HaCaT cells were dissolved in 200 µL rehydration buffer (7 M urea, 2 M thiourea, 3.5% CHAPS, 0.6% DTT, 0.5% IPG-buffer 4-7) and the proteins were absorbed into IPG-strips for 24 h at room temperature. IEF to a total of 55 kVh was done at 20 °C, and focused strips were equilibrated two times at room temperature for 15 min with SDS-equilibration buffer containing 6 M urea, 50 mM TrisHCl, pH 6.8, 2% SDS, 20% glycerol, and 10 mg/mL DTT for the first equilibration, and 25 mg/mL iodoacetamide for the second equilibration. SDS-PAGE was run at 200 V using a Criterionelectrophoresis unit (Bio-Rad) at 4 °C. Gels were stained with SYPRO Ruby protein gel stain (Sigma or Bio-Rad) according to the manufacturer’s instructions. Spot detection, matching and intensity-based quantitation were performed using ImageMaster 2D Platinum version 6.0. Three and four biological replicates for mitochondrial polyI:C- and control-samples, respectively, and six biological replicates for the cytoplasmic samples were used in the analysis. Statistical significance (p < 0.05) of the quantitation results was evaluated with Student t test. Spots with at least 2-fold difference in expression between control and polyI:C-transfected samples were considered differentially expressed. MS. For identification, proteins were detected with MScompatible silver staining.16 The differentially expressed proteins were in-gel digested with trypsin and the resulting peptides were analyzed by peptide-mass fingerprinting (PMF) as previously described17,18 or by nano-LC-MS/MS. For PMF the mass spectra were acquired using an Ultraflex TOF/TOF instrument (Bruker Daltonik, Bremen, Germany) in positive ion reflector mode, and the instrument was externally calibrated using a standard peptide mixture from Bruker. LC-MS/MS analysis was performed using an Ultimate 3000 nano-LC (Dionex) and a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization. The LC-MS/MS samples were first loaded on a ProteCol C18 trap column (10 mm × 150 µm, 3 µm, 120 Å) (SGE), followed by peptide separation on a PepMap100 C18 analytical column (15 cm × 75 µm, 5 µm, 100 Å) (LC Packings/Dionex) at 200 nL/ min. The separation gradient consisted of 0-50% B in 20 min, 50% B for 3 min, 50-100% B in 2 min and 100% B for 3 min (buffer A: 0.1% formic acid; buffer B: 0.08% formic acid in 80% acetonitrile). MS data were acquired using Analyst QS 2.0 software. Information-dependent acquisition method consisted of a 0.5 s TOF-MS survey scan of m/z 400-1400. From every survey scan two most abundant ions with charge states +2 to +4 were selected for product ion scans. Once an ion was selected for MS/MS fragmentation, it was put on an exclusion list for 60s. Protein Identification. The PMF spectra were processed with FlexAnalysis version 3.0. Spectra were internally calibrated using trypsin peaks. The raw spectra were baseline subtracted with algorithm Median, and peak detection was done using parameters: Algorithm Snap, S/N > 7, Snap average composition averagine, and peak width 0.75 m/z. The original MALDI spectra and peak lists are provided as Supplementary Data S1-S3, Supporting Information. Database searches with the obtained MALDI-MS data were done with publicly available Mascot search engine against the NCBInr database (http:// www.matrixscience.com). The LC-MS/MS data was searched with the in-house Mascot version 2.2 through the ProteinPilot 2.0.1 interface against the NCBInr database (version 20080331). The search criteria for both Mascot searches were: Humanspecific taxonomy, trypsin digestion with one missed cleavage

Subcellular Proteome Study of dsRNA-Stimulated Keratinocytes allowed, carbamidomethyl modification of cysteine as a fixed modification and oxidation of methionine as a variable modification. For the PMF spectra the maximum peptide mass tolerance was (50 ppm. Reported protein identifications have Mowse scores greater than 66 (p < 0.05). For the LC-MS/MS spectra both the maximum precursor ion mass tolerance and MS/MS fragment ion mass tolerance were 0.2 Da, and peptide charge state of +1, +2, or +3 was used. To consider the identification reliable, a minimum of two peptides with an ions score at least 40 was required. To eliminate the redundancy of proteins that appear in the database under different names and accession numbers, the single protein member with the highest protein score (top rank) was selected from the multiprotein family to the identification results in Tables 1 and 2. Western Blotting. Cells were lyzed with lysis buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, and 25% ethylene glycol supplemented with Complete mini protease inhibitor mixture (Roche Diagnostics) and homogenized by ultrasound sonication. Proteins (7.5 µg) were separated with SDS-PADE and transferred onto PVDF-membrane. Alternatively, HaCaT cells were fractionated into mitochondrial and cytoplasmic fractions, and the fractions were dissolved in 100 µL Laemmli sample buffer. Five µL of samples were run on SDS-PAGE, and the proteins were visualized with silver staining. For Western blotting an equal protein loading was determined from the silver stained gel. Membranes were blocked with 5% nonfat milk and stained with an antibody against R/β-tubulin, HSP90, phosphor-Ser 14-3-3 binding motif (Cell Signaling Technology), eIF4A, cytokeratin 18, caspase-3 (H-277), 14-3-3σ, pan14-3-3, or HSP27 (Santa Cruz Biotechnology), followed by staining with peroxidase-conjugated secondary antibodies and detection by the ECL system (Perkin-Elmer). Quantitative Real-Time RT-PCR. Total RNA was isolated from cell samples with RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen). cDNA was synthesized with TaqMan reverse transcription reagents (Applied Biosystems). Quantitative real-time PCR was performed with ABI PRISM 7500 Sequence Detection System applying TaqMan chemistry (Applied Biosystems). ELISA. Human IL-29 ELISAs were purchased from eBioscience and performed according to manufacturer’s instructions. Immunofluorescence Staining and Confocal Microscopy. For indirect immunofluorescence staining, HaCaT cells were grown on coverslips. Cells were transfected with YFP plasmid having mitochondrial targeting sequence followed by stimulation with cytosolic polyI:C. The cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% TritonX100 for 1 min. Cells were treated with specific antibodies for 1 h at room temperature followed by treatment with secondary, species-specific antibodies conjugated with Alexa594 (Molecular Probes) for 1 h. The samples were mounted in Mowiol and viewed under a Leica TCS SP5 confocal microscopy. A HCX APO 63×/1.30 (glycerol) objective was used and images were processed using the LAS AF program (Leica Application Suite Advanced Fluorescence) and Adobe Photoshop.

Results Effect of Activation of Cytosolic dsRNA Recognition Pathway on Subcellular Proteomes of Human Keratinocytes. Viral dsRNA is the most important viral structure involved in activation of innate immune response. Here we have used subcellular proteomics to characterize the signaling pathways activated by cytosolic dsRNA recognition pathway in human keratinocytes.

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HaCaT cells were transfected with a mimetic of dsRNA, polyI: C, for 18 h after which the cells were fractionated into cytoplasmic and mitochondrial fractions. Purity of mitochondrial fraction was determined with specific mitochondrial marker proteins, MAVS and VDAC (Figure 1A). Fractionated proteins were separated by 2-DE and visualized with fluorescent dye. Analysis of the 2-DE gel images showed that a total of 239 protein spots were reproducibly differentially expressed after dsRNA stimulation (Figure 1B and S4, arrows); 101 spots were in the cytoplasmic fraction and 138 in the mitochondrial fraction. From these 239 protein spots, we could identify proteins from 176 spots (Figure 1B numbered arrows and Tables 1 and 2). More than one protein was identified from 20 protein spots, so altogether we identified 200 proteins. These proteins include 133 distinct proteins and 10 proteins that occur both as whole length proteins and fragments. Functional Classification of the Identified Proteins. The identified proteins were classified based on different biological processes using the UniProt Knowledgebase (Swiss-Prot/ TrEMBL) and published literature. The distribution of the functionally classified proteins is shown in Figure 2 and listed in Tables 1 and 2. More than 70% of the proteins in each category (over/under-expressed mitochondrial and over/underexpressed cytoplasmic proteins) were mainly involved in four different biological classes: structural proteins, signaling proteins, protein destination, and RNA/DNA/Protein synthesis. Structural Proteins. Both mitochondrial and cytoplasmic fractions were found to contain a large number of differentially expressed cytoskeleton proteins. In the mitochondrial fraction, 42% (38 proteins) of the overexpressed proteins and 86% (13 proteins) of the under-expressed proteins were classified as structural proteins. In the cytoplasmic fraction the distribution was 21% (16 proteins) overexpressed and 29% (10 proteins) under-expressed protein during dsRNA stimulation. In particular, many cytokeratins were identified (cytokeratin 6C, 7, 8, 9, 13, 14, 15, 17, 18, 19). Interestingly, in control samples cytokeratins were observed as full-length proteins, but in dsRNA-stimulated samples cytokeratins were found mainly as fragments. Also several other structural proteins, such as actin and lamin, were found as fragments in dsRNA-stimulated 2Dgels. In addition, we identified R- and β-tubulin from several protein spots in mitochondrial fractions that had been prepared from polyI:C-stimulated cells. This observation was confirmed by Western blotting with specific R/β-tubulin antibody. Tubulin was detected only in the cytoplasmic fraction in control cells, and a clear mitochondrial translocation of tubulin was observed upon dsRNA stimulation (Figure 3A). Similar result was seen in keratinocytes infected with EMCV or VSV, which activate MDA-5 and RIG-I signaling pathways, respectively. An equal protein loading in gels was determined by silver staining (Figure 3D). Signaling Proteins. Our data shows that many signaling proteins are overexpressed upon dsRNA stimulation: 17% (15 proteins) of the proteins in the mitochondrial fraction and 28% (21 proteins) of the proteins in the cytoplasmic fraction. Altogether 8 protein spots were identified as different isoforms of 14-3-3 protein family. The 14-3-3 proteins are involved in many biological processes such as apoptosis, signal transduction and cell cycle control.19 In mammalian seven 14-3-3 isoforms (β, γ, η, θ, σ, ε, and ζ) and their phosphorylated forms have been identified. In our experiments, we identified six different isoforms (β, η, θ, σ, ε, ζ) that were more abundant in Journal of Proteome Research • Vol. 9, No. 3, 2010 1551

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Table 1. Identification Results from the Differentially Expressed Protein Spots in the Mitochondrial Fraction of dsRNA Stimulated Human Keratinocytesa

protein name

access no. fold spot difference number Swiss-Prot NCBInr

no. of identification peptides rms sequence Mowse type matchedb error (ppm) coverage score

theoretical MW

pI

Structural proteins actin actin cytokeratin 13 (C-term. frag.) cytokeratin 13 (frag.) cytokeratin 13 (frag.) cytokeratin 17 cytokeratin 17 (frag.) cytokeratin 17 (N-term. frag.) cytokeratin 17 (N-term. frag.) cytokeratin 18 (N-term. frag.) keratin 13 isoform A (C-term. fragment) keratin 2 (frag.) tubulin, beta 2C tubulin, beta chain actin (N-term. frag.) cytokeratin 14 (frag.) cytokeratin 17 (C-term. frag) cytokeratin 18 (N-term. frag.) myosin heavy chain 9 (C-term. frag.)g cytokeratin 14 (N-term. frag) tubulin, alpha 1A cytokeratin 13 (N-term. frag) keratin 19 (N-term. frag) tropomodulin 3 cytokeratin 13 (frag.) myosin heavy chain 9 (C-term. frag.)g tropomyosin 3 isoform 2 tubulin, alpha myosin heavy chain 9 (C-term. frag.)g lamin A/C (N-term. frag.) tubulin, beta 2C tubulin, beta chain (N-term. frag.) actin (frag.) actin tubulin, beta 2C cytokeratin 7 cytokeratin 9 (C-term. frag.) cytokeratin 15 cytokeratin 18 lamin B1 cytokeratin 19 cytokeratin 15 cytokeratin 14 keratin 13 isoform A cytokeratin 19 cytokeratin 13 cytokeratin 19 cytokeratin 17 cytokeratin 6C cytokeratin 8

c

Overexpressed 4501885e LC-MS/MS 4501885 LC-MS/MS 34033 LC-MS/MS 34033 LC-MS/MS 30377 LC-MS/MS 4557701 MALDI 4557701 LC-MS/MS 4557701 MALDI 4557701 LC-MS/MS 30311 LC-MS/MS 131412225 LC-MS/MS

54 59 88 73d 74d 37 84 83 82 79 87

P60709e P60709 P13646 P13646 P13646 Q04695 Q04695 Q04695 Q04695 P05783 A1A4E9

21.0 18.6 14.6 14.6 14.5

76 41 43 70 85 86 81 19

P35908 P68371 P07437 P60709 P02533 Q04695 P05783 P35579

47132620 5174735 18088719 4501885 15431310 4557701 30311 12667788

MALDI LC-MS/MS LC-MS/MS LC-MS/MS LC-MS/MS MALDI-LIFT MALDI LC-MS/MS

16/84 10 9 10 6 6/38 18/78 9

12.7 12.4 12.0 10.0 9.8 9.1 9.0

77 33 75 90 45 89 18

P02533 Q71U36 P13646 P08727 Q9NYL9 P13646 P35579

15431310 37492 30377 90111766 6934244 6016411 12667788

LC-MS/MS MALDI MALDI MALDI LC-MS/MS LC-MS/MS MALDI

12 11/8 18/27 17/34 9 12 22/35

7.6 7.1 6.8

69 32 17

P06753-2 Q71U36 P35579

24119203 LC-MS/MS 37492 MALDI 189036 MALDI

18 12/37 23/44

5.5 3.9 3.7

78 31 63

Q5TCJ4 P68371 P07437

55957496 LC-MS/MS 23958133 MALDI 18088719 LC-MS/MS

11 14/42 5

2.7 2.4 2.4 2.3 2.1

57 44 30 29 53

P60709e P60709e P68371 P08729 P35527

4501885e 4501885e 23958133 67782365 435476

LC-MS/MS LC-MS/MS MALDI MALDI MALDI

9 15 17/52 22/72 14/

7 12 3 16 8 5 9 14 4 13 10 6 11

P19012 P05783 P20700 P08727 P19012 P02533 A1A4E9 P08727 P13646 P08727 Q04695 P48668 P05787

Under-expressed 125081 LC-MS/MS 30311 MALDI 5031877 LC-MS/MS 90111766 LC-MS/MS 125081 LC-MS/MS 15431310 MALDI 131412225 LC-MS/MS 90111766 MALDI 118835468 MALDI 90111766 LC-MS/MS 4557701 MALDI 155969697 MALDI 181573 MALDI

25 24/31 16 16 28 13/10 13 13/14 19/30 22 26/37 10/5 21/39

c c c c c c c c c c

c c c

c

7.0 5.6 4.8 4.6 4.1 4.1 3.3 2.8 2.8 2.6 2.5 2.1

2 9 6 11 11 13/29 5 9/20 9 8 7

5% 21% 13% 22% 22% 33% 11% 19% 19% 15% 13%

83 96 79 142 372 108 174 85 114 156 277

41737 41737 49956 49956 46179 48361 48361 48361 48361 47305 49869

5.29 5.29 4.87 4.87 4.83 4.97 4.97 4.97 4.97 5.27 4.91

33% 23% 17% 24% 12% 15% 31% 5%

74 233 187 403 155 102 131 197

65678 50255 50096 41737 51875 48361 47305 227646

8.07 4.79 4.75 5.29 5.09 4.97 5.27 5.5

13

25% 34% 29% 40% 27% 24% 15%

1763 142 187 172 156 363 88

51875 50810 49956 44065 39727 49898 227646

5.09 5.02 4.87 5.04 5.08 4.91 5.5

8 16

49% 34% 24%

484 101 109

29243 4.75 50810 5.02 145623 5.23

21% 31% 13%

77 114 72

53219 6.13 50264 4.83 50096 4.75

25% 40% 48% 40% 25%

115 268 147 152 79

42052 42052 50264 51411 62320

5.29 5.29 4.83 5.4 5.19

48% 45% 26% 31% 50% 29% 30% 35% 36% 52% 57% 20% 46%

545 229 310 319 1315 155 172 161 179 562 258 127 163

49365 47305 66653 44065 49365 51875 49586 44065 49586 44065 48361 60273 53529

4.71 5.27 5.11 5.04 4.71 5.09 4.91 5.04 4.91 5.04 4.97 8.09 5.52

6 22

12

5 13

15 10 14

11

19 10 17

33

16 15 11 16 27 13

Signaling proteins 14-3-3 protein sigma (epithelial cellmarker 1/stratifin) 14-3-3 protein sigma (epithelial cellmarker 1/stratifin) annexin Ig cell cycle protein p38-2G4 homologue HSP 90 betag

1552

c

73d

P31947

Overexpressed 187302 LC-MS/MS

10

32%

216

27873 4.72

c

d

P31947

187302 LC-MS/MS

12

39%

444

27873 4.72

4502101 LC-MS/MS 2697005 LC-MS/MS

5 6

13% 14%

200 109

38918 6.57 44127 6.13

20149594 LC-MS/MS

6

8%

94

83554 4.97

c c

c

74

68 40

P04083 Q9UQ80

20

P08238


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Subcellular Proteome Study of dsRNA-Stimulated Keratinocytes Table 1. Continued

protein name

myosin light polypeptide 6 HSP 90 betag myosin heavy chain 9 (C-term. frag.)g myosin heavy chain 9 (C-term. frag.)g HSP 90 betag HSP27 (heat shock protein beta-1) myosin heavy chain 9 (C-term. frag.)g G protein beta subunit (frag.) PP2A, subunit A, PR65-alpha isoformg heat shock 70 kDa protein 1Ag

access no. fold spot difference number Swiss-Prot NCBInr c


19.2 14.5

93 22f 19

P60660 P08238 P35579

48146145 LC-MS/MS 20149594 MALDI 12667788 LC-MS/MS

2 15/38 9

9.0

18

P35579

12667788 MALDI

8.8 8.5

21 92

P08238 P04792

6.8

17

2.8 2.5 2.5

theoretical MW

pI

14

19% 22% 5%

98 88 197

17090 4.56 83554 4.97 227646 5.5

22/35

13

15%

88

227646 5.5

20149594 MALDI 4504517 MALDI

20/48 6/18

14 9

30% 29%

109 71

83554 4.97 22826 5.98

P35579

189036 MALDI

23/44

16

24%

109

145623 5.23

64 25

P62873 P30153

306785 LC-MS/MS 178663 LC-MS/MS

18% 20%

183 313

38061 5.75 66039 5

23f

P08107

18%

71

78018 5.97

62089222 MALDI

6 12 11/27

35

Protein destination 26S proteasome ATPase subunit 2 26S proteasome non-ATPase regulatory subunit 11 heat shock protein 60 heat shock protein 60 peptidase (mitochondrial processing) alpha 26S proteasome subunit 11 heat shock 70 kDa protein 5g HSP 90 betag HSP 90 betag HSP 90 betag heat shock 70 kDa protein 1Ag

c

48

P35998


9

20%

342

49002 5.71

c

39

O00231

28872725 LC-MS/MS

6

13%

92

47646 6.08

c

28 34 36

P10809 P10809 Q10713

31542947 LC-MS/MS 31542947 LC-MS/MS 24308013 LC-MS/MS

15 15 5

24% 29% 8%

270 429 147

61187 5.7 61187 5.7 58729 6.45

56 24 20 22f 21 23f

Q9UNM6 P11021 P08238 P08238 P08238 P08107

3746882 2506545 20149594 20149594 20149594 62089222

4 13 6 15/38 20/48 11/27

9% 22% 8% 22% 30% 18%

131 245 94 88 109 71

43188 72492 83554 83554 83554 78018

12% 23%

74 62

34423 5.71 40076 6.09

47%

137

46353 5.32

c c

3.7 3.6 c

19.2 8.8 2.5

LC-MS/MS LC-MS/MS LC-MS/MS MALDI MALDI MALDI

14 14 35

5.53 5.07 4.97 4.97 4.97 5.97

RNA/DNA/Protein synthesis 60S acidic ribosomal protein P0 eukaryotic translation initiation factor 3 subunit 3 eukaryotic translation initiation factor 4A isoform 1 eukaryotic translation initiation factor 4A isoform 1 human elongation factor-1-delta 40S ribosomal protein SA RuvB-2 eukaryotic translation elongation factor 1 beta 60S acidic ribosomal protein P0 SYNCRIP protein ribosome binding protein 1

c

Overexpressed 4506667 LC-MS/MS 4503515 LC-MS/MS

66 60

P05388 O15372

c

38

P60842

4503529 MALDI

c

46

P60842

4503529 LC-MS/MS

9

21%

337

46154 5.32

c

61

P29692

38522 LC-MS/MS

4

20%

124

31316 4.95

6.3 4.0 3.8

52 35 72

P08865 Q9Y265 P24534

20% 23% 13%

120 343 63

31888 4.84 51296 5.49 24919 4.5

3.0 2.6

67 47d

P05388 Q05CK9

4506667 LC-MS/MS 33874520 LC-MS/MS

6 5

31% 12%

111 142

34423 5.71 46942 5.85

A1A5C4

Under-expressed 110611218 LC-MS/MS

22

22%

491

108989 5.45

c

c

1, 2

34234 MALDI-LIFT 4506753 LC-MS/MS 4503477 LC-MS/MS

4 9 16/38

5/36 12 3

12

22

Metabolism S-adenosylhomochysteine hydrolase S-adenosylhomochysteine hydrolase ornithine aminotransferase pyrophosphatase 1 ATP-specific succinyl-CoA synthetase beta subunit puryvate dehydrogenase, beta subunit

c

50

P23526


6

13%

243

48255 5.92

c

49

P23526

9951915 LC-MS/MS

5

11%

90

48255 5.92

6.0 5.3 2.6

51 65 47d

P04181 Q15181 Q9P2R7

1168056 LC-MS/MS 11056044 LC-MS/MS 3766197 LC-MS/MS

5 3 7

11% 13% 14%

148 57 85

48847 6.39 33095 5.54 46732 5.84

2.7

15

P11177

Under-expressed 189754 LC-MS/MS

9

30%

193

39566 6.2

13% 45% 22%

200 86 245

38918 6.57 23569 5.43 72492 5.07

Cell rescue and death annexin Ig glutathione S-transferase P heat shock 70 kDa protein 5g

c

6.4 3.6

68 91 24

P04083 P09211 P11021

Overexpressed 4502101 LC-MS/MS 4504183 MALDI 2506545 LC-MS/MS

5 7/21 13

5

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Table 1. Continued

protein name

plasminogen activator inhibitor 2 PP2A, subunit A, PR65-alpha isoformg



theoretical MW

pI

2.8

58

P05120

189545

LC-MS/MS

10

22%

256

46939 5.46

2.5

25

P30153

178663

LC-MS/MS

12

20%

313

66039 5


12

37%

358

48089 4.95

18

41%

563

56691 5.12

Energy mitochondrial ATP synthase subunit beta (frag.)

4.8

42

Q0QEN7

Other interferon-induced protein with tetratricopeptide repeats 3 (IFIT3)

2.1

27

O14879


a The original MALDI spectra and peak lists used for database searches are provided as supplementary data S1 and S3, Supporting Information. MALDI identifications: matched/unmatched peptides. c The spots were present only in one experimental condition. d More than one protein was identified from the spot. e P63261/14501887 also possible. f Mascot search done with peptide tolerance maximum (100 ppm. g Proteins having more than one biological process. b

the cytoplasmic fraction and one (σ) in the mitochondrial fraction after dsRNA stimulation. Protein Destination. Proteins in this category are involved in protein folding, stabilization, modification and degradation. In the present study several heat shock proteins (HSPs) including HSP90R and β, HSP70, HSP60, and HSP27 were more abundant after dsRNA stimulation. Under normal conditions they work as molecular chaperones, assisting the correct folding and stabilization of proteins, but during cell stress they also regulate apoptosis, inflammatory signaling pathways, and antigen presentation. After stimulation, HSP90 was found in several spots in both cytoplasmic and mitochondrial 2-DE gels, whereas the expression of HSP27, HSP60, and HSP70 was increased only in mitochondria. The expression profiles of HSP90 and HSP27 in mitochondrial and cytoplasmic fractions were also analyzed by Western blotting (Figure 3B). HSP90 was seen only in the cytoplasmic fraction in untreated HaCaT cells. During dsRNA stimulation or VSV/EMCV-virus infection, the protein level of HSP90 increased in the cytoplasm, and mitochondrial translocation of HSP90 was clearly observed. In addition, Western blot analysis reveals that HSP27 was localized both in cytoplasm and mitochondria in untreated cells, and the increased expression was seen only in the mitochondrial cell fraction (Figure 3B). The ubiquitin-26S proteosome-dependent proteolytic pathway has a major role in protein degradation, and is also involved in a variety of cellular processes, such as apoptosis and modulation of immune response.20 We identified five clearly overexpressed protein spots (three in mitochondria and two in cytoplasm) as subunits of 26S proteosome. Interestingly, 26S proteosome subunits 2 and 11 were less abundant in cytoplasmic fraction and more abundant in mitochondria, indicating that the subunits translocate from the cytoplasm to the mitochondria during dsRNA stimulation. RNA/DNA/Protein Synthesis. The identified proteins in this category include ribosomal proteins and eukaryotic translation initiation factors. Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family form a structurally diverse group of RNA binding proteins that shuttle between the nucleus and the cytoplasm having various roles in mRNA export, cytoplasmic mRNA localization, translational regulation, and cytoplasmic mRNA degradation.21 In this study, hnRNP A/B, hnRNP K and hnRNP R were clearly overexpressed in the 1554


cytoplasmic fraction suggesting translocation of these proteins from the nucleus to the cytosol upon dsRNA stimulation. In addition, the expression of SYNCRIP, a cytoplasmic counterpart of hnRNP R, was increased both in the cytoplasm and in the mitochondrial fraction. In contrast, hnRNP F was downexpressed in the cytosol after dsRNA stimulation. Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into 80S ribosome at the initiation codon of mRNA. We identified several overexpressed eIFs in the cytoplasmic fraction (eIF3S1, eIF3S4, eIF2S1), and surprisingly, also in the mitochondrial fraction (eIF4A, eIF3S3, eIF1). The expression profile of eIF4A was also studied by Western blotting. This analysis strengthens the data from 2Danalysis, showing that eIF4A expression was increased during viral infection in the mitochondrial fraction but not in the cytoplasm (Figure 3C). In addition, the expression level of 40S ribosomal protein SA and 60S acidic ribosomal protein P0 was decreased in the cytoplasmic fraction and increased in the mitochondrial fraction, indicating that these proteins translocate from the cytoplasm to the mitochondria during dsRNA stimulation. These results suggest that dsRNA stimulation has a strong influence on the cellular translation initiation factors affecting protein synthesis. IFN-Inducible Proteins. Overexpression of IFN-stimulated genes during viral infection is essential to the activation of antiviral response. Surprisingly, we found only two IFNinducible proteins in our 2-DE analysis, namely IFIT3 and IRF6. To confirm normal IFN production in response to viral dsRNA in HaCaT cells, we measured type III IFN, IL-29 (also known as IFN-γ1), mRNA expression upon dsRNA stimulation. Transfection of polyI:C into the cells strongly activated IL-29 gene expression already at 2 h after stimulation (Figure 4A). Similarly, significant secretion of IL-29 protein was detected in the growth medium of polyI:C-transfected cells 6 h post stimulation (Figure 4B). We further analyzed the IFN response by determining the expression levels of two well-known IFN-inducible proteins, IFN-induced GTP-binding protein MxA and IFN-induced 15 kDa protein (ISG15) using Western blot analysis. dsRNA stimulation clearly induced the expression of these IFNinducible proteins (Figure 4C). The results show that HaCaT cells have normal IFN production in response to cytoplasmic

research articles

Subcellular Proteome Study of dsRNA-Stimulated Keratinocytes

Table 2. Identification Results from the Differentially Expressed Protein Spots in the Cytoplasm of dsRNA Stimulated Human Keratinocytesa

protein name



theoretical MW

pI

Structural proteins actin beta-spectrin (C-term. frag.) calponin 3 F-actin capping protein beta subunit filamin A filamin B lamin A/C (N-term frag.) lamin A/C (N-term. frag.) lamin B1 (N-term. frag.) tubulin, beta chain (frag.) tropomodulin 3 tropomyosin 4 alpha II spectrin lamin A/C (N-term frag.) lamin B2 (N-term. frag.) cytokeratin 18 (N-term frag.) cytokeratin 18 CAPZB (F-actin capping protein beta) cytokeratin 13 tubulin, beta 2 actin actin F-actin-capping protein subunit alpha cytokeratin 19 tubulin, beta chain cytokeratin 13

142 124d 151 159

P60709e Q01082 Q15417 P47756

Overexpressed 4501885e LC-MS/MS 338443 LC-MS/MS 4502923 LC-MS/MS 4826659 LC-MS/MS

8.6 6.9 5.3 4.7

123d 123d 169d 172 168 139d 140 155 122 171f 170 173

P21333 O75369 Q5TCJ4 Q5TCJ4 P20700 P07437 Q9NYL9 P67936 Q13813 Q5TCJ4 Q03252 P05783

53791219 38257363 55957496 55957496 5031877 18088719 6934244 4507651 1805280 55957496 27436951 30311

6.1 4.4

105 119

4.0 4.0 3.0 2.9 2.6 2.2 2.1 2.1

c c c c

c c c c c c c c

12 14 7 10

40% 5% 20% 30%

392 155 132 296

42052 275259 36562 30952

5.29 5.41 5.69 5.69

LC-MS/MS LC-MS/MS LC-MS/MS MALDI MALDI LC-MS/MS LC-MS/MS LC-MS/MS MALDI MALDI MALDI MALDI

20 8 12 13 19 10 3 19 50/38 19/31 22/26 17/83

30 20 23 11

8% 3% 20% 29% 27% 22% 9% 52% 20% 27% 29% 26%

365 120 516 109 131 226 133 575 202 107 161 92

280096 280188 53219 53219 66653 50096 39727 28619 285689 53219 67762 47305

5.7 5.49 6.13 6.13 5.11 4.75 5.08 4.67 5.22 6.13 5.29 5.27

P05783 P47756

Under-expressed 30311 MALDI 19352984 LC-MS/MS

17/11

16 4

42% 22%

217 83

47305 5.27 21264 7.93

99d 99d 113 104d 114d

P13646 Q13885 P60709e P60709e P47755

118835468 4507729 4501885e 4501885e 433308

MALDI MALDI MALDI MALDI MALDI

19/45 12/45 14/19 19/34 8/43

10 18 19 16 15

50% 30% 41% 49% 31%

137 80 160 119 73

49898 50274 42052 42052 32955

112 100d 100d

P08727 P07437 P13646

90111766 5174735 6016411

MALDI MALDI MALDI

30/39 29/45 13/45

10 15 11

75% 54% 34%

337 213 68

44065 5.04 50255 4.79 49898 4.91

10 10 6

39% 41% 20%

146 249 94

28082 4.76 26912 4.92 27873 4.72

4.91 4.78 5.29 5.29 5.58

Signaling proteins 14-3-3 protein beta 14-3-3 protein epsilon 14-3-3 protein sigma (epithelial cellmarker 1/stratifin) 14-3-3 protein sigma (epithelial cellmarker 1/stratifin) 14-3-3 protein zeta/delta annexin A4g COP9 signalosome subunit 6 HSP 90 alphag HSP 90 alphag HSP 90 alpha (N-term. frag.)g HSP 90 beta (N-term. frag.)g nitrilase homologue 2 p68 TRK-T3 (N-term. frag.) ras GTPase-activating protein-binding protein (C-term. frag.) serine/threonine-protein kinase PAK2 (N-term. frag)g S-phase kinase-associated protein 1 14-3-3 protein theta annexin A7 14-3-3 protein epsilon 14-3-3 protein beta 14-3-3 protein eta

c

167d 167d 163

P31946 P62258 P31947

c

164

P31947

c

167d 160d 154 124d 125 145 146 161 178 144

c

c

c c

c c c c c c c c c

4.6 4.5 4.1 2.0 2.0

Overexpressed 67464627 LC-MS/MS 67464424 LC-MS/MS 187302 LC-MS/MS 187302

LC-MS/MS

8

28%

135

27873 4.72

P63104 P09525 Q7L5N1 P07900 P07900 P07900 P08238 Q9NQR4 P04629 Q13283

52000887 1703319 34147637 83699649 83699649 83699649 20149594 9910460 1006668 5031703

LC-MS/MS LC-MS/MS MALDI LC-MS/MS MALDI MALDI MALDI LC-MS/MS LC-MS/MS LC-MS/MS

6 5 9/21 4 22/38 8/18 9/11 11 6 8

28% 16% 30% 4% 26% 14% 10% 39% 5% 15%

111 273 98 77 121 73 96 208 186 158

27745 36088 36482 98652 98652 98652 83554 30988 66323 52189

169d

Q13177

32483399

LC-MS/MS

10

15%

359

58291 5.69

174

P63208

1017813

LC-MS/MS

4

14%

85

17432 4.41

165 137 166 179d 179d

P27348 P20073 P62258 P31946 Q04917

71042776 55584155 67464424 67464627 83754683

MALDI LC-MS/MS MALDI LC-MS/MS LC-MS/MS

21/79 16 22/73 7 2

51% 26% 60% 28% 9%

165 187 127 192 70

29405 52991 26912 28082 28459

5.17 5.52 4.92 4.76 4.76

38% 4% 26% 14% 10%

339 77 121 73 96

53088 98652 98652 98652 83554

5.58 5.07 5.07 5.07 4.97

21 8 13 14

10 7

4.73 5.84 5.47 5.07 5.07 5.07 4.97 6.82 5.85 5.36

Protein destination CNDP dipeptidase 2 HSP 90 alphag HSP 90 alphag HSP 90 alpha (N-term. frag.)g HSP 90 beta (N-term. frag.)g

c c c c c

129 124d 125 145 146

Q96KP4 P07900 P07900 P07900 P08238

Overexpressed 8922699 LC-MS/MS 83699649 LC-MS/MS 83699649 MALDI 83699649 MALDI 20149594 MALDI

19 4 22/38 8/18 9/11

8 13 14


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¨ hman et al. O

Table 2. Continued

protein name

proteasome 26S ATPase subunit 3 isoform 2 serpin B8 T-complex protein 1 subunit gamma ubiquitin carboxyl-terminated esterase L3 (UCH-L3) proteasome subunit alpha type-1 proteasome activator subunit 3 protein phosphatase methylesterase 1 ubiquitin-activating enzyme 5 isoform 1 26S proteasome subunit 11 cathepsin D (heavy chain-fragment) cathepsin D (heavy chain-fragment) 26S proteasome ATPase subunit 2



theoretical MW

pI

c

132

B2R8V1

109106425

LC-MS/MS

22

41%

373

45507 5.39

c c

133 127d

P50452 P49368

1709895 671527

LC-MS/MS LC-MS/MS

8 8

16% 13%

134 95

43328 5.43 60862 6.23

c

167d

P15374

5174741

LC-MS/MS

8

25%

162

26337 4.84

162

P25786

190447

LC-MS/MS

10

33%

327

30493 6.51

115 108d

P61289 Q9Y570

Under-expressed 30410796 MALDI 7706645 LC-MS/MS

7/24 8

35% 20%

91 287

31038 5.79 42687 5.67

103

Q9GZZ9

13376212

LC-MS/MS

4

10%

153

45291 4.79

4.8 3.0

109 117

O00231 P07339

20978543 4503143

LC-MS/MS LC-MS/MS

25 8

55% 18%

863 103

47719 6.08 45037 6.1

2.9

116

P07339

4503143

LC-MS/MS

17%

110

45037 6.1

2.0

106

P35998

4506209

MALDI

46%

154

49002 5.71

2.7

c c c

8

9 17/31

13

RNA/DNA/Protein synthesis eukaryotic translation initiation factor 3, subunit 4 delta eukaryotic translation initiation factor, subunit 1 alpha heterogeneous nuclear ribonucleoprotein A/B heterogeneous nuclear ribonucleoprotein R (frag.) eukaryotic translation initiation factor 2, subunit 1 alpha heterogeneous nuclear ribonucleoprotein K SYNCRIP protein SYNCRIP protein purine-rich element binding protein B Under-expressed lysyl-tRNA synthetase lysyl-tRNA synthetase TAR DNA binding protein heterogeneous nuclear ribonucleoprotein F eukaryotic translation initiation factor 4A isoform 1 40S ribosomal protein SA 60S acidic ribosomal protein P0 eukaryotic translation initiation factor 5A

c

158

O75821


9

24%

272

35874 5.87

c

156

O75822

83281438

LC-MS/MS

8

32%

254

29159 4.72

c

143

Q99729

55956919

LC-MS/MS

3

11%

91

36059 6.49

c

128

O43390

5031755

LC-MS/MS

4

6%

164

71184 8.23

c

d

P05198

4758256

LC-MS/MS

5

14%

122

36374 5.02

23%

369

42009 5.43

47% 26% 22%

166 198 140

46942 5.85 46942 5.85 33392 5.35

114 112 240 120

68461 68461 45053 45985

152

c

d

138

Q5T6W2

55958547

LC-MS/MS

10

c

136 135 149d

Q05CK9 Q05CK9 Q96QR8

33874520 33874520 15147219

MALDI LC-MS/MS LC-MS/MS

19/72 11 8

97 96 108d 104d

Q15046 Q15046 Q13148 P52597

5031815 5031815 6678271 4826760

LC-MS/MS LC-MS/MS LC-MS/MS MALDI

16 12 7 19/34

14

18% 14% 18% 48%

2.9

d

104

P60842

4503529

MALDI

17/34

15

41%

91

46353 5.32

2.8 2.6 2.2

110 114d 121

P08865 P05388 P63241

9845502 12654583 4503545

MALDI MALDI LC-MS/MS

13/36 7/43 7

15 15

48% 35% 30%

168 88 182

32947 4.79 34424 5.42 17049 5.08


3

11%

95

26975 9.65

c c

c c c

2.9

16

5.94 5.94 5.85 5.38

Metabolism deoxyuridine 5′-triphosphate nucleotidohydrolase (frag.) deoxyuridine 5′-triphosphate nucleotidohydrolase, isoform 2 PAP-inositol-1,4-phosphate S-adenosylmonochysteine hydrolase spermine synthase transaldolase 1 transaldolase 1 (C-term. frag.)

c

176

P33316

c

177

P33316-2

4503423

LC-MS/MS

9

66%

440

17748 6.15

c

141 138d

O95861 P23526

6688197 9951915

LC-MS/MS LC-MS/MS

6 3

19% 6%

90 157

33743 5.46 48255 5.92

139d 150 149d

P52788 P37837 P37837

791051 5803187 5803187

LC-MS/MS LC-MS/MS LC-MS/MS

8 9 8

20% 25% 22%

132 173 145

41852 4.87 37688 6.36 37688 6.36

spermine synthase dihydrolipoamide dehydrogenase-binding protein

c

111d 102

P52788 O00330

Under-expressed 791051 LC-MS/MS 2316040 LC-MS/MS

4 7

9% 13%

55 72

41852 4.87 54337 8.61

16% 48%

273 129

36088 5.84 26795 5.15

15%

359

58291 5.69

59%

134

22826 5.98

c c c c

c

Cell rescue and death annexin A4g EF-hand domain-containing protein D2 (Swiprosin-1) serine/threonine-protein kinase PAK2 (N-term. frag)g HSP27 (heat shock protein beta-1)

1556

c

160d 157

P09525 Q96C19

Overexpressed 1703319 LC-MS/MS 20149675 MALDI

c

169d

Q13177

32483399

120

Q96EI7

4504517

c

2.5


LC-MS/MS MALDI

5 12/22

4

10 13/50

19

research articles

Subcellular Proteome Study of dsRNA-Stimulated Keratinocytes Table 2. Continued

protein name



theoretical MW

pI

Transport adaptor-related prot. complex2, beta1 subunit (C-term. frag.) archain (C-term frag.) Ras-related protein Rab-11A Sec1 family domain-containing protein translin-associated factor X Under-expressed arsenical pump-driving ATPase DDX19-like protein


c

147

P63010

c

153 175 126

P48444 P62491 Q8WVM8

773575 4758984 5138928

MALDI LC-MS/MS LC-MS/MS

9/29 7 5

c c

8 15

8%

348

104553 5.22

25% 28% 7%

83 215 177

53375 5.46 24492 6.12 72379 5.89

c

160d

Q99598

5174731

LC-MS/MS

6

21%

230

33206 6.1

c

111d 98

O43681 Q9NUU7

6647417 8922886

LC-MS/MS LC-MS/MS

7 4

21% 8%

63 78

39224 4.81 54397 6.19

Q14554

Overexpressed 1710248 MALDI

30%

68

46512 4.95

12%

92

53666 5.18

c

Redox protein disulfide isomerase-related protein 5

c

131

9/51

36

Transcription factor interferon regulatory factor 6 (IRF6, frag.)

c

148

O14896



5

Other echinoderm microtubule associated protein like 2 plasminogen activator inhibitor (C-term. frag.) protein phosphatase 1, regulatory subunit 7 annexin A8 BSCv mitochondrial inner membrane protein (mitofilin) mutS homologue 2 peflin

c

127d

O95834

5

9%

121

71603 5.86

c

152d

P05120

189545

LC-MS/MS

10

17%

174

46939 5.46

c

130

Q15435

4506013

LC-MS/MS

7

19%

365

41653 4.84

Under-expressed 60821642 MALDI 9836652 LC-MS/MS 48145703 LC-MS/MS

14/43 10 11

44% 22% 12%

81 246 132

37112 5.56 47887 5.78 84027 6.08

19% 17%

205 168

105418 5.58 30646 6.1

2.6 c c

2.6 2.6

114d 107 95

P13928 Q9HDC9 Q16891

94 118

P43246 Q9UBV8

4557761 6912582

LC-MS/MS LC-MS/MS

22 7

17

a The original MALDI spectra and peak lists used for database searches are provided as supplementary data S2 and S3, Supporting Information. MALDI identifications: matched/unmatched peptides. c The spots were present only in one experimental condition. d More than one protein was identified from the spot. e P63261/14501887 also possible. f Mascot search done with peptide tolerance maximum ( 100 ppm. g Proteins having more than one biological process. b

dsRNA stimulation despite that we identified only two known IFN-inducible proteins in our proteome analysis. Cytokeratin-18 Is Cleaved during Virus Infection and Fragments Are Localized onto Mitochondria. Our 2-DE analysis revealed that numerous cytokeratins were cleaved during dsRNA stimulation (Figure 1B and Tables 1 and 2). We selected one member of cytokeratins, cytokeratin-18, for more detailed studies. HaCaT cells were treated with Lipofectamine as control, transfected with polyI:C or infected with VSV or EMCV for 18 h. Cells were fractionated into mitochondrial and cytoplasmic fractions and examined by Western blot analysis using a specific cytokeratin-18 antibody. In the control cells, cytokeratin-18 was detected as one band with estimated molecular weight of 45 kDa, which corresponds to the full-length protein (Figure 5A). After polyI:C transfection, cytokeratin was clearly fragmented into approximately 25 kDa-fragments. Similar degradation of cytokeratin-18 was seen in EMCV- and VSVinfected keratinocytes. Unexpectedly, cytokeratins were almost exclusively found in the mitochondrial fraction in these experiments. Therefore, to confirm the subcellular localization of cytokeratin-18 during dsRNA stimulation we performed confocal microscopy (Figure 5B). In control cells, immunostaining analysis revealed a filamentous distribution of cytokeratin-18 in cytoplasm. A clear accumulation of mitochondria into surrounding the nucleus can be detected during dsRNA stimulation. And importantly, cytokeratin network was totally

disintegrated and accumulated into same area with mitochondria. Thus, our results show that dsRNA stimulation results in the rearrangement of cytokeratins in keratinocytes. A similar response was also detected in A549 lung epithelial cells that were transfected with polyI:C (Figure 5C). Forced Expression of RIG-I and MAVS Induces Cytokeratin18 Fragmentation. It has been previously shown that overexpression of RIG-I or MAVS initiates antiviral response by activating IFN production.10,14,22 To determine whether these molecules also mediate signaling pathways that lead to cytokeratin fragmentation, we transfected HaCaT cells with full length RIG-I, with a constitutively active form of RIG-I, ∆RIGI, and with MAVS. As shown in Figure 5D, RIG-I and ∆RIG-I expression evidently induces cytokeratin-18 fragmentation. Similarly, overexpression of MAVS also activates the cleavage of cytokeratin. In contrast, overexpression of TLR3 did not lead to cytokeratin-18 fragmentation. In conclusion, our results show that activation of RIG-I/MAVS signaling pathway results in cytokeratin fragmentation. Cytokeratin-18 Fragmentation Is Dependent on Caspase Activation. The cytokeratin network is known to act as an early target for caspase cleavage during apoptosis.23 In addition, cytolasmic polyI:C has recently been shown to activate caspase-3 and apoptosis in HaCaT cells.8 The proteolytic processing of pro-caspase-3 into active caspase-3 was observed as early as 4 h after transfection (Figure 6A). At the same time point, Journal of Proteome Research • Vol. 9, No. 3, 2010 1557

research articles

¨ hman et al. O

Figure 1. Effect of activation of cytosolic dsRNA recognition pathway on subcellular proteomes of human keratinocytes. HaCaT cells were left treated with Lipofectamine as control or transfected with a mimic of dsRNA, polyI:C, for 18 h, after which the mitochondrial and cytosolic fractions were prepared. (A) Purity of mitochondrial fraction was determined with specific mitochondrial marker proteins, MAVS and VDAC. mt ) mitochondrial fraction, cyt ) cytoplasmic fraction. (B) Fractionated proteins were separated using 11-cm pI 4-7 linear IPG strips in the first dimension and by 8-16% gradient SDS-PAGE in the second dimension. Gels were stained with SYPRO Ruby protein stain and analyzed using ImageMaster. A total of 239 protein spots were reproducibly differentially expressed after dsRNA stimulation (marked as arrows). Numbers indicate the successfully identified protein spots. The under-expressed protein spots are marked to the 2-DE images from control cells and the overexpressed protein spots are marked in the 2-DE images of polyI:C-transfected cells. The boxed areas are shown enlarged in Figure 7. The identification results are shown in Tables 1 and 2.

cleaved cytokeratin-18 fragment was observed (Figure 6B). This indicates that dsRNA induces caspase-dependent cytokeratin cleavage in human keratinocytes. To further investigate whether caspase activation is required for cytokeratin-18 fragmentation, we pretreated HaCaT cells with the general caspase inhibitor z-VAD before polyI:C transfection. Pretreatment of cells with z-VAD totally inhibited the proteolytic processing of procaspase-3 and fragmentation of cytokeratin-18 in response to dsRNA stimulation (Figure 6C). These results suggest that apoptosis-related caspases are involved in disruption of the cytokeratin network during viral infection in human keratinocytes. Virus Infection Increases the Level of 14-3-3 Adapter Proteins Both in Mitochondrial and Cytoplasmic Fractions. 14-3-3 proteins are involved in several cellular events, but the role of 14-3-3 proteins in viral infection has not been elucidated. A major finding of our 2-DE analysis was that multiple 14-3-3 proteins are more abundant upon dsRNA stimulation in keratinocytes. A cluster of proteins localizing in the pI 4-side of mitochondrial and cytoplasmic 2-DE-gels were identified as 14-3-3 protein family members (Figure 1, selected areas). 1558


Protein identities are pointed out in Figure 7A. We further analyzed the expression levels of 14-3-3 proteins by Western blot with general anti-14-3-3 protein antibody. In addition, one member of the 14-3-3 protein family, the 14-3-3σ (also known as stratifin) was selected for more detailed analysis as it is expressed only by epithelial cells. HaCaT cells were transfected with polyI:C for different time periods, and total cell lysates were prepared. The protein amount of 14-3-3 was induced already 4 h after transfection, and a weak but distinct lower band in 14-3-3 became visible after 6 h transfection (Figure 7B). Similar result was seen with 14-3-3σ isoform. Next, we characterized the subcellular localization of 14-3-3 during dsRNA stimulation. HaCaT cells were stimulated with dsRNA or infected with VSV or EMCV for 18 h, fractionated into mitochondrial and cytoplasmic fractions, and analyzed with Western blot. In control cells 14-3-3 proteins were localized mainly in the cytoplasm, but interestingly, the level of 14-3-3 protein clearly increased both in cytoplasm and mitochondria during stimulation (Figure 7C). In addition, the lower band of 14-3-3 was seen in both mitochondrial and cytoplasmic frac-


research articles

Figure 2. Functional classification of the differentially expressed proteins in dsRNA stimulated human keratinocytes. The identified proteins were classified based on different biological processes using the UniProt Knowledgebase (Swiss-Prot/TrEMBL) and published literature. The identified proteins were mostly involved in four different biological classes: structural proteins, signaling proteins, protein destination, and RNA/DNA/Protein synthesis.

tions. A recent report has shown that several C-terminal amino acids are removed from 14-3-3 during apoptosis in a partially caspase-dependent manner.24 Therefore, we next determined if 14-3-3 fragmentation during dsRNA stimulation is caspasedependent. HaCaT cells were treated with a general caspase inhibitor z-VAD-fmk before polyI:C transfection, after which the cells were fractioned into mitochondrial and cytoplasmic fractions and analyzed by Western blotting. Interestingly, pretreatment of HaCaT cells with z-VAD totally inhibited the 14-3-3 truncation in response to dsRNA stimulation (Figure 7D). Thus, the results show that caspases are also involved in the regulation of 14-3-3 proteins during viral infection. Viral dsRNA Stimulates Phosphorylation of 14-3-3 Binding Proteins. More than 300 cellular proteins have been reported to interact with 14-3-3 proteins so far. 14-3-3 binding to most of its targets has been shown to depend on the phosphorylation of the target protein. Two different phospho-Ser-containing motifs are found within nearly all known 14-3-3 binding proteins.25 To determine whether dsRNA stimulation induces 14-3-3 target protein phosphorylation, we used an antibody that recognizes the phosphorylated peptide motif of 14-3-3 target protein. HaCaT cells were transfected with polyI:C for different time periods, and total cell lysates were analyzed. PolyI:C transfection clearly induced the phosphorylation of 14-3-3 target proteins at 4 h post-transfection (Figure 7E). Importantly, the amount of 14-3-3 proteins increased at the same time point (Figure 7B). Phosphorylation is usually a dynamic event, and also here the amount of phosphorylation began to decrease at 6 h time point and totally disappeared 15 h after transfection. Taken together, our results show that viral infection activates 14-3-3-mediated signaling pathways in human keratinocytes.

Discussion In this report, we have used subcellular 2-DE-based proteomics to characterize activation of innate immunity in human keratinocytes. Subcellular fractionation and proteomics represent a powerful combination to identify the molecular composition of intracellular organelles and intracellular shuttling events. In addition, this strategy decreases sample complexity in comparison with whole cell proteomic analysis enabling more detailed characterization of the proteomes. However, it is becoming clear that a physical association of various organelles makes it almost impossible to completely separate the organelles and obtain pure fractions, and therefore the possibility of contamination cannot be totally excluded. 2-DE has so far been the most widely used technique in expression proteomics. It suffers from certain limitations, e.g. proteins with extreme pI’s or hydrophobicities are underrepresented or absent in 2-DE gels, and the sensitivity of the methods is not always enough to identify low-abundant proteins. However, one of the great advantages of 2-DE-based proteomics comes from its ability to distinguish between distinct protein isoforms with quite similar molecular weights and pIs as well as protein fragments from their full-length forms. Therefore, the information provided with subcellular 2-DE analysis has an important role in understanding the details of cellular signaling mechanisms. Intermediate filaments (such as keratins, vimentin and lamins), together with actin microfilaments and microtubulins (tubulins), are basic constituents of the cytoskeleton in eukaryotic cells. During apoptosis the cell undergoes dramatic changes in morphology due to a complete reorganization of its cytoplasmic and nuclear skeleton.26 One of the major Journal of Proteome Research • Vol. 9, No. 3, 2010 1559

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Figure 3. Expression level of R/β-tubulin, HSP90, HSP27, and eIF4A protein is increased in mitochondrial fractions upon viral infection. HaCaT cells were left untreated, transfected with polyI:C (t-pI:C) or infected with VSV or EMCV (MOI of 2) for 18 h. After this the cells were fractioned into mitochondrial and cytoplasmic fractions, and (A) R/β-tubulin, (B) HSP90 and HSP27, and (C) eIF4A protein expression was analyzed by Western blotting. (D) Equal protein loading in each sample was determined by silver staining.

findings of our analysis is a significant subcellular rearrangement of cytoskeletal proteins during dsRNA stimulation. We show that in control keratinocytes several structural proteins, such as cytokeratins, actin and tubulins, are in a full-length form. Data from 2-DE and Western blotting analysis suggested that cytokeratins almost exclusively occur in the mitochondrial fraction. However, cytokeratins are known to interact with many organelles, including mitochondria,27 and it is possible that cytokeratins accumulate onto mitochondrial fraction during cell fractionation. Therefore, cytoplasmic localization of keratins in control cells was confirmed by confocal microscopy. In contrast, fragmented structural proteins were mainly concentrated to mitochondrial fraction upon dsRNA stimulation and viral infection. Type I cytokeratins have previously been demonstrated to undergo degradation during apoptosis.28 This apoptosis-associated keratin cleavage finally results in the total collapse of the cytokeratin filaments into large aggregates and coincides with loss of intracellular contacts and detachment of cells from their substrates.29,30 In our experiments, cytokeratin-18 fragmentation during dsRNA stimulation was dependent on caspase activity and was associated with apoptosis 1560


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Figure 4. Viral dsRNA stimulation induces the expression of IFNs and IFN-inducible proteins. HaCaT cells were transfected with polyI:C for the time periods indicated. (A) Total RNA was extracted, cDNA was synthesized, and IL-29 mRNA expression was analyzed. The data are represented as relative units (RU), which is a fold change in gene expression that is normalized to an endogenous reference gene and is relative to NTC-calibrator. (B) Secreted IL-29 was determined from cell culture supernatants by ELISA. The experiments were done twice with similar results. (C) HaCaT cells were transfected with polyI:C for 18 h and cell lysates were analyzed by Western blotting with specific anti-MxA and anti-ISG15 antibodies.

of the cells. The mitochondrial translocation of cleaved cytokeratins during dsRNA stimulation has not previously been shown. Recently, we have shown that influenza A virus infection of human primary macrophages leads to significant mitochondrial translocation and caspase-dependent cleavage of actin microfilaments, and it was suggested that actin network interacts with mitochondria to regulate both antiviral and cell death signals.13 Therefore, it is possible that reorganization of cytokeratin network is also involved in the regulation of cell death during viral infection. The cytokeratin network has been proposed to have a regulatory function in apoptosis by anchoring of the effector molecules of the death receptor pathways,


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Figure 6. Cytokeratin fragmentation during dsRNA stimulation is dependent on caspase activity. (A) and (B) HaCaT keratinocytes were transfected with polyI:C for the time periods indicated, after which whole cell lysates were prepared and analyzed by Western blotting with caspase-3 p19/17 or cytokeratin-18 specific antibodies. ns ) nonspecific band. (C) HaCaT cells were left untreated or transfected with polyI:C for 18 h in the presence or absence of 25 µM z-VAD-fmk, and the cells were fractionated into mitochondrial and cytoplasmic fractions. The expression of cytokeratin-18 was analyzed by Western blotting.

Figure 5. Cytokeratins are fragmented, and fragments are translocated onto mitochondria upon viral infection in human keratinocytes. (A) HaCaT cells were left untreated, transfected with polyI:C (t-pI:C) or infected with VSV or EMCV (MOI of 2) for 18 h, after which the cells were fractionated into mitochondrial and cytoplasmic fractions, and cytokeratin-18 protein expression was analyzed by Western blotting. (B) Subcellular distribution of cytokeratin-18 was determined by confocal microscopy. HaCaT cells were transfected with YFP plasmid having mitochondrial targeting sequence (green) followed by stimulation with cytosolic polyI:C for 10 h. The cells were fixed with 4% paraformaldehyde and stained with cytokeratin-18 antibody (red). The colocalization is shown as yellow merge color. (C) A549 epithelial cells were left untreated or transfected with polyI:C (t-pI:C) for 18 h, after which the cells were fractionated into mitochondrial and cytoplasmic fractions, and cytokeratin-18 protein expression was analyzed. (D) HaCaT cells were transfected with full length RIGI, with a constitutively active form of RIG-I (∆RIG-I), and with MAVS. Cell lysates were prepared and cytokeratin-18 expression was studied by Western blotting.

such as DEDD or TRADD.31-33 Therefore it is tempting to speculate that cytokeratin fragmentation and mitochondrial

translocation is the mechanism how these molecules are released from the cytokeratin network resulting in induction of cell death. In addition to cytokeratins, the intermediate filaments nuclear lamins are also cleaved by caspases during apoptosis. A- and B-type lamins are the major components of nuclear lamina and they are key proteins for the maintenance of the nuclear organization. In our experiments, fragments of A- and B-type lamins were highly overexpressed in the cytoplasm, indicating that lamins are cleaved and released into the cytoplasm upon dsRNA stimulation. The cleavage of lamins probably serves to disassemble the lamina during apoptosis and this disassembly may be essential for chromatin condensation.34,35 The 14-3-3 proteins are phospho-serine/threonine binding proteins that can interact with a diverse array of cellular proteins, and thereby participate in a variety of cellular processes including metabolism, protein trafficking, apoptosis, signal transduction, and cell-cycle regulation.36,37 However, the role of 14-3-3 proteins in virus infection has remained uncharacterized. 14-3-3 family consists of seven distinct gene products in human cells (β, γ, , η, σ, t, and ζ). In our experiments, expression of six 14-3-3 isoforms was overexressed in the cytoplasmic fraction after viral dsRNA stimulation. In addition, 14-3-3 isoform σ was found in the mitochondrial proteome after dsRNA stimulation. Moreover, we show that dsRNA clearly induces phosphorylation of 14-3-3 target proteins. These results Journal of Proteome Research • Vol. 9, No. 3, 2010 1561

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Figure 7. Cytosolic dsRNA recognition pathway activates 14-3-3 protein mediated signaling in keratinocytes. (A) Enlarged areas from Figure 1 showing 14-3-3 proteins in mitochodrial and cytoplasmic cell fractions. (B) HaCaT cells were transfected with polyI:C for the time periods indicated and total cell lysates were analyzed by Western blotting with general 14-3-3 antibody and 14-3-3σ specific antibody. (C) HaCaT cells were transfected with polyI:C or infected with VSV or EMCV (MOI of 2) for 18 h, the cells were fractionated into mitochondrial and cytoplasmic fractions, and 14-3-3 protein expression was analyzed by Western blotting. (D) HaCaT cells were left untreated or transfected with polyI:C for 18 h in the presence or absence of 25 µM z-VAD-fmk, and the cells were fractionated into mitochondrial and cytoplasmic fractions. The expression of 14-3-3s was analyzed. (E) HaCaT cells were transfected with Lipofectamine alone as control or with polyI:C for different time periods, and total cell lysates were analyzed with pSer 14-3-3 binding motif antibody. An equal protein loading was determined by silver staining.

show that viral infection activates 14-3-3-mediated signaling pathways in human keratinocytes. It is known that the 14-3-3 proteins have a major function in the control of apoptosis through interactions with core components of the mitochondrial apoptotic machinery, such as BAD (BCL-2 antagonist of cell death).38 The 14-3-3-BAD interaction keeps BAD in the cytoplasm preventing BAD dimerization with BCL-XL on the mitochondria thereby suppressing apoptosis.39,40 It is likely that the 14-3-3 proteins regulate apoptotic signaling pathways also during virus infection. In addition, 14-3-3 proteins, especially isoform σ, associate with several intermediate filaments, such as keratins.41,42 This phosphorylation-dependent interaction is important in keratin filament organization,41 but also in protein synthesis, cell growth and cell cycle progression.43 1562


Although several 14-3-3 protein targets are known, there is little evidence about how the binding activity of 14-3-3 proteins is regulated. Most of the 14-3-3 isoforms, with the exception of σ isoform which is restricted to epithelial cells, are ubiquitously expressed and they bind to their targets with similar affinity.37 Therefore the binding properties of 14-3-3 proteins have to be regulated differently, for example, through posttranslational modifications. Here we observed 14-3-3 processing during dsRNA stimulation. A very recent study showed that similar 14-3-3 modification during apoptosis was at least partially due to caspase cleavage.24 In our experiments, the general caspase inhibitor z-VAD-fmk fully prevented 14-3-3 processing during dsRNA stimulation, suggesting that 14-3-3s undergo caspase-dependent truncation upon virus infection.

Subcellular Proteome Study of dsRNA-Stimulated Keratinocytes This further highlights the regulatory role of caspases in viral response and emphasizes the role of post-translational modifications during innate immune response. In conclusion, we provide novel information how the cytoskeleton network is disrupted in a caspase-dependent manner upon dsRNA stimulation and virus infection in human keratinocytes. These results indicate that RIG-I/MDA-5 RNA recognition pathway has an important function, in addition to activating antiviral response, in triggering cell death in virus-infected cells. Also, our results suggest a role for 14-3-3 proteins in innate immune response.

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Acknowledgment. We thank Niina Ahonen for the assistance with 2-DE analysis and Rauna Tanskanen for the help with immunofluorescence staining and confocal microscopy. The YFP plasmid having the mitochondrial targeting sequence was a kind gift from Dr. Tero Ahola. Financial support: This work was supported by the Academy of Finland (grant 114437), and grants from the Sigrid Juse´lius Foundation, the Lundbeck Foundation and the Aarhus University Research Foundation.

Supporting Information Available: S1: The original MALDI spectra of mitochondrial protein spots. S2: The original MALDI spectra of cytoplasmic protein spots. S3: The original MALDI peak lists used for database searches. S4: A higher resolution figure of Figure 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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