Proteomic Analysis of Human U937 Cell Line Activation Mediated by Haemophilus influenzae Type b P2 Porin and Its Surface-Exposed Loop 7 Valeria Severino,†,# Angela Chambery,*,†,# Mariateresa Vitiello,‡ Marco Cantisani,§,| Stefania Galdiero,§,| Massimiliano Galdiero,‡ Livia Malorni,⊥ Antimo Di Maro,† and Augusto Parente† Department of Life Science, Second University of Naples, Via Vivaldi 43, I-81100 Caserta, Italy, Department of Experimental Medicine, Second University of Naples, Via De Crecchio 7, I-80138 Napoli, Italy, Department of Biological Sciences, University of Naples “Federico II”, Via Mezzocannone 16, I-80134, Napoli, Italy, Institute of Biostructure and Bioimaging, C.N.R., Via Mezzocannone 16, I-80134, Napoli, Italy, and Proteomic and Biomolecular Mass Spectrometry Center, Institute of Food Science and Technology, C.N.R., Via Roma 52 A-C, I-83100 Avellino, Italy Received October 16, 2009
The virulence of Haemophilus influenzae type b (Hib) has been attributed to a variety of potential factors associated with its cell surface, including lipopolysaccharides (LPS) and major outer membrane proteins (OMPs). P2 porin, one of the best-characterized porins in terms of its functional characteristics, is the most abundant OMP in Hib and has also been shown to possess proinflammatory activity. To characterize the role played by bacterial surface components in disease onset and development, the proteomic profiling of human U937 cell line activated by H. influenzae type b P2 porin and its most active surface-exposed loop (L7) was performed by means of two-dimensional electrophoresis and mass spectrometry. The study provided a list of candidate proteins with potential relevance in the host immune and inflammatory response. Most of the differentially expressed proteins are involved in metabolic processes, remodelling of cytoskeleton, stress response and signal transduction pathways. The results constitute the basis for dissecting signal transduction cascades activated by P2 stimulation and gain insights into the molecular events involved in the modulation of pathogen-host cell interactions. Keywords: Haemophilus influenzae type b • proteomic profiling • porin • immune response • pathogen-host interaction
Introduction Haemophilus influenzae type b (Hib) is a pathogenic Gramnegative bacterium responsible for pleiotropic infections, including pneumonia, bronchitis and meningitis, in both children and adults. Hib can be classified into typeable and nontypeable strains depending on the presence or absence of encapsulation, respectively. Since the early 1990s, Hib diseases are preventable due to the availability of highly effective vaccines that dramatically reduced their incidence in the United States and Europe. However, significant health risks remain in developing countries and undervaccinated regions of western societies. Indeed, * To whom correspondence should be addressed: Mailing address: Dipartimento di Scienze della Vita, Seconda Universita` di Napoli, Via Vivaldi 43, I-81100 Caserta, Italy, Phone: +39 0823 274535. Fax: +39 0823 274571. E-mail:
[email protected]. † Department of Life Science, Second University of Naples. # These authors contributed equally to this work. ‡ Department of Experimental Medicine, Second University of Naples. § Department of Biological Sciences, University of Naples “Federico II”. | Institute of Biostructure and Bioimaging, C.N.R. ⊥ Institute of Food Science and Technology, C.N.R.
1050 Journal of Proteome Research 2010, 9, 1050–1062 Published on Web 12/31/2009
according to the World Health Organization, Hib is estimated to be still responsible for near 400 000 deaths per year, and almost all victims are children under the age of five.1 Surprisingly, one of the main obstacles to Hib prevention resides in the information shortage, largely due to the difficulty of diagnosing Hib disease. In addition, high costs of Hib vaccine prevent planning of infant immunization programmes in several developing countries. In this framework, it is undoubted that a better understanding of the molecular mechanisms related to Hib pathogenesis could provide the basis for developing new therapeutic strategies for Hib disease prevention, diagnosis and treatment. Understanding the cellular and molecular events that occur during the interaction of individual pathogenic components with host cells is critical for preventing both infection and the resulting tissue damage. In this regard, bacterial surfaces are of utmost importance when considering the interaction with host cells in the context of pathogenesis and immunity to infections.2 Indeed, several bacterial components, including the outer membrane of Gram-negative bacteria, embedded with 10.1021/pr900931n
2010 American Chemical Society
Proteomic Analysis of Human U937 Cell Line Activation many protein and lipopolysaccharide (LPS) molecules, are involved in the modulation of pathogen-host cell interactions. One of the best characterized components of the outer cell wall of Gram-negative bacteria, in terms of structural and functional properties, is the LPS. In several experimental systems, it has been demonstrated that cellular functional responses to LPS stimulation evoke transcriptional and translational events not restricted to cytokine release as previously believed.3,4 Transcriptomic analysis has been proven to be a powerful tool to unravel mechanisms underlying cells activation by various bacterial stimuli, including LPS.5,6 Nevertheless, this approach often fails to provide a comprehensive understanding of molecules involved in such process, due to the susceptibility of mRNA to degradation and to the discrepancy between mRNA and protein expression levels.5 Over the past few years, proteomic profiling has become a powerful complementary approach for surveying global protein expression changes in cellular response to external stimuli. In particular, there has been a growing interest in proteomic analysis of LPS stimulated cells, focused to unravel the regulatory networks of pathogen-host cell interaction process.5,7,8 Beyond LPS, bacterial porins, being a relevant component of the Gram-negative bacterial outer membrane, play an important role in pathogenesis of bacterial infections by stimulating the release of several cytokines with proinflammatory activity triggering the immunological response.9-11 Nevertheless, the mechanisms by which porins alter host gene transcription are only recently beginning to be studied and appear to be extremely complex. Bacterial porins activate signaling pathways essential for the infection of target cells.12-15 Porin P2 of Hib, one of the best-characterized porins in terms of its functional characteristics, is the most abundant outer membrane protein (OMP) in both typeable and nontypeable Hib. From a structural point of view, similarly to other bacterial porins, P2 folding pattern is constituted by a β-barrel containing 16 antiparallel β-strands crossing the outer membrane. Eight large loops of variable length connect the β-strands on the external surface of the bacterial membrane and eight short turns protrude on the periplasmic membrane side. Recently, it has been demonstrated that synthetic peptides corresponding to loops 5, 6, and 7 can mimic the role of intact P2 by inducing the activation of the mitogen-activated protein kinase (MAPK) cascade, essentially JNK and p38.16 In addition, the ability to induce the release of TNF-alpha and IL-6 has been also demonstrated for these surface-exposed loops. Loop 7 (L7) was found to be the most active peptide in both MAPK activation and TNF-alpha/IL-6 release.16,17 These evidence suggest that the L7 sequence can be considered as a microbial structure that initiate the innate immune response. In the present study, the differential protein expression of Hib P2- and L7-stimulated U937 human monocytic cell line was analyzed by a proteomic approach with the aim to compare the molecular response induced by the whole porin and its L7. In addition, LPS-activated U937 cells were also used for comparative purposes to investigate similarities and differences between P2 and L7 priming and the effects induced by the well-studied LPS response.
Experimental Section Materials. Chemical reagents and TPCK-treated trypsin were from Sigma (Milan, Italy). Immobilized pH gradient (IPG) buffers, IPG strips and electrophoresis apparatus were purchased from Amersham-Biosciences (Milan, Italy). Electro-
research articles phoresis reagents, including acrylamide, N,N′-methylenebisacrylamide, N,N,N′,N′-tetramethylethylenediamine, ammonium persulfate and sodium dodecylsulfate (SDS), were from BioRad (Milan, Italy). All tissue culture reagents were from Invitrogen Corporation (Carlsbad, CA). All other reagents were of analytical grade. Cell Cultures. U937 monocytes (ATCC CRL-1593.2) were grown and differentiated as previously described.14 Briefly, cells were grown at 37 °C in 5% CO2 in RPMI 1640 with HEPES supplemented with 10% heat-inactivated fetal calf serum, glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 U/mL) in 150 cm2 tissue culture flasks. Before treatments, the serum concentration was reduced to 5% for 24 h at 37 °C and then further reduced to zero for at least 10 h. Cells were tested every 2 weeks by a PCR-based detection assay for mycoplasma contamination. Bacteria, Growth Conditions, and Preparation of Hib P2 and LPS. H. influenzae type b (Hib) was obtained from the American Type Culture Collection (ATCC 9795) and grown in CY medium for 18-24 h at 37 °C.18 Bacteria were harvested at the end of the exponential growth phase. P2 porin was isolated and purified from Hib as previously described.17 Briefly, the bacterial envelopes were treated with Triton X-100 buffer for 2 h at 37 °C with rotary shaking, dissolved in 0.1 M sodium phosphate pH 7.2 containing 4% SDS, and applied to an Ultragel ACA34 column equilibrated with 0.25% SDS sodium azide buffer. The protein fraction, identified by measuring the absorption at 280 nm, was extensively dialyzed and analyzed by 12% SDS-PAGE. The protein concentration of the porin preparation was determined according to Lowry et al.19 Possible traces of LPS were revealed on SDS-PAGE gels stained with silver nitrate and by the Limulus amoebocyte lysate assay.20 LPS contamination in the porin preparation was estimated to be about 0.001%, compared with a standard Hib LPS solution. The pore-forming ability of P2 preparation was checked by a functional liposome-swelling assay after incorporation into proteoliposomes.21 Hib LPS was extracted by the phenol-chloroform-petroleum ether method of Galanos et al.22 Peptides Synthesis. Peptide corresponding to L7 was prepared by standard 9-fluorenylmethoxycarbonyl polyamine solid-phase synthesis, using a PSSM8 multispecific peptide synthesizer (Shimadzu Corporation Biotechnology Instruments Department, Kyoto, Japan). Synthesis, purification, and characterization of this peptide have been previously described.16 Cell Stimulation for Proteomic Analysis. U937 cells (30 × 106 cells) were stimulated with P2 Hib porin (5 µg/mL) or L7 of P2 Hib porin (130 nmol/mL). Both untreated U937 cells and LPS-activated (1 µg/mL) U937 were used for comparative purposes. After cell stimulation, incubation was carried out with continuous rotation (4 h, 37 °C) both in the presence and absence of stimuli. Three independent cell cultures preparations were pooled to address biological variation. 2D Sample Preparation. Monolayer cultures of stimulated and untreated U937 cells were harvested and, after three washes in ice-cold PBS, incubated with a solution containing trypsin (0.5 g/L) and EDTA (0.2 g/L). After centrifugation at 2000 rpm for 5 min at 4 °C in a JA14 rotor (Beckman centrifuge GS-15R; Beckman Coulter, Inc., CA), cell pellets were washed three times with PBS and lysed with 60 µL of lysis buffer (40 mM Tris-Cl pH 8.0 containing 8 M urea, 4% CHAPS, 65 mM DTT and 1 mM PMSF). Total proteins were extracted by freeze-thawing several times the cell pellet in liquid nitrogen. Samples were further centrifuged at 17 500g for 15 min at 4 Journal of Proteome Research • Vol. 9, No. 2, 2010 1051
research articles °C, to eliminate cellular debris and sonicated into an ultrasonic bath for 15 min. Samples were then centrifuged at 17 500g for 15 min at 4 °C to eliminate cellular debris. The supernatant was collected and protein concentration determined by the Bradford method, according to manufacturer’s instructions (Biorad, Milan, Italy). Lysates were aliquoted and stored at -80 °C until use. Two-Dimensional Gel Electrophoresis. A total amount of 700 µg of protein per gel was analyzed in triplicate, according to previously reported procedures.23 Samples to be processed by isoelectrofocusing (IEF) were mixed with the rehydration buffer (8 M urea, 0.5% CHAPS, 0.2% DTT, 0.5% IPG ampholytes and 0.002% bromophenol blue) to a final volume of 340 µL. The precast IPG strips (3-10 linear pH gradient, 18 cm long, Amersham Biosciences, Milan, Italy), used for the first dimension, were passively rehydrated and loaded with the sample at room temperature for 12 h under low-viscosity paraffin oil. IEF was then performed using an IPGphor isoelectric focusing cell (Amersham Biosciences, Milan, Italy), according to the following protocol: 500 V for 700 Vh, 1000 V for 1400 Vh, 8000 V for 34 500 Vh. Strips were then equilibrated twice for 15 min with gentle shaking in the equilibration solution (6 M urea, 50 mM Tris-Cl buffer pH 8.8, 30% glycerol, 2% SDS, 0.002% bromophenol blue) containing 1% DTT to reduce disulfide bonds, in the first equilibration step, and 2.5% iodoacetamide to alkylate thiols, in the second. The second-dimension separation was performed in an Ettan DALTsix Electrophoresis Unit on homogeneous polyacrylamide gels (12% T, 1% C). The equilibrated strips were sealed to the top of the vertical gel with agarose solution (0.5% agarose and 0.002% bromophenol blue dissolved in SDS/Tris running buffer) and electrophoresis was carried out at constant power (2.5 W/gel for 30 min followed by 100 W/gel for about 4 h and 30 min) and temperature (20 °C), until the blue dye reached the bottom of the gel. At the end of the electrophoresis, the protein spots were visualized, after washing gels in 50% methanol/10% acetic acid for 2 h, by incubating gels with colloidal Coomassie blue stain (2% phosphoric acid, 10% ammonium sulfate, 20% methanol, 0.1% Coomassie brilliant blue G-250) overnight, followed by three individual 2 h washes with deionized water. Gel Scanning and Image Analysis. Resulting 2D gels were imaged and analyzed for comparison, to provide data on matching spots.23 Gels were scanned using a Molecular Dynamics densitometer, model 375-557 (Amersham Biosciences, Milan, Italy). Gel images were processed for spot detection, background subtraction and matching using ImageMaster 2D Elite 3.1 software (Amersham Biosciences, Milan, Italy). Spot intensity was quantified by calculation of the spot volume after normalization of the image using the total spot volume normalization method according to the manufacturer’s instructions. Briefly, relative intensity (RI ) vi/vt) of each spot was calculated by dividing the volume of the spot by the total volume of the detected spots on the gels, multiplied by the total area of all spots as scaling factor. These were considered differentially expressed when they were either present or absent in comparison with the reference gel, and quantitatively different to corresponding control when their normalized total volume values differed significantly at P e 0.05, based on ANOVA analysis. In addition, only proteins whose differences in spot intensity were consistently observed in two or more experiments were considered for further analyses. Expression intensity ratiostimulated/control values higher than 1.5 (P e 0.05) or smaller than 0.6 (P e 0.05) were set as a threshold indicating 1052
Journal of Proteome Research • Vol. 9, No. 2, 2010
Severino et al. significant changes. Further gel processing procedures were applied as previously reported.23 Protein Identification by MALDI-TOF Mass Spectrometry. Protein identification by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed according to Chambery et al.23 Briefly, selected protein spots were excised from gels and destained by washing twice with 100 µL aliquots of water, performing a further washing step with 50% acetonitrile. The gel pieces were then dried in a SpeedVac Vacuum (Savant Instruments, Holbrook, NY) and rehydrated with 10 µL of 50 mM ammonium bicarbonate. Digestions were performed by adding 5 µL of a 70 ng/ mL TPCK-treated porcine trypsin solution and incubating samples at 37 °C for 3 h. Further amounts of buffer solution without trypsin were added when necessary to keep the gel pieces wet during the digestion. Peptides were extracted in two steps by sequential addition of 1% trifluoroacetic acid (TFA) and then of 2% TFA/50% acetonitrile for 5 min in a sonication bath. The combined supernatants were concentrated in the SpeedVac Vacuum for mass spectrometry (MS) analysis. After in situ tryptic digestion, proteins were identified by peptide mass fingerprint by MALDI-TOF MS. Tryptic peptides were mixed with an equal volume of saturated R-cyano-4-4-hydroxycinnamic acid matrix solution [10 mg/mL in 50% ethanol in water, containing 0.1% TFA] and spotted onto a MALDI-TOF target plate. The droplet was dried at room temperature. Once the liquid was completely evaporated, the sample was loaded into the mass spectrometer and analyzed. Peptide spectra were collected in positive ion reflectron mode on a MALDI-TOF micro MX (Waters Co., Manchester, U.K.), equipped with a pulsed nitrogen laser (λ ) 337 nm). The instrument source voltage was set at 12 kV. The pulse voltage was optimized at 1999 V, and the detector and reflectron voltages were set at 5200 and 2350 V, respectively. Measurements were performed in the mass range m/z 800-3000 with a suppression mass gate set at m/z 500 to prevent detector saturation from matrix cluster peaks and an extraction delay of 600 ns. The instrument was externally calibrated using a tryptic alcohol dehydrogenase digest (Waters Co., Manchester, U.K.) as standard. A mass accuracy near to the nominal (50 ppm) was achieved for each standard. The protonated monoisotopic mass of ACTH peptide (m/z 2465.199) was used as internal lock mass to further improve the peptide mass accuracy. All spectra were processed and analyzed using the MassLynx 4.1 software (Waters Co., Manchester, U.K.). The obtained spectra were used to identify proteins in the Swiss-Prot protein sequence database (release 56.0; 392 667 entries) by using Protein Lynx Global Server 2.3 software. The following searching parameters were used: mass tolerance 100 ppm; allowed number of missed cleavage sites up to 1; cysteine residue modified as carbamidomethyl-cys; minimum number of matched-peptides 3. Immunoblotting. For Western blot analysis, aliquots corresponding to equal amounts (20 µg of proteins) from cell lysates were resolved by 12% SDS-PAGE and transferred onto nitrocellulose membrane (Sartorius, Go¨ttingen, Germany) with an electroblot apparatus (Bio-Rad, Milan, Italy), according to the manufacturer’s instructions. The membrane was blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at 37 °C and washed with PBTS (0.1% Tween-20 in PBS). Subsequently, it was incubated with anti-NDPK (FL-152, sc-28829, Santa Cruz Biotechnology, CA) diluted 1:500 in PBTS for 1 h at 37 °C and then, after washing with PBTS, with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG;
Proteomic Analysis of Human U937 Cell Line Activation
research articles
Figure 1. Representative 2D-PAGE maps obtained from untreated (A) and LPS-stimulated (B) U937 cells. 2D gels were obtained using a pH linear range of 3-10 in the first dimension and 12% SDS-PAGE in the second.
Figure 2. Representative 2D-PAGE maps obtained from P2- (A) and L7-stimulated (B) U937 cells. 2D gels were obtained using a pH linear range of 3-10 in the first dimension and 12% SDS-PAGE in the second.
Sigma, Milan, Italy) diluted 1:10 000 in PBTS for 1 h at 37 °C. Immunoreactive protein bands were visualized by the enhanced chemiluminescence (ECL) detection system (Sigma, Milan, Italy) according to the manufacturer’s instructions. Gene Ontology Analysis. Enriched Gene Ontology (GO) categories under biological process classification were evaluated for differentially expressed proteins following P2, L7 and LPS stimulation by using the WebGestalt software package developed by Bioinformatics Resource Center at Vanderbilt University (http://bioinfo.vanderbilt.edu/webgestalt). The entire human genome was used as reference set by using the hypergeometric test (P < 0.01).24 Pathway Analysis. The pathway analysis for differentially identified proteins was performed using the Ingenuity Pathways Analysis (IPA) software, version 6.3 (Ingenuity Systems, www. ingenuity.com). The lists of differentially expressed proteins were imported into the IPA platform for batch analysis and identification of the canonical pathways that differ between the stimuli as reported.25
Results Total cell proteins were extracted from untreated and P2-, L7-, and LPS-stimulated U937 cells and separated by 2D-PAGE using IPG strips with a linear 3-10 pH gradient as described in the Experimental Section.
For a reliable analysis of protein expression, 2D gel maps of each cell line were performed and analyzed in triplicate. Representative 2D-PAGE maps obtained from untreated U937 and LPS-stimulated cells are reported in Figure 1, while 2D maps of proteins derived from P2- and L7-stimulated cells are reported in Figure 2. An overview of the differentially expressed proteins was obtained by comparing the 2D gels of the three stimulated cells to the untreated U937 map. Selected protein spots showing differential expression were excised from gels, in situ digested with trypsin, and identified by MALDI-TOF mass spectrometry and database search. Gel image and mass spectrometry analysis yielded a list of 44 differentially expressed proteins (0.6 g fold change g 1.5), listed according to their common (Table 1) and noncommon (Table 2) regulation trend. In particular, a common regulation trend for P2, L7 and LPS primed U937 with respect to control cells was revealed for 14 proteins (Table 1). In addition, several spots were uniquely revealed under a given condition (Table 3). Protein identification revealed that several spots corresponded to the same protein, suggesting the presence of different isoforms, the occurrence of post-translational modifications (e.g., phosphorylation, glycosylation, etc.), and/or protein degradation events. The 44 differentially expressed proteins are represented by Venn diagrams in Figure 3, showing that the protein sets common to the three conditions for up- (Figure 3A) and downJournal of Proteome Research • Vol. 9, No. 2, 2010 1053
research articles
Severino et al.
Table 1. Significantly Regulated (0.6 g fold change g 1.5) Proteins with a Common Regulation Trend in Hib P2 (P)-, L7 (L)-, and LPS (S)-Exposed U937 Cell Line with Respect to Control (C) Cellsa spot no.
accession no.
2 201 116
P06733 P27797 P33316
86 85 62 7
Q04760 Q04760 P35232 P07237
35
P12324
4 61
P06733 P04792
122
P09651
71
P22626
44 66
P14618 P00938
description
Th. Mr
Th. pI
no. peptides
I. Common Up-Regulated Proteins Alpha enolase 47.0 7.2 30 Calreticulin 48.1 4.1 25 Deoxyuridine 5 triphosphate 26.7 9.8 22 nucleotidohydrolase Lactoylglutathione lyase 20.5 5.1 16 Lactoylglutathione lyase 20.5 5.1 18 Prohibitin 29.8 5.4 15 Protein disulfide isomerase 57.0 4.6 43 precursor PDI Tropomyosin 29.0 4.6 40 II. Common Down-Regulated Proteins 47.0 7.2 30 22.8 6.0 15
Alpha enolase Heat shock 27 kDa protein HSP 27 Heterogeneous nuclear ribonucleoprotein A1 Heterogeneous nuclear ribonucleoproteins A2 Pyruvate kinase M1 isozyme Triosephosphate isomerase
coverage (%)
Pr
change L/C
change P/C
change S/C
63.7 53.5 77.8
100.0 99.5 99.7
2.3 2.9 2.7
5.1 4.0 2.9
2.0 6.1 3.2
52.5 51.9 67.3 74.2
99.7 100.0 100.0 98.2
1.7 1.5 26.1 6.8
2.1 >2* 4.7 1.7
>2* >2* 11.8 6.5
83.1
100.0
2.3
3.2
3.5
58.2 62.0
100.0 100.0
0.3 0.3
0.4 0.5
0.5 0.2
38.7
9.5
25
58.8
100.0
0.3
0.0
0.0
37.4
9.2
27
57.8
100.0
0.1
0.5
0.5
57.7 26.5
7.5 6.5
35 20
55.5 76.6
50.9 100.0
0.2 0.4
0.1 0.5
0.2 0.5
a Protein theoretical relative masses (Th. Mr) and pI (Th. pI) are reported along with the number of matching peptides, the coverage percentages and probability scores (Pr) for identifications. Fold changes not quantifiable due to protein pattern distribution (i.e., high amounts and/or gel streaking) are indicated by asterisks.
(Figure 3B) regulated proteins contained 8 and 6 proteins, respectively. The assessment of significantly enriched categories was performed by using the WebGestalt software package on the basis of hypergeometric test as described in the Experimental Section. The significantly enriched gene ontology categories under biological process of differentially expressed proteins revealed that most of the protein changes were involved into metabolic pathways and regulation of cellular processes (Figure 3C). In addition, a significant number of differentially regulated proteins were found to be involved in cell death events. Several differentially expressed proteins play key roles in cytoskeleton remodelling, stress response, and signal transduction pathways. In addition, proteins involved in nucleotide metabolic pathways and modulation of splice site selection (e.g., several splicing factor arginine serine rich and heterogeneous nuclear ribonucleoproteins) and many molecular chaperones and folding catalysts were differentially expressed following P2- and L7-activation. In particular, several members of heat shock proteins (HSPs), including HSP70, HSP27, and HSP60 (Supporting Information, Figure 1S), were differentially regulated. A difference between LPS and P2 stimulation was revealed regarding the HSP70 expression levels. Indeed, differently from the up-regulated levels of HSP70 in the LPS condition, a drastic decrease in the expression levels was detected following P2 stimulation (Figure 1SA). Higher levels of HSP27 were also detected in P2-primed U937 cells (Figure 1SB), while an overall decrease of regulation levels was revealed for the acidic form of HSP27 in all the analyzed conditions (Figure 1SC). A different response between LPS and P2 priming was revealed for the expression levels of HSP60 (Figure 1SD). Following LPS stimulation, the up-regulation of HSP60 expression levels was detected for spots 20 and 21, whereas a marked down-regulation was revealed for spot 19. On the other hand, the main form of HSP60 (spot 20) slightly decreased in both P2- and L7-stimulated condition, together with changes revealed in the overall pattern of HSP60 distribution (Figure 1SD). A differential expression of several protein forms has been revealed also for cyclophilin A, a cytoplasmic protein secreted 1054
Journal of Proteome Research • Vol. 9, No. 2, 2010
under inflammatory conditions, including those involved in response to oxidative stress and LPS stimulation. Similarly, different spots corresponding to nucleophosmin (NMP), a known modulator of the cellular oxidative stress response and NF-κB activity, were detected following cell activation with LPS, P2, and L7 (Supporting Information, Figure 2S). The upregulation of other molecular chaperones (e.g., calreticulin and PDI) was revealed following P2, L7, and LSP priming (Supporting Information, Figure 3S). In particular, spot 201, corresponding to calreticulin, was found to be strongly up-regulated in LPS and L7 samples. The corresponding spot 201 in P2 sample, also identified as calreticulin by MALDI-TOF analysis, showed a lower molecular weight. Several proteins have been found to be potentially involved in the activation of proinflammatory cytokines through the p38 and JNK MAPK, as previously demonstrated by complementary approaches for Hib P2.17 Interestingly, the differential protein expression of dUTP deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and nucleoside diphosphate kinase A (NDPKA) was revealed (Figure 4). A significant up-regulation of dUTPase expression levels was revealed in P2-, L7-, and LPS-stimulated cells, while a decrease of NDPKA expression levels, differently from the LPS response, was observed in P2 and L7 activated U937, also confirmed by Western blot analysis (Figure 4C). To better understand the mutual interactions of identified proteins, network maps were constructed using the Ingenuity Pathway Analysis software. The graphical view of the resulting network revealed that several differentially regulated proteins following P2 priming were clustered on the basis of their reciprocal interactions. The two pathways obtained for P2stimulated cells are reported in Figures 5 and 6. Several proteins differentially expressed were found to converge on the JNK, ERK, and NF-κΒ complex (Figure 5). In addition, a second network was found to converge on the TNF signaling pathway (Figure 6). Notably, the differentially expressed proteins identified following L7- and LPS-stimulation focused on the same pathways attesting the occurrence of common activation
research articles
Proteomic Analysis of Human U937 Cell Line Activation
Table 2. Significantly Differentially Regulated (0.6 g Fold Change g 1.5) Proteins with a Noncommon Regulation Trend in Hib P2 (P)- and L7 (L)-Exposed U937 Cell Line versus Untreated U937 Control Cells (C)a spot no.
accession no.
21
P10809
20
P10809
19
P10809
200
P05387
1 3 15
P06733 P06733 O60664
80
P09211
82
P09211
60
P04792
166
P11142
70
P22626
204
P12268
161
P40121
207 104 115
P06748 P06748 P15531
127
P05092
130
P05092
123
P05092
129
P05092
157
P30086
10
Q15365
118 46
Q9UHV9 Q01130
93
P23152
94
P23152
45
Q16629
202
P38646
14
P31948
description
Th. Mr
Th. pI
no. peptides
coverage (%)
Pr
change L/C
change P/C
change S/C
60 kDa heat shock protein mitochondrial 60 kDa heat shock protein mitochondrial 60 kDa heat shock protein mitochondrial 60S acidic ribosomal protein P2 Alpha enolase Alpha enolase Cargo selection protein TIP47 Glutathione S transferase P Glutathione S transferase P Heat shock 27 kDa protein HSP 27 Heat shock cognate 71 kDa protein Heterogeneous nuclear ribonucleoproteins A2 Inosine 5 monophosphate dehydrogenase 2 Macrophage capping protein Nucleophosmin Nucleophosmin Nucleoside diphosphate kinase A Peptidyl prolyl cis trans isomerase A Peptidyl prolyl cis trans isomerase A Peptidyl prolyl cis trans isomerase A Peptidyl prolyl cis trans isomerase A Phosphatidylethanolamine binding protein Poly rC binding protein 1 hnRNP E1 Prefoldin subunit 2 Splicing factor arginine serine rich 2 Splicing factor arginine serine rich 3 Splicing factor arginine serine rich 3 Splicing factor arginine serine rich 7 Stress 70 protein mitochondrial precursor 75 kDa Stress induced phosphoprotein 1
61.0
5.6
52
75.0
100.0
1.3 T
No Match
4.3 v
61.0
5.6
56
71.9
100.0
0.7 T
0.6 V
2.3 v
61.0
5.6
53
66.7
100.0
0.6 V
No Match
0.1 V
11.7
4.2
9
75.7
92.1
No Match
10.1 v
No Match
47.0 47.0 47.0
7.2 7.2 5.1
40 25 21
73.4 52.9 62.7
100.0 100.0 96.4
0.9 T 0.6 V 1.5 v
No Match 0.7 T 3.3 v
0.6 V 0.8 T 0.6 V
23.2
5.3
11
70.8
99.6
0.5 V
No Match
1.8 v
23.2
5.3
11
67.9
94.3
3.1 v
No Match
2.4 v
22.8
6.0
14
56.6
100.0
0.9 T
2.3 v
1.1 T
70.8
5.2
31
41.2
100.0
0.3 V
1.8 v
0.4 V
37.4
9.2
31
67.4
100.0
2.8 v
1.1 T
0.5 V
55.8
6.5
29
46.5
100.0
1.7 v
No Match
1.9 v
38.5
5.8
16
55.7
100.0
1.2 T
0.9 T
2.0 v
32.5 32.5 17.1
4.4 4.4 5.8
22 14 16
56.5 35.7 80.9
70.8 99.2 100.0
0.4 V 0.7 T 0.1 V
4.6 v 0.6 V 0.0 V
1.6 v 0.6 V 0.9 T
17.9
7.9
19
84.1
97.4
1.2 T
1.9 v
0.8 T
17.9
7.9
16
78.0
99.5
0.5 V
0.7 T
0.0 V
17.9
7.9
15
69.5
78.7
No Match
0.2 V
No Match
17.9
7.9
19
84.8
99.5
1.8 v
0.1 V
1.7 v
20.9
7.4
14
65.6
99.7
0.8 T
0.4 V
1.1 T
37.5
6.7
28
66.0
100.0
0.6 V
No Match
1.0 T
16.6 25.5
6.2 12.3
15 12
76.0 32.1
99.8 79.9
0.8 T 1.0 T
0.0 V 1.0 T
1.0 T 0.6 V
19.3
12.0
9
54.3
91.8
1.4 T
1.2 T
0.4 V
19.3
12.0
9
47.0
61.1
2.3 v
1.5 v
0.7 T
27.3
12.2
13
40.8
100.0
1.2 T
No Match
2.3 v
73.7
5.9
69
77.0
100.0
1.5 v
0.1 V
2.7 v
62.6
6.4
39
64.3
99.9
1.1 T
0.4 V
2.3 v
a Protein regulation levels of lipopolysaccharide (S)-activated U937 cells are also reported for comparison. The regulation trend is indicated by arrows. Protein theoretical relative masses (Th. Mr) and pI (Th. pI) are reported along with the number of matching peptides, the coverage percentages and probability scores (Pr) for identifications.
signaling cascades (Supporting Information, Figure 4S and Figure 5S, respectively).
Discussion Bacterial porins trigger signaling pathways essential for the infection of target cells and/or the stimulation of an innate cellular immune response.12 Therefore, understanding the structure and function of Gram-negative surface components, such as porins, is essential for elucidating the microbial virulence that causes infection and the development of disease. This study represents the first attempt to identify, using a mass spectrometry proteomic approach, the post-transcriptional alterations induced by P2 porin from Hib and its surface exposed loop L7 in the U937 experimental system. The differentially expressed proteins were preliminary mapped in the
context of known signal transduction pathways involved in the inflammatory response. Some differentially expressed proteins have been grouped into functional protein families such as those of HSPs, known to be potent activators of the innate immune system by inducing the production of proinflammatory cytokines.26,27 Numerous studies have shown that HSPs are essential for the survival of the cell when it has been exposed to stressful situations.28 For example, in human blood monocytes, exposure to heat or physiological stress during phagocytosis increases the synthesis of HSPs.29 Although heat shock proteins have been studied for decades, new intracellular and extracellular functions in a variety of diseases continue to be discovered. Indeed, HSPs are involved in multiple protein networks for their capability to rapidly modulate cellular responses under Journal of Proteome Research • Vol. 9, No. 2, 2010 1055
research articles
Severino et al. a
Table 3. Proteins Uniquely Detected to the Hib P2 (P2)-, L7 (L7)- and LPS-Stimulated U937 Cell Line spot no.
accession
description
Th. Mr
Th. pI
peptides
coverage (%)
Pr
16
P17980
49.0
4.9
19
44.6
82.4
P2/L7/LPS
22
P11021
72.2
4.9
20
28.1
97.0
Control
205 206 136
P02571 P02571 P06576
41.8 41.8 56.5
5.2 5.2 5.0
29 32 38
58.1 68.8 56.1
51.9 55.3 100.0
P2/L7/LPS P2/L7/LPS Control
137
P06576
56.5
5.0
25
51.4
100.0
Control
13
O43175
56.6
6.3
18
37.9
99.8
Control
8 53
P16930 P04406
46.3 35.9
6.5 8.7
29 34
59.4 68.9
100.0 86.5
51
P04406
35.9
8.7
19
45.8
86.3
Control
26
P11142
70.8
5.2
22
32.8
92.7
Control
27
P11142
70.8
5.2
22
36.1
99.9
Control
69
P08238
83.0
4.8
27
34.3
99.9
Control
77
P31943
49.2
5.9
25
53.0
96.2
Control
72
P22626
37.4
9.2
28
58.9
99.9
P2/L7
9
O75874
46.7
6.4
24
47.1
99.0
Control/LPS
5
P30101
56.7
5.9
39
65.9
79.8
P2/L7/LPS
6
P30101
56.7
5.9
27
53.9
100.0
P2/L7/LPS
11
Q99832
26S protease regulatory subunit 6A 78 kDa glucose regulated protein precursor GRP 78 Actin Actin ATP synthase beta chain mitochondrial ATP synthase beta chain mitochondrial D 3 phosphoglycerat e dehydrogenase Fumarylacetoacetase Glyceraldehyde 3 phosphate dehydrogenase Glyceraldehyde 3 phosphate dehydrogenase Heat shock cognate 71 kDa protein Heat shock cognate 71 kDa protein Heat shock protein HSP 90 beta Heterogeneous nuclear ribonucleoprotein H Heterogeneous nuclear ribonucleoproteins A2 Isocitrate dehydrogenase Protein disulfide isomerase A3 Protein disulfide isomerase A3 T complex protein 1 eta subunit
59.3
7.5
41
58.7
100.0
Control/LPS
a
condition
Control/LPS Control
Proteins detected only in untreated U937 control cells are also reported. See Table 1 for headings.
Figure 3. Venn diagrams of up- (A) and down-regulated (B) proteins differentially expressed (0.6 g fold change g 1.5) in LPS-, P2-, and L7-stimulated U937 cell line with respect to control cells (see Tables 1 and 2 for details). (C) Bar chart of the enriched gene ontology categories under Biological Process (p < 0.01) of proteins differentially regulated following P2- and L7-stimulation. The entire human genome has been used as reference set.
stress conditions without requiring new protein synthesis. As previously mentioned, extensive work in the last 10 years has 1056
Journal of Proteome Research • Vol. 9, No. 2, 2010
suggested that HSPs may be potent activators of the innate immune system. It has been reported that HSP60, HSP70,
Proteomic Analysis of Human U937 Cell Line Activation
research articles
Figure 4. Magnification of spots corresponding to dUTPase and NDPKA (A) and relative expression changes (B). Western blot analysis of NDPKA in P2, LPS, and L7 stimulated U937 protein extracts is reported in panel C.
HSP90, and gp96 are capable of inducing the production of proinflammatory cytokines by the monocyte-macrophage system and the activation and maturation of dendritic cells (antigen-presenting cells) in a manner similar to the effects of LPS and bacterial lipoprotein via CD14/Toll-like receptor2 (TLR2) and CD14/TLR4 receptor complex-mediated signal transduction pathways.26,27 In our study, higher expression levels of HSP27 in P2 primed U937 were detected. On the contrary, an overall decrease of regulation levels was revealed for an acidic form of HSP27 in all the analyzed conditions. HSP27 phosphorylation is mediated by MAPK cascades. Indeed, heat stress activates the p38 kinase cascade and regulates HSP27 phosphorylation by means of two downstream MAPK (i.e., MAPKAPK2 and MAPKAP3). In addition, cytokines such as TNF and IL-1 can also regulate HSP27 phosphorylation through the same MAPK cascade.30,31 It has been also largely reported that the increased expression of HSP27 protects cells against cellular stress and cytotoxic responses, mainly by modulating intracellular reactive oxygen species content, and preventing apoptotic cell death triggered by a variety of stimuli, including TNF-alpha.32,33 In addition, HSP27 has been shown to interact with the actin cytoskeleton.34 The reorganization of cytoskeleton during immune interactions is critical for the generation of immune response. Indeed, the actin cytoskeleton reorganization mediated by porins and LPS in a variety of cells including macrophages is a well-known phenomenon.6,35-39 Consistently, we found an increased expression of high molecular weight actin upon U937 stimulation. Other HSPs were found to be differentially regulated following P2 and L7 stimulation, such as HSP60. It has been reported that human HSP60 exhibits immunoregulatory properties,40 primarily by inducing proinflammatory responses in innate immune cells, by macrophages activation via TLR4, a key mediator of the
innate immune response.41-43 HSP60 expression is upregulated during inflammation and stimulates macrophages to produce cytotoxic and proinflammatory mediators including nitric oxide, TNF-alpha, IL-6, and IL-12. In addition, HSP60 activated p38 MAPK and NFkB, the c-Jun-NH(2)-terminal kinase (JNK), and the extracellular signal-regulated kinase 1/2 (ERK).44,45 A defined region of HSP60 is also involved in LPS binding, thereby implicating a physiological role of HSP60 as LPS-binding protein.46 Accordingly, we found increased levels of the most abundant form of HSP60 (spot 20) in LPSstimulated U937, while the same protein spot slightly decrease in both P2 and L7 stimulated samples together with the changes revealed in the pattern of HSP60 distribution. Several molecular chaperones (e.g., prohibitin, calreticulin, poly(C) BP1, cyclophylin, protein disulfide isomerase), for which an essential role in cell survival under various stress conditions, including those related to immune response, has been largely demonstrated, were also found differentially regulated. In particular, a significant increase of protein disulfide isomerase (PDI) was revealed following P2, L7, and LPS stimulation. It has been reported that PDI can inhibit LPSinduced proinflammatory cytokine production in macrophages, thus, being considered as an intracellular anti-inflammatory molecule.47 Similarly, during our stimulation with P2 and L7 from Hib, the up-regulation of PDI may protect cells against apoptotic death. Another strongly up-regulated chaperone following LPS, P2, and L7 priming was identified as calreticulin, known to be involved in several autoimmune processes, including molecular mimicry, epitope spreading, complement inactivation and stimulation of inflammatory mediators, such as nitric oxide production.48 Interestingly, following P2 stimulation, we detected a spot corresponding to calreticulin with a lower molecular mass, whose nature needs to be further Journal of Proteome Research • Vol. 9, No. 2, 2010 1057
research articles
Severino et al.
Figure 5. Protein interaction networks obtained for the differentially expressed proteins following P2 U937 stimulation converging on the JNK, ERK, and NF-κB core molecules. The up- and down-regulated input proteins are colored in red and green, respectively. The color intensity is directly proportional to fold change values reported in Tables 1 and 2. The pathway components identified by the algorithm and not detected in the analysis are reported in white.
investigated in order to understand the molecular events responsible for this finding. Of particular interest is the differential expression of dUTPase and NDPKA, enzymes involved in the homeostasis of the thymine/uracil exchange, a fundamental process determining cell survival or death. Indeed, most DNA polymerases cannot distinguish between thymine and uracil bases and the regulation of dUTP/dTTP ratio constitutes the only mechanism to keep uracil out of DNA.49 dUTPase and NDPK are responsible for this task by controlling dUTP levels and providing the precursor for dTMP biosynthesis (Figure 6). It has been reported that dUTPase is essential for viability in all living organisms as lower dUTPase levels lead to the occurrence of highly uracil-substituted DNA which, upon excision repair, is excessively fragmented causing cell death.49 In addition, new roles for dUTPase are emerging beyond its metabolic activity. Several studies brought evidence that introduction of uracil bases in viral DNA during genome replication might be a defense mechanism against some viruses.50 As countermeasures, several viruses have evolved in encoding their own dUTPase, supporting the hypothesis that viruses are sensitive to the presence of uracil residues in their genome. For example, Epstein-Barr Virus (EBV)-encoded dUTPase up-regulates the expression of cytokines by NFκB activation.51-53 Recent works report that, in human monocyte1058
Journal of Proteome Research • Vol. 9, No. 2, 2010
derived macrophages, EBV-encoded dUTPase was able to upregulate the expression of several proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6 and IL-8), as well as the production of the antiinflammatory cytokine IL-10.51-53 The dUTPasemediated induction of TNF-alpha and IL-6 was dependent upon NFkB activation through TLR2 and requires the recruitment of the adaptor molecule MyD88.51 These evidence demonstrate that a nonstructural viral encoded protein can potentially contribute to the pathophysiology associated with diseases caused by EBV. In light of these considerations, the increase of dUTPase levels observed in this study is very intriguing as it could be hypothesized that also endogenous dUTPase may be endowed with the same properties of EBV enzyme. However, it is difficult to interpret the observed dUTPase increase until more data are obtained. NDPKs also play a major role in cell metabolism by catalyzing the transfer of the terminal phosphate of a nucleoside triphosphate to a nucleoside diphosphate, thus, equilibrating, together with dUTPase, the NDP and NTP cellular pools (Figure 7). In addition to its metabolic activity, NDPKs regulate a fascinating variety of cellular responses, including signal transduction, proliferation, development, differentiation, control of the cell adhesion machinery by regulating cell surface expression of integrin receptors and matrix metallo-proteases. Among
Proteomic Analysis of Human U937 Cell Line Activation
research articles
Figure 6. Protein interaction networks obtained for the differentially expressed proteins following P2 U937 stimulation converging on the TNF signaling pathway. The up- and down-regulated input proteins are colored in red and green, respectively. The color intensity is directly proportional to fold change values reported in Tables 1 and 2. The pathway components identified by the algorithm and not detected in the analysis are reported in white.
Figure 7. Involvement of dUTPase and NDPKs in the homeostasis of the cellular thymine/uracil exchange. The regulation of dUTP/ dTTP ratio is essential in determining cell survival (A) or cell death (B).
these pleiotropic effects, it has been demonstrated that NDPK directly interact with the Macrophage Migration Inhibitory Factor (MIF), a pluripotent proinflammatory cytokine identified as a central regulator of innate immunity and inflammation, implicated in several host disease states including tumorigen-
esis, arthritis, atherosclerosis, and septic shock.54 Considering these novel functions, we believe that the NDPKA differential expression upon P2 and LPS priming needs further studies in order to verify its potential implication in MIF signal transduction pathways. Journal of Proteome Research • Vol. 9, No. 2, 2010 1059
research articles Several proteins involved in the modulation of oxidative stress were also found as differentially expressed. Among them, several spots corresponding to nucleophosmin (NMP), likely due to post-translational modifications, were revealed following LPS, P2 and L7 cell activation. A recent study demonstrated that NPM may have potent biological activities that contribute to systemic inflammation, including the induction of MAPKs phosphorylation and the release of several proinflammatory cytokines.55 A differential expression of protein forms was revealed also for cyclophilin A (CyPA), a cytoplasmic protein secreted under inflammatory conditions, also known as peptidyl-prolyl cis-trans isomerase.56 CypA induced the expression of cytokine/chemokines such as TNF-alpha, IL-8, and IL-1beta and matrix metalloproteinase (MMP)-9 through a pathway dependent on NF-κB activation.57 Cyclophilins can also induce leukocyte chemotaxis and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. In addition, human recombinant CyPA activated MAPKs ERK1/2, JNK and p38 in cultured human umbilical vein EC.58 One of the main limits of proteomic analysis in delineating molecular pathways associated with a cellular response to a given stimulus mainly deals with the difficulty to detect proteins whose expression levels are below the sensitivity limits of the adopted technique and/or whose physicochemical properties (e.g., limited pH gradient of the 2D gels, solubility, extreme Mw and pI) prevent their detection. Likely for these reasons, proteins directly involved in the control of the oxidative stress, known to play an important role in the immune response against pathogens attack, were not detected in our study. However, bioinformatic approaches can support the experimental work by providing algorithms for pathway analysis that aims at connecting individual genes or proteins identified in “omics” surveys. To better understand the mutual interactions of identified proteins, a network analysis was performed using the Ingenuity Pathway Analysis software. The view that emerges from this pathway analysis following P2 stimulation is that several differentially expressed proteins converge on the JNK, ERK and NF-κB complex and, as expected, on the TNF signaling pathway. More interestingly, the differentially expressed proteins identified following L7 and LPS stimulation converge on the same pathways showing common activation signaling cascades. An additional point of convergence of networks was found to be the activation of the caspase complex, thus, confirming the results of the significantly enriched gene ontology categories analysis that highlighted the involvement of a significant number of differentially regulated proteins in cell death events. These findings are not surprising considering that the innate immune response plays a key role as the primary host defense against invading pathogens. As a consequence, the immune response uniquely combines proinflammatory features with a proapoptotic potential, thus, regulating the cell fate between life and death. Indeed, the TNF signaling that mediate the proliferative and inflammatory response through the recruitment of transcription factors, including NFκB, is also able to activate the caspase cascade, containing the major effector molecules critically involved in apoptosis, thus, mediating the opposite cellular response. As network data need to be further investigated, information derived from the analysis of these pathways provides useful suggestions for future experimental works. 1060
Journal of Proteome Research • Vol. 9, No. 2, 2010
Severino et al.
Conclusions This study represents the first attempt to identify, by a proteomics approach, the post-transcriptional alterations in the Hib P2 and L7-exposed U937 cells. The proteomic analysis provided a list of qualitative and quantitative changes with potential relevance in the host immune and inflammatory response upon Hib P2 and L7 activation. Among the differentially expressed proteins, many are described as directly or indirectly involved in immune and inflammatory responses. For several differentially expressed proteins, a similar behavior was observed following activation by the whole P2 porin and by L7. Furthermore, a common response was also observed for several proteins following P2, L7 and LPS activation. The perspectives of the present research will be focused on further investigation on proteins found to be differentially expressed with the aim to provide the basis for better understanding of the signal transduction cascades activated upon porin stimulation and to understand the molecular events involved in the modulation of pathogen-host cell interactions.
Acknowledgment. This study was supported by funds from the Second University of Naples. Supporting Information Available: Magnifications and relative expression changes of spots corresponding to HSP70, HSP27 and its acidic form; nucleophosmin in untreated, L7, P2 and LPS stimulated U937 cells; and calreticulin and protein disulphide isomerase in untreated, L7, P2 and LPS stimulated U937 cells. Protein interaction networks obtained for the differentially expressed proteins following L7 U937 stimulation and LPS U937 stimulation performed with the Ingenuity Pathway Analysis software. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Watt, J. P.; Wolfson, L. J.; O’Brien, K. L.; Henkle, E.; Deloria-Knoll, M.; McCall, N.; Lee, E.; Levine, O. S.; Hajjeh, R.; Mulholland, K.; Cherian, T. Burden of disease caused by Haemophilus influenzae type b in children younger than 5 years: global estimates. Lancet 2009, 374 (9693), 903–11. (2) Dufrene, Y. F. Using nanotechniques to explore microbial surfaces. Nat. Rev. Microbiol. 2004, 2 (6), 451–60. (3) McDonald, P. P.; Bald, A.; Cassatella, M. A. Activation of the NFκB pathway by inflammatory stimuli in human neutrophils. Blood 1997, 89 (9), 3421–33. (4) Cassatella, M. A. The production of cytokines by polymorphonuclear neutrophils. Immunol. Today 1995, 16 (1), 21–6. (5) Fessler, M. B.; Malcolm, K. C.; Duncan, M. W.; Worthen, G. S. A genomic and proteomic analysis of activation of the human neutrophil by lipopolysaccharide and its mediation by p38 mitogen-activated protein kinase. J. Biol. Chem. 2002, 277 (35), 31291– 302. (6) Fessler, M. B.; Malcolm, K. C.; Duncan, M. W.; Worthen, G. S. Lipopolysaccharide stimulation of the human neutrophil: an analysis of changes in gene transcription and protein expression by oligonucleotide microarrays and proteomics. Chest 2002, 121 (3), 75S–76S. (7) Gadgil, H. S.; Pabst, K. M.; Giorgianni, F.; Umstot, E. S.; Desiderio, D. M.; Beranova-Giorgianni, S.; Gerling, I. C.; Pabst, M. J. Proteome of monocytes primed with lipopolysaccharide: analysis of the abundant proteins. Proteomics 2003, 3 (9), 1767–80. (8) Zhang, X.; Kuramitsu, Y.; Fujimoto, M.; Hayashi, E.; Yuan, X.; Nakamura, K. Proteomic analysis of macrophages stimulated by lipopolysaccharide: Lipopolysaccharide inhibits the cleavage of nucleophosmin. Electrophoresis 2006, 27 (8), 1659–68. (9) Achouak, W.; Heulin, T.; Pages, J. M. Multiple facets of bacterial porins. FEMS Microbiol. Lett. 2001, 199 (1), 1–7. (10) Galdiero, F.; de L’ero, G. C.; Benedetto, N.; Galdiero, M.; Tufano, M. A. Release of cytokines induced by Salmonella typhimurium porins. Infect. Immun. 1993, 61 (1), 155–61.
research articles
Proteomic Analysis of Human U937 Cell Line Activation (11) Galdiero, M.; Cipollaro de L’ero, G.; Donnarumma, G.; Marcatili, A.; Galdiero, F. Interleukin-1 and interleukin-6 gene expression in human monocytes stimulated with Salmonella typhimurium porins. Immunology 1995, 86 (4), 612–9. (12) Galdiero, M.; Vitiello, M.; Galdiero, S. Eukaryotic cell signaling and transcriptional activation induced by bacterial porins. FEMS Microbiol. Lett. 2003, 226 (1), 57–64. (13) Galdiero, M.; D’Isanto, M.; Vitiello, M.; Finamore, E.; Peluso, L.; Galdiero, M. Monocytic activation of protein tyrosine kinase, protein kinase A and protein kinase C induced by porins isolated from Salmonella enterica serovar Typhimurium. J. Infect. 2003, 46 (2), 111–9. (14) Galdiero, M.; Tortora, A.; Damiano, N.; Vitiello, M.; Longanella, A.; Galdiero, E. Induction of cytokine mRNA expression in U937 cells by Salmonella typhimurium porins is regulated by different phosphorylation pathways. Med. Microbiol. Immunol. 2005, 194 (1-2), 13–23. (15) Galdiero, M.; D’Amico, M.; Gorga, F.; Di Filippo, C.; D’Isanto, M.; Vitiello, M.; Longanella, A.; Tortora, A. Haemophilus influenzae porin contributes to signaling of the inflammatory cascade in rat brain. Infect. Immun. 2001, 69 (1), 221–7. (16) Galdiero, S.; Capasso, D.; Vitiello, M.; D’Isanto, M.; Pedone, C.; Galdiero, M. Role of surface-exposed loops of Haemophilus influenzae protein P2 in the mitogen-activated protein kinase cascade. Infect. Immun. 2003, 71 (5), 2798–809. (17) Galdiero, S.; Vitiello, M.; Amodeo, P.; D’Isanto, M.; Cantisani, M.; Pedone, C.; Galdiero, M. Structural requirements for proinflammatory activity of porin P2 Loop 7 from Haemophilus influenzae. Biochemistry 2006, 45 (14), 4491–501. (18) Coulton, J. W.; Wan, D. T. The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins. Can. J. Microbiol. 1983, 29 (2), 280–7. (19) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193 (1), 265–75. (20) Yin, E. T.; Galanos, C.; Kinsky, S.; Bradshaw, R. A.; Wessler, S.; Luderitz, O.; Sarmiento, M. E. Picogram-sensitive assay for endotoxin: gelation of Limulus polyphemus blood cell lysate induced by purified lipopolysaccharides and lipid A from Gram-negative bacteria. Biochim. Biophys. Acta 1972, 261 (1), 284–9. (21) Nikaido, H.; Rosenberg, E. Y. Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J. Bacteriol. 1983, 153 (1), 241–52. (22) Galanos, C.; Luderitz, O.; Westphal, O. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 1969, 9 (2), 245– 9. (23) Chambery, A.; Farina, A.; Di Maro, A.; Rossi, M.; Abbondanza, C.; Moncharmont, B.; Malorni, L.; Cacace, G.; Pocsfalvi, G.; Malorni, A.; Parente, A. Proteomic analysis of MCF-7 cell lines expressing the zincfinger or the proline-rich domain of retinoblastoma-interacting-zincfinger protein. J. Proteome Res. 2006, 5 (5), 1176–85. (24) Zhang, B.; Kirov, S.; Snoddy, J. WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res. 2005, 33 (Web Server issue), W741–8. (25) Mendelsohn, B. A.; Malone, J. P.; Townsend, R.; Gitlin, J. Proteomic analysis of anoxia tolerance in the developing zebrafish embryo. Comp. Biochem. Physiol., Part D 2009, 4 (1), 21–31. (26) Tsan, M. F.; Gao, B. Cytokine function of heat shock proteins. Am. J. Physiol.: Cell Physiol. 2004, 286 (4), C739–44. (27) Tsan, M. F.; Gao, B. Heat shock proteins and immune system. J. Leukocyte Biol. 2009, 85 (6), 905–10. (28) Jaattela, M. Heat shock proteins as cellular lifeguards. Ann. Med. 1999, 31 (4), 261–71. (29) Barreto, A.; Gonzalez, J. M.; Kabingu, E.; Asea, A.; Fiorentino, S. Stress-induced release of HSC70 from human tumors. Cell Immunol. 2003, 222 (2), 97–104. (30) Niwa, M.; Hotta, K.; Kanamori, Y.; Hatakeyama, D.; Hirade, K.; Katayama, M.; Hara, A.; Mori, H.; Ito, H.; Kato, K.; Matsuno, H.; Uematsu, T.; Kozawa, O. Involvement of p38 mitogen-activated protein kinase in heat shock protein 27 induction in human neutrophils. Eur. J. Pharmacol. 2003, 466 (3), 245–53. (31) Engel, K.; Ahlers, A.; Brach, M. A.; Herrmann, F.; Gaestel, M. MAPKAP kinase 2 is activated by heat shock and TNF-alpha: in vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade. J. Cell Biochem. 1995, 57 (2), 321–30. (32) Jaattela, M.; Wissing, D. Heat-shock proteins protect cells from monocyte cytotoxicity: possible mechanism of self-protection. J. Exp. Med. 1993, 177 (1), 231–6. (33) Haddad, J. J.; Land, S. C. Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activa-
(34)
(35)
(36) (37)
(38) (39) (40) (41) (42)
(43)
(44)
(45)
(46) (47)
(48)
(49) (50) (51)
(52)
(53)
(54)
tion and MAPK-mediated TNF-alpha biosynthesis. Br. J. Pharmacol. 2002, 135 (2), 520–36. Lavoie, J. N.; Hickey, E.; Weber, L. A.; Landry, J. Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J. Biol. Chem. 1993, 268 (32), 24210–4. Sancho, D.; Vicente-Manzanares, M.; Mittelbrunn, M.; Montoya, M. C.; Gordon-Alonso, M.; Serrador, J. M.; Sanchez-Madrid, F. Regulation of microtubule-organizing center orientation and actomyosin cytoskeleton rearrangement during immune interactions. Immunol. Rev. 2002, 189, 84–97. Yin, H. L.; Hartwig, J. H. The structure of the macrophage actin skeleton. J. Cell Sci. Suppl. 1988, 9, 169–84. Eswarappa, S. M.; Pareek, V.; Chakravortty, D. Role of actin cytoskeleton in LPS-induced NF-kappaB activation and nitric oxide production in murine macrophages. Innate Immun. 2008, 14 (5), 309–18. De Martino, L.; Nazzaro, C.; Concilio, S.; Galdiero, M. Morphological changes induced in HEp-2 cells by Salmonella typhimurium porins. J. Submicrosc. Cytol. Pathol. 1995, 27 (4), 445–9. Galdiero, M.; Palomba, E.; De, L.; Vitiello, M.; Pagnini, P. Effects of the major Pasteurella multocida porin on bovine neutrophils. Am. J. Vet. Res. 1998, 59 (10), 1270–4. Habich, C.; Burkart, V. Heat shock protein 60: regulatory role on innate immune cells. Cell. Mol. Life Sci. 2007, 64 (6), 742–51. Ohashi, K.; Burkart, V.; Flohe, S.; Kolb, H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 2000, 164 (2), 558–61. Vabulas, R. M.; Ahmad-Nejad, P.; da Costa, C.; Miethke, T.; Kirschning, C. J.; Hacker, H.; Wagner, H. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J. Biol. Chem. 2001, 276 (33), 31332–9. Cohen-Sfady, M.; Nussbaum, G.; Pevsner-Fischer, M.; Mor, F.; Carmi, P.; Zanin-Zhorov, A.; Lider, O.; Cohen, I. R. Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway. J. Immunol. 2005, 175 (6), 3594–602. Kim, Y. S.; Koh, J. M.; Lee, Y. S.; Kim, B. J.; Lee, S. H.; Lee, K. U.; Kim, G. S. Increased circulating heat shock protein 60 induced by menopause, stimulates apoptosis of osteoblast-lineage cells via upregulation of toll-like receptors. Bone 2009, 45 (1), 68–76. Zhao, Y.; Yokota, K.; Ayada, K.; Yamamoto, Y.; Okada, T.; Shen, L.; Oguma, K. Helicobacter pylori heat-shock protein 60 induces interleukin-8 via a Toll-like receptor (TLR)2 and mitogen-activated protein (MAP) kinase pathway in human monocytes. J. Med. Microbiol. 2007, 56 (2), 154–64. Habich, C.; Kempe, K.; van der Zee, R.; Rumenapf, R.; Akiyama, H.; Kolb, H.; Burkart, V. Heat shock protein 60: specific binding of lipopolysaccharide. J. Immunol. 2005, 174 (3), 1298–305. Zhou, M.; Jacob, A.; Ho, N.; Miksa, M.; Wu, R.; Maitra, S. R.; Wang, P. Downregulation of protein disulfide isomerase in sepsis and its role in tumor necrosis factor-alpha release. Crit. Care 2008, 12 (4), R100. Ghebrehiwet, B.; Peerschke, E. I. cC1q-R (calreticulin) and gC1qR/p33: ubiquitously expressed multi-ligand binding cellular proteins involved in inflammation and infection. Mol. Immunol. 2004, 41 (2-3), 173–83. Vertessy, B. G.; Toth, J. Keeping uracil out of DNA: physiological role, structure and catalytic mechanism of dUTPases. Acc. Chem. Res. 2009, 42 (1), 97–106. Sire, J.; Querat, G.; Esnault, C.; Priet, S. Uracil within DNA: an actor of antiviral immunity. Retrovirology 2008, 5, 45. Ariza, M. E.; Glaser, R.; Kaumaya, P. T.; Jones, C.; Williams, M. V. The EBV-encoded dUTPase activates NF-kappa B through the TLR2 and MyD88-dependent signaling pathway. J. Immunol. 2009, 182 (2), 851–9. Glaser, R.; Padgett, D. A.; Litsky, M. L.; Baiocchi, R. A.; Yang, E. V.; Chen, M.; Yeh, P. E.; Klimas, N. G.; Marshall, G. D.; Whiteside, T.; Herberman, R.; Kiecolt-Glaser, J.; Williams, M. V. Stress-associated changes in the steady-state expression of latent Epstein-Barr virus: implications for chronic fatigue syndrome and cancer. Brain Behav. Immun. 2005, 19 (2), 91–103. Waldman, W. J.; Williams, M. V., Jr.; Lemeshow, S.; Binkley, P.; Guttridge, D.; Kiecolt-Glaser, J. K.; Knight, D. A.; Ladner, K. J.; Glaser, R. Epstein-Barr virus-encoded dUTPase enhances proinflammatory cytokine production by macrophages in contact with endothelial cells: evidence for depression-induced atherosclerotic risk. Brain Behav. Immun. 2008, 22 (2), 215–23. Jung, H.; Seong, H. A.; Ha, H. Direct interaction between NM23H1 and macrophage migration inhibitory factor (MIF) is critical
Journal of Proteome Research • Vol. 9, No. 2, 2010 1061
research articles for alleviation of MIF-mediated suppression of p53 activity. J. Biol. Chem. 2008, 283 (47), 32669–79. (55) Nawa, Y.; Kawahara, K. I.; Tancharoen, S.; Meng, X.; Sameshima, H.; Ito, T.; Masuda, Y.; Imaizumi, H.; Hashiguchi, T.; Maruyama, I. Nucleophosmin may act as an alarmin: implications for severe sepsis. J. Leukocyte Biol. 2009, 86 (3), 645–53. (56) Arora, K.; Gwinn, W. M.; Bower, M. A.; Watson, A.; Okwumabua, I.; MacDonald, H. R.; Bukrinsky, M. I.; Constant, S. L. Extracellular cyclophilins contribute to the regulation of inflammatory responses. J. Immunol. 2005, 175 (1), 517–22.
1062
Journal of Proteome Research • Vol. 9, No. 2, 2010
Severino et al. (57) Kim, H.; Kim, W. J.; Jeon, S. T.; Koh, E. M.; Cha, H. S.; Ahn, K. S.; Lee, W. H. Cyclophilin A may contribute to the inflammatory processes in rheumatoid arthritis through induction of matrix degrading enzymes and inflammatory cytokines from macrophages. Clin. Immunol. 2005, 116 (3), 217–24. (58) Jin, Z. G.; Lungu, A. O.; Xie, L.; Wang, M.; Wong, C.; Berk, B. C. Cyclophilin A is a proinflammatory cytokine that activates endothelial cells. Arterioscler., Thromb., Vasc. Biol. 2004, 24 (7), 1186–91.
PR900931N
