Proteasomal Serine Hydrolases Are Up-Regulated by and Required

Article pubs.acs.org/jpr

Proteasomal Serine Hydrolases Are Up-Regulated by and Required for Influenza Virus Infection Md Shahiduzzaman,† Peyman Ezatti,‡ Gang Xin,†,§ and Kevin M. Coombs*,†,‡,∥ †

Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada Manitoba Center for Proteomics and Systems Biology and ∥Manitoba Institute for Child Health, University of Manitoba, Winnipeg, Manitoba R3E 3P4, Canada § Department of Microbiology and Immunology, Shantou University Medical College, Shantou, Guangdong 515041, China ‡

ABSTRACT: Interactions between viruses and their host cells are important determinants of virus replication and of immune responses to the virus. However, these interactions and resulting consequences of these interactions remain poorly defined. Numerous recent quantitative proteomic approaches have measured host proteins affected by virus infection. Here, we used activity-based protein profiling (ABPP) to measure functional alterations in host serine hydrolases after influenza A virus infection of Madin-Darby canine kidney and human A549 lung cells. We identified 62 serine proteases. We then combined the ABPP approach with stable isotope labeling to directly measure how serine hydrolase activities were affected by virus infection. Differentially regulated SHs mapped into a few key cellular pathway systems, most notably the proteasomal system. The specific serine protease inhibitors Aprotinin and Pefablock and specific proteasomal inhibitors Bortezomib and MG132 significantly inhibited influenza virus growth. Some inhibitors also down-regulated activities of several proteasomal proteins, including PSMA1, PSMA2, and PMSB3. Genetic knockdown of PMSA2 also attenuated influenza virus replication. These findings further our understanding of enzymatic cellular processes affected by influenza virus and may be beneficial in the search for additional antiviral therapeutic targets. KEYWORDS: RNA virus, virus infection, host cell alterations, enzyme activity, proteomics, mass spectrometry, liquid chromatography, bioinformatics

■

INTRODUCTION Influenza A virus (IAV) causes infection in a variety of organisms including mammals. IAVs have been responsible for many yearly epidemics and global pandemics, including the 1918 H1N1, 1957 H2N2, 1968 H3N2, and most recently, the A/H1N1pdm2009 pandemics. IAV remain of high concern due to their ability to mutate to escape antiviral drugs, vaccines, and host immunity. IAVs are obligatory intracellular parasites that depend on many cellular functions to complete their life cycle. Thus, an alternate antiviral strategy would be to better understand and interfere with or modulate host factors that are influenced by and required by the virus for its efficient propagation. These interactions are complex and often poorly understood. Gene array studies have been performed to assess IAV replication.1,2 However, gene identifications alone offer only limited insights into the biochemical pathways that determine virus, cell, and tissue function. Since proteins are involved in complex metabolic and signaling networks and functionally regulated through post-translational modifications, understanding the cellular proteins in these complex processes would provide deeper insight into the molecular pathways in IAV−host interaction. However, the contribution of cellular proteins to this process has only been partially elucidated. Application of © 2014 American Chemical Society

relatively new quantitative proteomic methodologies allowed some global analyses of host protein quantities3−5 but do not provide information about host proteins’ functional activities. Thus, alternative global analyses of protein functional status are warranted to complement quantitative studies and provide better understanding of host responses to IAV infection. Serine hydrolases (SHs) are one of the largest and most diverse classes of enzymes in higher eukaryotes. SHs are involved in a variety of physiological and pathological processes. Because of their biological importance, SHs have been targeted for development of drugs against diseases such as obesity,6 diabetes,7 microbial infections,8 and Alzheimer’s disease.9 Host serine proteases are essential for the influenza virus life cycle because the viral hemeagglutinin (HA) is synthesized as a precursor that requires proteolytic maturation.10−12 Similarly, the Flavivirus nonstructural 3 protease (NS3, a chymotrypsin-like serine protease) plays a pivotal role in viral replication.13−17 Therefore, NS3 is one of the most promising targets for drug development against Flavivirus infections.18,19 Development of several protease inhibitors against Dengue virus and West Nile virus NS320,21 are Received: August 7, 2013 Published: March 26, 2014 2223

dx.doi.org/10.1021/pr5001779 | J. Proteome Res. 2014, 13, 2223−2238

Journal of Proteome Research

Article

Figure 1. Schematic representations. (A) Activity-based protein profiling probes, with FP-TAMRA (top) or Biotin (bottom) tag (left), central linker region, and active-site-specific ‘warhead’ (right). (B) General scheme for ABPP utilization and detection by SDS-PAGE analysis. Cultures of influenza virus PR8-infected MDCK or A549 cells are harvested, lysed, treated with FP-TAMRA-labeled ABPP reagent, and resolved by SDS-PAGE. (C) General scheme for gel-free comparative ABPP-SILAC, affinity purification, and detection by mass spectrometry. One set of MDCK or A549 cell samples (top, pink) are isotopically labeled with 12C6-lysine and 12C6/14N4-arginine (Light), and the other set of samples (bottom, blue) are isotopically labeled with 13C6-Lysine and 13C6/15N4-arginine (Heavy) amino acids. In some experiments, one set of cells is infected with influenza PR8 virus and the other cell set is mock-infected, whereas in other experiments both sets of cells are infected but one set is treated with SH inhibitors. After infection cells are combined in a 1:1 ratio and lysed, and the extracts are treated with FP-desthiobiotin (db-FP). Extracts are then affinity purified with Avidin beads, washed, and trypsin-digested, and peptides are resolved and measured by LC−MS.

considered effective and showed good success rates22 in antiHCV clinical trials. Other serine proteases involved in pathogenesis and various virus life cycles are being considered as chemotherapy targets. The catalytic activity of the herpes simplex virus type 1 serine protease is essential for viral nucleocapsid formation and for viral replication.23 A trypsin-like serine protease is involved in pseudorabies viral penetration of the basement membrane during mucosal invasion,24 and serine protease inhibitors impede pseudorabies virus invasion. A vaccinia virus serine protease inhibitor prevents virus-induced cell fusion.25 Serine protease inhibitors 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF) and p-aminobenzamidine (pAB) significantly reduce influenza A virus replication in mouse models.26 In addition to known roles in IAV HA processing, SHs likely play further pivotal roles in IAV replication. Thus, identification of active SHs and their functional characterization are necessary for better under-

standing the molecular pathogenesis and development of antiviral strategies. Activity based protein profiling (ABPP) is a chemical strategy that is currently used to identify functional states of proteins or enzymes. For the current study, we combined ABPP27,28 with SILAC (stable isotope labeling by amino acids in cell culture)29,30 as an alternate quantitative approach to examine SH functional alterations that occur in IAV-infected host cells to identify additional potential antiviral targets. To demonstrate the utility of ABPP-SILAC to quantify alterations in SHs after infection, comparative functional mass-spectrometry-based proteomic analyses were performed on infected and mockinfected MDCK (Madin-Darby Canine Kidney) and human A549 (adenocarcinomal human alveolar basal epithelial) lung cells, both of which are routinely used for IAV propagation and analysis.31,32 Several proteasomal SHs were found to be affected and were further specifically assessed by RNAi and Western blot analyses. 2224



■

Article

EXPERIMENTAL PROCEDURES

cells were mixed. The mixed cells were subjected to protein extraction and quantification as described below. Experiments were performed a minimum of two separate times and in some cases were performed in triplicate.

Cell Culture, Virus, and Infection

Canine MDCK and human A549 lung cells were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Grand Island, NY, USA) supplemented with L-glutamine, Na-Pyruvate, nonessential amino acids, and 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO2/95% air atmosphere as described.4 Cells were passaged 3 times weekly. The virus strain used was IAV strain A/Puerto-Rico/8/34 (H1N1) “PR8”, a laboratory attenuated nonpathogenic strain that has been used in our previous quantitative proteomic analyses.4,33 Virus titers were determined by plaque assay on MDCK cells.34 A549 and MDCK cells at approximately 95% confluency were infected with PR8 at a multiplicity of infection (MOI) of 0.01 or 3−5 plaque-forming units (PFU)/cell depending upon the experiment and as detailed below. Infection was maintained in the above completed DMEM, but lacking serum and supplemented with penicillin/streptomycin and amphotericin-B for various periods of time. To determine effects of serine hydrolase (SH) inhibitors on IAV replication, sets of cells were pretreated with various concentrations of Aprotinin or Pefabloc SC Plus (Roche Applied Science, Germany) or with proteasomal inhibitors Bortezomib or MG132 (Selleck Chemicals, Houston, Texas) for 4 h, infected with PR8 at MOI = 0.01, and incubated for an additional 42 h in the presence of inhibitor. Supernatants were then harvested, and virus yield was determined by plaque assay. For knockdown (KD) studies, sets of A549 cells at ∼25% confluency were treated with 25 nM of Dharmacon SMARTPool siRNA for 72 h in OptiMEM according to manufacturer’s instructions. The siRNAs targeted the PSMA2 gene or were scrambled nonsilencing (N-Si) control. MDCK cells were similarly treated but with 75 nM siRNA. Sets of KD cells were then infected or mock-infected with PR8 at an MOI of 3 and incubated 24 h in OptiMEM lacking FBS. Supernatants were harvested for virus titration and cells were processed for Western blotting as detailed below. Cell viabilities were determined by the WST-1 assay according to manufacturer’s instructions after cells were treated with various SH inhibitors, proteasomal inhibitors, or DMSO vehicle for 48 h.

Protein Extraction

At the end of cell culture and infection, cells were harvested by scraping and centrifuged at 500 × g for 5 min. Harvested cells were washed twice with ice-cold phosphate-buffered saline (PBS; pH 7.2) and resuspended in ice-cold PBS. The samples were sonicated on ice (3× for 10 s) and centrifuged at 500 × g for 5 min. The supernatants were collected in new tubes (tube 1). The remaining pellets were resuspended in lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol) and kept on ice for 30 min. The samples were centrifuged at high speed (16,000 × g for 5 min), and the supernatants were added to corresponding tube 1 tubes. The protein content of every sample was measured by BCA (bicinchoninic acid) assay (Pierce; Rockford, IL). Protein Labeling with Serine Hydrolase Probe

Two strategies were used for ABPP analysis of IAV-infected cells (outlined in Figure 1). Protein samples of 50 μL (100 μg) were labeled with 2 μM fluorophosphonate (FP)-TAMRA probe (Activ X TAMRA-FP, ThermoScientific, Pierce Biotechnology, USA) for 1 h at room temperature. The probe reaction was stopped by adding NuPAGEP sample loading buffer (Invitrogen, CA). DTT (50 mM) was added to the sample and boiled for 5 min before labeled proteins were resolved in SDS-PAGE. Differences in SH expression levels between infection and mock control were determined by fluorescent gel scanning, using an Alpha Innotech FluorChemQ MultiImage III instrument. To confirm serine hydrolase FP probe specificity, some samples were denatured by boiling for 5 min before adding the FP probe, and some samples were not treated with probe prior to electrophoresis. Alternatively, Activ X Desthiobiotin-FP (db-FP) serine hydrolase probe (ThermoScientific, Pierce Biotechnology, USA) was used to label all active SH present in the cell lysates. Approximately 22 μM concentration of db-FP probe was added to a total of 5 mg of protein (1:1 of L:H) for each sample and incubated for 1 h at room temperature. An equal volume of 12 M urea/lysis buffer was added to the probelabeled proteins to terminate the reactions. The samples were reduced by using 5 mM DTT, incubated at 65 °C for 30 min, and alkylated by incubating in 40 mM iodoacetamide at room temperature in the dark for 30 min. To remove excess salt and free probe, samples were passed through desalting columns (Zeba Spin ThermoScientific, Rockford, USA). The labeled proteins were then enriched on PBS-prewashed streptavidin beads (Dynabeads MyOne Streptavidin T1, Invitrogen Dynal, Norway). After 1 h of incubation at room temperature, the beads were washed 3× with wash buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 4% urea), 3× with PBS, and 3× with distilled water. On-bead digestion of peptides was performed with 1 μg of sequence grade trypsin (Promega, Madison, WI) dissolved in 100 mM ammonium bicarbonate at 37 °C overnight. Trypsin reaction was stopped by adding 1% TFA. Peptides were collected from the supernatant and dried by Speed Vac. The lyophilized peptides were stored at −20 °C until analyzed as detailed below.

SILAC Assay

The relative functional activities of serine hydrolases in IAVinfected samples were determined by SILAC analyses of differentially labeled samples, essentially as described,4,33 but with modifications to include ABPP selection. Briefly, fresh SILAC “Light” and “Heavy” media were prepared separately with DMEM supplemented with 10% dialyzed FBS (GIBCO, Grand Island, NY, USA) and nonessential amino acids. 12C6Lys and 12C614N4-Arg (“Light”; L) were added to light media and 13C6-Lys and 13C615N4-Arg (“Heavy”; H) were added to heavy media. Sets of cells were separately grown in SILAC L and H media through 6 doublings to allow >99% incorporation of the L and H amino acids into respective cells (>99% incorporation was confirmed by mass spectrometric analysis). Light cells were infected with PR8 at an MOI of 5, and an equivalent number of H cells were mock-infected as the control. The infections were maintained for various periods of time with or without various SH inhibitors. The cells were counted after harvesting, and equivalent numbers of H and L 2225



Article

Figure 2. Cells were infected with PR8 at a MOI of 5, incubated at 37 °C for the indicated times (hours postinfection indicated above lanes), harvested, and lysed, and FP-TAMRA-labeled proteomes were resolved by SDS-PAGE and visualized by fluorescence image documentation system. (A) Probe specificity analysis. Cell extracts were reacted as indicated with or without probe or heat-denatured before probe reaction. (B) Differential expression of SH (arrows) in MDCK cells. (C) Differential expression of SHs (arrows) in A549 cells. 0 corresponds to a mock-infection treated with DMSO vehicle.

In-Gel Digestion of FP-TAMRA-Labeled Proteins

range of 100−1600 (total cycle time 2.3 s). Switching criteria were set to ions greater than mass to charge ratio (m/z) 400 and smaller than m/z 1250 with charge state of 2−5 and an abundance threshold of more than 250 counts. Former target ions were excluded for 8 s. A sweeping collision energy setting of 37 ± 15 eV was applied to all precursor ions for collisioninduced dissociation. For both in-gel and in-solution digestion, peptide digests were desalted and purified off-line using ZipTip C18 Pipette Tips (Millipore), frozen at −80 °C, and dried using a speed vacuum.

Differentially expressed visible FP-TAMRA-labeled protein bands in gels were excised and placed in separate 1.5-mL microfuge tubes. Gel slices were washed 2× with 200 μL 100 mM ammonium bicarbonate, 3 × 15 min in 50:50 acetonitrile/ 100 mM ammonium bicarbonate with periodic vortexing, and 2× in 50 μL 100% acetonitrile to dry the gel slices. Acetonitrile was removed, and the dried gel slices were incubated in 20 μL of 100 mM ammonium bicarbonate containing 10 ng/μL sequence grade trypsin (Promega, USA) overnight. The supernatants wre transferred to new tubes and dried in a SpeedVac. Tryptic peptides were then resolved and identified by mass spectrometry as detailed below.

Database Searching and Protein Identification

Wiff. files from Analyst TF 1.5 software were submitted simultaneously to Protein Pilot 4.0 (Applied Biosystems) for identification and relative quantification and protein identification of each of the replicate samples using the Paragon algorithm as the search engine. Peptides were identified by searching each MS/MS spectrum against a database of human protein sequences (NCBI ftp released March (ftp://ftp.ncbi. nih.gov/refseq/H_sapiens/mRNA_Prot/; 37,391 entries)) and Canis protein sequences (ftp://ftp.ncbi.nih.gov/genomes/ Canis_lupus_familiaris/protein/; 21,965 entries). Protein ratios were quantified by calculating the area under both the light and heavy peaks of the identified peptides. The search parameters allowed for cysteine modification by iodoacetic acid and biological modifications programmed in the algorithm (i.e., phosphorylations, amidations, semitryptic fragments, etc.). The threshold for detecting proteins (unused protscore (confidence)) in the software was set to 2.0 to achieve 99% confidence with a false discovery rate of 1%, and identified proteins were grouped by the ProGroup algorithm (Applied Biosystems, Foster City, CA) to minimize redundancy. The bias correction option was used to correct for small pipetting errors.

Quantitative Protein Identification by nanoRP-LC−MS/MS

Samples were analyzed by nano-RPLC−MS/MS using a splitless Ultra 2D Plus (Eksigent, Dublin, CA) system coupled to a high speed Triple TOF 5600 mass spectrometer (AB SCIEX, Concord, Canada). Peptides were injected via a PepMap100 trap column (0.3 × 5 mm, 5 μm, 100 Å, Dionex, Sunnyvale, CA) and a 100 μm × 150 mm analytical column packed with 5-μm Luna C18(2) prior to MS/MS analysis. Both eluents A (water) and B (99% acetonitrile) contained 0.1% formic acid as an ion-pairing modifier. The tryptic digest was analyzed with a 60-min gradient that consisted of eluent B from 0 to 35% over 48 min, 35% to 85% in 1 min, and then keep at 85% for 5 min at a flow rate of 500 nL/min. Key parameter settings for the TripleTOF 5600 mass spectrometer were as follows: ionspray voltage floating (ISVF) 3000 V, curtain gas (CUR) 25, interface heater temperature (IHT) 150, ion source gas 1 (GS1) 25, declustering potential (DP) 80 V. All data were acquired using information-dependent acquisition (IDA) mode with Analyst TF 1.5 software (AB SCIEX, USA). For IDA parameters, 0.25 s MS survey scans in the mass range of 400− 1250 were followed by 20 MS/MS scans of 100 ms in the mass 2226



Article

Table 1. Serine Hydrolases Identified in A549 and MDCK Cells serine hydrolasea Abhydrolase domain-containing protein 10 Abhydrolase domain-containing protein 14B Acyl-coenzyme A thioesterase 1 Acyl-coenzyme A thioesterase 2 Acyl-coenzyme A thioesterase 9 Acyl-coenzyme A thioesterase 9, mitochondrial-like isoform 2 Acyl-protein thioesterase 1 Acyl-protein thioesterase 2 Acyl-protein thioesterase 2-like Cytosolic acyl coenzyme A thioester hydrolase Cytosolic phospholipase A2 Dipeptidyl peptidase 4 Fatty acid synthase Fatty-acid amide hydrolase 1 Isoamyl acetate-hydrolyzing esterase 1 homologue Lactamase, beta 2 isoform 1 Liver carboxylesterase 1 Lon protease homologue Lon protease homologue, mitochondrial isoform 1 Lysophospholipase-like protein 1 Lysosomal protective protein Monoacylglycerol lipase ABHD12 Neuropathy target esterase Neutral cholesterol ester hydrolase 1 Ovarian cancer-associated gene 2 protein Platelet-activating factor acetylhydrolase IB β Platelet-activating factor acetylhydrolase IB γ Pro-cathepsin H Prolyl endopeptidase Protease, serine, 3 isoform 3 Proteasome subunit alpha type-1 Proteasome subunit alpha type-2 Proteasome subunit alpha type-3 Proteasome subunit alpha type-4 Proteasome subunit alpha type-5 Proteasome subunit alpha type-5-like Proteasome subunit alpha type-6 isoform 2 Proteasome subunit alpha type-7 Proteasome subunit alpha type-7-like Proteasome subunit alpha type-7-like Proteasome subunit beta type-1 Proteasome subunit beta type-2 Proteasome subunit beta type-3 Proteasome subunit beta type-4 Proteasome subunit beta type-5 Proteasome subunit beta type-5-like Proteasome subunit beta type-6 Proteasome subunit beta type-7 Proteasome subunit beta type-9 Protein disulfide-isomerase Protein disulfide-isomerase A3 Protein disulfide-isomerase A4 isoform 3 Protein disulfide-isomerase A6 Protein phosphatase methylesterase 1 Putative ATP-dependent Clp protease proteo sub Retinoid-inducible serine carboxypeptidase SEC23-interacting protein Serine beta-lactamase-like protein Serine protease HTRA2 S-formylglutathione hydrolase Signal peptidase complex catalytic subunit

gene symbol ABHD10 ABHD14B ACOT1 ACOT2 ACOT9 LOC100856631 LYPLA1 LYPLA2 LOC100855579 ACOT7 PLA2G4A DPP4 FASN FAAH IAH1 LACTB2 CES1 LONP1 LONP1 LYPLAL1 CTSA ABHD12 PNPLA6 NCEH1 OVCA2 PAFAH1B2 PAFAH1B3 CTSH PREP PRSS3 PSMA1 PSMA2 PSMA3 PSMA4 PSMA5 LOC100856348 PSMA6 PSMA7 LOC100856395 PSMA8 PSMB1 PSMB2 PSMB3 PSMB4 PSMB5 LOC100855659 PSMB6 PSMB7 PSMB9 P4HB PDIA3 PDIA4 PDIA6 PPME1 CLPP SCPEP1 SEC23IP LACTB HTRA2 ESD SEC11A 2227

accession no. (A549) gi|34222621 gi|50428913 gi|269849771 gi|224471815 gi|41017274 gi| 41017276 gi|28381347 gi|317373312 gi|269849686

accession no (MDCK) gi|345796194 gi|345786899 gi|73964277 gi|359324143 gi|345793234 gi|359318941 gi|345800657 gi|345797236 gi|73964695 gi|73977880

gi|121941741 gi|73999212 gi|119576 gi|12644239 gi|73987072 gi|74762275 gi|20178316 gi|345789124 gi|150403921 gi|74737782 gi|74731010 gi|55977294 gi|3024344 gi|215273868 gi|130848 gi|130850 gi|130859 gi|130861 gi|38258905 gi|46397659 gi|12643540

gi|345799902 gi|57036489 gi|345798093 gi|345778584 gi|57097397 gi|345787836 gi|73981673 gi|73963050

gi|359319848 gi|73962756 gi|359319980

gi|108936006 gi|130853 gi|1709762 gi|20532411 gi|116242733 gi|187608890 gi|20532407 gi|17380263 gi|417529 gi|2507460 gi|2507461 gi|119530 gi|2501205 gi|47606055 gi|3023512

gi|73974057 gi|345780641 gi|356461018 gi|345782656 gi|359320183 gi|73955410

gi|73964749 gi|345794865 gi|359321459 gi|73980394

gi|73966651 gi|55584014 gi|46397478 gi|17376879 gi|544254 gi|54039634 dx.doi.org/10.1021/pr5001779 | J. Proteome Res. 2014, 13, 2223−2238


Article

Table 1. continued serine hydrolasea

gene symbol

Tripeptidyl-peptidase 1 precursor a

accession no. (A549)

TPP1

accession no (MDCK) gi|62078829

Serine hydrolases identified with a minimum of 2 peptides

Table 2. Identified Serine Hydrolases and Their SILAC Ratios (Infected:Mock) in MDCK Cells Infected with PR8 serine hydrolasea Proteasome subunit beta type-6 Acyl-protein thioesterase 2-like Protein disulfide-isomerase Proteasome subunit beta type-3 b Proteasome subunit alpha type-2 isoform 1 Proteasome subunit beta type-5-like Fatty acid synthase Protein disulfide-isomerase A6 Prolyl endopeptidase isoform 2 Protein disulfide-isomerase A4 isoform 3 Proteasome subunit alpha type-3 isoform 1b Proteasome subunit alpha type-6 isoform 2 Monoacylglycerol lipase ABHD12 Proteasome subunit beta type-2 isoform 2 Protein disulfide-isomerase A3 Cytosolic acyl coenzyme A thioester hydrolase Proteasome subunit beta type-1 Pro-cathepsin H a

accession no.

gene symbol

gi|73955410 gi|359318941 gi|73964749 gi|356461018 gi|73981673 gi|359320183 gi|73964695 gi|73980394 gi|345778584 gi|359321459 gi|73963050 gi|73962756 gi|345789124 gi|345780641 gi|345794865 gi|345800657 gi|73974057 gi|345798093


■

Gene ontology enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool (http://david.abcc. ncifcrf.gov). The GI numbers were converted to official gene symbols by DAVID. All identified SHs were overlaid with proteins or genes that we have previously identified in global RNAi35 or as significantly affected by quantitative SILAC,4 screens in IAV-infected A549 cells, and a network was constructed by the STRING online functional protein associate network tool (http://string.embl.de//). Noninteracting proteins were removed from networks. The resultant genes were used to search KEGG reference pathway database in DAVID to obtain reference pathways for these proteins.

PSMB6 LOC100855579 P4HB PSMB3 PSMA2 LOC100855659 FASN PDIA6 PREP PDIA4 PSMA3 PSMA6 ABHD12 PSMB2 PDIA3 ACOT7 LOC100688526 CTSH

L/H ratio mean ± SD 14.49 2.58 2.27 2.03 1.50 1.44 1.36 1.31 1.28 1.15 1.15 1.07 1 0.96 0.95 0.84 0.75 0.74

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.09 1.13 0 0.56 0.37 0.24 0.16 0.14 0 0.13 0.36 0.33 0 0.19 0.22 0 0 0

RESULTS

Serine Hydrolase Activities during IAV Infection

IAV infections of MDCK and A549 cells were maintained until cytopathic effect (CPE) was observed microscopically. When infected at an MOI of 5, CPE became apparent at 16 hpi in MDCK cells and at 48 h in A549 cells. Extracts of the infections were taken at different time points up until CPE became apparent to globally assess SH activity. Extracts were reacted with FP-TAMRA and resolved by SDS-PAGE. Probe labeling specificity was confirmed by lack of signal when probe was not added and by a very different pattern if cell extracts had been predenatured (Figure 2A). This analysis revealed one noticeable difference between the 0 and 3 hpi samples (Figure 2B, upper arrow), with most other detectable changes occurring after 6 h in MDCK cells (Figure 2B, lower arrows). SH profile changes were consistently observed in all MDCK infected samples after this time point. Some SH profile alterations were observed by 24 hpi in A549 cells, although maximum SH alterations were seen after 42 hpi (Figure 2C, arrows), indicating that IAV infection-associated SH alterations occur more rapidly in MDCK cells than in A549 cells. Gel-based approaches offer a means to rapidly screen global SH alterations, but protein identification may be a challenge, particularly when nonlabeled high-abundant proteins comigrate with FP-TAMRA-labeled low-abundant SHs. Thus, to more accurately identify IAV-altered SHs, we harvested IAVinfected and mock-infected MDCK cells at 16 hpi and A549 cells at 48 hpi, reacted the cell extracts with db-FP, affinitypurified the bound SHs, digested them with trypsin, and identified SHs by mass spectrometry (Table 1). A total of 62 SHs were identified from A549 and MDCK cells (Table 1) of which 21 were common to both cell types. Affinity purification of non-db-FP-treated samples identified only fatty acid synthase (FASN), suggesting all other proteins were specifically

Western Blotting for Proteasomal Components

Sets of mock-infected and PR8-infected cells that had been pretreated or not with various inhibitors or siRNA molecules were harvested at defined time points post-treatment or postinfection. Cells were washed 3× with ice-cold PBS and lysed in 0.5% NP-40/sonication as described earlier. Protein concentrations were determined, samples were supplemented with Laemmli electrophoresis sample buffer, and proteins were resolved by mini-SDS-PAGE. Proteins were transferred to PVDF membranes, and the membranes were briefly stained with Ponceau-S to confirm protein transfer. Membranes were blocked with 5% skim milk and probed with various antibodies. Primary antibodies were rabbit anti-PSMA2 (Cell Signaling, catalog no. 2455) or mouse anti-Actin (Cell Signaling, no. 3700). HRP-conjugated goat anti-rabbit or goat anti-mouse antibody was used as secondary. HRP was detected by enhanced chemiluminescence, and band intensities were measured with an Alpha Innotech FluorChemQ MultiImage III instrument. 2228



Article

Table 3. Identified Serine Hydrolases and Their SILAC Ratios (Infected:Mock) in A549 Cells Infected with PR8 serine hydrolasesa Protein disulfide-isomerase S-Formylglutathione hydrolase Proteasome subunit alpha type-2 Cytosolic acyl coenzyme A thioester hydrolase Signal peptidase complex catalytic subunit Protein disulfide-isomerase A4 Serine beta-lactamase-like protein Liver carboxylesterase 1 Protein disulfide-isomerase A6 Prolyl endopeptidase Lon protease homologue Cytosolic phospholipase A2 Protein disulfide-isomerase A3 Fatty acid synthase Acyl-protein thioesterase 2 Proteasome subunit beta type-2 Platelet-activating factor acetylhydrolase IB subunit beta Neutral cholesterol ester hydrolase 1 Acyl-protein thioesterase 1 Platelet-activating factor acetylhydrolase IB subunit gamma Serine protease HTRA2 a

accession no. gi|2507460 gi|544254 gi|130850 gi|28381347 gi|54039634 gi|119530 gi|46397478 gi|119576 gi|2501205 gi|215273868 gi|12644239 gi317373312 gi|2507461 gi|269849686 gi|41017276 gi|1709762 gi|55977294 gi|74737782 gi|41017274 gi|3024344 gi|17376879

gene symbol P4HB ESD PSMA2 ACOT7/ BACH SEC11A/ SPC18 PDIA4 LACTB CES1 PDIA6 PREP LONP1 PLA2G4A PDIA3 FASN LYPLA2 PSMB2 PAFAH1B2 NCEH1 LYPLA1 PAFAH1B3 HTRA2

L/H ratio mean ± SD 2.47 2.28 2.25 1.67 1.5 1.47 1.35 1.32 1.32 1.07 1.06 1.06 0.7 0.48 0.45 0.45 0.40 0.36 0.34 0.22 0.21

± 0.5 ± 0.75

± 0.11

± ± ± ±

0.08 0.5 0.16 0.07


strongly represented by “catabolism, metabolism, and physiological process”, and the up-regulated A549 SHs belonged to “physiological process and metabolism”. There were significant differences in the numbers and proportions of identified A549 and MDCK proteins constituting the “regulation of metabolic process, lipid metabolic process, positive regulation of biological process, and positive regulation of cellular process” GOTERMs (Figure 3A). MDCK cells also were assigned to numerous molecular functions, of which “hydrolase, endopeptidase and peptidase activities” were the main ones, and cellular components, of which “cell, cytoplasm, intracellular, cytosol, and nucleus” were dominant. Identified A549 molecular functions included many involving “hydrolyse activity and catalytic activity” and cellular components were strongly represented by “cell, cytoplasm and intracellular”. Several of these categories also were significantly differently represented between the A549 and MDCK cells (Figure 3B,C). Overall, the Proteasome was the major cellular pathway identified in both A549 and MDCK cells, with a Benjamimi P value of 7.75 × 10−5 in MDCK cells and a P value of 0.05 in A549 cells. Additional top scoring cellular pathways identified in A549 cells were Ether lipid metabolism (P = 0.036) and Glycerophospholipid metabolism (P = 0.065).

identified by the probe. We then labeled cells with SILAC and performed additional affinity purifications and quantitation. The SILAC-based proteomic screen was expected to identify fewer SHs because success relies on identification and measurement of both cognate L and H peptides rather than simply identification of a single peptide. Nevertheless, we successfully identified many proteins and quantified their functional changes as a result of infection. For determining the level of alteration, the SHs were classified into three groups: up-regulated (L:H ≥ 1.45), down-regulated (L:H ≤ 0.65), and not regulated (L:H = 0.65−1.45). These cutoffs were determined by statistical analyses of the data set and correspond to p < 0.05. A total of 18 SHs were identified in MDCK cells. Five of these (PSMB6, P4HB, LOC100855579, PSMB3, and PSMA2) were up-regulated 1.5-fold or more, but no significantly down-regulated (to 1.5fold down-regulation) SHs were observed (Table 2). Among the altered SHs, PSMB6 is highly (14.5×) up-regulated. A total of 21 SHs were identified and measured in A549 cells; 6 were up-regulated 1.45-fold or more (P4HB, ESD, PSMA2, ACOT7, SEC11A, and PDIA4), and 8 were down-regulated to < 0.5-fold (i.e., >2-fold down-regulation) (FASN, LYPLA2, PSMB2, PAFAH1B2, NCEH1, LYPLA1, PAFAH1B3, and HTRA2) (Table 3). PSMA2 (2.25×) ESD (2.28×) and P4HB (2.47×) were strongly up-regulated in A549 cells. Pronounced downregulation was observed for HTRA2 (−5×) and PAFAH1B3 (−4.7×). PSMA2 and P4HB were up-regulated in both cell types. The alterations of common SHs between the cell lines did not differ significantly: FASN (p > 0.02), PSMB2 (p > 0.06), and PDIA4 (p > 0.07) (p value obtained from z score analysis).

Functional and Pathway Analyses

Functional network analysis by STRING showed that upregulated SHs are predicted to interact with other serine hydrolases, some of which we also identified as altered, and non-SH proteins that we had identified in previous SILAC4 and RNAi35 screens (Figure 4). PMSB6 and PSMB3 are predicted to interact within the proteasome subunits, and PSMA2 additionally interacts with INS (insulin precursor), EXOSC4 (exosome component 4 involved in RNA degradation), SBDS (Shwachman-Bodian-Diamond syndrome), RPS19 (ribosomal protein S19), and RPL15 (ribosomal protein L15). P4HB is predicted to strongly interact with PDIA6, NNT (nicotinamide nucleotide transhydrogenase), and VCP (valosin-containing protein). PSMA2 strongly interacts with other proteasomes and

Gene Ontology and Enrichment Analysis

Gene ontology analyses of the up-regulated and downregulated SHs were performed to map the genes involved in different cellular processes including biological and functional events (Figure 3). A large number of biological process GO terms were identified. The up-regulated MDCK SHs were 2229



Article

Figure 3. Gene ontology categories for ABPP-identified proteins. (A) Biological process; (B) molecular function; (C) cellular component. The number of proteins identified is indicated on the x-axes. GO categories represented by two or fewer proteins were filtered out. Proteins found significantly different between the A549 and MDCK cells are indicated: *p < 0.05; &p < 0.02; †p < 0.01.

Functional Characterization of SHs by Specific Inhibitors

VCP in the network. ESD is a strongly up-regulated SH predicted to interact with other SHs such as CES1 and NCEH1, which is strongly down-regulated. HTRTA2 is a pronounced down-regulated SH that interacts with LACTB and UCHI1 (ubiquitin thiolesterase). Differentially expressed SHs were analyzed by using the KEGG pathway to determine cellular pathways impacted by IAV. The proteasome was the most significant pathway regulated differentially by infection in both MDCK and A549 cells. Ether lipid metabolism, proteasome, and glycerophospholipid metabolism were more highly down-regulated in A549 cells infected with PR8 except for PSMA2, which was upregulated. Strong up-regulation of the proteasome was only found in MDCK cells.

The above results suggest multiple SHs are activated by influenza virus infection. We then assessed the roles that SH inhibitors might have on these SHs and on IAV infection. Since we were examining SH inhibitors and the SH trypsin is routinely used in cell culture to facilitate viral HA maturation, we tested replication of our virus in the presence and absence of both trypsin and DMSO (Figure 5A). These assays confirmed that MDCK cells possess sufficient proteases to allow PR8 replication in the absence of exogenously added protease. We used two SH inhibitors, Aprotinin (Roche Applied Science, Germany) and Pefabloc SC Plus (Roche Applied Science; AEBSF). Cell viability assays, using the WST1 reagent, indicated that MDCK cells tolerated doses of Aprotinin up to 1 mg/mL with no apparent cytotoxicity (Figure 5B). An inhibitory concentration (IC50) value could 2230



Article

Figure 4. Overlay of ABPP, RNAi, and SILAC results from influenza virus-infected A549 cells. The network was constructed using STRING (default mode). Red arrows indicate genes whose knockdown protects infected cells from influenza virus infection, as identified in refs 35 and 85. STRING does not provide overlay regulation data, but significantly regulated serine hydrolase activities are indicated by red text if up-regulated ≥ 2-fold or in green text if down-regulated ≥ 2-fold and circled in red, as determined by SILAC-ABPP (see Table 4). Noninteracting proteins and a few proteins at the periphery of the interactions were removed from this map for clarity.

not be determined for Aprotinin because no achievable Aprotinin concentration demonstrated toxicity. MDCK cells also tolerated relatively high doses of Pefablock (Figure 5C). Cell viability was not reduced below 100% until the Pefablock concentration exceeded 100 μg/mL. The Pefablock IC50 was estimated as ∼200 μg/mL. However, these concentrations had dramatic effects upon IAV replication, with the higher doses reducing IAV replication by 2(log10) or more. Comparisons of the cellular IC50 values with the IAV inhibition indicated that some of the viral inhibitory effects, particularly those induced by Pefablock, could be attributable to cellular toxicity (Figure 5D). We then assessed SH activity levels in Aprotinin- and Pefablock-treated cells. Global alterations were determined by visualizing FP-TAMRA-labeled bands after SDS-PAGE resolution (Figure 6A). Labeled bands that were clearly present in the nontreated, infected samples or in the low-dose Aprotinintreated samples, but absent in the higher-dose Pefablock-treated samples, were excised from the nontreated samples and identified as PSMA1, PSMA2, PSMA3, DPP4, and LONP1 by LC−MS. Levels of inhibition were then quantified by ABPPSILAC as above (Table 4). PSMA2 and PSMB3 inhibitions were 16.67- and 11.94-fold compared to nontreated DMSO

controls. LONP1 and DPP4, identified in the gel-based approach, were not detected by ABPP-SILAC. Knockdown of Proteasomal Components

The above results point to the probable importance of the proteasome. We then assessed the roles that proteasomal inhibitors might have on IAV infection and on selected proteasomal components. We used two proteasomal inhibitors, Bortezomib (Selleck Chemicals, Houston, Texas) and MG132 (Selleck Chemicals). Since both inhibitors are poorly soluble in aqueous solutions and stocks must be dissolved in DMSO vehicle, we initially assessed the viability of MDCK cells in DMSO. MDCK cells tolerated DMSO concentrations up to 2%, with an estimated IC50 value of ∼3.3% DMSO (Figure 5E). Thus, stock dilutions of these inhibitors were prepared such that the final DMSO concentrations did not exceed 1%. PR8 replication was unaffected by Bortezomib concentrations up to 0.01 μM but was inhibited >100-fold by concentrations of 0.1 μM (Figure 5E). However, the compound also was highly toxic to MDCK cells, with an IC50 estimated to be 8.8 nM. Thus, Bortezomib appears to have no practical utility to inhibit IAV replication in this in vitro system. By contrast, MDCK cells tolerated higher MG132 doses. The MDCK IC50 was estimated 2231



Article

Figure 5. Virus replication in MDCK cells treated with (A) trypsin and/or DMSO compared to cells not treated. The amount of input virus is indicated by the dashed horizontal line. Effect of (B) Aprotinin, (C) Pefablock, (E) Bortezomib, and (F) MG132 on production of infectious PR8 virus in MDCK cells. MDCK cells were pretreated with the indicated concentrations of SH or proteasomal inhibitors for 4 h before infection with PR8 at MOI = 0.01. After virus adsorption, cells were overlaid with media that contained the indicated concentrations of inhibitor and incubated at 37 °C. Virus was harvested at 42 hpi, and viral titers were determined. Results are displayed as a relative titer, with infectious progeny virus produced at each inhibitor concentration expressed as a proportion of virus produced in the untreated controls (indicated by ∇). Cell viability, determined by WST-1 assay after 48 h of treatment with indicated concentrations of inhibitors is indicated by ●, and cell viability of DMSO-treated controls is indicted by ○ in panel E. The data represent the average of a minimum of two experiments (for virus replication) or five data points (for WST assay), and the error bars represent standard errors of the means. Selectivity indices (ratio of virus inhibition to cell toxicity) were calculated for SH inhibitors (D) and proteasomal inhibitors (G). 2232



Article

Figure 6. (A) MDCK cells infected with PR8 at MOI of 5 for indicated periods of time (top) were treated with indicated SH inhibitors (top), harvested, and lysed, and FP-TAMRA-labeled proteomes were resolved by SDS-PAGE and visualized by fluorescence image documentation system. The indicated inhibited SHs (indicated by arrows) were identified by LC−MS. (B) Western blot analyses of PSMA2 after treatment of MDCK cells with indicated concentrations of SH and proteasomal inhibitors for 48 h. (C) Densitometric quantitation of PMSA2 after treatment with indicated SH and proteasomal inhibitors was performed with an Alpha Innotech FluorChem Imaging System and normalized to the β-actin signal in the respective lanes. (D) Western blot analyses of PSMA2 after treatment of A549 cells with nothing (None) or 25 nM concentrations of indicated Dharmicon SMARTPool siRNAs for 48 h and infected, or not, with PR8 at MOI = 3 for 24 h. MDCK cells were similarly treated, but with 75nM siRNAs. (E) β-Actin-normalized densitometric quantitation of the amount of PSMA2 protein in Nontreated cells either Mock-infected or infected with PR8 (from gel images as in panel D, left-most pair of lanes). (F) β-Actin-normalized densitometric quantitation of PMSA2 KD (from images as in panel D, center and right-most pairs of lanes) and virus production, compared to N-Si controls.

∼95% nucleotide identity. We initially confirmed our antiPSMA2 antibodies would recognize canine proteins (Figure 6B left-most lanes and data not shown). We chose various doses of each inhibitor, corresponding approximately to little or no effect upon cell viability or virus replication (low dose); a dose near the cell IC50 or, in the case of Aprotinin, that reduced PR8 replication ∼10-fold (medium dose); or a dose that reduced cell viability below 20% or, in the case of Aprotinin, that attenuated PR8 replication >100-fold (high dose). None of the tested Aprotinin or Bortezomib concentrations significantly

as 0.57 μM, a concentration that inhibits PR8 replication more than 30-fold (Figure 5F,G). The above activity-based studies indicated that several proteasomal SH enzymatic activities were significantly reduced by SH inhibitors. To determine whether this reduction was caused by overall reduction in quantities of enzymes or whether the enzymes were less active, we assessed the amount of various proteasomal subunits by quantitative Western blotting after treatment with various inhibitors. BLAST analyses indicated the canine and human PSMA2 protein shares 100% identity and 2233



Article

based approach provides for rapid screening, assessment of activity levels is hampered by the fact that low-abundant SHs are usually masked by co-migrating higher abundant proteins. To overcome this, a general modification is to apply avidin- or streptavidin-bead-based selective purification schemes to reduce or eliminate the higher abundant proteins. We then coupled this strategy with SILAC-labeling for sensitive and quantitative detection and measurement of active SHs in both cell types. Functional and network analysis of altered SHs identified a large list of cell metabolic and biological pathways that were affected by IAV infection. By studying the difference in networks generated from human and nonhuman cell lines, we examined how the host cell’s response to infection changed in two nonrelated host environments. The top functions that were most affected by the differentially regulated proteins in both cell lines included catalytic and hydrolytic activity and catabolism and metabolism in various biological processes. Many of these SHs interact with numerous other non-SH proteins identified as either up-regulated or down-regulated, indicating that these types of approaches are complementary and can serve to focus subsequent analyses on a small, manageable number of key molecules. Most of the identified up-regulated and down-regulated SHs were of low molecular weight (24−30 kDa). The proteasome was revealed as one of the most significant pathway systems differentially regulated by IAV infection in both MDCK and A549 cells. Several protein disulfide isomerases and other SHs were also differentially functionally regulated.

Table 4. Identified Serine Hydrolases and Their SILAC Ratios (Infected:Mock) in PR8-Infected MDCK Cells ± Pefablock Treatment serine hydrolase Fatty acid synthase Pro-cathepsin H Proteasome subunit alpha type-2 Proteasome subunit alpha type-3 Proteasome subunit alpha type-6 isoform 2 Proteasome subunit beta type-3 Protein disulfide-isomerase A4 isoform 3 Protein disulfide-isomerase A6

gene symbol

no inhibition controla

Pefablocka (fold inhibition)

FASN CTSH PSMA2

1.36 0.74 1.5

0.18 (7.56) 0.39 (1.89) 0.09 (16.67)

PSMA3

1.15

0.25 (4.6)

PSMA6

1.07

0.15 (7.13)

PSMB3

2.03

0.17 (11.94)

PDIA4

1.15

0.21 (5.47)

PDIA6

1.31

0.38 (3.45)

a

Values represent SILAC Infected:Mock ratios under indicated inhibition conditions

affected the quantities of PSMA2, whereas medium MG132 and high Pefablock concentrations reduced PSMA2 >2.5-fold (Figure 6C). To more specifically assess the role of PSMA2, it was knocked down with siRNA (Figure 6D). When non-siRNAtreated cells were infected with PR8, there was a slight increase of about 45% in the amount of PSMA2 protein in the infected cells (Figure 6E). Preliminary titration experiments indicated that ≥10nM was sufficient to knock down PSMA2 in A549 cells >5-fold, whereas ≥50nM was needed to KD the protein in MDCK cells. Thus, concentrations of 25 and 75nM were used for A549 and MDCK cells, respectively. These concentrations resulted in 10-fold KD in A549 and almost 5-fold KD in MDCK cells, respectively, compared to N-Si controls (Figure 6F). These KD levels also resulted in 2.5-fold attenuation of PR8 replication in A549 cells and about 5-fold attenuation of PR8 replication in MDCK cells (Figure 6F).

Proteasomal Subunits Are Affected by IAV Infection

The proteasomal alpha and beta subunits are involved in numerous virus−host interactions (reviewed in refs 38−41). The apparent quantity of PSMA2 was only moderately upregulated (∼45%) by infection in both A549 and MDCK cells. However, the activity-based screen suggested a larger increase in the relative activity of the enzyme. Similarly, treatment of MDCK cells with 250 μM Pefablock caused approximately a 3fold decrease in the amount of PSMA2 (Figure 6B,C) but a 16fold reduction in FP-TAMRA-measurable activity (Figure 6A, Table 4). Thus, ABPP and immunoblotting provide complementary approaches to assess the relative activities and amounts of the classes of enzymes targeted by the probes. PSMA2 is upregulated by a variety of other viral infectious diseases, including viral infections of the central nervous system, orthopoxvirus, measles, cytomegalovirus infection, and disease due to lentivirus.42 The ubiquitin−proteasome system is the primary mechanism for intracellular, extralysosomal protein degradation. It plays a key role in a variety of cell functions, including cell cycle regulation, antigen processing, apoptosis, signal transduction, and transcriptional regulation.43 Members of multiple families of both RNA and DNA viruses have been shown to modulate the ubiquitin−proteasome system to their advantage for a variety of reasons, including immune evasion, viral entry or release, transcriptional regulation, and apoptosis inhibition.44−46 The ubiquitin−proteasome system has been shown to facilitate entry or transport of incoming influenza virus,47 mouse minute virus,48 and murine coronavirus.49 Additionally, proteasome inhibition markedly reduces coxsackievirus group B3 viral RNA and protein synthesis levels, resulting in a decrease in the release of progeny virus.50 Inhibition of proteasome activity was shown to impair West Nile Virus genome amplification.51 Another report indicated that coxsackievirus-induced activation of the extracellular signal-

■

DISCUSSION A growing number of studies have used quantitative proteomic approaches to assess how proteins are affected by various stressors. For example, quantitative methods such as 2DIGEMS/MS,36 iTRAQ,37 and SILAC4,33 have been used to measure absolute quantities of various host proteins after IAV infection of various cell types. However, while these types of studies provide some indication of how the quantities of host proteins may be modulated by virus infection, they do not provide information about the relative functional activities of various proteins. Thus, the main goal of this study was to monitor and measure changes in host cell SH activities during IAV infection. The global screening of altered SHs after PR8 infection was performed in MDCK cells, in which IAV generally grows very well, and in A549 lung cells, the generally accepted most relevant cultured human cell line, in order to better define functional host cellular proteome responses. The FP-TAMRAbased gel approach provided a quick screen for determining alteration of host cell SHs after virus infection. Most alterations were observed after 6 h in MDCK cells and by 42 h in A549 cells at MOI of 5 indicating SH activity changes at a very late stage of IAV infection in human cells. In previous studies, quantitative proteome alterations were reported as early as 12 hpi and 48 hpi for MDCK and A549 cells.3,4 While the gel2234



Article

multifunctional proteins with thiol-disulfide redox-isomerase activities that catalyze the rearrangement of -S-S- bonds in proteins. PDI inhibitors significantly reduced HIV-1 entry in human monocyte-derived macrophages70 and lymphocytes.71 PDIA1 and PDIA4 activity might catalyze cysteine oxidation and disulfide bond isomerization of HCV proteins and is essential for HIV infectivity.72 Esterase D (ESD), or S-formyl glutathione hydrolase, is strongly up-regulated in A549 cells but slightly down-regulated in MDCK cells. An ESD mutant of Neisseria gonorrheae is susceptible to nitrite and to Snitrosoglutathione.73 Thus, ESD may play roles in intracellular pathogen regulation. Acyl-coenzyme A thioester hydrolase (ACOT 7), which plays important cellular roles in mammalian fatty acid metabolism through modulation of cellular concentrations of activated fatty acyl-CoAs,74 and Signal peptidase complex catalytic subunit (SPC18), which regulates growth of Plasmodium falciparum,75 were also up-regulated. HTRA2 is a serine hydrolase that promotes or induces cell death.76 Cytomegalovirus modulates this protein in late phase replication events,77 and viral proteins can inhibit HTRA2 in a cytokine-induced cell death response.78 Acyl-protein thioesterase 1 and 2 were down-regulated in A549 cells. Acyl protein thioesterases are involved in cellular fatty acid metabolism process. Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43, which is induced in Herpes simplex virus infection.79 Platelet activating factor acetylhydrolase (PAF-AH) beta and gamma subunits were down-regulated in A549 cells. PAF-AH activity is found to be significantly lower in HCV patients.80 Collectively, these assays have identified numerous SHs that are affected by IAV infection. These proteins may be attractive therapeutic targets for anti-influenza strategies and additional work to delineate their roles in IAV infection is warranted.

regulated kinase (ERK) pathway, which is required for its replication,52 is reduced following proteasome inhibition,53 suggesting a possible mechanism of proteasome action. The ubiquitin−proteasome pathway also appears to be a critical factor in the lifecycle of HIV. Efficient processing of Gag polyprotein and subsequent release and maturation of HIV particles is dependent upon ubiquitin−proteasome activities.54,55 Many of these proteasomal proteins interact with numerous other proteins that also play key roles in cell regulation and in viral infection. For example, PSMA2 interacts with insulin signaling pathways and MAPK signaling pathways. Insulinmediated PI3K/Akt and MAPK signaling pathways inhibit TLR3-mediated human bronchial epithelial cell apoptosis in viral infection.56 An increasing number of viruses have been found to gain control of key cellular signaling pathways including PI3K/Akt and MAPK/Erk1/2. For example, the PI3K/Akt pathway is found to be required for the efficient replication of influenza A virus, human cytomegalovirus, and Juniń virus.57−59 Furthermore, activation of MAPK/Erk1/2 pathway occurred during infections by coxsackievirus B3, HIV1, hepatitis B virus, Kaposi’s sarcoma-associated herpesvirus, and human cytomegalovirus.60,61 Some of these proteins also interact with VCP, the ATPase p97/valosin-containing protein. The activity of VCP is reported to be required for proteasomal degradation of adenovirus62 where VCP may mediate ATP hydrolysis-driven disassembly and/or partial unfolding of the adenovirus capsid, enabling the 19S regulatory particle to pass capsid components (and the associated antibody) into the 20S core particle for degradation. VCP is essential for TRIM21mediated virus neutralization.62 In poliovirus infection VCP is required for viral RNA replication among membrane trafficking proteins and provides a novel link between cellular protein secretion and viral RNA replication.63 PSMB3 strongly interacts with USP14 (ubiquitin specific peptidase 14), a deubiquitinating enzyme. Proteasome activity is enhanced while inhibiting USP14 by a small-molecule inhibitor of USP14,64 resulting in antiviral effects.65,66 Cell culture studies suggest that inhibiting USP14 could help prevent RNA virus infection. Ubiquitin C-terminal hydrolase-L1 and -L5 (UCH-L1 and UCH-L5) are deubiquitinating enzymes that catalyze the hydrolysis of polyubiquitin precursors and small ubiquitin adducts. UCH-L1 has been detected in a variety of malignant and metastatic tumors and is up-regulated upon infection of B lymphocytes with Epstein−Barr virus.67,68 UCH-L1 is upregulated in coxsackievirus infection, and application of a proteasome inhibitor attenuates CVB3-induced myocardial injury in mice.69 However, the recent studies provided a pivotal role of the ubiquitin−proteasome system in viral infectivity. Alltogether it is speculated that ubiquitin− proteasome systems involving any or some of these proteins may be attractive therapeutic targets for anti-influenza strategies. However, more experiments are required to confirm the differential activities and to reveal their molecular mechanisms and biological functions. Further characterization of these enzymes will be required to confirm their differential activities and reveal their potential roles during IAV replication and infectivity.

SH Inhibitors Modulate IAV Infection

FP profiling also was used to select for specific inhibitors of SHs that can be further used in chemical knockout assays to address the biochemical and biological roles of the targeted hydrolases. Such an approach has been successfully applied to elucidate the biochemical function of the human serine hydrolase KIAA1363 in lipid metabolism.81 Our SH inhibition assay demonstrated that PR8 replication was strongly inhibited by both Aprotinin and Pefablock in MDCK cells at concentrations lower than the agents’ respective IC50 doses (Figure 5). In addition, PSMA1 and PSMA2 were strongly inhibited by Pefablock (Figure 6; Table 4). The inhibition of PSMB3 was not identified by the gel-based approach, but the complementary SILAC-ABPP approach indicated strong down-regulation (11.94-fold; Table 4). PSMA1 and PSMB6 were found to be up-regulated in the nontreated control but not present after inhibition, which suggests strong inhibition by Pefablock. On the basis of our studies and analysis it is speculated that manipulation of the ubiquitin−proteasome system involving PSMA1, PSMA2, PSMB6, and/or PSMB3 might result in inhibition of IAV reproduction. This was confirmed by using proteasomal inhibitors and by genetic knockdown. Indeed, the approach of treating influenza infections by enzyme inhibitors such as AEBSF,82 pAB,83 ε-aminocaproic acid or aprotinin84 has been reported. As proteasomes have chymotrypsin-like activity, the question of whether our SH inhibitors are directly involved in HA cleavage, or may be indirectly involved in activating zymogen(s) (pre- or pro-enzymes) that are supporting HA

Other Serine Hydrolases Also Are Affected by IAV Infection

Several other SHs were identified and will only be briefly discussed. Protein disulphide isomerases (PDIs) were also identified and measured in both cell types. The isomerases are 2235



Article

(5) Kroeker, A. L.; Ezzati, P.; Halayko, A. J.; Coombs, K. M. Response of primary human airway epithelial cells to Influenza infection − A quantitative proteomic study. J. Proteome Res. 2012, 11, 4132−4136. (6) Henness, S.; Perry, C. M. Orlistat: A review of its use in the management of obesity. Drugs 2006, 66 (12), 1625−1656. (7) Thornberry, N. A.; Weber, A. E. Discovery of JANUVIA (TM) (Sitagliptin), a selective dipeptidyl peptidase IV inhibitor for the treatment of type2 diabetes. Curr. Top. Med. Chem. 2007, 7 (6), 557− 568. (8) Kluge, A. F.; Petter, R. C. Acylating drugs: redesigning natural covalent inhibitors. Curr. Opin. Chem. Biol. 2010, 14 (3), 421−427. (9) Racchi, M.; Mazzucchelli, M.; Porrello, E.; Lanni, C.; Govoni, S. Acetylcholinesterase inhibitors: novel activities of old molecules. Pharmacol. Res. 2004, 50 (4), 441−451. (10) Bosch, F. X.; Garten, W.; Klenk, H. D.; Rott, R. Proteolytic cleavage of influenza-virus hemagglutinins - Primary structure of the connecting peptide between Ha1 and Ha2 determines proteolytic cleavability and pathogenicity of avian influenza-viruses. Virology 1981, 113 (2), 725−735. (11) Bottcher-Friebertshauser, E.; Freuer, C.; Sielaff, F.; Schmidt, S.; Eickmann, M.; Uhlendorff, J.; Steinmetzer, T.; Klenk, H. D.; Garten, W. Cleavage of influenza virus hemagglutinin by airway proteases TMPRSS2 and HAT differs in subcellular localization and susceptibility to protease inhibitors. J. Virol. 2010, 84 (11), 5605− 5614. (12) Garten, W.; Klenk, H. D. Cleavage activation of the influenza virus hemagglutinin and its role in pathogenesis. In Avian Influenza: Monographs in Virology; Karger: Basel, Switzerland, 2008; Vol. 27. (13) Falgout, B.; Pethel, M.; Zhang, Y. M.; Lai, C. J. Both nonstructural proteins Ns2b and Ns3 are required for the proteolytic processing of Dengue virus nonstructural proteins. J. Virol. 1991, 65 (5), 2467−2475. (14) Mukhopadhyay, S.; Kuhn, R. J.; Rossmann, M. G. A structural perspective of the Flavivirus life cycle. Nat. Rev. Microbiol. 2005, 3 (1), 13−22. (15) Chappell, K. J.; Stoermer, M. J.; Fairlie, D. P.; Young, P. R. Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies. J. Biol. Chem. 2006, 281 (50), 38448−38458. (16) Failla, C.; Tomei, L.; Defrancesco, R. Both Ns3 and Ns4a are required for proteolytic processing of hepatitis-C virus nonstructural proteins. J. Virol. 1994, 68 (6), 3753−3760. (17) Meylan, E.; Curran, J.; Hofmann, K.; Moradpour, D.; Binder, M.; Bartenschlager, R.; Tschopp, R. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 2005, 437 (7062), 1167−1172. (18) Chappell, K. J.; Stoermer, M. J.; Fairlie, D. P.; Young, P. R. West Nile Virus NS2B/NS3 protease as an antiviral target. Curr. Med. Chem. 2008, 15 (27), 2771−2784. (19) Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M. Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3 ′ nontranslated region are essential for virus replication in vivo. J. Virol. 2000, 74 (4), 2046−2051. (20) Cregar-Hernandez, L.; Jiao, G. S.; Johnson, A. T.; Lehrer, A. T.; Wong, T. A.; Margosiak, S. A. Small molecule pan-dengue and West Nile virus NS3 protease inhibitors. Antiviral Chem. Chemother. 2011, 21, 209−217. (21) Steuer, C.; Gege, C.; Fischl, W.; Heinonen, K. H.; Bartenschlager, R.; Klein, C. D. Synthesis and biological evaluation of alpha-ketoamides as inhibitors of the Dengue virus protease with antiviral activity in cell-culture. Bioorg. Med. Chem. 2011, 19 (13), 4067−4074. (22) Lee, L. Y.; Tong, C. Y. W.; Wong, T.; Wilkinson, M. New therapies for chronic hepatitis C infection: a systematic review of evidence from clinical trials. Int. J. Clin. Pract. 2012, 66 (4), 342−355. (23) Gao, M.; Matusickkumar, L.; Hurlburt, W.; Ditusa, S. F.; Newcomb, W. W.; Brown, J. C.; Mccann, P. J.; Deckman, I.; Colonno, R. J. The Protease of herpes-simplex virus type-1 is essential for

cleavage, or other as yet unknown steps in IAV replication will require further studies. Further characterization of these enzymes is required to confirm the differential activities and to reveal their molecular mechanisms and biological functions during IAV replication and infectivity. In conclusion, we have identified differentially activated enzyme activities of serine hydrolases in influenza virus-infected cells, measured the up- and down-regulation of many of these enzymatic activities, and identified several specific proteasomal subunits, including PSMA1, PSMA2, PSMB3, and PSMB6, that may be inhibited and that appear to modulate influenza virus replication. Further analysis of these targets may lead to additional antiviral therapeutic strategies.

■

AUTHOR INFORMATION

Corresponding Author

*Ph: 204.789.3976. Fax: 204.480.1362. E-mail: kevin.coombs@ med.umanitoba.ca. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS

This work was supported by grants MT-11630 and PAN-83159 from the Canadian Institutes of Health Research to K.M.C. The authors thank Neil Salter and Rakesh Patel for expert technical assistance, Dr. James House, Director, Animal Sciences for embryonated hens eggs in which some influenza virus stocks were grown, and members of the laboratory for critically reviewing the work.

■

ABBREVIATIONS ABPP, activity-based protein profiling; BCA, bicinchoninic acid; CPE, cytopathic effect; db-FP, desthiobiotin-fluorophosphonate; D-MEM, Dulbecco’s modified Eagle’s medium; FP, fluorophosphonate; H, SILAC Heavy media/label; hpi, hours post infection; IAV, influenza A virus; L, SILAC Light media/ label; MDCK, Madin-Darby canine kidney; MOI, multiplicity of infection; pAB, p-aminobenzamidine; PBS, phosphate buffered saline; PR8, influenza virus strain A/PR/8/34(H1N1); SH, serine hydrolase; SILAC, stable isotope labeling by amino acids in cell culture

■

REFERENCES

(1) Karlas, A.; Machuy, N.; Shin, Y.; Pleissner, K. P.; Artarini, A.; Heuer, D.; Becker, D.; Khalil, H.; Ogilvie, L. A.; Hess, S.; Maurer, A. P.; Muller, E.; Wolff, T.; Rudel, T.; Meyer, T. F. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 2010, 463, 818−822. (2) Konig, R.; Stertz, S.; Zhou, Y.; Inoue, A.; Hoffmann, H. H.; Bhattacharyya, S.; Alamares, J. G.; Tscherne, D. M.; Ortigoza, M. B.; Liang, Y.; Gao, Q.; Andrews, S. E.; Bandyopadhyay, S.; De Jesus, P.; Tu, B. P.; Pache, L.; Shih, C.; Orth, A.; Bonamy, G.; Miraglia, L.; Ideker, T.; Garcia-Sastre, A.; Young, J. A. T.; Palese, P.; Shaw, M. L.; Chanda, S. K. Human host factors required for influenza virus replication. Nature 2010, 463, 813−817. (3) Vester, D.; Rapp, E.; Gade, D.; Genzel, Y.; Reichl, U. Quantitative analysis of cellular proteome alterations in human influenza A virusinfected mammalian cell lines. Proteomics 2009, 9 (12), 3316−3327. (4) Coombs, K. M.; Berard, A.; Xu, W.; Krokhin, O.; Meng, X.; Cortens, J. P.; Kobasa, D.; Wilkins, J.; Brown, E. G. Quantitative proteomic analyses of influenza virus-infected cultured human lung cells. J. Virol. 2010, 84 (20), 10888−10906. 2236



Article

functional capsid formation and viral growth. J. Virol. 1994, 68 (6), 3702−3712. (24) Glorieux, S.; Favoreel, H. W.; Steukers, L.; Vandekerckhove, A. P.; Nauwynck, H. J. A trypsin-like serine protease is involved in pseudorabies virus invasion through the basement membrane barrier of porcine nasal respiratory mucosa. Vet. Res. 2011, DOI: 10.1186/ 1297-9716-42-58. (25) Law, K. M.; Smith, G. L. A vaccinia serine protease inhibitor which prevents virus-induced cell-fusion. J. Gen. Virol. 1992, 73, 549− 557. (26) Bahgat, M. M.; Blazejewska, P.; Schughart, K. Inhibition of lung serine proteases in mice: a potentially new approach to control influenza infection. Virol. J. 2011, DOI: 10.1186/1743-422X-8-27. (27) Cravatt, B. F. Activity-based proteomics: Applications for enzyme and inhibitor discovery. Mol. Cell. Proteomics 2009, S29−S29. (28) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 2010, 468 (7325), 790−795. (29) Everley, P. A.; Krijgsveld, J.; Zetter, B. R.; Gygi, S. P. Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research. Mol. Cell. Proteomics 2004, 3 (7), 729−35. (30) Graumann, J.; Hubner, N. C.; Kim, J. B.; Ko, K.; Moser, M.; Kumar, C.; Cox, J.; Scholer, H.; Mann, M. Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins. Mol. Cell. Proteomics 2008, 7 (4), 672−683. (31) Reina, J.; FernandezBaca, V.; Blanco, I.; Munar, M. Comparison of Madin-Darby canine kidney cells (MDCK) with a green monkey continuous cell line (Vero) and human lung embryonated cells (MRC5) in the isolation of influenza A virus from nasopharyngeal aspirates by shell vial culture. J. Clin. Microbiol. 1997, 35 (7), 1900−1901. (32) Feng, S. Z.; Jiao, P. R.; Qi, W. B.; Fan, H. Y.; Liao, M. Development and strategies of cell-culture technology for influenza vaccine. Appl. Microbiol. Biotechnol. 2011, 89 (4), 893−902. (33) Kroeker, A. L.; Ezzati, P.; Coombs, K. M.; Halayko, A. J. Influenza A infection of primary human airway epithelial cells upregulates proteins related to purine metabolism and ubiquitin-related signaling. J. Proteome Res. 2013, 12, 3139−3151. (34) Brown, E. G. Increased virulence of a mouse-adapted variant of Influenza A/Fm/1/47 virus is controlled by mutations in genome segments 4, 5, 7, and 8. J. Virol. 1990, 64 (9), 4523−4533. (35) Tran, A. T.; M.N., R.; Ranadheera, C.; Kroeker, A. L.; Cortens, J. P.; Opanubi, K. J.; Wilkins, J. A.; Coombs, K. M. Knockdown of specific host factors protects against influenza virus-induced cell death. Cell Death Dis. 2013, 4, e769. (36) Ohman, T.; Rintahaka, J.; Kalkkinen, N.; Matikainen, S.; Nyman, T. A. Actin and RIG-I/MAVS signaling components translocate to mitochondria upon influenza A virus infection of human primary macrophages. J. Immunol. 2009, 182 (9), 5682−5692. (37) Lietzen, N.; Ohman, T.; Rintahaka, J.; Julkunen, I.; Aittokallio, T.; Matikainen, S.; Nyman, T. A. Quantitative subcellular proteome and secretome profiling of influenza A virus-infected human primary macrophages. PLoS Pathog. 2011, 7 (5), e1001340. (38) Dielen, A. S.; Badaoui, S.; Candresse, T.; German-Retana, S. The ubiquitin/26S proteasome system in plant-pathogen interactions: a never-ending hide-and-seek game. Mol. Plant Pathol. 2010, 11 (2), 293−308. (39) Gustin, J. K.; Moses, A. V.; Fruh, K.; Douglas, J. L. Viral takeover of the host ubiquitin system. Front. Microbiol. 2011, 2, 161. (40) Choi, A. G.; Wong, J.; Marchant, D.; Luo, H. L. The ubiquitinproteasome system in positive-strand RNA virus infection. Rev. Med. Virol. 2013, 23 (2), 85−96. (41) Fehr, A. R.; Yu, D. Control the host cell cycle: Viral regulation of the anaphase-promoting complex. J. Virol. 2013, 87, 8818−8825. (42) Disease Atlas PMSA2 Gene, 2013 (http://www.ebi.ac.uk/gxa/ gene/P25787).

(43) Ciechanover, A. The ubiquitin-proteasome proteolytic pathway. Cell 1994, 79 (1), 13−21. (44) Banks, L.; Pim, D.; Thomas, M. Viruses and the 26S proteasome: hacking into destruction. Trends Biochem. Sci. 2003, 28 (8), 452−459. (45) Gao, G.; Luo, H. L. The ubiquitin-proteasome pathway in viral infections. Can. J. Physiol. Pharmacol. 2006, 84 (1), 5−14. (46) Shackelford, J.; Pagano, J. S. Targeting of host-cell ubiquitin pathways by viruses. Essays Biochem. 2005, 41, 139−156. (47) Khor, R.; McElroy, L. J.; Whittaker, G. R. The ubiquitinvacuolar protein sorting system is selectively required during entry of influenza virus into host cells. Traffic 2003, 4 (12), 857−868. (48) Ros, C.; Kempf, C. The ubiquitin-proteasome machinery is essential for nuclear translocation of incoming minute virus of mice. Virology 2004, 324 (2), 350−360. (49) Yu, G. Y.; Lai, M. A. C. The ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry. J. Virol. 2005, 79 (1), 644−648. (50) Luo, H. L.; Zhang, J. C.; Cheung, C.; Suarez, A.; McManus, B. M.; Yang, D. C. Proteasome inhibition reduces coxsackievirus B3 replication in murine cardiomyocytes. Am. J. Pathol. 2003, 163 (2), 381−385. (51) Gilfoy, F.; Fayzulin, R.; Mason, P. W. West Nile virus genome amplification requires the functional activities of the proteasome. Virology 2009, 385 (1), 74−84. (52) Luo, H. L.; Yanagawa, B.; Zhang, J. C.; Luo, Z. S.; Zhang, M.; Esfandiarei, M.; Carthy, C.; Wilson, J. E.; Yang, D. C.; McManus, B. M. Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. J. Virol. 2002, 76 (7), 3365−3373. (53) Wong, J.; Zhang, J. C.; Si, X. N.; Gao, G.; Luo, H. L. Inhibition of the extracellular signal-regulated kinase signaling pathway is correlated with proteasome inhibitor suppression of coxsackievirus replication. Biochem. Biophys. Res. Commun. 2007, 358 (3), 903−907. (54) Patnaik, A.; Chau, V.; Wills, J. W. Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (24), 13069−13074. (55) Strack, B.; Calistri, A.; Accola, M. A.; Palu, G.; Gottlinger, H. G. A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (24), 13063−13068. (56) Numata, T.; Araya, J.; Fujii, S.; Hara, H.; Takasaka, N.; Kojima, J.; Minagawa, S.; Yumino, Y.; Kawaishi, M.; Hirano, J.; Odaka, M.; Morikawa, T.; Nishimura, S. L.; Nakayama, K.; Kuwano, K. Insulindependent phosphatidylinositol 3-kinase/Akt and ERK signaling pathways inhibit TLR3-mediated human bronchial epithelial cell apoptosis. J. Immunol. 2011, 187 (1), 510−519. (57) Shin, Y. K.; Liu, Q.; Tikoo, S. K.; Babiuk, L. A.; Zhou, Y. Effect of the phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation. J. Gen. Virol. 2007, 88, 942−950. (58) Johnson, R. A.; Wang, X.; Ma, X. L.; Huong, S. M.; Huang, E. S. Human cytomegalovirus up-regulates the phosphatidylinositol 3-kinase (PI3-K) pathway: Inhibition of PI3-K activity inhibits viral replication and virus-induced signaling. J. Virol. 2001, 75 (13), 6022−6032. (59) Linero, F. N.; Scolaro, L. A. Participation of the phosphatidylinositol 3-kinase/Akt pathway in Junin virus replication in vitro. Virus Res. 2009, 145 (1), 166−170. (60) Yang, X. Y.; Gabuzda, D. Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway. J. Virol. 1999, 73 (4), 3460−3466. (61) Zheng, Y. Y.; Li, J.; Johnson, D. L.; Ou, J. H. Regulation of hepatitis B virus replication by the Ras-mitogen-activated protein kinase signaling pathway. J. Virol. 2003, 77 (14), 7707−7712. (62) Hauler, F.; Mallery, D. L.; McEwan, W. A.; Bidgood, S. R.; James, L. C. AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (48), 19733−19738. (63) Arita, M.; Wakita, T.; Shimizu, H. Valosin-containing protein (VCP/p97) is required for poliovirus replication and is involved in 2237



Article

cellular protein secretion pathway in poliovirus infection. J. Virol. 2012, 86 (10), 5541−5553. (64) Lee, B. H.; Lee, M. J.; Park, S.; Oh, D. C.; Elsasser, S.; Chen, P. C.; Gartner, C.; Dimova, N.; Hanna, J.; Gygi, S. P.; Wilson, S. M.; King, R. W.; Finley, D. Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 2010, 467 (7312), 179− 184. (65) Nag, D. K.; Finley, D. A small-molecule inhibitor of deubiquitinating enzyme USP14 inhibits Dengue virus replication. Virus Res. 2012, 165 (1), 103−106. (66) Perry, J. W.; Ahmed, M.; Chang, K. O.; Donato, N. J.; Showalter, H. D.; Wobus, C. E. Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response. PLoS Pathog. 2012, 8, No. e1002783. (67) Ovaa, H.; Kessler, B. M.; Rolen, U.; Galardy, P. J.; Ploegh, H. L.; Masucci, M. G. Activity-based ubiquitin-specific protease (USP) profiling of virus-infected and malignant human cells. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (8), 2253−2258. (68) Rolen, U.; Freda, E.; Xie, J. J.; Pfirrmann, T.; Frisan, T.; Masucci, M. G. The ubiquitin C-terminal hydrolase UCH-L1 regulates B-cell proliferation and integrin activation. J. Cell. Mol. Med. 2009, 13 (8B), 1666−1678. (69) Gao, G.; Zhang, J. C.; Si, X. N.; Wong, J.; Cheung, C.; McManus, B.; Luo, H. L. Proteasome inhibition attenuates coxsackievirus-induced myocardial damage in mice. Am. J. Physiol.: Heart Circ. Physiol. 2008, 295 (1), H401−H408. (70) Stantchev, T. S.; Paciga, M.; Lankford, C. R.; Schwartzkopff, F.; Broder, C. C.; Clouse, K. A. Cell-type specific requirements for thiol/ disulfide exchange during HIV-1 entry and infection. Retrovirology 2012, DOI: 10.1186/1742-4690-9-97. (71) Fenouillet, E.; Barbouche, R.; Courageot, J.; Miquelis, R. The catalytic activity of protein disulfide isomerase is involved in human immunodeficiency virus envelope- mediated membrane fusion after CD4 cell binding. J. Infect. Dis. 2001, 183 (5), 744−752. (72) Singaravelu, R.; Blais, D. R.; McKay, C. S.; Pezacki, J. P. Activitybased protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe. Proteome Sci. 2010, DOI: 10.1186/1477-5956-8-5. (73) Potter, A. J.; Kidd, S. P.; Edwards, J. L.; Falsetta, M. L.; Apicella, M. A.; Jennings, M. P.; McEwan, A. G. Esterase D is essential for protection of Neisseria gonorrhoeae against nitrosative stress and for bacterial growth during interaction with cervical epithelial cells. J. Infect. Dis. 2009, 200 (2), 273−278. (74) Kirkby, B.; Roman, N.; Kobe, B.; Kellie, S.; Forwood, J. K. Functional and structural properties of mammalian acyl-coenzyme A thioesterases. Prog. Lipid Res. 2010, 49 (4), 366−377. (75) Tuteja, R.; Pradhan, A.; Sharma, S. Plasmodium falciparum signal peptidase is regulated by phosphorylation and required for intraerythrocytic growth. Mol. Biochem. Parasitol. 2008, 157 (2), 137−147. (76) Bartke, T.; Pohl, C.; Pyrowolakis, G.; Jentsch, S. Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase. Mol. Cell 2004, 14 (6), 801−811. (77) McCormick, A. L.; Roback, L.; Wynn, G.; Mocarski, E. S. Multiplicity-dependent activation of a serine protease-dependent cytomegalovirus-associated programmed cell death pathway. Virology 2013, 435 (2), 250−257. (78) Ma, X.; Kalakonda, S.; Srinivasula, S. M.; Reddy, S. P.; Platanias, L. C.; Kalvakolanu, D. V. GRIM-19 associates with the serine protease HtrA2 for promoting cell death. Oncogene 2007, 26 (33), 4842−4849. (79) Henken, D. B.; Goldstein, M. E.; Zhang, Q. L.; Curtis, R. Neuronal sprouting in mouse sensory ganglia infected with herpessimplex virus type-2 (Hsv-2) - Induction of growth-associated protein (Gap-43) and ultrastructural evidence. J. Neurovirol. 1995, 1 (2), 157− 164. (80) Caini, P.; Guerra, C. T.; Giannini, C.; Giannelli, F.; Gragnani, L.; Petrarca, A.; Solazzo, V.; Monti, M.; Laffi, G.; Zignego, A. L. Modifications of plasma platelet-activating factor (PAF)-acetylhydrolase/PAF system activity in patients with chronic hepatitis C virus infection. J. Viral Hepatitis 2007, 14 (1), 22−28.

(81) Kaschani, F.; Gu, C.; Niessen, S.; Hoover, H.; Cravatt, B. F.; van der Hoorn, R. A. L. Diversity of serine hydrolase activities of unchallenged and Botrytis-infected Arabidopsis thaliana. Mol. Cell. Proteomics 2009, 8 (5), 1082−1093. (82) Walsmann, P.; Richter, M.; Markward, F Inactivation of trypsin and thrombin by 4-amidinobenzolsulfofluoride and 4-(2-aminoethyl)benzolsulfofluoride. Acta Biol. Med. Ger. 1972, 28 (4), 577−585. (83) Llanos, M.; Vigil, P.; Salgado, A. M.; Morales, P. Inhibition of the acrosome reaction by trypsin-inhibitors and prevention of penetration of spermatozoa through the human zona-pellucida. J. Reprod. Fertil. 1993, 97 (1), 173−178. (84) Zhirnov, O. P.; Ovcharenko, A. V.; Bukrinskaya, A. G. Suppression of influenza-virus replication in infected mice by protease Inhibitors. J. Gen. Virol. 1984, 65 (Jan), 191−196. (85) Tran, A. T.; Cortens, J. P.; Du, Q.; Wilkins, J. A.; Coombs, K. M. Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation. J. Virol. 2013, 87 (2), 1049−1060.

2238


Proteasomal Serine Hydrolases Are Up-Regulated by and Required

Recommend Documents