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Systems Biology reveals NS4B-Cyclophilin A interaction: a new target to inhibit YFV replication Alessandra Vidotto, Ana T. S. Morais, Milene Rocha Ribeiro, Carolina C. Pacca, Ana C. B. Terzian, Laura H. V. G. Gil, Ronaldo Mohana-Borges, Philippe Gallay, and Mauricio L Nogueira J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00933 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 20, 2017

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Journal of Proteome Research

Systems biology reveals NS4B-Cyclophilin A interaction: a new target to inhibit YFV replication

Alessandra Vidotto1; Ana T. S. Morais1; Milene R. Ribeiro1; Carolina C. Pacca1; Ana C. B. Terzian1; Laura H. V. G. Gil2; Ronaldo Mohana-Borges3; Philippe Gallay4, Mauricio L. Nogueira1* 1

Laboratório de Virologia, Faculdade de Medicina de José do Rio Preto, São José do Rio Preto,

São Paulo 15090-000, Brazil. 2

Departamento de Virologia, Centro de Pesquisa Aggeu Magalhães, Fundação Oswaldo Cruz

(FIOCRUZ) - Recife, Pernambuco 50740-465, Brazil. 3

Laboratório de Genômica Estrutural, Instituto de Biofísica Carlos Chagas Filho, Universidade

Federal do Rio de Janeiro - UFRJ, Rio de Janeiro, RJ 21941-902, Brazil. 4

Department of Immunology & Microbial Science - The Scripps Research Institute - La Jolla,

San Diego, California 92037, United States. *e-mail: [email protected]

ABSTRACT Yellow fever virus (YFV) replication is highly dependent upon host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here, interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein which has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. Fifty-nine proteins were found to be significantly increased when compared to a negative control, and CypA

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exhibited the greatest difference, with a 22-fold change. Fisher’s exact test was significant for 58 proteins, and the p-value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified sixteen pathways, and this analysis indicated Sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using Sirolimus and Cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction. Keywords: Yellow Fever Virus, NS4B, Proteomics, Protein Interactions, Systems Biology, Cyclophilin A, Cyclosporine A and Sirolimus.

INTRODUCTION Flavivirus replication is highly dependent upon host cell factors

1-2

. However, the

mechanisms of cellular response to flavivirus infection and the proteomic profile of the infection are not completely known. Proteomics can be used to identify and quantify cellular and possibly viral proteins, and the protein profile seems to be altered in infected cells in vitro and in vivo in several cellular pathways 3-9. Proteomics have been utilized to study the interaction between virus and host cell proteins, as well as to determine differences in expression in infected cells

10-36

. The most

significant challenge of analyzing high-throughput screening data, as in proteomics, is to understand the data generated by discovery-driven studies. Nevertheless, this approach has produced scan data on the interactions between the YFV and the human host.


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Yellow fever is a disease caused by YFV, a prototype of the Flavivirus genus. YFV is composed of a single, 11-kb positive-strand RNA genome that encodes a single polyprotein, which is cleaved into three structural proteins - capsid (C), membrane (M) and envelope (E) -, and seven nonstructural proteins - NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 37-40. We focus on the NS4B protein due to its potential role in viral replication 41-43. The NS4B protein appears to play an important role in the formation of viral replication complexes and in neutralizing innate immune responses, including type I IFN signaling

44

, the formation of stress

granules 45, the unfolded protein response 46-47, and RNA interference 48. Studies indicate that NS4B may be involved in interferon inhibition in Dengue, West Nile, and Yellow Fever infections

49-50

. NS4B inhibits interferon (IFN)-induced STAT1

phosphorylation and nuclear translocation, thereby preventing the establishment of a cellular antiviral state by blocking the IFN-alpha/beta pathway

51

. This protein also participates in the

formation of hepatic tumors in infections caused by hepatitis C virus (HCV)

52

and in the

modulation of NS3 helicase activity 53. Liang et al found that, after the infection of human fetal neural stem cells, the ZIKV NS4B protein inhibits the Akt-mTOR signaling pathway, disrupting neurogenesis and inducing autophagy 54 . The NS4B protein has also been found to induce a specific membrane alteration that serves as a scaffold for the virus replication complex. This membrane alteration gives rise to what is known as the endoplasmic reticulum-derived membranous web, which contains the replication complex 55-56. NS4B is an hydrophobic protein with five integral transmembrane segments

57

and a

molecular mass of approximately 27 kDa 58 located in the perinuclear region of the endoplasmic reticulum membrane of cells infected by flavivirus

59-60

. Studies involving interactions between

NS4B and cellular proteins may help to understand the involvement of this protein in viral



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replication and in the pathogenesis of flavivirus infections, and they may eventually become a new target for drug design development. Although there is a vaccine against YFV, it remains a serious public health threat in endemic countries. The ongoing outbreak in Africa was detected in Angola in late December 2015, and has spread to Democratic Republic of Congo. Meanwhile China and Kenya have also recorded imported cases

61

. Thus, the NS4B protein may be an excellent target for the selective

inhibition of flavivirus replication. In order to identify host partners and pathways in which NS4B is involved, GST pulldown studies were performed challenging GST-NS4B against cellular extracts. Using onedimensional gel electrophoresis (1-DE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), the proteins identified were classified by their cellular roles. Importantly, we identified Cyclophilin A (CypA) or Peptidyl-prolyl cis-trans isomerase A (PPIA), as putative NS4B partners using systems biology. Supporting the hypothesis that NS4BCypA interactions play an important role in YFV replication, we found that the two CypA inhibitors - Sirolimus and Cyclosporine A (CsA) - efficiently reduce viral infection.

METHODS Cloning and Plasmid Construction After several attempts to work with the full NS4B, we divided the protein into cytoplasmic domains: Cit1 and Cit2-NS4B. The Cit1-NS4B plasmid was generated by cloning the region encoding NS4B cytoplasmic domains from pGBKT-7 (Clontech Laboratories, Mountain View, Califórnia, EUA) into the prokaryote expression vector pGEX-5X-1 (GE Healthcare, Little Chalfont, United Kingdom, UK) by PCR using the primer 5`- AAA CTC GAG CTC ATA AAG GGA AGG -3` (reverse, NS4B-Cit1-PGEX5X_XhoI). The Cit2-NS4B primer


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was 5`- TAT GGA TCC TCA CCG GCG TCC AGT TTT -3` (NS4B-BamHI-R). The PCR products were digested and cloned into the corresponding sites of the pGEX-5X-1 plasmid. All amplicons were sequenced using Big Dye v3.1 in an ABI3130 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

Protein Expression and Solubilization E. coli BL21(DE3) cells (Novagen) transformed with the plasmids Cit1-NS4B-pGEX-5X1, Cit1-NS4B-pGEX-5X-1, and pGEX-5X-1 (for Glutathione S-Transferase [GST] production) were incubated in 500 mL of Luria-Bertani (LB) medium in the presence of ampicillin (2.0 µg/mL) and chloramphenicol (1.5 µg/mL) at 37ºC. The cultures at were induced with isopropyl1-thio-β-D-galactopyranoside (IPTG) when IPTG reached an optical density of 0.6 (OD600) and at a final concentration of 1 mM for 16 h at 37oC. The harvested cells were centrifuged at 3000 g for 10 min at 4°C, washed in PBS 1X (pH 7.4), and suspended in lysis buffer (0.01 M Tris-HCl; 0.15 M NaCl; 0.001 M EDTA; pH 8.0) containing 1.4% sarkosyl, protease inhibitors (Complete Mini - Roche, Basileia, Switzerland), and 0.01 M DTT. The cell suspensions were lysed by adding lysozyme (Sigma-Aldrich, St. Louis, Missouri, USA) at a final concentration of 0.25 mg/mL for 30 min at 4°C and by sonication in a D100 sonic dismembrator (Fischer Scientific, Hampton, Nova Hampshire, USA) for 2 min at 0°C. After sonication, the lysates were incubated for 1 h at 0°C under gentle agitation. The lysates were then centrifuged at 16,000 g for 20 min at 4°C, and the samples were analyzed using 1-DE or SDS-PAGE.



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Purification Procedure and GST Pull-Down Assay Cit1-NS4B-pGEX-5X-1, Cit2-NS4B-pGEX-5X-1, and GST (pGEX-5X-1) were purified using a Glutathione SepharoseTM 4B affinity column (GE Healthcare, Little Chalfont, United Kingdom, UK). Beads were washed three times with PBS 1X (pH 7.4), and elution was performed with glutathione elution buffer. Eluted samples were analyzed using SDS-PAGE. The Cit1-NS4B-pGEX-5X-1, Cit2-NS4B-pGEX-5X-1, and GST fractions were dialyzed separately with PBS 1X (pH 7.4) and concentrated using an Amicon Ultra-15 centrifugal filter unit with Ultracel-10 membrane (Millipore, Billerica, MA, USA). Samples were quantified based on the Bradford method 62. Two mg of Cit1-NS4B-pGEX-5X-1 and 1 mg of Cit2-NS4B-pGEX-5X-1, which were matched with the equivalent amounts of GST, were incubated with 1.33 mL of Glutathione Sepharose 4B at 50% concentration (GE Healthcare, Little Chalfont, United Kingdom, UK). Two mg of proteins from HeLa cell extracts were mixed with GST beads and pulled down for 16 hours at 0oC under gentle agitation. Beads were then centrifuged at 1000 g for 5 min at 4°C and washed three times with PBS 1X (pH 7.4). In the initial portion of this study, we performed a GST-pull down assay on a smaller scale, with low NS4B, GST and HeLa cell production. In both experiments, bound material was eluted with glutathione elution buffer and analyzed using 1-DE.

In-Gel Protein Digestion and Mass Spectrometry (MS) After visual inspection of the SDS-PAGE and the comparison of the bands from the Cit1NS4B-GST and HeLa to those from the Cit2-NS4B-GST + HeLa and to those from the GST and HeLa cell extract we removed the differentially precipitated bands and gel slices for screening. A total of 61 sequential slices (25 squares and 36 lanes) were manually cut out from the gels. Most lines and squares were removed from the SDS-PAGE for protein screening, because we did not


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know what could be identified in the samples. But we removed the bands corresponding to GST, Cit1, and Cit2-NS4B for confirmation by mass spectrometry. Gel samples were destained in 250 µL of 50% acetonitrile (ACN)/25 mM ammonium bicarbonate and dehydrated with 50 µL of ACN for 15 min. The ACN was discarded, and the gel pieces were dried in a Speed-Vac concentrator (ThermoFisher, Waltham, MA, USA) for 30 min. A trypsin solution was added to each gel piece, and the sample was incubated for 24 h at 37oC. Peptides were extracted with 50 µL 1% trifluoroacetic acid (TFA) for 12 h and 50 µL 1% TFA/50% ACN for 2 h. Supernatants were mixed and concentrated in a vacuum centrifuge to 5-10 µL. Digested samples that had been removed from the one-dimensional gel were applied to a C18 RP-HPLC column coupled with a nanoUPLC (nanoAcquity)-electrospray tandem MS on a Q-TOF Ultima mass spectrometer (Waters Corporation, Milford, MA, USA) at a flow rate of 0.6 mL/min. The gradient was 0-50% ACN in 0.1% formic acid over 60 min. The instrument was operated in the “top three” mode, in which one MS spectrum is acquired, followed by MS/MS of the top three most intense peaks detected. The raw data files from the UPLC-tandem MS runs were processed using Mascot Distiller, version 2.3.2.0 and Mascot Daemon, version 2.3.0 (Matrix Science Ltd., London, UK). The parameters for spectrum acquisition were set as follows: Homo sapiens taxonomy; trypsin enzyme; NCBInr Database, one missed cleavage site; carbamidomethylation of cysteine and oxidation of methionine as modifications; peptide tolerance of 0.1 Da; MS/MS tolerance of 0.1 Da; monoisotopic masses; +2, +3 and +4 peptide charge; mascot generic data format; and an ESI-QUAD-TOF instrument. Uniprot Databases was used to assign biological process terms to differentially pulled-down proteins.


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Bioinformatics Analysis After obtaining the list of proteins identified from the 61 slices from SDS-PAGE, the data were organized using matching between samples. Two lists of differentially precipitated proteins were obtained by comparing the proteins identified in the pull-down Cit1-NS4B-pGEX-5X-1 + HeLa vs GST + HeLa and Cit2-NSAB-pGEX-5X-1 + HeLa vs GST + HeLa. In order to achieve subsequent functional studies, the biological processes of the identified proteins were analyzed, as were their functions and cellular locations. The Gene and Protein Databases

63-64

and the

Uniprot Database 51 were used to this end.

Scaffold 3.06 Software The spectra were acquired using the MassLynx software, version 4.1 (Waters Corporation, Milford, MA, USA), and the raw data files were converted to a peak list format (mgf) in the Mascot Distiller software, version 2.2.1.0 (Matrix Science Ltd., London, UK). After the search in the NCBI non-redundant database (NCBInr) using Mascot Daemon, version 2.2.0 (Matrix Science Ltd., London, UK), .dat files were obtained. These files are used for protein identification based on the Homo sapiens taxonomy. The search results of 1-DE were exported to the Scaffold software, version 3.06 (Proteome Software Inc., Portland, OR, USA) and visualized using a filter. The parameters were protein identification probability of > 99% and a minimum of two peptides. Briefly, proteins from the 61 gel slices from each pull down were grouped in three categories: Cit1-NSAB-pGEX-5X-1 + HeLa, Cit2-NSAB-pGEX-5X-1 + HeLa, and GST + HeLa. The normalized spectral counts were obtained for each protein, and proteins with a fold change of at least 1.5 were considered with differential abundance between the categories. Fold change was calculated using the statistical tests in the Scaffold 3.06 Software.


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Ingenuity Pathway Analysis (IPA) We use the IPA core analysis to organize and understand the large amount of data we obtained by MS. Using this approach within Ingenuity Systems, we identified 16 pathways with the list of proteins identified as differentially precipitated. The IPA Upstream Regulator can identify the cascade of upstream transcriptional regulators that can explain the observed changes in gene expression in a dataset and illuminate the biological activities occurring in the tissues or cells under study 65.

Virus and Viral Infection With the consent of Animal Studies Committee, newborn Swiss mice were obtained from the Aldolfo Lutz Institute of São Paulo. The YFV vaccine strain 17DD (FIOCRUZ, Brazil) were inoculated via intracerebral injection, and the newborn mice were monitored for 7 days.

Immunofluorescence Analysis After Ingenuity Systems indicated Sirolimus as a potential inhibitor of CypA in the upstream analysis, we performed the interaction validation of CypA with YFV NS4B. We also used CsA, a drug well known as a ligand for CypA, to validate the CypA-NS4B interaction. Vero E6 cells (8x104) were placed onto 24-well plates with cover glasses and incubated at 37oC with 5% CO2 for 24 h and supplemented with 10% fetal bovine serum (FBS). After incubation, the cells were challenged with YFV 17DD at a multiplicity of infection (MOI) of 10 and incubated at 37oC with 5% CO2 for 24 h and FBS-starved for 2 h. We tested five Sirolimus concentrations from 5 µM to 20 mM

66-70

and used CsA at 10 and 20 µM. Cells were washed

three times with PBS 1X and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then washed and incubated with PBS 1X containing 1% Triton X-100 (JT Baker,



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Phillipsburg, NJ, USA) for 5 min. Cells were blocked with PBS 1X containing 10% normal goat serum (Zymed Laboratories Inc, South San Francisco, CA, USA), incubated with an anti-NS4AB antibody (provided by Charles Rice, Rockfeller University) at a final dilution of 1:2000, and washed three times with PBS 1X. Cells were then incubated with a conjugated anti-rabbit antibody (AlexaFluor 488 Goat Anti-rabbit IgG, Molecular Probes, USA) at a dilution of 1:2000, washed with PBS 1X and ultrapure water, and incubated with DAPI for 5 min. Fluorescently labeled cells were visualized using a reflected fluorescence system (Olympus, Shinjuku, Tokyo, Japan), and images were processed in the Image-Pro Plus software (Media Cybernetics, Rockville, Maryland, USA).

Viral Plaque Reduction Assay We performed a viral plaque reduction assay to test the inhibitory action of Sirolimus and CsA in YFV infection of VERO E6 cells as previously described

71

, with minor modifications.

Vero E6 cells were plated into six-well microplates with 8x105 cells/well. After 24 h, cells were infected with 50 and 100 PFU of YFV 17DD in the presence of various concentrations of Sirolimus and CsA. After 1 h of adsorption at 37oC, the medium was removed. The cells were then overlaid with MEM and 1% carboxymethyl cellulose (CMC - Sigma-Aldrich, St. Louis, Missouri, USA) and incubated for 5 days at 37oC. The overlay medium was the removed, the cells were fixed and stained with crystal violet, and the plaques were counted.

BHK-21-rep-YFV17D-LucNeoIres and Luciferase Activity Assay BHK-21-rep-YFV17D-LucNeoIres cells contain a bicistronic dual-reporter YFV replicon expressing neomycin resistance and luciferase reporter genes. In this cell, the plasmid containing the YFV 17D strain replicon (pBSC-repYFV-17D) was handled for heterologous expression of


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the firefly luciferase reporter gene (repYFV-17D-Luc) 72.

Therefore, when this cell divides, the

YFV replicon is produced with emission of luciferase. Here,

BHK-21-rep-YFV17D-LucNeoIres

cells

were

incubated

with

various

concentrations of Sirolimus and CsA for 24 h. To measure luciferase activity, BHK-21-repYFV17D-LucNeoIres cells were washed with PBS and lysed through the addition of cell culture lysis buffer (Promega Corporation, Madison, Wisconsin, EUA) according to the manufacturer's instructions. Cell lysate was then centrifuged, and 20 µL of the resulting supernatant was mixed with 100 µL of luciferase substrate (Luciferase 1000 Assay System - Promega Corporation, Madison, Wisconsin, EUA) prior to the measurement of luciferase activity using a Sirius luminometer, version 3.2 (Berthold Detection Systems, Pforzheim, Germany).

RESULTS AND DISCUSSION Overexpression, Purification and GST Pull Down After several failed attempts to express and purify full-length NS4B, we divided the protein into cytoplasmic domains, which were cloned into a pGEX-5X-1 expression vector, successfully overexpressed in E. coli BL21 (DE3), and purified. In the GST-pull down assay, we observed various protein bands derived from HeLa cells, which were pulled down by cytoplasmic regions 1 and 2 of NS4B, but not by GST (Figure 1). The GST-pull down assay was performed in two steps: one experiment with low Cit1, Cit2-NS4B-pGEX-5X-1, GST, and HeLa cell production, and another GST-pull down assay with high protein production. We performed two GST-pull down assays with different amounts of protein mass for protein profile confirmation and for more effective identification by mass spectrometry.



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Bioinformatics Analysis We identified 136 differentially precipitated proteins in the manual matching performed in the test Cit1-NSAB-pGEX-5X-1 + HeLa compared to GST protein + HeLa. Of these, 130 were human proteins. In the pull down of Cit2-NSAB-pGEX-5X-1 + HeLa compared to GST protein + HeLa, 109 differentially precipitated proteins were identified, 100 of which were from Homo sapiens. Some of the proteins were found to coincide in both assays, as Cit1 and Cit2 are both cytoplasmic regions within the NS4B protein. In addition, there was an overlap between some identified protein families, which included histones, keratins, ubiquitins, tubulins, and ribosomal proteins. Therefore, there may have been false-positive identifications. An important finding was the identification of the YFV polyprotein, in addition to E. coli proteins, a finding which validated the experiment and encourages additional studies. According to the Gene Database, some proteins have been classified by their cellular roles, such as entry into host cell, initiation of viral infection, regulation of viral genome replication, viral transcription, cell-cell signaling, interferon signaling pathway, virus uncoating, response to the virus, blood coagulation signal transduction, defense response, platelet activation, RNA processing, translation, protein maturation, post-Golgi vesicle-mediated transport, cell proliferation, cell cycle checkpoint, DNA repair, actin cytoskeleton organization, apoptosis, protein ubiquitination, proteolysis, fatty acid transport, and glycolysis (Figure 2).



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In this first organization of the MS data, the taxonomy was not limited. A search of data in the non-redundant NCBI (NCBInr) was performed with "all entries." In this search, we identified human proteins, E. coli proteins (certainly nonspecific), and the YFV polyprotein. This result was important for validating our study, but we decided to perform a more refined search to identify only human proteins. Thus, for human protein identification, the MS/MS data were searched against the NCBInr using Mascot Distiller, version 2.2.1.0, and Mascot Daemon, version 2.2.0 (Matrix Science Ltd., London, UK). The parameters for spectrum acquisition were set as follows: Homo sapiens taxonomy; trypsin enzyme; one missed cleavage site; carbamidomethylation of cysteine and oxidation of methionine as modifications; peptide tolerance of 0.1 Da; MS/MS tolerance of 0.1 Da; and monoisotopic masses. The criterion for positive identification of proteins was the individual ion scores at p

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