[This corrects the article DOI: 10.1371/journal.pone.0000312.].

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, are chronic diseases of the gastrointestinal tract, with an unknown etiology, that affect over 6.8 million people worldwide. To characterize disease pathogenesis, proteomic and bioinformatic analyses were performed on colon biopsies collected during diagnostic endoscopy from 119 treatment-naïve pediatric patients, including from 78 IBD patients and 41 non-IBD patients who served as controls. Due to the presence of noninflamed and/or inflamed regions in IBD patients, up to two biopsies were obtained from IBD patients as compared to a single noninflamed biopsy from non-IBD pediatric control patients. Additional biopsies were obtained and analyzed from 33 of the IBD patients after IBD-directed therapeutic intervention for comparison of pre- and post-treatment proteomes. SuperSILAC was utilized to perform quantitative analysis of homogenized tissues, which were processed by filter-aided sample preparation. Hierarchical clustering and principal component analyses revealed proteomic patterns that distinguished inflamed from noninflamed tissues independent of therapy. Gene ontology revealed that proteins downregulated in inflammation are associated with metabolism, whereas upregulated proteins contribute to protein processing. A comparison of pre- and post-treatment proteomes from CD patients identified over 100 proteins that are significantly different between patients who responded and those who did not respond to therapy, including creatine kinase B and basigin.

Alterations in gut microbiota have been implicated in the pathogenesis of inflammatory bowel disease (IBD), however factors that mediate the host-microbiota interactions remain largely unknown. Here we collected mucosal-luminal interface samples from a pediatric IBD inception cohort and characterized both the human and microbiota proteins using metaproteomics. We show that microbial proteins related to oxidative stress responses are upregulated in IBD cases compared to controls. In particular, we demonstrate that the expression of human proteins related to oxidative antimicrobial activities is increased in IBD cases and correlates with the alteration of microbial functions. Additionally, we reveal that many of these human proteins are present and show altered abundance in isolated free extracellular vesicles (EVs). Therefore, our study suggests that the alteration of intestinal EV proteomes is associated with the aberrant host-microbiota interactions in IBD.

OBJECTIVE:Improved biomarkers are an unmet clinical need for suspected inflammatory bowel disease (IBD). Need is greatest for children, since current biomarkers suffers from low specificity, particularly in this population; thus, invasive testing methods, with the accompanying risk of complications, are necessary. Additionally, current biomarkers do not delineate disease extent assessment for ulcerative colitis (UC), a factor involved in therapeutic decisions. METHODS:Intestinal mucosal-luminal interface (MLI) aspirates from the ascending colon (AC) and descending colon (DC) were collected during diagnostic colonoscopy from treatment-naïve children. The MLI proteomes of 18 non-IBD and 42 IBD patients were analyzed by liquid chromatography mass spectrometry. Analyses of proteomic data generated protein panels distinguishing IBD from non-IBD and pancolitis from non-pancolitis (UC disease extent). Select protein biomarkers were evaluated in stool samples by enzyme-linked immunosorbent assay (n = 24). RESULTS:A panel of four proteins discriminated active IBD from non-IBD (discovery cohort) with a sensitivity of 0.954 (95% confidence interval (CI): 0.772-0.999) and >0.999 (95% CI: 0.824-1.00) for the AC and DC, respectively, and a specificity of >0.999 (AC, 95% CI: 0.815-1.00; DC, 95% CI:0.692-1.00) for both the AC and DC. A separate panel of four proteins distinguished pancolitis from non-pancolitis in UC patients with sensitivity >0.999 (95% CI: 0.590-1.00) and specificity >0.999 (95% CI: 0.715-1.00). Catalase (p < 0.0001) and LTA4H (p = 0.0002) were elevated in IBD stool samples compared to non-IBD stool samples. CONCLUSION:This study identified panels of proteins that have significantly different expression levels and contribute to accurate IBD diagnosis and disease extent characterization in children with UC. Biomarkers identified from the MLI demonstrate transferable results in stool samples.

Objective Accurate differentiation between Crohn's disease (CD) and UC is important to ensure early and appropriate therapeutic intervention. We sought to identify proteins that enable differentiation between CD and UC in children with new onset IBD.Design Mucosal biopsies were obtained from children undergoing baseline diagnostic endoscopy prior to therapeutic interventions. Using a super-stable isotope labeling with amino acids in cell culture (SILAC)-based approach, the proteomes of 99 paediatric control and biopsies of patients with CD and UC were compared. Multivariate analysis of a subset of these (n= 50) was applied to identify novel biomarkers, which were validated in a second subset (n= 49).Results In the discovery cohort, a panel of five proteins was sufficient to distinguish control from IBD-affected tissue biopsies with an AUC of 1.0 (95% CI 0.99 to 1.0); a second panel of 12 proteins segregated inflamed CD from UC within an AUC of 0.95 (95% CI 0.86 to 1.0). Application of the two panels to the validation cohort resulted in accurate classification of 95.9% (IBD from control) and 80% (CD from UC) of patients. 116 proteins were identified to have correlation with the severity of disease, four of which were components of the two panels, including visfatin and metallothionein-2.Conclusions This study has identified two panels of candidate biomarkers for the diagnosis of IBD and the differentiation of IBD subtypes to guide appropriate therapeutic interventions in paediatric patients.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTProteomic and Metaproteomic Approaches to Understand Host–Microbe InteractionsAmanda E. Starr*†, Shelley A. Deeke†, Leyuan Li†, Xu Zhang†, Rachid Daoud†, James Ryan†, Zhibin Ning†, Kai Cheng†, Linh V. H. Nguyen†, Elias Abou-Samra†, Mathieu Lavallée-Adam†, and Daniel Figeys*†‡§View Author Information† Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada‡ Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada§ Molecular Architecture of Life Program, Canadian Institute for Advanced Research, Toronto, Ontario, M5G 1M1, Canada*E-mail: [email protected]*E-mail: [email protected]Cite this: Anal. Chem. 2018, 90, 1, 86–109Publication Date (Web):October 23, 2017Publication History Published online9 November 2017Published inissue 2 January 2018https://doi.org/10.1021/acs.analchem.7b04340Copyright © 2017 American Chemical SocietyRIGHTS & PERMISSIONSACS AuthorChoiceArticle Views7700Altmetric-Citations31LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-AlertsSUBJECTS:Bacteria,Biological databases,Peptides and proteins,Protein identification,Proteomics Get e-Alerts

The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites) to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015) [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number http://www.ebi.ac.uk/pride/archive/projects/PXD002265.

Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-heavy protein/peptide ratio measurements were obtained using a high-resolution mass spectrometer and the quantitative proteomic software tool Census. We subsequently employed our approach to study the in vitro modulating effects of fructo-oligosaccharide and five different monosaccharides on the microbiota. Our methodology improves the accuracy of quantitative intestinal metaproteomics, which would promote the application of proteomics for functional studies of intestinal microbiota.

Intestinal microbial dysbiosis is associated with Crohn's disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host-microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis.

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6′ in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1′ (with a ~ 32–93% preference for leucine depending on the MMP), and basic and small residues in P2′ and P3′, respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1′ leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1′-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1′. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.

The lower risk of coronary artery disease in premenopausal women than in men and postmenopausal women implicates sex steroids in cardioprotective processes. β‐Estradiol upregulates liver low‐density lipoprotein receptor (LDLR), which, in turn, decreases circulating levels of low‐density lipoprotein, which is a risk factor for coronary artery disease. Conversely, LDLR protein is negatively regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9). Herein, we investigated PCSK9 regulation by β‐estradiol and its impact on LDLR in human hepatocarcinoma HuH7 cells grown in the presence or absence of β‐estradiol. Immunoblot analysis showed upregulation of LDLR at 3 μm β‐estradiol (140%), and the upregulation reached 220% at 10 μm β‐estradiol; only at the latter dose was an increase in LDLR mRNA detected by qPCR, suggesting post‐translational regulation of LDLR. No changes in PCSK9 mRNA or secreted protein levels were detected by qPCR or ELISA, respectively. β‐estradiol‐conditioned medium devoid of PCSK9 failed to upregulate LDLR. Similarly, PCSK9 knockdown cells showed no upregulation of LDLR by β‐estradiol. Together, these results indicate a requirement for PCSK9 in the β‐estradiol‐induced upregulation of LDLR. A radiolabeling assay showed a significant, dose‐dependent decrease in the ratio of secreted phosphoPCSK9 to total secreted PCSK9 with increasing β‐estradiol levels, suggesting a change in the functional state of PCSK9 in the presence of β‐estradiol. Our results indicate that the protein upregulation of LDLR at subtranscriptionally effective doses of β‐estradiol, and its supratranscriptional upregulation at 10 μm β‐estradiol, occur through an extracellular PCSK9‐dependent mechanism.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTBottom-Up Proteomics (2013–2015): Keeping up in the Era of Systems BiologyJanice Mayne, Zhibin Ning, Xu Zhang, Amanda E. Starr, Rui Chen, Shelley Deeke, Cheng-Kang Chiang, Bo Xu, Ming Wen, Kai Cheng, Deeptee Seebun, Alexandra Star, Jasmine I. Moore, and Daniel Figeys*View Author Information Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada, K1H8M5*Phone: 613-562-5800 ext. 8674. Fax: 613-562-5655. E-mail: [email protected]Cite this: Anal. Chem. 2016, 88, 1, 95–121Publication Date (Web):November 11, 2015Publication History Published online24 November 2015Published inissue 5 January 2016https://pubs.acs.org/doi/10.1021/acs.analchem.5b04230https://doi.org/10.1021/acs.analchem.5b04230review-articleACS PublicationsCopyright © 2015 American Chemical SocietyRequest reuse permissionsArticle Views3926Altmetric-Citations53LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Bacteria,Biological databases,Peptides and proteins,Protein identification,Proteomics Get e-Alerts

Cysteine cathepsins are primarily lysosomal proteases involved in general protein turnover, but they also have specific proteolytic functions in antigen presentation and bone remodeling. Cathepsins are most stable at acidic pH, although growing evidence indicates that they have physiologically relevant activity also at neutral pH. Post-translational proteolytic processing of mature chemokines is a key, yet underappreciated, level of chemokine regulation. Although the role of selected serine proteases and matrix metalloproteases in chemokine processing has long been known, little has been reported about the role of cysteine cathepsins. Here we evaluated cleavage of CXC ELR (CXCL1, -2, -3, -5, and -8) and non-ELR (CXCL9-12) chemokines by cysteine cathepsins B, K, L, and S at neutral pH by high resolution Tris-Tricine SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Whereas cathepsin B cleaved chemokines especially in the C-terminal region, cathepsins K, L, and S cleaved chemokines at the N terminus with glycosaminoglycans modulating cathepsin processing of chemokines. The functional consequences of the cleavages were determined by Ca2+ mobilization and chemotaxis assays. We show that cysteine cathepsins inactivate and in some cases degrade non-ELR CXC chemokines CXCL9-12. In contrast, cathepsins specifically process ELR CXC chemokines CXCL1, -2, -3, -5, and -8 N-terminally to the ELR motif, thereby generating agonist forms. This study suggests that cysteine cathepsins regulate chemokine activity and thereby leukocyte recruitment during protective or pathological inflammation.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTFine Tuning of Proteomic Technologies to Improve Biological Findings: Advancements in 2011–2013Janice Mayne, Amanda E. Starr, Zhibin Ning, Rui Chen, Cheng-Kang Chiang, and Daniel Figeys*View Author Information Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada K1H8M5*E-mail: [email protected]. Tel: 613-562-5800, ext. 8674. Fax: 613-562-5655.Cite this: Anal. Chem. 2014, 86, 1, 176–195Publication Date (Web):November 5, 2013Publication History Published online22 November 2013Published inissue 7 January 2014https://pubs.acs.org/doi/10.1021/ac403551fhttps://doi.org/10.1021/ac403551freview-articleACS PublicationsCopyright © 2013 American Chemical SocietyRequest reuse permissionsArticle Views2222Altmetric-Citations15LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Mass spectrometry,Peptides and proteins,Plasma,Protein identification,Proteomics Get e-Alerts

Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8-/- versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and validated evidence for the existence of the protease web, a network that affects the activity of most proteases and thereby influences the functional state of the proteome and cell activity.

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12−/− mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation.

Leukocyte migration and activation is orchestrated by chemokines, the cleavage of which modulates their activity and glycosaminoglycan binding and thus their roles in inflammation and immunity. Early research identified proteolysis as a means of both activating or inactivating CXC chemokines and inactivating CC chemokines. Recent evidence has shown activating cleavages of the monocyte chemoattractants CCL15 and CCL23 by incubation with synovial fluid, although the responsible proteases could not be identified. Herein we show that CCL15 is processed in human synovial fluid by matrix metalloproteinases (MMPs) and serine proteases. Furthermore, a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines revealed that each is precisely cleaved by one or more MMPs. By MALDI-TOF-MS, 149 cleavage sites were sequenced including the first reported instance of CCL1, CCL16, and CCL17 proteolysis. Full-length CCL15-(1-92) and CCL23-(1-99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini, respectively. Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding. Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.