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C4 RP-HPLC separation of A9 (retention time, 13.1 min; peak 1) and A9* (retention time 14 min; peak 2) from neutrophil cytosol. A, shown are the first 20 N-terminal amino acid residues of A9 and A9* protein sequences, depicting the start Met (Met 1 and Met 5 ). B, a representative chromatogram of one of five donors is shown. The deconvoluted mass spectrum of peak 1 (C) indicated a major component of 13,152 Da, corresponding to A9. The major component of the deconvoluted mass spectrum of peak 2 (D) indicated a molecular mass of 12,690 Da, corresponding to A9*. Components with masses of 13,169 Da and 12,705 Da likely correspond to oxidation of a single Met residue in A9 and A9*, respectively.
Source publication
Reactive oxygen species generated by activated neutrophils can cause oxidative stress and tissue damage. S100A8 (A8) and S100A9
(A9), abundant in neutrophil cytoplasm, are exquisitely sensitive to oxidation, which may alter their functions. Murine A8
is a neutrophil chemoattractant, but it suppresses leukocyte transmigration in the microcirculation...
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Background/Aim
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Methods
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Study Design
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Methods
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Citations
... When coexpressed, S100A8 and S100A9 spontaneously form homodimers and the heterodimer S100A8/A9 inside cells, with the latter having been the preferred form (10,11). There are two isoforms of human S100A9, full-length and truncated (D5-S100A9), with the latter generated by translation from an alternate start site at methionine 5. Truncated S100A9 accounts for ∼30% of total S100A9 in neutrophils and does not form heterodimers with S100A8 (13). Structural studies of S100 proteins indicate at least three recognition sites within two distinct surfaces that accommodate multiple binding partners, including the hinge domain between the Ca 21 -binding regions (14). ...
The study of S100A9 in viral infections has seen increased interest since the COVID-19 pandemic. S100A8/A9 levels were found to be correlated with the severity of COVID-19 disease, cytokine storm, and changes in myeloid cell subsets. These data led to the hypothesis that S100A8/A9 proteins might play an active role in COVID-19 pathogenesis. This review explores the structures and functions of S100A8/9 and the current knowledge on the involvement of S100A8/A9 and its constituents in viral infections. The potential roles of S100A9 in SARS-CoV-2 infections are also discussed.
... Human S100A9 is encoded by a single copy gene with two isoforms-full-length and -truncated S100A9-and is translated from an alternate start site at codon 4 of the full-length form and lacks the single Cys 3 residue, making it less susceptible to oxidation. In the studies of Lim and colleagues [112,113] the many pro-inflammatory functions described for S100A8 and S100A9, as well as their anti-inflammatory roles in wound healing and protection against excessive oxidative tissue damage, are discussed, and an explanation that oxidative modifications may act as a regulatory switch for the disparate functional roles of S100A8 and S100A9 is suggested. ...
In this review, we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. In the beginning, we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section, the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteomes. Three sections are dedicated to the description of glycation and enzymatic glycosylation. Citrullination and N- and C-terminal post-translational modifications (PTMs) and miscellaneous other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed throughout the sections of the manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.
... In the studies of Lim and coll. [112,113] the many pro-inflammatory functions described for S100A8 and S100A9, as well as its antiinflammatory roles in wound-healing and protec tion against excessive oxidative tissue damage, are discussed and an explanation that ox idative modifications may act as a regulatory switch for the disparate, functional roles of S100A8 and S100A9 is suggested. ...
In this review we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. At the beginning we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteome. Three sections are dedicated to the description of the glycation and enzymatic glycosylation. Citrullination and N- and C- terminal PTMs and a miscellaneous of other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed along the sections of manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.
... S100A8, S100A9 and MCP1 have the specific chemotactic activation, mediate intracellular inflammatory signal transduction pathways, 30 and play an important role in the regulatory of inflammation. 31 Moreover, they had extensive biological functions and participated in multiple biological processes. Hence, they are important proinflammatory cytokines. ...
Purpose
Our recent studies found a splice region mutation in C3 accompanied by a significantly increased C3 in psoriatic peripheral blood. Mesenchymal stem cells (MSCs) are a key immunological suppression cell. We further investigate the regulation of MSCs on C3 in psoriasis.
Patients and Methods
We analyzed the C3 and its upstream S100A9, S100A8 and downstream MCP1 in psoriatic and control skin, and in normal human epidermal keratinocytes (NHEKs) co-cultured with psoriatic versus control dermal-derived mesenchymal stem cells (DMSCs) by mRNA, iTRAQ (isobaric tags for relative and absolute quantitative) and simple Western analysis.
Results
The mRNA and Simple Western analysis showed that the expression of C3, S100A8 and S100A9 are upregulated in psoriatic lesion (C3: mRNA, 9.23-fold, p = 0.0092; protein, 3.56-fold, p = 0.0244. S100A8: mRNA, 28.35-fold, p = 0.0015; protein, 4.68-fold, p = 0.0215. S100A9: mRNA, 79.45-fold, p = 0.0066; protein, 12.42-fold, p > 0.05). Moreover, the iTRAQ showed that C3 and S100A9 were significantly increased in NHEKs after co-cultured with psoriatic DMSCs compared to that of control DMSCs (C3: 3.40-fold, p = 0, FDR = 0; S100A9: 2.30-fold, p = 9.86E-241, FDR = 6.50E-239), verified by Simple Western. However, the expression of S100A8 and MCP1 was slightly different between the two groups.
Conclusion
Our results suggest that psoriatic DMSCs contribute to the increased C3 expression in psoriatic lesion via upregulating S100A9, providing the theoretical basis for the role of C3 and DMSCs in the pathogenesis of psoriasis.
... Besides transcriptional regulation as discussed above, posttranslational modifications have been implicated in the function of S100a8 and/or S100a9. Post-translational modifications, including S-nitrosylation, S-gluthathionylation and phosphorylation, have been proposed to be differentially associated with pro-and antiinflammatory functions of S100a9 (64)(65)(66). Recent studies have identified dysregulated miR-146a-5p and miR-155-5p miRNAs as drivers of phosphorylated S100a8/a9 heterodimers and the production of proinflammatory cytokines in female rheumatoid arthritis patients (67). Both of these microRNAs have also been found to be dysregulated in SLE patients (68,69). ...
Systemic lupus erythematosus (SLE) is an autoimmune disorder disproportionally affecting women. A similar sex difference exists in the murine New Zealand Black/White hybrid model (NZBWF1) of SLE with all females, but only 30-40% of males, developing disease within the first year of life. Myeloid-derived suppressor cells (MDSCs) are prominent in NZBWF1 males and while depletion of these cells in males, but not females, promotes disease development, the mechanism of suppression remains unknown. S100a9, expressed by neutrophils and MDSCs, has previously been shown to exert immunosuppressive functions in cancer and inflammation. Here we investigated if S100a9 exerts immunosuppressive functions in NZBWF1 male and female mice. S100a9 +/+, S100a9 +/- and S100a9 -/- NZBWF1 mice were followed for disease development for up to 8 months of age. Serum autoantibody levels, splenomegaly, lymphocyte activation, glomerulonephritis and proteinuria were measured longitudinally or at the time of harvest. In accordance with an immunosuppressive function of MDSCs in male mice, S100a9-deficient male NZBWF1 mice developed accelerated autoimmunity as indicated by increased numbers of differentiated effector B and T cells, elevated serum autoantibody levels, increased immune-complex deposition and renal inflammation, and accelerated development of proteinuria. In contrast, female mice showed either no response to S100a9-deficiency or even a slight reduction in disease symptoms. Furthermore, male, but not female, S100a9-/- NZBWF1 mice displayed an elevated type I interferon-induced gene signature, suggesting that S100a9 may dampen a pathogenic type I interferon signal in male mice. Taken together, S100a9 exerts an immunosuppressive function in male NZBWF1 mice effectively moderating lupus-like disease development via inhibition of type I interferon production, lymphocyte activation, autoantibody production and the development of renal disease.
... Extracellular activity of S100A8 is controlled by oxidation and nitrosylation on its cysteine residue, leading to the formation of covalent bonds between monomers (27,28). A number of reports demonstrate that oxidized S100A8 exerts anti-inflammatory effects by inhibiting mast cell degranulation and cytokine secretion induced by FcεR cross-linking (28,30,70,71). Thus, we propose a model in which S100A8 is rapidly secreted during inflammatory reactions, thereby enhancing inflammatory responses until its oxidization by reactive oxygen species is produced at the site of inflammation. ...
High concentrations of the damage-associated molecular patterns S100A8 and S100A9 are found in skin and serum from patients suffering from psoriasis, an IL-17-related disease. Notably, although the expression of these proteins correlates with psoriatic disease severity, the exact function of S100A8 and S100A9 in psoriasis pathogenesis remains unclear. In this study, we investigated the role of S100A8 and S100A9 in psoriasis-associated skin hyperplasia and immune responses using S100a8-/- and S100a9-/- mice in an imiquimod-induced model of psoriasis. We found that S100a8-/- and S100a9-/- psoriatic mice exhibit worsened clinical symptoms relative to wild-type mice and increased expression of S100A9 and S100A8 proteins in keratinocytes, respectively. In addition, the loss of S100A8 enhances proliferation of keratinocytes and disrupts keratinocyte differentiation. We further detected elevated production of IL-17A and -F from CD4+ T cells in the absence of S100A8 and S100A9, as well as increased infiltration of neutrophils in the skin. In addition, treatment with anti-IL-17A and -F was found to reduce psoriasis symptoms and skin hyperplasia in S100a8-/- and S100a9-/- mice. These data suggest that S100A8 and S100A9 regulate psoriasis by inhibiting production of IL-17A and -F, thereby, to our knowledge, providing new insights into their biological functions.
... The first was found decreased in gingivitis patients compared to the healthy controls, while the latter was found increased [24]. It is common knowledge that GCF contains significant levels of reduced glutathione which is responsible for the local antioxidant capacity, typical of periodontally healthy subjects [83] and that glutathionylation of S100-A9 alters its ability to form complexes with S100-A8, to bind endothelial cells, and limits neutrophil migration in inflammatory lesions [84]. Based on these results, our group speculated that the glutathionylation in S100-A9 may result in a protective effect against oxidative process at the site of inflammation thus providing a coherent explanation to the observed reversed ratio between the m/z = 13,458 (both glutathionylated and acetylated) and the m/z = 13,153 (acetylated only) forms of S100-A9 peptide in gingivitis patients compared to healthy subjects [24]. ...
Given its intrinsic nature, gingival crevicular fluid (GCF) is an attractive source for the discovery of novel biomarkers of periodontal diseases. GCF contains antimicrobial peptides and small proteins which could play a role in specific immune-inflammatory responses to guarantee healthy gingival status and to prevent periodontal diseases. Presently, several proteomics studies have been performed leading to increased coverage of the GCF proteome, however fewer efforts have been done to explore its natural peptides. To fill such gap, this review provides an overview of the mass spectrometric platforms and experimental designs aimed at GCF peptidome profiling, including our own data and experiences gathered from over several years of matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) based approach in this field. These tools might be useful for capturing snapshots containing diagnostic clinical information on an individual and population scale, which may be used as a specific code not only for the diagnosis of the nature or the stage of the inflammatory process in periodontal disease, but more importantly, for its prognosis, which is still an unmet medical need. As a matter of fact, current peptidomics investigations suffer from a lack of standardized procedures, posing a serious problem for data interpretation. Descriptions of the efforts to address such concerns will be highlighted.
... Translation of proteomic biomarkers into clinical practice requires precise and specific analytical methods for accurate in vivo quantification in plasma 20 , which involves assay validations and investigations of circulating protein forms 21,22 . Among DAMPs, S100A8 and S100A9 proteins have molecular weights of 10.8 and 13.2 kDa, respectively, and show high structural diversity [23][24][25][26][27][28][29] . Several intracellular complexes and proteoforms 21 have been described in neutrophils and monocytes, where the S100A8/S100A9 complexes account for up to 40% of the detergent-soluble cytosolic protein content 24 . ...
... Isolated and purified S100A8 and S100A9 from human granulocytes are observed predominantly as noncovalently associated complexes, either in heterodimeric or heterotetrameric forms depending on bivalent cations 23,25,26 . In addition, post-translational modifications have been reported, including S-nitrosylated S100A8 in human neutrophils 27 , S-glutathionylated S100A9 in neutrophils 28 and phosphorylated S100A9 in monocytes 29 . In human plasma or serum, S100A8 and S100A9 are low-abundance 4 proteins and few reports have addressed the exact extracellular and circulating forms 30,31 . ...
... Part of the present study was dedicated to the identification and relative quantification of S100A8 and S100A9 proteoforms in plasma from septic shock patients cohort. It has been shown in vitro and in vivo, but not in plasma, that S100A8 and S100A9 may be post-translationally modified by S-nitrosylation, oxidation, S-glutathionylation and phosphorylation 27,28,29 . ...
Well-characterized prognostic biomarkers and reliable quantitative methods are key in sepsis management. Among Damage-Associated Molecular Patterns, S100A8/S100A9 complexes are reported to be markers for injured cells and to improve the prediction of death in septic shock patients. In view of the structural diversity observed for the intracellular forms, insight into circulating complexes and proteoforms is required to establish prognostic biomarkers. Here, we developed top-down and bottom-up proteomics to characterize the association of S100A8 and S100A9 in complexes and major circulating proteoforms. An antibody-free method was developed for absolute quantification of S100A8/S100A9 in a cohort of 49 patients to evaluate the prognostic value the first day after admission for septic shock. The predominant circulating identified forms by top-down proteomics were S100A8, mono-oxidized S100A8, truncated acetylated S100A9 and S-nitrosylated S100A9. S00A8, truncated acetylated S100A9 and mono-oxidized S100A8 were found to be discriminating for septic shock prognosis, along with total S100A8/S100A9 measured by the antibody-free bottom-up method. Overall, new insights into circulating S100A8/S100A9 and confirmation of its prognostic value in septic shock are crucial in qualification of this biomarker. Also, the simple antibody-free assay would support the harmonization of S100A8/S100A9 measurements
... However, it is important to note that in this study, the effect of S100A8/A9 was not tested and recombinant S100A8 and S100A9 proteins were used, which were devoid of post-translational modifications. Indeed, there is no doubt that the involvement of S100A8 and S100A9 in the regulation of inflammatory functions depends, among other things, on the monomeric and oligomeric forms of S100A8 and S100A9 [34] as well as their post-translational modifications [35,36]. In support of this affirmation, it has been proposed that oxygen derivatives induce post-translational modifications of S100A8/A9, through which the protein complex can scavenge released ROS and thus limit the propagation of inflammation [37,38]. ...
The release of cytokines by neutrophils constitutes an essential process in the development of inflammation by recruiting and activating additional cells. Neutrophils are also able to secrete a complex of S100A8 and S100A9 proteins (S100A8/A9), which can amplify the general inflammatory state of the host and is involved in the pathogenesis of several chronic inflammatory diseases, such as rheumatoid arthritis (RA). S100A8/A9 have received renewed attention due to their susceptibility to several function-altering post-translational modifications. In that context, it has been recently demonstrated that only the phosphorylated form of S100A8/A9 (S100A8/A9-P) is able to induce the secretion of several cytokines in neutrophils. Here, we investigate the mechanism by which this post-translational modification of S100A8/A9 can regulate the extracellular activity of the protein complex and its impact on the inflammatory functions of neutrophils. We found that S100A8/A9-P are present in large amounts in the synovial fluids from RA patients, highlighting the importance of this form of S100A8/A9 complex in the inflammation process. Using miRNA-sequencing on S100A8/A9-P-stimulated differentiated HL-60 cells, we identified a dysregulation of miR-146a-5p and miR-155-5p expression through TRL4 signaling pathways. Our data reveal that overexpression of these miRNAs in neutrophil-like cells reduces S100A8/A9-P-mediated secretion of pro-inflammatory cytokines.
... For instance, S-thiolation has been described for S100A9 and cystatin B playing a role in inflammation and development [114][115][116]. Cysteine can also undergo S-acylation reactions and post-translational modification consisting in the covalent attachment of an acyl chain to a cysteine residue of the target protein. ...
More than 300 different protein post‐translational modifications are currently known, but only a few have been extensively investigated because modified proteoforms are commonly present in sub‐stoichiometry amount. For this reason, improvement of specific enrichment techniques are particularly useful for the proteomic characterization of post‐translationally modified proteins. Enrichment proteomic strategies could help the researcher in the challenging issue to decipher the complex molecular cross‐talk existing between the different actors influencing the cellular pathways. In this review the state of art of the platforms applied for the enrichment of specific and most common post‐translational modifications, such as glycosylation and glycation, phosphorylation, sulfation, redox modifications (i.e. sulphydration and nitrosylation), methylation, acetylation and ubiquitinylation, are described. Enrichments strategies applied to characterize less studied post‐translational modifications are also briefly discussed. This article is protected by copyright. All rights reserved
