The hetero-oligomeric complex of the S100A8/S100A9 protein is extremely protease resistant

S100A8, S100A9 and S100A12 proteins are associated with inflammation and tissue remodelling, both processes known to be associated with high protease activity. Here, we report that homo-oligomeric forms of S100A8 and S100A9 are readily degraded by proteases, but that the preferred hetero-oligomeric S100A8/A9 complex displays a high resistance even against proteinase K degradation. S100A12 is not as protease resistant as the S100A8/A9 complex. Since specific functions have been assigned to the homo- and heterooligomeric forms of the S100A8 and A9 proteins, this finding may point to a post-translational level of regulation of the various functions of these proteins in inflammation and tissue remodelling.     

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.     

S100A8/A9 in COVID-19 pathogenesis: Impact on clinical outcomes

Coronavirus disease 2019 (COVID-19) has a broad range of clinical manifestations, highlighting the need for specific diagnostic tools to predict disease severity and improve patient prognosis. Recently, calprotectin (S100A8/A9) has been proposed as a potential biomarker for COVID-19, as elevated serum S100A8/A9 levels are associated with critical COVID-19 cases and can distinguish between mild and severe disease states. S100A8/A9 is an alarmin that mediates host proinflammatory responses during infection and it has been postulated that S100A8/A9 modulates the cytokine storm; the hallmark of fatal COVID-19 cases. However, it has yet to be determined if S100A8/A9 is a bona-fide biomarker for COVID-19. S100A8/A9 is widely implicated in a variety of inflammatory conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disorder (COPD), as well as pulmonary infectious diseases, including tuberculosis and influenza. Therefore, understanding how S100A8/A9 levels correlate with immune responses during inflammatory diseases is necessary to evaluate its candidacy as a potential COVID-19 biomarker. This review will outline the protective and detrimental roles of S100A8/A9 during infection, summarize the recent findings detailing the contributions of S100A8/A9 to COVID-19 pathogenesis, and highlight its potential as diagnostic biomarker and a therapeutic target for pulmonary infectious diseases, including COVID-19.     

S100A8、S100A9在幽门螺杆菌相关胃癌发生发展中的作用研究进展

S100家族是由20余个结构相似但功能各异的成员组成。该家族成员广泛参与感染、促炎、自身免疫等各种病理过程。近年来,越来越多学者发现S100家族成员在肿瘤的发展过程中也有不同程度的表达失调,且具有特异性。胃癌是我国常见的恶性肿瘤之一,国家癌症中心统计数据表明,2015年我国胃癌新发病率为679／10万,死亡率为498／10万[1],位居所有恶性肿瘤第2位。幽门螺杆菌（H. pylori）作为胃癌的Ⅰ类危险因子,目前其与胃癌的密切关系也得到了广大学者的认可。研究发现,S100家族成员——S100A8、S100A9在H. pylori感染相关胃炎、胃癌患者病理组织中表达显著上调,因此其在胃癌发生发展中的作用受到了学者的关注。本文主要就S100A8、S100A9在H. pylori相关胃癌发生发展中的作用作一综述。     

Oncogenic Kras expression in postmitotic neurons leads to S100A8-S100A9 protein overexpression and gliosis

Previous studies suggest that up-regulation of Ras signaling in neurons promotes gliosis and astrocytoma formation in a cell nonautonomous manner. However, the underlying mechanisms remain unknown. To address this question, we generated compound mice (LSL Kras G12D/+;CamKII-Cre) that express oncogenic Kras from its endogenous locus in postmitotic neurons after birth. These mice developed progressive gliosis, which is associated with hyperactivation of Ras signaling pathways. Microarray analysis identified S100A8 and S100A9 as two secreted molecules that are significantly overexpressed in mutant cortices. In contrast to their usual predominant expression in myeloid cells, we found that overexpression of S100A8 and S100A9 in the mutant cortex is primarily in neurons. This neuronal expression pattern is associated with increased infiltration of microglia in mutant cortex. Moreover, purified S100A8-S100A9 but not S100A8 or S100A9 alone promotes growth of primary astrocytes in vitro through both TLR4 and receptor of advanced glycation end product receptors. In summary, our results identify overexpression of S100A8-S100A9 in neurons as an early step in oncogenic Kras-induced gliosis. These molecules expressed in nonhematopoietic cells may be involved in tumorigenesis at a stage much earlier than what has been reported previously.     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.     

S100A8/A9-alarmin promotes local myeloid-derived suppressor cell activation restricting severe autoimmune arthritis

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Induction of alarmin S100A8/A9 mediates activation of aberrant neutrophils in the pathogenesis of COVID-19

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The expression of S100A8 in pancreatic cancer-associated monocytes is associated with the Smad4 status of pancreatic cancer cells

The cross-talk between tumour cells and the surrounding supporting host cells (stroma) is a key regulator of cancer growth and progression. By undertaking 2-DE analysis of laser capture microdissected malignant and stromal components of pancreatic tumours and benign ductal elements, we have identified high levels of S100A8 and S100A9 in tumour-associated stroma but not in benign or malignant epithelia. Immunohistochemical analysis (n = 71 patients) revealed strong expression of both proteins in stromal myeloid cells, subsequently identified as CD14(+)/CD68(- )monocytes/macrophages. Co-immunofluorescence revealed that S100A8 was expressed in a subset of S100A9-positive cells. Correlation of the expression of S100A8 and S100A9 to patient parameters revealed that the microenvironments of tumours which lacked expression of the tumour suppressor protein, Smad4, had significantly reduced numbers of S100A8-immunoreactive (p = 0.023) but not S100A9-immunoreactive (p = 0.21) cells. The ratio of S100A8- to S100A9-positive cells within individual tumours was significantly lower in Smad4-negative tumours than in Smad4-positive tumours (p<0.003). Pancreatitic specimens also contained S100A8- and S100A9-expressing cells, although this was not observed in regions displaying extensive fibrosis. In conclusion, our study provides an extensive analysis of S100A8 and S100A9 in pancreatic disease and highlights a potentially important relationship between pancreatic cancer cells and their surrounding microenvironment.     

Inflammation: a novel mechanism for the transport of extracellular nucleotide-induced arachidonic acid by S100A8/A9 for transcellular metabolism

Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide-mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5-lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5-lipoxygenase-mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide-induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B(4)(LTB(4)), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.     

外周血单核细胞CHOP、NLRP3、S100A8/A9 mRNA表达水平与脓毒症患者炎症反应和预后的关系分析

摘要 目的：探讨外周血单核细胞（PBMC）中CCAAT/增强子结合蛋白同源蛋白（CHOP）、核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）、S100A8/A9 信使核糖核酸（mRNA）表达水平与脓毒症患者炎症反应和预后的关系。方法：选取2020年1月～2022年1月新疆维吾尔自治区人民医院收治的170例脓毒症患者为脓毒症组,另选取同期68名健康体检者为对照组。采用酶联免疫吸附法检测血清白介素（IL）-6、IL-10、IL-17、IL-23、转化生长因子-β（TGF-β）水平,实时荧光定量PCR（qRT-PCR）法检测PBMC中CHOP、NLRP3、S100A8/A9 mRNA表达水平,比较脓毒症组与对照组上述指标差异。采用Pearson/Spearman相关系数分析脓毒症患者PBMC中CHOP、NLRP3、S100A8/A9 mRNA表达水平与炎症因子水平的相关性。脓毒症患者根据28d预后差异分为死亡组和存活组,收集临床资料,采用多因素Logistic回归分析脓毒症患者预后的影响因素。结果：脓毒症组PBMC中CHOP、NLRP3、S100A8/A9 mRNA相对表达量和血清IL-6、IL-10、IL-17、IL-23、TGF-β水平均明显高于对照组（P＜0.05）。Pearson/Spearman相关性分析显示,脓毒症患者PBMC中CHOP、NLRP3、S100A8/A9 mRNA表达水平与血清IL-6、IL-10、IL-17、IL-23、TGF-β水平均呈正相关（P＜0.05）。不同预后脓毒症患者比较,死亡组年龄大于存活组,脓毒性休克、多器官功能障碍综合征（MODS）、ICU住院时间≥10 d、机械通气时间≥3 d患者比例、脓毒症相关器官衰竭评估（SOFA）评分、血乳酸、血清IL-6、IL-10、IL-17、IL-23、TGF-β水平以及PBMC中CHOP、NLRP3、S100A8/A9 mRNA相对表达量均高于存活组,而氧合指数低于存活组（P＜0.05）。多因素Logistic回归分析显示,校正其他因素后,PBMC中CHOP mRNA、NLRP3 mRNA、S100A8/A9 mRNA表达水平升高是脓毒症患者预后不良的危险因素（P＜0.05）。结论：脓毒症患者PBMC中CHOP、NLRP3、S100A8/A9 mRNA呈高表达,且表达水平与炎症反应及患者预后密切相关。     

S100A8通过自噬促进B细胞淋巴瘤耐药性的机制研究

B细胞淋巴瘤是起源于淋巴造血系统的恶性肿瘤,是由分化过程中淋巴细胞恶性转化的复杂过程引起的.B细胞淋巴瘤细胞的耐药性是制约B细胞淋巴瘤治疗的关键因素.自噬是细胞成分降解和再循环的重要细胞生物学过程,近年来其与肿瘤耐药性的相关性受到越来越多的关注.S100A8是钙结合蛋白S100家族的重要成员,其在淋巴瘤的的耐药调控中发...     

Structure/function studies of S100A8/A9

Summary The functional importance of members of the S100 Ca²⁺-binding protein family is recently emerging. A variety of activities, several of whcih are apparently opposing, are attributed to S100A8, a protein implicated in embryogenesis, growth, differentiation, and immune and inflammatory processes. Murine (m) S100A8 was initially described as a chemoattractant (CP-10) for myeloid cells. It is coordinately expressed with mS100A9 (MRP14) in neutrophils and the non-covalent heterodimer is presumed to be the functional intracellular species. The extracellular chemotactic activity of mS100A8, however, is not dependent on mS100A9 and occurs at concentrations (10⁻¹³-10⁻¹¹ M) at which the non-covalent heterodimer would probably dissociate. This review focuses on the structure and post-translational modifications of mS100A8/A9 and their effects on function, particularly chemotaxis.     

Structure/function studies of S100A8/A9

The functional importance of members of the S100 Ca²⁺-binding protein family is recently emerging. A variety of activities, several of which are apparently opposing, are attributed to S100A8, a protein implicated in embryogenesis, growth, differentiation, and immune and inflammatory processes. Murine (m) S100A8 was initially described as a chemoattractant (CP-10) for myeloid cells. It is coordinately expressed with mS100A9 (MRP14) in neutrophils and the non-covalent heterodimer is presumed to be the functional intracellular species. The extracellular chemotactic activity of mS100A8, however, is not dependent on mS100A9 and occurs at concentrations (10^-13–10^-11 M) at which the non-covalent heterodimer would probably dissociate. This review focuses on the structure and post-translational modifications of mS100A8/A9 and their effects on function, particularly chemotaxis.     

Review of S100A9 biology and its role in cancer

S100A9 is a calcium binding protein with multiple ligands and post-translation modifications that is involved in inflammatory events and the initial development of the cancer cell through to the development of metastatic disease. This review has a threefold purpose: 1) describe the S100A9 structural elements important for its biological activity, 2) describe the S100A9 biology in the context of the immune system, and 3) illustrate the role of S100A9 in the development of malignancy via interactions with the immune system and other cellular processes.     

Crosslinking of the CD69 molecule enhances S100A9 production in activated neutrophils

Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.     

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC–tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.     

S100 A9 is expressed and secreted by the oviduct epithelium,interacts with gametes and affects parameters of human sperm capacitation in vitro

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [³⁵S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [³⁵S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.     

Effects of S100A9 in a rat model of asthma and in isolated tracheal spirals

S100A9 is a member of the S100 family of proteins that contain two EF-hand calcium-binding motifs. We previously reported that S100A9 was differentially expressed during the early airway response phase of asthma and can be regulated by acupuncture. To understand the possible role of S100A9 in asthma, the effects of the S100A9 were investigated in a rat model of asthma and in isolated tracheal spirals. The pulmonary function and isometric tension were measured after the administration of purified recombinant S100A9. The results of in vivo experiments showed that S100A9 (0.1 μg/kg) significantly decreased the pulmonary resistance and increased the dynamic compliance. The in vitro experimental results showed that S100A9 (100, 200, 400, or 800 ng/ml, final concentrations) significantly reduced the isometric tension of isolated tracheal spirals. These results suggest that S100A9 elicits dose-dependent anti-asthmatic effects and may provide further insight into the treatment of asthma.     

S100 proteins in cartilage: Role in arthritis

S100 proteins are low molecular weight calcium binding proteins expressed in vertebrates. The family constitutes 21 known members that are expressed in several tissues and cell types and play a major role in various cellular functions. Uniquely, members of the S100 family have both intracellular and extracellular functions. Several members of the S100 family (S100A1, S100A2, S100A4, S1008, S100A9, S100A11, and S100B) have been identified in human articular cartilage, and their expression is upregulated in diseased tissue. These S100 proteins elicit a catabolic signaling pathway via receptor for advanced glycation end products (RAGE) in cartilage and may promote progression of arthritis. This review summarizes our current understanding of the role of S100 proteins in cartilage biology and in the development of arthritis.