Search Results (17)

Cytophaga is a genus of Gram-negative bacteria occurring in soil and the gut microbiome. It is closely related to pathogenic Flavobacterium spp. that cause severe diseases in fish. Cytophaga strain L43-1 secretes cytophagalysin (CPL1), a 137 kDa peptidase with reported collagenolytic and gelatinolytic activity. We performed highly-confident structure prediction calculations for CPL1, which identified 11 segments and domains, including a signal peptide for secretion, a prosegment (PS) for latency, a metallopeptidase (MP)-like catalytic domain (CD), and eight immunoglobulin (Ig)-like domains (D3–D10). In addition, two short linkers were found at the D8–D9 and D9–D10 junctions, and the structure would be crosslinked by four disulfide bonds. The CPL1 CD was found closest to ulilysin from Methanosarcina acetivorans, which assigns CPL1 to the lower-pappalysin family within the metzincin clan of MPs. Based on the structure predictions, we aimed to produce constructs spanning the full-length enzyme, as well as PS+CD, PS+CD+D3, and PS+CD+D3+D4. However, we were successful only with the latter three constructs. We could activate recombinant CPL1 by PS removal employing trypsin, and found that both zymogen and mature CPL1 were active in gelatin zymography and against a fluorogenic gelatin variant. This activity was ablated in a mutant, in which the catalytic glutamate described for lower pappalyins and other metzincins was replaced by alanine, and by a broad-spectrum metal chelator. Overall, these results proved that our recombinant CPL1 is a functional active MP, thus supporting the conclusions derived from the structure predictions. Full article

Neutrophil-derived proteases are critical to the pathology of many inflammatory lung diseases, both chronic and acute. These abundant enzymes play roles in key neutrophil functions, such as neutrophil extracellular trap formation and reactive oxygen species release. They may also be released, inducing tissue damage and loss of tissue function. Historically, the neutrophil serine proteases (NSPs) have been the main subject of neutrophil protease research. Despite highly promising cell-based and animal model work, clinical trials involving the inhibition of NSPs have shown mixed results in lung disease patients. As such, the cutting edge of neutrophil-derived protease research has shifted to proteases that have had little-to-no research in neutrophils to date. These include the cysteine and serine cathepsins, the metzincins and the calpains, among others. This review aims to outline the previous work carried out on NSPs, including the shortcomings of some of the inhibitor-orientated clinical trials. Our growing understanding of other proteases involved in neutrophil function and neutrophilic lung inflammation will then be discussed. Additionally, the potential of targeting these more obscure neutrophil proteases will be highlighted, as they may represent new targets for inhibitor-based treatments of neutrophil-mediated lung inflammation. Full article

The minor secreted proteinase of B. pumilus 3-19 MprBp classified as the unique bacillary adamalysin-like enzyme of the metzincin clan. The functional role of this metalloproteinase in the bacilli cells is not clear. Analysis of the regulatory region of the mprBp gene showed the presence of potential binding sites to the transcription regulatory factors Spo0A (sporulation) and DegU (biodegradation). The study of mprBp activity in mutant strains of B. subtilis defective in regulatory proteins of the Spo- and Deg-systems showed that the mprBp gene is partially controlled by the Deg-system of signal transduction and independent from the Spo-system. Full article

Metalloproteinases (MPs) belong to the superfamily of zinc endopeptidases, which are called metzincins. Three families of MPs have been studied in healthy and diseased conditions in humans. The first study on MPs was published in 1962, and following that, several studies characterized their activity and their effect on health and disease. Several diseases have been related to MPs, such as cardiovascular disease, cancer, inflammatory diseases, gynecological disorders, and others. Moreover, tissue inhibitors of metalloproteinases (TIMPs) have been widely studied, and drugs and artificial molecules that could target MP activity have been evaluated. MPs are relevant to public health because of their role in several diseases and, most of all, their role as biomarkers that also impact the quality of life and the psychosocial dimension of affected patients. In this context, new pathways to precision health and precision medicine have been opened in the area of MPs. This review describes, from the initial studies, the complex dimensions of MPs and related issues centered on health and disease dimensions. Full article

Standard chemotherapy of Glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to acquired chemoresistance. Tumor-associated macrophages and microglia (TAMs) as major immune cell population in the tumor microenvironment are potential modulators of TMZ response. However; little is known about how TAMs participate in TMZ induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important mediators of cellular communication in the tumor microenvironment. A qPCR screening was performed to identify potential targets within the ADAM and MMP family members in GBM cells. In co-culture with macrophages ADAM8 was the only signature gene up-regulated in GBM cells induced by macrophages under TMZ treatment. The relationship between ADAM8 expression and TAM infiltration in GBM was determined in a patient cohort by qPCR; IF; and IHC staining and TCGA data analysis. Moreover; RNA-seq was carried out to identify the potential targets regulated by ADAM8. CCL2 expression levels were determined by qPCR; Western blot; IF; and ELISA. Utilizing qPCR; IF; and IHC staining; we observed a positive relationship between ADAM8 expression and TAMs infiltration level in GBM patient tissues. Furthermore; ADAM8 induced TAMs recruitment in vitro and in vivo. Mechanistically; we revealed that ADAM8 activated HB-EGF/EGFR signaling and subsequently up-regulated production of CCL2 in GBM cells in the presence of TMZ treatment; promoting TAMs recruitment; which further induced ADAM8 expression in GBM cells to mediate TMZ chemoresistance. Thus; we revealed an ADAM8 dependent positive feedback loop between TAMs and GBM cells under TMZ treatment which involves CCL2 and EGFR signaling to cause TMZ resistance in GBM. Full article

Background: We have previously shown that metzincin protease ADAMTS-4 accompanies renal fibrogenesis, as it appears in the blood of hemodialysis patients. Methods: Native kidney (NKB) and kidney transplant (TXCI) biopsy samples as well as plasma from patients with various stages of CKD were compared to controls. In paired analysis, 15 TXCI samples were compared with their zero-time biopsies (TX0). Tissues were evaluated and scored (interstitial fibrosis and tubular atrophy (IFTA) for NKB and Banff ci for TXCI). Immunohistochemical (IHC) staining for ADAMTS-4 and BMP-1 was performed. Plasma ADAMTS-4 was detected using ELISA. Results: ADAMTS-4 IHC expression was significantly higher in interstitial compartment (INT) of NKB and TXCI group in peritubular capillaries (PTC) and interstitial stroma (INT). Patients with higher stages of interstitial fibrosis (ci > 1 and IFTA > 1) expressed ADAMTS-4 in INT more frequently in both groups (p = 0.005; p = 0.013; respectively). In paired comparison, TXCI samples expressed ADAMTS-4 in INT and PTC more often than TX0. ADAMTS-4 plasma concentration varied significantly across CKD stages, being highest in CKD 2 and 3 compared to other groups (p = 0.0064). Hemodialysis patients had higher concentrations of ADAMTS-4 compared to peritoneal dialysis (p < 0.00001). Conclusion: ADAMTS-4 might have a significant role in CKD as a potential novel diagnostic indicator. Full article

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens. Full article

Prostate cancer remains a leading cause of cancer-related morbidity in men. Potentially important regulators of prostate cancer progression are members of the metzincin superfamily of proteases, principally through their regulation of the extracellular matrix. It is therefore timely to review the role of the metzincin superfamily in prostate cancer and its progression to better understand their involvement in this disease. A systematic-like search strategy was conducted. Articles that investigated the roles of members of the metzincin superfamily and their key regulators in prostate cancer were included. The extracted articles were synthesized and data presented in tabular and narrative forms. Two hundred and five studies met the inclusion criteria. Of these, 138 investigated the role of the Matrix Metalloproteinase (MMP) subgroup, 34 the Membrane-Tethered Matrix Metalloproteinase (MT-MMP) subgroup, 22 the A Disintegrin and Metalloproteinase (ADAM) subgroup, 8 the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) subgroup and 53 the Tissue Inhibitor of Metalloproteinases (TIMP) family of regulators, noting that several studies investigated multiple family members. There was clear evidence that specific members of the metzincin superfamily are involved in prostate cancer progression, which can be either in a positive or negative manner. However, further understanding of their mechanisms of action and how they may be used as prognostic indicators or molecular targets is required. Full article

Meprin α and β are zinc-dependent proteinases implicated in multiple diseases including cancers, fibrosis, and Alzheimer’s. However, until recently, only a few inhibitors of either meprin were reported and no inhibitors are in preclinical development. Moreover, inhibitors of other metzincins developed in previous years are not effective in inhibiting meprins suggesting the need for de novo discovery effort. To address the paucity of tractable meprin inhibitors we developed ultrahigh-throughput assays and conducted parallel screening of >650,000 compounds against each meprin. As a result of this effort, we identified five selective meprin α hits belonging to three different chemotypes (triazole-hydroxyacetamides, sulfonamide-hydroxypropanamides, and phenoxy-hydroxyacetamides). These hits demonstrated a nanomolar to micromolar inhibitory activity against meprin α with low cytotoxicity and >30-fold selectivity against meprin β and other related metzincincs. These selective inhibitors of meprin α provide a good starting point for further optimization. Full article

Meprin α is a zinc metalloproteinase (metzincin) that has been implicated in multiple diseases, including fibrosis and cancers. It has proven difficult to find small molecules that are capable of selectively inhibiting meprin α, or its close relative meprin β, over numerous other metzincins which, if inhibited, would elicit unwanted effects. We recently identified possible molecular starting points for meprin α-specific inhibition through an HTS effort (see part I, preceding paper). Here, in part II, we report further efforts to optimize potency and selectivity. We hope that a hydroxamic acid meprin α inhibitor probe will help define the therapeutic potential for small molecule meprin α inhibition and spur further drug discovery efforts in the area of zinc metalloproteinase inhibition. Full article

Matrix metalloproteinases (MMPs) and A disintegrin and Metalloproteinase (ADAMs) are zinc-dependent endopeptidases belonging to the metzincin superfamily. Upregulation of metzincin activity is a major feature in many serious pathologies such as cancer, inflammations, and infections. In the last decades, many classes of small molecules have been developed directed to inhibit these enzymes. The principal shortcomings that have hindered clinical development of metzincin inhibitors are low selectivity for the target enzyme, poor water solubility, and long-term toxicity. Over the last 15 years, a novel approach to improve solubility and bioavailability of metzincin inhibitors has been the synthesis of carbohydrate-based compounds. This strategy consists of linking a hydrophilic sugar moiety to an aromatic lipophilic scaffold. This review aims to describe the development of sugar-based and azasugar-based derivatives as metzincin inhibitors and their activity in several pathological models. Full article

Tumor-cell infiltration is a major obstacle to successful therapy for brain tumors. Membrane-type matrix metalloproteinases (MT-MMPs), a metzincin subfamily of six proteases, are important mediators of infiltration. The cellular source of MT-MMPs and their role in glioma biology, however, remain controversial. Thus, we comprehensively analyzed the expression of MT-MMPs in primary brain tumors. All MT-MMPs were differentially expressed in primary brain tumors. In diffuse gliomas, MT-MMP1, -3, and -4 were predominantly expressed by IDH1^mutated tumor cells, while macrophages/microglia contributed significantly less to MT-MMP expression. For functional analyses, individual MT-MMPs were expressed in primary mouse p53^−/− astrocytes. Invasion and migration potential of MT-MMP-transduced astrocytes was determined via scratch, matrigel invasion, and novel organotypic porcine spinal slice migration (OPoSSM) and invasion assays. Overall, MT-MMP-transduced astrocytes showed enhanced migration compared to controls. MMP14 was the strongest mediator of migration in scratch assays. However, in the OPoSSM assays, the glycosylphosphatidylinositol (GPI)-anchored MT-MMPs MMP17 and MMP25, not MMP14, mediated the highest infiltration rates of astrocytes. Our data unequivocally demonstrate for the first time that glioma cells, not microglia, are the predominant producers of MT-MMPs in glioma and can act as potent mediators of tumor-cell infiltration into CNS tissue. These proteases are therefore promising targets for therapeutic interventions. Full article

The metalloproteinase (MP) family of zinc-dependent proteases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteases (ADAMs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) plays a crucial role in the extracellular matrix (ECM) remodeling and degradation activities. A wide range of substrates of the MP family includes ECM components, chemokines, cell receptors, and growth factors. Metalloproteinases activities are tightly regulated by proteolytic activation and inhibition via their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), and the imbalance of the activation and inhibition is responsible in progression or inhibition of several diseases, e.g., cancer, neurological disorders, and cardiovascular diseases. We provide an overview of the structure, function, and the multifaceted role of MMPs, ADAMs, and TIMPs in several diseases via their cellular functions such as proteolysis of other cell signaling factors, degradation and remodeling of the ECM, and other essential protease-independent interactions in the ECM. The significance of MP inhibitors targeting specific MMP or ADAMs with high selectivity is also discussed. Recent advances and techniques used in developing novel MP inhibitors and MP responsive drug delivery tools are also reviewed. Full article

Zinc-dependent matrix metalloproteinases (MMPs) belong to metzincins that comprise not only 23 human MMPs but also other metalloproteinases, such as 21 human ADAMs (a disintegrin and metalloproteinase domain) and 19 secreted ADAMTSs (a disintegrin and metalloproteinase thrombospondin domain). The many setbacks from the clinical trials of broad-spectrum MMP inhibitors for cancer indications in the late 1990s emphasized the extreme complexity of the participation of these proteolytic enzymes in biology. This editorial mini-review summarizes the Special Issue, which includes four review articles and 10 original articles that highlight the versatile roles of MMPs, ADAMs, and ADAMTSs, in normal physiology as well as in neoplastic and destructive processes in tissue. In addition, we briefly discuss the unambiguous involvement of MMPs in wound healing. Full article

The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish adamts5 during embryogenesis. This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated. Full article