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Molecular & Cellular Proteomics : MCP Jun 2023The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal...
The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr-Gly bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.
Topics: Cell Membrane; Hemopexin; Matrix Metalloproteinase 14; Peptides; Proteolysis; Humans
PubMed: 37169079
DOI: 10.1016/j.mcpro.2023.100566 -
Biomaterials Advances Sep 2023In the lung, pulmonary epithelial cells undergo mechanical stretching during ventilation. The associated cellular mechanoresponse is still poorly understood at the...
Mechanical activation of lung epithelial cells through the ion channel Piezo1 activates the metalloproteinases ADAM10 and ADAM17 and promotes growth factor and adhesion molecule release.
In the lung, pulmonary epithelial cells undergo mechanical stretching during ventilation. The associated cellular mechanoresponse is still poorly understood at the molecular level. Here, we demonstrate that activation of the mechanosensitive cation channel Piezo1 in a human epithelial cell line (H441) and in primary human lung epithelial cells induces the proteolytic activity of the metalloproteinases ADAM10 and ADAM17 at the plasma membrane. These ADAMs are known to convert cell surface expressed proteins into soluble and thereby play major roles in proliferation, barrier regulation and inflammation. We observed that chemical activation of Piezo1 promotes cleavage of substrates that are specific for either ADAM10 or ADAM17. Activation of Piezo1 also induced the synthesis and ADAM10/17-dependent release of the growth factor amphiregulin (AREG). In addition, junctional adhesion molecule A (JAM-A) was shed in an ADAM10/17-dependent manner resulting in a reduction of cell contacts. Stretching experiments combined with Piezo1 knockdown further demonstrated that mechanical activation promotes shedding via Piezo1. Most importantly, high pressure ventilation of murine lungs increased AREG and JAM-A release into the alveolar space, which was reduced by a Piezo1 inhibitor. Our study provides a novel link between stretch-induced Piezo1 activation and the activation of ADAM10 and ADAM17 in lung epithelium. This may help to understand acute respiratory distress syndrome (ARDS) which is induced by ventilation stress and goes along with perturbed epithelial permeability and release of growth factors.
Topics: Humans; Mice; Animals; Amyloid Precursor Protein Secretases; Lung; ADAM10 Protein; Membrane Proteins; Epithelial Cells; Ion Channels; Intercellular Signaling Peptides and Proteins; Metalloproteases; ADAM17 Protein
PubMed: 37348330
DOI: 10.1016/j.bioadv.2023.213516 -
American Journal of Respiratory and... Dec 2018
Topics: ADAM Proteins; Disintegrins; Humans; Membrane Proteins; Metalloproteases; Pulmonary Disease, Chronic Obstructive
PubMed: 29986153
DOI: 10.1164/rccm.201805-1012ED -
Respiratory Research Jul 2022Idiopathic pulmonary fibrosis (IPF) is a diffuse parenchymal lung disease characterized by exuberant deposition of extracellular matrix (ECM) proteins in the lung...
BACKGROUND
Idiopathic pulmonary fibrosis (IPF) is a diffuse parenchymal lung disease characterized by exuberant deposition of extracellular matrix (ECM) proteins in the lung interstitium, which contributes to substantial morbidity and mortality in IPF patients. Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases, many of which have been implicated in the regulation of ECM degradation in lung fibrosis. However, the roles of MMP-2 and -9 (also termed gelatinases A and B) have not yet been explored in lung fibrosis in detail.
METHODS
AdTGF-β1 was applied via orotracheal routes to the lungs of WT, MMP-2 KO, MMP-9 KO and MMP-2/-9 dKO mice on day 0 to induce lung fibrosis. Using hydroxyproline assay, FlexiVent based lung function measurement, histopathology, western blot and ELISA techniques, we analyzed MMP-2 and MMP-9 levels in BAL fluid and lung, collagen contents in lung and lung function in mice on day 14 and 21 post-treatment.
RESULT
IPF lung homogenates exhibited significantly increased levels of MMP-2 and MMP-9, relative to disease controls. Enzymatically active MMP-2 and MMP-9 was increased in lungs of mice exposed to adenoviral TGF-β1, suggesting a role for these metalloproteinases in lung fibrogenesis. However, we found that neither MMP-2 or MMP-9 nor combined MMP-2/-9 deletion had any effect on experimental lung fibrosis in mice.
CONCLUSION
Together, our data strongly suggest that both gelatinases MMP-2 and MMP-9 play only a subordinate role in experimental lung fibrosis in mice.
Topics: Animals; Extracellular Matrix Proteins; Idiopathic Pulmonary Fibrosis; Lung; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Mice
PubMed: 35804363
DOI: 10.1186/s12931-022-02105-7 -
Processing of Snake Venom Metalloproteinases: Generation of Toxin Diversity and Enzyme Inactivation.Toxins Jun 2016Snake venom metalloproteinases (SVMPs) are abundant in the venoms of vipers and rattlesnakes, playing important roles for the snake adaptation to different environments,... (Review)
Review
Snake venom metalloproteinases (SVMPs) are abundant in the venoms of vipers and rattlesnakes, playing important roles for the snake adaptation to different environments, and are related to most of the pathological effects of these venoms in human victims. The effectiveness of SVMPs is greatly due to their functional diversity, targeting important physiological proteins or receptors in different tissues and in the coagulation system. Functional diversity is often related to the genetic diversification of the snake venom. In this review, we discuss some published evidence that posit that processing and post-translational modifications are great contributors for the generation of functional diversity and for maintaining latency or inactivation of enzymes belonging to this relevant family of venom toxins.
Topics: Adaptation, Biological; Animals; Catalytic Domain; Enzyme Stability; Metalloproteases; Protein Processing, Post-Translational; Proteolysis; Snake Venoms; Snakes
PubMed: 27294958
DOI: 10.3390/toxins8060183 -
Journal of Thrombosis and Haemostasis :... Oct 2022ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor, is crucial for normal hemostasis. Acquired autoantibody-mediated deficiency of plasma ADAMTS13... (Review)
Review
ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor, is crucial for normal hemostasis. Acquired autoantibody-mediated deficiency of plasma ADAMTS13 results in a potentially fatal blood disorder, immune thrombotic thrombocytopenic purpura (iTTP). Plasma ADAMTS13 protease appears to exist in multiple conformations. Under physiological conditions, plasma ADAMTS13 exists predominantly in its "closed" conformation (or latent form), which may be activated by lowering pH, ligand binding, and binding of an antibody against the distal domains of ADAMTS13. In patients with iTTP, polyclonal antibodies target at various domains of ADAMTS13. However, nearly all inhibitory antibodies bind the spacer domain, whereas antibodies that bind the distal C-terminal domains may activate ADAMTS13 through removing its allosteric inhibition. Additionally, the anti-C-terminal antibodies may alter the potency of inhibitory antibodies towards ADAMTS13 activity. This review summarizes some of the most recent knowledge about the ADAMTS13 conformation and its mechanism of inhibition by its autoantibodies.
Topics: ADAM Proteins; ADAMTS13 Protein; Autoantibodies; Humans; Ligands; Purpura, Thrombocytopenic, Idiopathic; Purpura, Thrombotic Thrombocytopenic; Thrombosis; von Willebrand Factor
PubMed: 35842925
DOI: 10.1111/jth.15822 -
International Journal of Molecular... Jun 2019Parathyroid hormone-related protein (PTHrP), with isoforms ranging from 139 to 173 amino acids, has long been implicated in the development and regulation of multiple... (Review)
Review
Parathyroid hormone-related protein (PTHrP), with isoforms ranging from 139 to 173 amino acids, has long been implicated in the development and regulation of multiple tissues, including that of the skeleton, via paracrine and autocrine signaling. PTHrP is also known as a potent mediator of cancer-induced bone disease, contributing to a vicious cycle between tumor cells and the bone microenvironment that drives the formation and progression of metastatic lesions. The abundance of roles ascribed to PTHrP have largely been attributed to the N-terminal 1-36 amino acid region, however, activities for mid-region and C-terminal products as well as additional shorter N-terminal species have also been described. Studies of the protein sequence have indicated that PTHrP is susceptible to post-translational proteolytic cleavage by multiple classes of proteases with emerging evidence pointing to novel functional roles for these PTHrP products in regulating cell behavior in homeostatic and pathological contexts. As a consequence, PTHrP products are also being explored as potential biomarkers of disease. Taken together, our enhanced understanding of the post-translational regulation of PTHrP bioactivity could assist in developing new therapeutic approaches that can effectively treat skeletal malignancies.
Topics: Animals; Bone Neoplasms; Humans; Metalloproteases; Parathyroid Hormone-Related Protein; Protein Processing, Post-Translational; Proteolysis
PubMed: 31181800
DOI: 10.3390/ijms20112814 -
The EMBO Journal Apr 2018Death receptor 6 (DR6) is an orphan member of the TNF receptor superfamily and controls cell death and differentiation in a cell-autonomous manner in different cell...
Death receptor 6 (DR6) is an orphan member of the TNF receptor superfamily and controls cell death and differentiation in a cell-autonomous manner in different cell types. Here, we report an additional non-cell-autonomous function for DR6 in the peripheral nervous system (PNS). DR6-knockout (DR6 KO) mice showed precocious myelination in the PNS Using an myelination assay, we demonstrate that neuronal DR6 acts in on Schwann cells (SCs) and reduces SC proliferation and myelination independently of its cytoplasmic death domain. Mechanistically, DR6 was found to be cleaved in neurons by "a disintegrin and metalloprotease 10" (ADAM10), releasing the soluble DR6 ectodomain (sDR6). Notably, in the myelination assay, sDR6 was sufficient to rescue the DR6 KO phenotype. Thus, in addition to the cell-autonomous receptor function of full-length DR6, the proteolytically released sDR6 can unexpectedly also act as a paracrine signaling factor in the PNS in a non-cell-autonomous manner during SC proliferation and myelination. This new mode of DR6 signaling will be relevant in future attempts to target DR6 in disease settings.
Topics: ADAM10 Protein; Amyloid Precursor Protein Secretases; Animals; Cell Death; Cell Line; Cell Proliferation; Cytoplasm; Death Domain; Disintegrins; Female; HEK293 Cells; Humans; Hybridomas; Male; Membrane Proteins; Metalloproteases; Mice; Mice, Knockout; Myelin Sheath; Neurons; Paracrine Communication; Phenotype; Receptors, Tumor Necrosis Factor; Schwann Cells; Substrate Specificity
PubMed: 29459438
DOI: 10.15252/embj.201797390 -
Modulation of matrix metalloproteases by ciliary neurotrophic factor in human placental development.Cell and Tissue Research Oct 2022Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor complex containing a specific subunit, CNTF receptor α (CNTFRα). The two...
Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor complex containing a specific subunit, CNTF receptor α (CNTFRα). The two molecules are constitutively expressed in key structures for human placental growth and differentiation. The possible role of CNTF in enhancing cell proliferation and/or invasion during placental development and remodelling was investigated using HTR-8/SVneo and BeWo cells, taken respectively as cytotrophoblast and syncytiotrophoblast models. In both cell lines, treatment with human recombinant (hr) CNTF activated JAK2/STAT3 signalling and inhibited the ERK pathway. Interestingly, in HTR-8/SVneo cells, 50 ng hrCNTF induced significant downregulation of matrix metalloprotease (MMP)-1 and significant upregulation of MMP-9. Moreover, pharmacological inhibition of JAK2/STAT3 signalling by AG490 and curcumin resulted in MMP-9 downregulation; it activated the ERK signalling pathway and upregulated MMP-1 expression. Collectively, these data suggest a role for CNTF signalling in extravillous cytotrophoblast invasion through the modulation of specific MMPs.
Topics: Ciliary Neurotrophic Factor; Ciliary Neurotrophic Factor Receptor alpha Subunit; Curcumin; Cytokines; Female; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Placenta; Placentation; Pregnancy; Receptor, Ciliary Neurotrophic Factor
PubMed: 35794391
DOI: 10.1007/s00441-022-03658-1 -
Frontiers in Cellular and Infection... 2022Visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) affect most of the poor populations worldwide. The current treatment modalities include...
Visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) affect most of the poor populations worldwide. The current treatment modalities include liposomal formulation or deoxycholate salt of amphotericin B, which has been associated with various complications and severe side effects. Encouraged from the recent marked antimalarial effects from plant-derived glycosides, in this study, we have exploited a green chemistry-based approach to chemically synthesize a library of diverse glycoside derivatives (Gly1-12) and evaluated their inhibitory efficacy against the AG83 strain of . Among the synthesized glycosides, the inhibitory activity of Glycoside-2 (Gly2) (1.13 µM IC50 value) on promastigote demonstrated maximum cytotoxicity with ~94% promastigote death as compared to amphotericin B that was taken as a positive control. The antiproliferative effect of Gly2 on promastigote encouraged us to analyze the structure-activity relationship of Gly2 with Gp63, a zinc metalloprotease that majorly localizes at the surface of the promastigote and has a role in its development and multiplication. The result demonstrated the exceptional binding affinity of Gly2 toward the catalytic domain of Gp63. These data were thereafter validated through cellular thermal shift assay in a physiologically relevant cellular environment. Mechanistically, reduced multiplication of promastigotes on treatment with Gly2 induces the destabilization of redox homeostasis in promastigotes by enhancing reactive oxygen species (ROS), coupled with depolarization of the mitochondrial membrane. Additionally, Gly2 displayed strong lethal effects on infectivity and multiplication of amastigote inside the macrophage in the amastigote-macrophage infection model as compared to amphotericin B treatment. Gp63 is also known to bestow protection against complement-mediated lysis of parasites. Interestingly, Gly2 treatment enhances the complement-mediated lysis of promastigotes in serum physiological conditions. In addition, Gly2 was found to be equally effective against the clinical promastigote forms of PKDL strain (IC50 value of 1.97 µM); hence, it could target both VL and PKDL simultaneously. Taken together, this study reports the serendipitous discovery of Gly2 with potent antileishmanial activity and proves to be a novel chemotherapeutic prototype against VL and PKDL.
Topics: Amphotericin B; Antiprotozoal Agents; Glycosides; Humans; Leishmania donovani; Leishmaniasis, Visceral; Metalloproteases
PubMed: 35601095
DOI: 10.3389/fcimb.2022.803048