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Journal of Cellular and Molecular... May 2021Both Colony-stimulating factor 1 receptor (CSF1R) and triggering receptor expressed on myeloid cells-2 (TREM2) are trans-membrane receptors and are expressed in the...
Both Colony-stimulating factor 1 receptor (CSF1R) and triggering receptor expressed on myeloid cells-2 (TREM2) are trans-membrane receptors and are expressed in the brain primarily by microglia. Mutations in these two microglia-expressed genes associated with neurodegenerative disease have recently been grouped under the term "microgliopathy". Several literatures have indicated that CSF1R and TREM2 encounters a stepwise shedding and TREM2 variants impair or accelerate the processing. However, whether CSF1R variant affects the shedding of CSF1R remains elusive. Here, plasmids containing human CSF1R or TREM2 were transiently transfected into the human embryonic kidney (HEK) 293T cells. Using Western Blot and/or ELISA assay, we demonstrated that, similar to those of TREM2, an N-terminal fragment (NTF) shedding of CSF1R ectodomain and a subsequent C-terminal fragment (CTF) of CSF1R intra-membrane were generated by a disintegrin and metalloprotease (ADAM) family member and by γ-secretase, respectively. And the shedding was inhibited by treatment with Batimastat, an ADAM inhibitor, or DAPT or compound E, a γ-secretase inhibitor. Importantly, we show that the cleaved fragments, both extracellular domain and intracellular domain of a common disease associated I794T variant, were decreased significantly. Together, our studies demonstrate a stepwise approach of human CSF1R cleavage and contribute to understand the pathogenicity of CSF1R I794T variant in adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). These studies also suggest that the cleaved ectodomain fragment released from CSF1R may be proposed as a diagnostic biomarker for ALSP.
Topics: Amyloid Precursor Protein Secretases; HEK293 Cells; Humans; Leukoencephalopathies; Membrane Glycoproteins; Mutant Proteins; Mutation; Proteolysis; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Receptors, Immunologic
PubMed: 33783963
DOI: 10.1111/jcmm.16474 -
Journal of Virology Apr 2021Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme...
Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2. Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.
Topics: Ambroxol; Amino Acid Substitution; Angiotensin-Converting Enzyme 2; Bromhexine; COVID-19; Cell Line; Humans; Mutation, Missense; Proteolysis; Severe acute respiratory syndrome-related coronavirus; SARS-CoV-2; Serine Endopeptidases; Severe Acute Respiratory Syndrome; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 33608407
DOI: 10.1128/JVI.00002-21 -
Frontiers in Microbiology 2024HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion...
HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62L-shedding mediated viral release and its potential inhibition, we screened compounds specific for serine-, cysteine-, aspartyl-, and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Zn-metalloproteinase inhibitors, including BB-94, TAPI, prinomastat, GM6001, and GI25423X, suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L, we observed association between budding HIV-1 virions and CD62L by transmission electron microscope, and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally, these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release.
PubMed: 38572241
DOI: 10.3389/fmicb.2024.1385775 -
FEBS Open Bio Apr 2018The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic...
The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG, synthesized as a propeptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although (ADAM17) a disintegrin and metalloprotease 17 is a key sheddase of AREG, ADAM8-, ADAM15-, and batimastat (broad metalloprotease inhibitor)-sensitive metalloproteases have also been implicated in AREG shedding. In the present study, using a curly bare ( ) mouse model that shows loss-of-hair, enlarged sebaceous gland, and rapid cutaneous wound-healing phenotypes mediated by enhanced mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated mice lacking ADAM17 specifically in the skin and examined the above phenotypes of mice. We find that ADAM17 deficiency in the skin of mice restores a full hair coat, prevents sebaceous gland enlargement, and impairs the rapid wound-healing phenotype observed in mice. Furthermore, , stimulated shedding of AREG is abolished in mouse embryonic keratinocytes lacking ADAM17. Thus, our data support previous findings demonstrating that ADAM17 is the major ectodomain sheddase of AREG.
PubMed: 29632822
DOI: 10.1002/2211-5463.12407 -
3 Biotech May 2021Matrix metalloproteinases (MMPs) are the major proteolytic enzymes which assist in regulating the metastatic process by degrading the extracellular matrix proteins. In...
UNLABELLED
Matrix metalloproteinases (MMPs) are the major proteolytic enzymes which assist in regulating the metastatic process by degrading the extracellular matrix proteins. In this study, we have investigated the anti-metastatic potential of major bioactive compounds in the medicinal plant targeting matrix metalloproteinases (MMP2 & MMP9) and it's in silico pharmacokinetic profiles using computational studies. (Sivanar vembu in Tamil) is a renowned medicinal herb in traditional Indian medicine which contains indigocarpan, mucronulatol, indigocarpan diacetate, erythroxydiol X and erythroxydiol Y as the major constituents. The 3-dimensional structure of MMP2 and MMP9 was designed by using and Modeller and it was validated by PROCHECK. The structures of mucronulatol and indigocarpan have been retrieved from PubChem and indigocarpan diacetate, erythroxydiol X & Y were drawn by using Chemdraw Ultra 6.0. Batimastat was used as a positive control. Molecular docking was performed by using AutoDock 4.2 tools and AutoDock vina, an open-source program which signifies an effective interaction between the phytoligands and MMP2 & MMP9. From the results, AutoDock 4.2 have showed that indigocarpan possesses strong binding energy (ΔG) of - 7.68 kcal/mol towards MMP2 and - 6.35 kcal/mol towards MMP9, whereas batimastat showed binding energy (ΔG) of - 6.34 kcal/mol for MMP2 and - 5.66 kcal/mol for MMP9, meanwhile the results from AutoDock vina indicates that indigocarpan possesses strong binding energy (ΔG) of - 8.0 kcal/mol towards MMP2 and - 8.2 kcal/mol towards MMP9, whereas batimastat showed binding energy (ΔG) of - 7.2 kcal/mol for MMP2 and - 7.6 kcal/mol for MMP9. Also, the ADME and toxicity results suggest that the indigocarpan compound possesses a druggable pharmacokinetic potentiality and does not have carcinogenicity and Ames mutagenesis compared with other phytoligands. Hence, it is evident from our results that both AutoDock platforms strongly revealed that the phytoligand, indigocarpan possesses strong inhibitory activity against MMP2 and MMP9 to control cancer metastasis.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13205-021-02731-w.
PubMed: 33927994
DOI: 10.1007/s13205-021-02731-w -
The Journal of Biological Chemistry Jan 2020Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and...
Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.
Topics: Antineoplastic Agents; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Lysosomes; Neoplasms; Proteolysis; Proto-Oncogene Mas; Proto-Oncogene Proteins c-met; Thiosemicarbazones
PubMed: 31744884
DOI: 10.1074/jbc.RA119.011341 -
Oncotarget Aug 2018Matrix metalloproteinases (MMPs) may play a critical role in metastatic cancers, yet multiple human clinical trials targeting MMPs have surprisingly failed. Cancer cell...
Matrix metalloproteinases (MMPs) may play a critical role in metastatic cancers, yet multiple human clinical trials targeting MMPs have surprisingly failed. Cancer cell density changes dramatically during the early growth of a primary tumor and during the early seeding steps of secondary tumors and has been implicated in playing an important role in regulating metastasis and drug resistance. This study reveals that the expression of MMPs is tightly regulated by local tumor cell density through the synergistic signaling mechanism of Interleukin 6 (IL-6) and Interleukin 8 (IL-8) via the JAK2/STAT3 complex. Local tumor cell density also plays a role in the responsiveness of cells to matrix metalloproteinases inhibitors (MMPI), such as Batimastat, Marimastat, Bryostatin I, and Cipemastat, where different migratory phenotypes are observed in low and high cell density conditions. Cell density-dependent MMP regulation can be directly targeted by the simultaneous inhibition of IL-6 and IL-8 receptors via Tocilizumab and Reparixin to significantly decrease the expression of MMPs in mouse xenograft models and decrease effective metastasis. This study reveals a new strategy to decrease MMP expression through pharmacological intervention of the cognate receptors of IL-6 and IL-8 to decrease metastatic capacity of tumor cells.
PubMed: 30220965
DOI: 10.18632/oncotarget.25863 -
American Journal of Physiology. Lung... Jul 2003Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. We hypothesized that a previously found increase in collagenolytic activity of...
Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. We hypothesized that a previously found increase in collagenolytic activity of matrix metalloproteinases during hypoxia promotes pulmonary vascular remodeling and hypertension. To test this hypothesis, we exposed rats to hypoxia (fraction of inspired oxygen = 0.1, 3 wk) and treated them with a metalloproteinase inhibitor, Batimastat (30 mg/kg body wt, daily ip injection). Hypoxia-induced increases in concentration of collagen breakdown products and in collagenolytic activity in pulmonary vessels were inhibited by Batimastat, attesting to the effectiveness of Batimastat administration. Batimastat markedly reduced hypoxic pulmonary hypertension: pulmonary arterial blood pressure was 32 +/- 3 mmHg in hypoxic controls, 24 +/- 1 mmHg in Batimastat-treated hypoxic rats, and 16 +/- 1 mmHg in normoxic controls. Right ventricular hypertrophy and muscularization of peripheral lung vessels were also diminished. Batimastat had no influence on systemic arterial pressure or cardiac output and was without any effect in rats kept in normoxia. We conclude that stimulation of collagenolytic activity in chronic hypoxia is a substantial causative factor in the pathogenesis of pulmonary vascular remodeling and hypertension.
Topics: Animals; Chronic Disease; Collagen; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Metalloendopeptidases; Phenylalanine; Protease Inhibitors; Pulmonary Circulation; Rats; Rats, Wistar; Specific Pathogen-Free Organisms; Thiophenes
PubMed: 12665462
DOI: 10.1152/ajplung.00167.2002 -
Dental Materials Journal Aug 2023This study aimed to evaluate the effect of different matrix metalloproteinase inhibitors (MMPIs) on the microtensile bond strength (μTBS) and nanoleakage of universal...
This study aimed to evaluate the effect of different matrix metalloproteinase inhibitors (MMPIs) on the microtensile bond strength (μTBS) and nanoleakage of universal adhesives. One hundred twenty non-carious human molars were prepared and randomly assigned to two groups: Scotchbond Bond Universal (SBU) and Gluma Bond Universal (GBU). The samples in each group were assigned to five subgroups (n=12) based on one control (water) and four MMPIs (Benzalkonium-chloride (BAC), Batimastat (BB94), Chlorhexidine (CHX), and Epigallocatechin-gallate (EGCG)). Each adhesive was applied in self-etch (SE) mode or etch-and-rinse (ER) mode. Dentin/composite sticks were fabricated and subjected to the μTBS test after 24 h or 6 months. At 6 months, MMPIs did not affect the μTBS of the adhesives, regardless of etching mode. Nanoleakage was more pronounced in ER mode than in SE mode for all subgroups. All MMPIs, with the exception of CHX, decreased the nanoleakage of GBU in ER mode.
Topics: Humans; Acid Etching, Dental; Adhesives; Dental Bonding; Dental Cements; Dentin; Dentin-Bonding Agents; Materials Testing; Matrix Metalloproteinase Inhibitors; Resin Cements; Tensile Strength; Molar
PubMed: 37302822
DOI: 10.4012/dmj.2022-282 -
PloS One 2014UL16 binding proteins (ULBPs) are a family of cell surface proteins that are present in transformed and stressed cells and ligands for NKG2D. Soluble NKG2D ligands have...
UL16 binding proteins (ULBPs) are a family of cell surface proteins that are present in transformed and stressed cells and ligands for NKG2D. Soluble NKG2D ligands have been found in sera from cancer patients with their protein concentrations correlated with poor cancer prognosis. Here we show, for the first time, that human tumor cells lost their surface expression of ULBP2, but not ULBP1 and ULBP3, during NK cell-mediated cytolysis. In contrast to spontaneous shedding of NKG2D ligands, NK cytolysis-mediated shedding of ULBP2 was linked to target cell apoptosis, although both resulted from metalloproteinase cleavages. Inhibition of ULBP2 shedding by a metalloproteinase inhibitor BB-94 lead to reduced NK cell-mediated cytotoxicity and cytokine production. These results illustrate a regulation of NK cell effector functions through cytolysis-induced NKG2D ligand shedding. Consequently, compounds inhibiting NKG2D ligand shedding may offer therapeutic means to reduce excessive pathogenic NK cell activities.
Topics: Apoptosis; Cell Line, Tumor; Cell Membrane; Cytotoxicity, Immunologic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; Ligands; Metalloproteases; NK Cell Lectin-Like Receptor Subfamily K; Phenylalanine; Signal Transduction; Thiophenes
PubMed: 24614922
DOI: 10.1371/journal.pone.0091133