Implantation Serine Proteinase 1 Exhibits Mixed Substrate Specificity that Silences Signaling via Proteinase-Activated Receptors

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1–100 nM) or AP2 (10–100 μM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1–1 nM) or AP2 (1–300 μM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours. © 2001 Cancer Research Campaign http://www.bjcancer.co

The role of protease-activated receptor-2 (PAR2) in the modulation of beating of the mouse isolated ureter: lack of involvement of mast cells or sensory nerves

1. The localization of protease-activated receptor-2 (PAR2) and the effects of PAR2 activators were investigated in the mouse isolated ureter in order to test the hypothesis that PAR2 activation may initiate neuropeptide release from sensory nerve fibres and hence contribute to inflammation. 2. PAR2 was localized by fluorescence immunohistochemistry to both the smooth muscle and epithelium of the ureter. Macrophage-like cells in the adventitia of the ureter were also PAR2-immunoreactive. PAR2-immunoreactivity was not observed in mast cells or nerve fibres. 3. In circular muscle preparations of the ureter in which continuous rhythmic beating was induced by KCl (20 mM) and the thromboxane A(2) mimetic U46619 (0.3 μM), trypsin (0.3 U ml(−1)) reduced beat frequency to 84.6±2.0% of control rates. The PAR2-selective peptide agonist SLIGRL-NH(2) concentration-dependently (0.1–3.0 μM) slowed beat frequency to a maximum of 72.7±2.0%. 4. Histamine (1–300 μM) was more efficacious than SLIGRL-NH(2) in inhibiting ureter beat frequency in a concentration-dependent manner to a maximum (at 300 μM) of 7.9±2.5% of the control rate. 5. Pretreatment of preparations with capsaicin (10 μM for 30 min) markedly attenuated the inhibitory effect of histamine, but not that of SLIGRL-NH(2), indicating a role for sensory nerves in the inhibitory effect of histamine only. 6. The inhibitory effect of SLIGRL-NH(2) on ureter beat frequency was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 μM) or the cyclo-oxygenase inhibitor, indomethacin (3 μM). 7. In conclusion, PAR2 activation causes inhibition of beating in the mouse ureter that is not mediated by axon reflex release of inhibitory neuropeptides. This inhibitory effect of PAR2 appears to be mediated directly on smooth muscle cells, although the contribution of non-NO, non-prostanoid epithelium-derived factors cannot be ruled out

Proteinase-mediated cell signalling: targeting proteinase-activated receptors (PARs) by kallikreins and more

1431-673

Design and synthesis of thrombin receptor-derived nonpeptide mimetics utilizing a piperazine scaffold

Journal URL: http://www.sciencedirect.com/science/journal/0968089

Targeting a proteinase-activated receptor 4 (PAR4) carboxyl terminal motif to regulate platelet function

© 2017 by The American Society for Pharmacology and Experimental Therapeutics. Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand bindingsite antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and b-arrestin interactions. By disrupting this PAR4 calcium/b-arrestin signaling process with a novel cellpenetrating peptide, we were able to inhibit both thrombintriggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans

Myopia-inhibiting concentrations of muscarinic receptor antagonists block activation of alpha \u3csub\u3e2a\u3c/sub\u3e -adrenoceptors in vitro

© 2018 The Authors. PURPOSE. Myopia is a refractive disorder that degrades vision. It can be treated with atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, but the mechanism is unknown. Atropine may block α-adrenoceptors at concentrations ≥0.1 mM, and another potent myopia-inhibiting ligand, mamba toxin-3 (MT3), binds equally well to human mAChR M 4 and α 1A -and α 2A -adrenoceptors. We hypothesized that mAChR antagonists could inhibit myopia via α 2A -adrenoceptors, rather than mAChR M 4 . METHODS. Human mAChR M 4 (M 4 ), chicken mAChR M 4 (cM 4 ), or human α 2A -adrenergic receptor (hADRA2A) clones were cotransfected with CRE/promoter-luciferase (CRE-Luc; agonist-induced luminescence) and Renilla luciferase (RLuc; normalizing control) into human cells. Inhibition of normalized agonist-induced luminescence by antagonists (ATR: atropine; MT3; HIM: himbacine; PRZ: pirenzepine; TRP: tropicamide; OXY: oxyphenonium; QNB: 3-quinuclidinyl benzilate; DIC: dicyclomine; MEP: mepenzolate) was measured using the Dual-Glo Luciferase Assay System. RESULTS. Relative inhibitory potencies of mAChR antagonists at mAChR M 4 /cM 4 , from most to least potent, were QNB \u3e OXY ≥ ATR \u3e MEP \u3e HIM \u3e DIC \u3e PRZ \u3e TRP. MT3 was 56☓ less potent at cM 4 than at M 4 . Relative potencies of mAChR antagonists at hADRA2A, from most to least potent, were MT3 \u3e HIM \u3e ATR \u3e OXY \u3e PRZ \u3e TRP \u3e QNB \u3e MEP; DIC did not antagonize. CONCLUSIONS. Muscarinic antagonists block hADRA2A signaling at concentrations comparable to those used to inhibit chick myopia (≥0.1 mM) in vivo. Relative potencies at hADRA2A, but not M 4 /cM 4 , correlate with reported abilities to inhibit chick form-deprivation myopia. mAChR antagonists might inhibit myopia via α 2 -adrenoceptors, instead of through the mAChR M 4 /cM 4 receptor subtype

Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2

© 2017 John Wiley & Sons Ltd Background: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. Objectives: We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. Methods: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. Findings: Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. Conclusions and Relevance: We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma