IRSD, INSERM (U1220), INRA, ENVT, UPS, Université de Toulouse, Toulouse, France
Protease-activatedreceptors (PARs) belong to the G protein-coupled receptor (GPCR) family. Compared to other GPCRs, the specificity of the four PARs is the lack of physiologically soluble ligands able to induce their activation. Indeed, PARs are physiologically activated after proteolytic cleavage of their N-terminal domain by proteases. The resulting N-terminal end becomes a tethered activation ligand that interact with the extracellular loop 2 domain and thus induce PAR signal. PARs expression is ubiquitous and these receptors have been largely described in chronic inflammatory diseases and cancer. In this review, after describing their discovery, structure, mechanisms of activation, we then focus on the roles of PARs in the intestine and the two main diseases affecting the organ, namely inflammatory bowel diseases and cancer.
assembled GPCRs-G proteins complexes (Galés et al., 2005; Galés et al., 2006; Nobles et al., 2005).
Protease-activatedreceptors (PARs) are a family of 4 different receptors that are activated by various proteases (Hollenberg and Compton, 2002; Macfarlane et al., 2001). PAR1, PAR3 and PAR4 respond to highly selective group of serine proteases that include thrombin, plasmin, the factor Xa and the activated protein C (Cottrell et al., 2002; Macfarlane et al., 2001). In contrast, PAR2 receptor can be activated by trypsin, tryptase and the coagulation factors VIIa and Xa (Macfarlane et al., 2001; Cottrell et al., 2002; Myatt and Hill, 2005). The activation mechanism of these receptors involved the cleavage of their N-terminal segment by the protease, unmasking a new N-terminus that acts as a tethered ligand, directly activating the transmembrane core of the receptor (Coughlin, 1999). Of interest, PARs can also be activated by synthetic peptides that mimic the tethered ligand, without the requirement of protease cleavage of receptors (Al-Ani et al., 2002; Chen et al., 1994; Vu et al., 1991). Such an activation mechanism leads to the constitutive activation of the receptor by proteases, followed by the rapid desensitization, internalization and degradation of the cleaved receptor (Chen et al., 2004; Cottrell et al., 2002; Trejo, 2003). This desensitization process involved the well characterized phosphorylation of the receptor by GRKs and the recruitment of arrestins (Paing et al., 2002; Trejo, 2003). PARs are involved in a number of physiological processes such as, thrombosis (Chung et al., 2002), vascular biology (Barnes et al., 2004), inflammation (Kannan, 2002) and the regulation of cell proliferation and tumorigenesis (Boire et al., 2005) and represent important new targets for the treatment of a number of pathologies.
Secretory serine proteases have long been known to play an important role in several physiological processes such as hemostasis (primary hemostasis, coagulation and fibrinolysis) and immune responses to infection . Increasing interactions between these two systems are being discovered, including during viral infection . Serine proteases of the coagulation cascade, such as thrombin, are able to activate complement components [27,28] and to regulate cell function through the activation of a family of G-protein coupled receptors called protease-activatedreceptors (PAR) [29–33]. PARs have a unique mechanism of activation in which irreversible proteolytic cleavage within the extracellular N-terminus of the receptor exposes a new N-terminal sequence that acts as a tethered ligand. The latter interacts with the second extracellular loop of the receptor, hereby activating the receptor [30,34,35]. Four PARs have been characterized until now (PAR1, PAR2, PAR3 and PAR4). They are all activated by thrombin except PAR2 for which the main activating protease is trypsin [30,35].
Thrombin is a multifunctional serine protease that plays a central role in hemostasis by converting soluble fibrinogen into an insoluble fibrin clot and by promoting platelet aggregation (11). Thrombin has also a cytokine-like activity exerted via specific cell surface receptors called Protease-ActivatedReceptors (PAR). PARs belong to the seven transmembrane domain G-protein-coupled receptor superfamily. There are altogether four PARs, 3 of them, PAR-1, PAR-3 and PAR-4, being thrombin receptors. All PARs are activated by cleavage of the amino terminal sequence of the extracellular exodomain, resulting in a new amino terminal sequence which acts as a tethered ligand and initiates signal transduction (11). PAR-1 is expressed by several human cellular types like platelets, endothelial cells, fibroblasts, smooth muscle cells and T lymphocytes (12, 14, 34). Besides its procoagulant effects, many studies have shown the implication of thrombin and PAR-1, its main receptor, on inflammation, fibrogenesis and extracellular matrix remodeling in several organs and particularly in kidney and liver. Thus, we and others have shown that PAR-1 was overexpressed in human liver fibrosis (27, 34). In vitro, thrombin via PAR-1 stimulates rat (16, 26) and human (24) hepatic stellate cell proliferation, it regulates their migration (18) and their capacity to synthesize extracellular matrix components (17) and protease inhibitors (30).
We then investigated whether the proinflammatory effects of thrombin administered into the colon of mice were dependent on the activation of PAR1, the first identified thrombin receptor. 27 , 28
We first used a pharmacological approach, treating mice orally with the PAR1 antagonist vorapaxar. We observed that all the thrombin-induced signs of inflammation [increased macroscopic damage score, wall thickness and bacterial translocation] were sig- nificantly inhibited by treating mice orally with the PAR1 antagonist vorapaxar [ Figure 2G–J ]. The disrupted ZO1 staining and the dis- rupted claudin-1 staining observed in mouse colons after exposure to thrombin were also ameliorated by vorapaxar treatment of mice [ Supplementary Figure 5 ]. We then employed a genetic approach, using mice deficient for the two signalling thrombin receptors: PAR1 and PAR4. We administered thrombin intracolonically as described above, in WT as well as in PAR1- or PAR4-deficient mice. In accord- ance with the data described above, we observed that in WT mice that thrombin caused signs of inflammation characterised by an in- creased macroscopic damage score, increased colonic wall thickness, and increased bacterial translocation [ Figure 3A–C ]. In addition, we followed MPO activity, apoptosis, and permeability [as measured by the passage of FITC dextran] [ Figure 3D–F ]. All of these inflamma- tory parameters were significantly increased in WT mice that received an intracolonic administration of thrombin [100 U/ml per day per mouse] compared with mice that received intracolonic saline alone [ Figure 3 ]. In comparison with the WT animals, PAR1-deficient mice did not show increased bacterial translocation, apoptosis, or perme- ability in response to intracolonic thrombin [ Figure 3C , E , and F ], but still exhibited significant macroscopic damage, increased colonic
Toxoplasma gondii is a widespread intracellular parasite, which naturally enters the
organism via the oral route and cross the intestinal barrier to disseminate. In addition to neuronal and ocular pathologies, this pathogen also causes gut inflammation in a number of animals. This infection-triggered inflammation has been extensively studied in the C57BL/6 mice, highlighting the importance of the immune cells and their mediators in the development of gut pathology. However, despite their importance in inflammation, the role of Protease-ActivatedReceptors (PAR) was never reported in the context of T.gondii-mediated small intestine inflammation. Using genetically modified mice, we show that PAR 2 plays a pathogenic role in the development
The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady- state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.
Figure 2: Expression of P2X4 and P2X7 receptors in isolated hepatocytes.
A: RT-PCR analysis showing similar levels of P2X4 and P2X7 receptor mRNAs in hepatocytes isolated from control (lane 1) and clodronate-treated rats (lane 2).
B: Western blot analysis demonstrating the presence of P2X4 and P2X7 proteins in hepatocytes. For P2X4 detection (left panel), rat salivary gland membrane proteins (lane 1) were run in parallel with hepatocyte lysates from control (lane 2) and clodronate-treated rats (lane 3). P2X7 (right panel) was detected at similar levels in hepatocytes isolated from control (lane 3) and clodronate-treated rats (lane 4). Lysates of P2X7-transfected HEK cells were used as a positive control (lane 2). P2X7 was not detected in wild-type HEK cells (lane 1). Results were confirmed in at least three independent experiments.
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[Abstract] The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the assessment of intestinal functions encompasses, among others, the monitoring of its integrity, its cellular renewing, its immune defenses, the production of enteroendocrine hormones and its digestive capacities. Here, we describe in detail how to assess the activity of the proteases secreted in the intestinal lumen of adult Drosophila melanogaster flies. This method can also be used for larval intestines. The present protocol is adapted and improved from the Sigma-Aldrich’s protocol proposed in the ‘Protease Fluorescent Detection Kit’ (Product code PF0100). Keywords: Drosophila melanogaster, Intestine, Opportunistic bacteria, Protease activity, Protein metabolism
5. Why is ADAM28 an ambivalent protease in cancer?
ADAM28 is overexpressed by carcinoma cells in multiple cancer types: breast cancer [ 9 , 12 , 13 ], renal cell carcinoma [ 58 ], non-small cell lung carcinoma (NSCLC) [ 59 , 60 ], leukemia [ 49 , 61 ], prostate cancer [ 29 ], chondrosarcoma [ 62 ], bladder cancer [ 63 ], pancreatic cancer [ 64 ] and head and neck carcinoma [ 65 ]. In experimental in vitro models, ADAM28 expression is induced by the protein kinase src in v-src-trans- formed epithelial cells and in various human carcinoma cells including PC-9 and MDA-MB231 cells [ 66 ]. Both PI3K/Akt and MEK/ERK signaling pathways are thought to be the downstream effectors of src and mediate ADAM28 expression. Likewise, PI3K/Akt signaling pathway has been shown to regulate ADAM28 expression in B-cell acute lymphoblastic leukemia [ 61 ]. In NSCLC, expression levels of ADAM28 are correlated with tumor size, lymph node metastasis and cell prolif- eration indicating an association between ADAM28 and cancer pro- gression [ 59,60,67 ]. The impact of ADAM28 expression has been studied on disease-free survival after operation and overall survival of patients with NSCLC [ 60 ]. According to ADAM28 immunodetection in carcinoma tissues, three groups of patients have been determined as the following: a high-expressing, a medium-expressing and a low-expressing group. Results indicate that disease-free survival after operation is significantly lower in the high-expressing group compared to both medium-expressing and low-expressing groups. Overall survival of pa- tients from the high-expressing group seems to be lower when compared to others groups although it is not statistically significant. In B-cell acute lymphoblastic leukemia (B-ALL) ADAM28 is a potential prognostic factor as its expression in bone marrow is associated with disease relapse which obviously compromises patient survival
Heterodimers can adopt various polarities when bound to different REs, and RXR can be positioned either upstream or downstream of the heterodimer partner. This relative orientation and its impact on the transcriptional activity of receptors has been dissected for RAR-RXR heterodimers. On DR2 and DR5 elements, RXR occupies the 5’ hexameric motif, whereas the RAR partner occupies the 3’ motif. The polarity is reversed on DR1 response elements. This structural arrangement has dramatic consequences on the transactivation properties of RXR-RAR heterodimers, as RAR agonists are unable to activate transcription from a DR1 RE. This relates to the allosteric control of NCoR assembly on these various DR REs [157,160,161] whose geometry imposes an important structural adaptation of receptor domains. In support of this, DNA binding of RXR-VDR dimers was shown to alter VDR H12 structure  . Crystallographic structures of isolated GR DBD bound to DNA identified the so-called “lever arm”, located between the two GR zinc fingers, which adopts different conformations according to the RE geometry and influences coactivator recruitment  . Other heterodimers such as PPAR"-RXR" bind to DNA similarly to RAR-RXR" and form a polar head-to-tail interaction with DR1, where RXR" binds exclusively to the 3' site [11,92] . For VDR assembled on a DR3, TR and LXR on a DR4 and NGFI-B on a NBRE, the RXR DBD was found to bind to the 5' upstream half-site [50,89,163] .
Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent proces- sive metalloprotease FtsH and cleaves MgtE, the major high- affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn 2+ /Zn 2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn 2+ and Zn 2+ ions and facilitates MgtE cleavage. Independently of its intrin- sic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolu- tionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of “ERAD” in bacteria and exempli- fies an ancestral function of rhomboid-superfamily proteins. Keywords ER-associated degradation; intramembrane protease; membrane transporter; proteostasis; rhomboid
The catalytic triad is part of an extensive hydrogen bonding network. It spans the active site cleft, with Ser195 on one side and Asp102 and His57 on the other (Figure 3). Generally, hydrogen bonds are shown between the Nδ1-H of His57 and Oδ1 of Asp102, also between the OH of Ser195 and the Nε2-H of His57, although the latter hydrogen bond is lost when His57 is protonated. The complexes of chymotrypsin and protein inhibitor eglin C are discussed to explain the mechanism in serine protease (Figure 4). The hydrogen bonds are observed throughout the catalytic cycle. Moreover, the OH of Ser214 forms a hydrogen bond with Oδ1 of Asp102, this hydrogen bond can be found in almost all chymotrypsin-like proteases. Accordingly, Ser214 was once considered the fourth member of the catalytic triad, although more recent evidence indicates that it is dispensable. Hydrogen bonds are also observed between the Oδ2 of Asp102 and the main chain NHs of Ala56 and His57. These hydrogen bonds are believed to orient Asp102 and His57. Additionally, a novel hydrogen bond is observed between the Cε1-H of His57 and the main chain carbonyl of Ser214, the carbonyl oxygen of Ser214 also plays a part in the polypeptide binding site, so it was considered to mediate the communication between substrate and the catalytic triad. Similar hydrogen bonds are also observed in subtilisin and other classes of serine hydrolases, which suggests that this interaction may be important for catalytic triad function (13, 18).
Chemokine receptors in food allergy
IgE-mediated food allergies induce various symptoms and can affect the skin, the intestine, and the lungs. They are trig- gered by food allergens, which are usually harmless for healthy people. The prevalence is very difficult to estimate because of the different tests used (skin prick tests, self- awareness, IgE concentration, etc.), but there are approxi- mately 4 –8% of food-allergic people among children and 1 –3% among adults (65). Within the lamina propria, the antigen-presenting cells are dendritic cells, particularly cells expressing CD103 (66). After antigen capture, they migrate to the mesenteric LN owing to the expression of CCR7 to activate na €ıve T cells. In the same manner, na€ıve T cells enter the LN through high endothelial venules and express CD62L and CCR7, which bind to their respective ligands on endothelial cells (GlyCAM-1, CCL21, and CCL19) (67). In the LN, CD103 + cells induce the homing molecules CCR9 and a4b7 on T cells, which allow them to migrate to the intestine. Regulatory T cells are also imprinted by dendritic cells in the mesenteric LN to maintain oral tolerance in the intestine. Thus, oral tolerance to food allergens can be impaired in mice lacking the gene for a4b7, MadCam1, or CCR9 (68, 69). However, these results are still subject to debate (67). Despite contradictory results, everyone agrees that food allergy is a breach in oral tolerance. The mecha- nisms are not clear, but the expression of the homing mole- cules is a crucial step in cell recruitment to the intestine. Moreover, the allergic response is characterized by Th2 pro- liferation, and Th2-associated cytokines and chemokines can be found in the serum of allergic patients. In a model of food allergy in mice, the receptor CCR8 and the ligands of CCR4, CCL17, and CCL22 were overexpressed in the intestine (70). Once again, the receptor CX3CR1 is also involved in food allergies. In CX3CR1 / mice, oral tolerance is impaired because of inhibition of the migration of regulatory T cells. The secretion of IL-10 was also decreased in this model (68). It was shown that Tregs leave the mesenteric LN, multiply among themselves, and migrate toward the lamina propria owing to CX3CR1-expressing myeloid cells, which also secrete IL-10 (67, 71). The receptor CCR6 is also involved in oral tolerance because IL-10-secreting Treg cells express CCR6 on their membrane. Mice lacking this gene displayed
CAN is modulated
by divalent cations, and (3) a high-frequency stimulation (HFS) synaptically generates a slow inward current through mGluRs, once glutamate and GABA ionotropic receptors have been blocked. This synaptic current demonstrates features specific to the CAN current; in particular, it is calcium-dependent (blocked by BAPTA) and G-protein-dependent (blocked by G-proteins inhibitors). Therefore, in CA1 pyramidal neurons, in addition to the well characterized fast excitatory currents resulting from the activation of ionotropic AMPA and NMDA receptors, brief tetani generate, via the activation of mGluRs, a long-lasting CAN current. Because of its unique features I CAN may play a major role
Membrane resistivity (ρ) is equal to the transmembrane potential (ΔE) divided by the intensity (I) of the current passing through a unit of membrane surface: ρ = ΔE × S/I (where S is the area of the membrane domain). The lower the resistivity is, the more intense is the transmembrane current generated by a given voltage gradient. The open- ing of channels, regardless if they mediate depolarizing or hyperpolarizing currents at the resting membrane potential, decreases membrane resistivity and promotes the dissipa- tion of action potentials, a phenomenon termed shunting inhibition. Indeed, it enables ions to respond to the electro- chemical gradient created by the action potential with local transmembrane flows instead of the expected longitudinal flows that are necessary for propagating the action potential to other membrane locations. Depolarizing currents medi- ated by GABA A receptors can interfere with action poten-
Both trypsin and tryptase have been shown to activate PAR 2 . 85 In all animal studies investigating by which mechanisms tryptase, trypsin or IBS patient biopsy supernatants were causing
increased permeability, neuron hyperexcitability or visceral hypersensitivity, proteases and/or PAR 2 activation were identi- ﬁed as the principal mechanism of action. 86 This suggests that PAR 2 antagonism could constitute a valid therapeutic option for the treatment of IBS. However, a study investigating the effects of IBS patient biopsy supernatants on human submucosal or myenteric neurons preparations has determined that PAR 1 rather than PAR 2 was activated in human tissues. 87 88 This sug- gests that in human, PAR 1 antagonists should be considered for the treatment of IBS symptoms. However, the most recent advances in the pharmacology of PARs has taught us that PARs have several ways to signal other than calcium mobilisation usually measured. 89 Adenylyl cyclase, MAPK and ERK signal- ling and β-arrestin recruitment would also have to be investi- gated in PAR 2 response of human neurons, before ruling out a possible involvement of PAR 2 . In addition, the most striking effect of PAR 2 activation was observed on visceral hypersensitiv- ity symptoms and in sensory primary afferents, which might respond differently from submucosal or myenteric neurons. Therefore, for the time being, both PAR 1 and PAR 2 antagonism should still be considered as potential therapeutic options for IBS treatment.