Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.
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PMID:Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis. 1615 85

Cigarette smoking is a risk factor for the development of airway hyperresponsiveness and chronic obstructive pulmonary disease. Little is known concerning the effect of cigarette smoking on the contractility of airway smooth muscle. The current study was performed to determine the responsiveness of bronchial smooth muscles isolated from rats that were subacutely exposed to mainstream cigarette smoke in vivo. Male Wistar rats were exposed to diluted mainstream cigarette smoke for 2 h/d every day for 2 wk. Twenty-four hours after the last cigarette smoke exposure, a marked airway inflammation (i.e., increases in numbers of neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid and peribronchial tissues) was observed. In these subacutely cigarette smoke-exposed animals, the responsiveness of isolated intact (nonpermeabilized) bronchial smooth muscle to acetylcholine, but not to high K+ -depolarization, was significantly augmented when compared with the air-exposed control group. In alpha-toxin-permeabilized bronchial smooth muscle strips, the acetylcholine-induced Ca2+ sensitization of contraction was significantly augmented in rats exposed to cigarette smoke, although the contraction induced by Ca2+ was control level. Immunoblot analyses revealed an increased expression of RhoA protein in the bronchial smooth muscle of rats that were exposed to cigarette smoke. Taken together, these findings suggest that the augmented agonist-induced, RhoA-mediated Ca2+ sensitization may be responsible for the enhanced bronchial smooth muscle contraction induced by cigarette smoking, which has relevance to airway hyperresponsiveness in patients with chronic obstructive pulmonary disease.
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PMID:Effect of cigarette smoke exposure in vivo on bronchial smooth muscle contractility in vitro in rats. 1616 43

The regulation of the two isoforms of phospholipase C-gamma, PLCgamma(1) and PLCgamma(2), by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCgamma isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCgamma may also be regulated by Rho GTPases. In this study, PLCgamma(1) and PLCgamma(2) were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCgamma activity. PLCgamma(2), but not PLCgamma(1), was markedly activated in intact cells by constitutively active Rac1(G12V), Rac2(G12V), and Rac3(G12V) but not by Cdc42(G12V) and RhoA(G14V). The mechanism of PLCgamma(2) activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCgamma(2)-activating protein-tyrosine kinases. Activation of PLCgamma(2) by Rac2(G12V) in intact cells coincided with a translocation of PLCgamma(2) from the soluble to the particulate fraction. PLCgamma isozyme-specific activation of PLCgamma(2) by Rac GTPases (Rac1 approximately Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5'-(3-O-thio)triphosphate caused a marked stimulation of PLCgamma(2) but had no effect on the activity of PLCgamma(1). PLCgamma(1) and PLCgamma(2) have previously been shown to be indiscriminately activated by PtdInsP(3) in vitro. Thus, the results suggest a novel mechanism of PLCgamma(2) activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP(3).
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PMID:Isozyme-specific stimulation of phospholipase C-gamma2 by Rac GTPases. 1617 25

The fawn-hooded rat (FHR) develops severe pulmonary hypertension (PH) when raised for the first 3-4 wk of life in the mild hypoxia of Denver's altitude (5,280 ft.). The PH is associated with sustained pulmonary vasoconstriction and pulmonary artery remodeling. Furthermore, lung alveolarization and vascularization are reduced in the Denver FHR. We have recently shown that RhoA/Rho kinase signaling is involved in both vasoconstriction and vascular remodeling in animal models of hypoxic PH. In this study, we investigated the role of RhoA/Rho kinase signaling in the PH of Denver FHR. In alpha-toxin permeabilized pulmonary arteries from Denver FHR, the contractile sensitivity to Ca2+ was increased compared with those from sea-level FHR. RhoA activity and Rho kinase I protein expression in pulmonary arteries of Denver FHR (10-wk-old) were higher than in those of sea-level FHR. Acute inhalation of the Rho kinase inhibitor fasudil selectively reduced the elevated pulmonary arterial pressure in Denver FHR in vivo. Chronic fasudil treatment (30 mg.kg-1.day-1, from birth to 10 wk old) markedly reduced the development of PH and improved lung alveolarization and vascularization in Denver FHR. These results suggest that Rho kinase-mediated sustained vasoconstriction, through increased Ca2+ sensitivity, plays an important role in the established PH and that RhoA/Rho kinase signaling contributes significantly to the development of PH and lung dysplasia in mild hypoxia-exposed FHR.
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PMID:Involvement of RhoA/Rho kinase signaling in pulmonary hypertension of the fawn-hooded rat. 1632 74

We studied the protease activated receptor-1 coupling to a serum response element (SRE)-dependent luciferase activity readout in transfected COS-7 cells. Thrombin, with a pEC50 of 10.5, was 3000-fold more potent than the peptide agonists SFLLR and its derived compound C721-40 in stimulating luciferase activity, although the three agonists exhibited similar efficacy at the maximal concentration tested. Interestingly, SFLLR- and C721-40-induced luciferase activity was biphasic, suggesting that at least two populations of G proteins couple to the receptor. Further pharmacological characterization of this system was performed using selective protease activated receptor-1 antagonists. SCH203099 and ER-112787 blocked SFLLR-induced luciferase activity with similar potencies (pK(B) of 7), slightly higher than that exhibited by an arylisoxazole derivative compound from Merck (pK(B) of 6.1). These values correlated with their affinities established by competition binding experiments using [3H]-C721-40 as radioligand for protease activated receptor-1. Transduction mechanisms of protease activated receptor-1 coupling to SRE-dependent luciferase activity were examined using specific inhibitors. The Ca2+ chelator BAPTA-AM, as well as the calmodulin inhibitors W-7 and ophiobolin A, robustly inhibited SFLLR-induced SRE activation. Overexpression of RGS2 and a dominant negative rhoA protein abolished the SFLLR signal in an additive manner, suggesting a major role of Gq and G12/13 proteins. Furthermore, inhibition of phospholipase C, MAP-kinases, phosphatidyl inositol-3 kinase, rho-kinase and Ca2+/calmodulin-dependent protein kinases, all downstream effectors of Gq and G12/13, partially blocked the SFLLR-induced luciferase signal. Taken together, this SRE-luciferase assay reveals a complex network of transduction pathways of protease activated receptor-1 in accordance with the pleiotrophic action of thrombin.
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PMID:Pharmacological characterization of protease activated receptor-1 by a serum responsive element-dependent reporter gene assay: major role of calmodulin. 1652 61

Endothelial hyperpermeability is a hallmark of an inflammatory reaction and contributes to tissue damage in severe infections. Loss of endothelial cell-cell adhesion leads to intercellular gap formation allowing paracellular fluid flux. Although Staphylococcus aureus alpha-toxin significantly contributed to staphylococci disease, little is known about its mechanism of endothelial hyperpermeability induction. Here, we demonstrate that in a model of isolated perfused rat ileum discontinuation of capillary vascular-endothelial-cadherin (VE-cadherin) was observed after bolus application of S. aureus alpha-toxin being inhibited by the endogenous peptide adrenomedullin (ADM). In vitro, alpha-toxin exposure induced loss of immunoreactivity of VE-cadherin and occludin in human cultured umbilical vein endothelial cells. Likewise, ADM blocked alpha-toxin-related junctional protein disappearance from intercellular sites. Additionally, cyclic AMP elevation was shown to stabilize endothelial barrier function after alpha-toxin application. Although no RhoA activation was noted after endothelial alpha-toxin exposure, inhibition of Rho kinase and myosin light chain kinase blocked loss of immunoreactivity of VE-cadherin and occludin as well as intercellular gap formation. In summary, stabilization of endothelial junctional integrity as indicated by interendothelial immunostaining might be an interesting approach to stabilize endothelial barrier function in severe S. aureus infections.
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PMID:Perturbation of endothelial junction proteins by Staphylococcus aureus alpha-toxin: inhibition of endothelial gap formation by adrenomedullin. 1659 65

Heterotrimeric GTP-binding (G) proteins transduce hormone-induced signals to their effector enzymes, which include several phospholipases. In particular, the G(o)/G(i) and G(q) protein families have been shown to couple signaling to phospholipase A(2) (PLA(2)), phospholipase C, and phospholipase D, while the G(12)/G(13) family has been linked to the activation of small GTPases of the Rho family, and hence, to phospholipase D activation. Here, we demonstrate that in CHO cells, the G(12)/G(13) family is also able to activate cPLA(2)alpha, through the activation of RhoA and, subsequently, ERK1/2. Hormone-induced arachidonic acid release increased as a consequence of Galpha(13) overexpression, and was inhibited through inhibition of Galpha(13) signaling. The Galpha(13)-mediated cPLA(2)alpha activation was inhibited by pharmacological blockade of ERK1/2 with either U0126 or PD98059, and by RhoA inactivation with C3 toxin or a dominant-negative RhoA (N19RhoA), and was stimulated by the serine-threonine phosphatase inhibitor calyculin A. Our data thus identify a pathway of cPLA(2)alpha regulation that is initiated by thrombin and purinergic receptor activation, and that signals through Galpha(13), RhoA and ERK1/2, with the involvement of a calyculin-sensitive phosphatase.
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PMID:Galpha13 mediates activation of the cytosolic phospholipase A2alpha through fine regulation of ERK phosphorylation. 1680 23

PACAP and its receptors are expressed in growth zones of the brain. By stimulating PAC(1)-receptors PACAP can enhance, as well as reduce, the proliferation rate in a cell-type dependent manner. PACAP can enhance the proliferation rate by activating phospholipase C and protein kinase C, although other signal transduction pathways may also be responsible. PACAP can suppress proliferation by inhibiting protein complexes of the cyclins D and E with the cyclin-dependent kinases 4/6 and 2, respectively, which are necessary for entry into the cell cycle. PACAP seems to exert these inhibitory effects by acting via the Sonic hedgehog glycoprotein and the small GTPase RhoA. Also, the activation of a cyclin-dependent kinase inhibitor has been suggested. The signal transduction pathways mediating the effects of PACAP on proliferation are discussed.
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PMID:The effects of PACAP on neural cell proliferation. 1701 42

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[14C]glucose.
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PMID:EhRho1, a RhoA-like GTPase of Entamoeba histolytica, is modified by clostridial glucosylating cytotoxins. 1705 97

Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca(2+) sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were approximately 15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTPgammaS was increased in KO compared with WT as shown using a pull-down assay. Following alpha-toxin permeabilization, no difference in Ca(2+) sensitivity or in Ca(2+) sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased alpha(1)-adrenergic contraction. Following inhibition of PKC, alpha(1)-adrenergic contraction was normalized. PDBu-induced Ca(2+) sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca(2+) sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca(2+) sensitivity.
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PMID:Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1. 1710 36


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