Post-translational allosteric activation of the P2X 7 receptor through glycosaminoglycan chains of CD44 proteoglycans

Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5′-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and Emax. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg2+. GAGs activated P2X7 receptor-mediated cytoplasmic Ca2+ influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation

P2X 7 receptor activity regulation: the role of CD44 proteoglycan GAG chains

P2X7 receptors have received special attention in the literature for their involvement in several diseases characterized by inflammatory processes such as cancer, arthritis, neurodegenerative pathologies and chronic pains

Coupling of vinculin to F-actin demands Syndecan-4 proteoglycan

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in orderto address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior. (C) 2016 Elsevier B.V. All rights reserved.CAPES (Coordenagdo de Aperfeicoamento de Pessoal de Nivel Superior)CNPq (Conselho Nacional de Desenvolvimento Cientffico e Tecnologico)FAPESP (Fundacao de Amparo a Pesquisa do Estado de sao Paulo), BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, Sao Paulo, SP, BrazilUniv Liverpool, Inst Integrat Biol, Dept Biochem, Liverpool, Merseyside, EnglandUniv Fed Sao Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Diadema, SP, BrazilUniv Houston, Coll Optometry, TOSI, Houston, TX USAUniv Fed Sao Paulo, Grp Interdisciplinar Ciencias Exatas Saude, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Diadema, SP, BrazilUniv Fed Sao Paulo, Grp Interdisciplinar Ciencias Exatas Saude, Sao Paulo, SP, BrazilFAPESP: 15/08782-3FAPESP: 15/03964-6Web of Scienc

Trisulfate disaccharide decreases calcium overload and protects liver injury secondary to liver ischemia/reperfusion

Background Ischemia and reperfusion (I/R) causes tissue damage and intracellular calcium levels are a factor of cell death. Sodium calcium exchanger (NCX) regulates calcium extrusion and Trisulfated Disaccharide (TD) acts on NCX decreasing intracellular calcium through the inhibition of the exchange inhibitory peptide (XIP). Objectives The aims of this research are to evaluate TD effects in liver injury secondary to I/R in animals and in vitro action on cytosolic calcium of hepatocytes cultures under calcium overload. Methods Wistar rats submitted to partial liver ischemia were divided in groups: Control: (n = 10): surgical manipulation with no liver ischemia; Saline: (n = 15): rats receiving IV saline before reperfusion; and TD: (n = 15): rats receiving IV TD before reperfusion. Four hours after reperfusion, serum levels of AST, ALT, TNF-α, IL-6, and IL-10 were measured. Liver tissue samples were collected for mitochondrial function and malondialdehyde (MDA) content. Pulmonary vascular permeability and histologic parameters of liver were determined. TD effect on cytosolic calcium was evaluated in BRL3A hepatic rat cell cultures stimulated by thapsigargin pre and after treatment with TD. Results AST, ALT, cytokines, liver MDA, mitochondrial dysfunction and hepatic histologic injury scores were less in TD group when compared to Saline Group (p<0.05) with no differences in pulmonary vascular permeability. In culture cells, TD diminished the intracellular calcium raise and prevented the calcium increase pre and after treatment with thapsigargin, respectively. Conclusion TD decreases liver cell damage, preserves mitochondrial function and increases hepatic tolerance to I/R injury by calcium extrusion in Ca2+ overload situations

ANTIHEMOSTATIC ACTIVITY of HEPARIN DISACCHARIDES and OLIGOSACCHARIDES OBTAINED BY CHEMICAL and ENZYMATIC FRAGMENTATION - REVERSAL of the HEMORRHAGIC ACTIVITY BY ATP and MYOSIN

ESCOLA PAULISTA MED,DEPT BIOQUIM,CP 20372,BR-04023 São Paulo,SP,BRAZILESCOLA PAULISTA MED,DEPT BIOQUIM,CP 20372,BR-04023 São Paulo,SP,BRAZILESCOLA PAULISTA MED,DEPT BIOQUIM,CP 20372,BR-04023 São Paulo,SP,BRAZILWeb of Scienc

First purification of heparan sulfate disaccharides with an amine resin used as solid support for peptide synthesis

For the first time, highly substituted benzhydryramine-resin (BHAR), a solid support used only for the peptide synthesis method was assayed successfully as an anion exchanger resin for fractionation of (-2)-, (-3)- and (-4)-charged disaccharides. (C) 2000 Elsevier Science B.V. All rights reserved.Universidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc

High molecular weight kininogen as substrate for cathepsin B

We investigated the influence of pH and divalent cations (Zn2+, Mg2+ and Ca2+) on high molecular weight kininogen processing by cathepsin B. At pH 6.3, high molecular weight kininogen is hydrolyzed by cathepsin B at three sites generating fragments of 80, 60 and 40 kDa. Cathepsin B has kininogenase activity at this pH which is improved in the absence of divalent cations. At pH 7.35, high molecular weight kininogen is slightly cleaved by cathepsin B into fragments of 60 kDa, and cathepsin B kininogenase activity is impaired. Our results suggest that high molecular weight kininogen is a substrate for cathepsin B under pathophysiological conditions.UNIFESP, EPM, Dept Bioquim, BR-04044020 São Paulo, BrazilUMC, Ctr Interdisciplinar Invest Bioquim, BR-08701970 Mogi Das Cruzes, SP, BrazilUNIFESP, EPM, Dept Biofis, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Bioquim, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc