Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.

Control of ion transport in mammalian airways by protease activated receptors type 2 (PAR-2)

Protease-activated receptors (PARs) are widely distributed in human airways. They couple to G-proteins and are activated after proteolytic cleavage of the N terminus of the receptor. Evidence is growing that PAR subtype 2 plays a pivotal role in inflammatory airway diseases, such as allergic asthma or bronchitis. However, nothing is known about the effects of PAR-2 on electrolyte transport in the native airways. PAR-2 is expressed in airway epithelial cells, where they are activated by mast cell tryptase, neutrophil proteinase 3, or trypsin. Recent studies produced conflicting results about the functional consequence of PAR-2 stimulation. Here we report that stimulation of PAR-2 receptors in mouse and human airways leads to a change in electrolyte transport and a shift from absorption to secretion. Although PAR-2 appears to be expressed on both sides of the epithelium, only basolateral stimulation results in inhibition of amiloride sensitive Na+ conductance and stimulation of both luminal Cl- channels and basolateral K+ channels. The present data indicate that these changes occur through activation of phospholipase C and increase in intracellular Ca2+, which activates basolateral SK4 K+ channels and luminal Ca2+-dependent Cl- channels. In addition, the present data suggest a PAR-2 mediated release of prostaglandin E2, which may contribute to the secretory response. In conclusion, these results provide further evidence for a role of PAR-2 in inflammatory airway disease: stimulation of these receptors may cause accumulation of airway surface liquid, which, however, may help to flush noxious stimuli away from the affected airways. ©2005 FASEB

Comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A(2) (IIa) with antiinflammatory activity

Few reported inhibitors of secretory phospholipase A(2) enzymes inhibit the IIa human isoform (hnpsPLA(2)-IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D-tyrosine was derivastised to give a series of potent new inhibitors of hnpsPLA(2)-IIa. A 2.2-Angstrom crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca2+ ion through carboxylate and amide oxygen atoms, H bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T-shaped aromatic-group-His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.

Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A(2) occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A(2)-alpha

Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.

Antifibrotic activity of an inhibitor of group IIA secretory phospholipase A(2) in young spontaneously hypertensive rats

The development of fibrosis in the chronically hypertensive heart is associated with infiltration of inflammatory cells and cardiac hypertrophy. In this study, an inhibitor of the proinflammatory enzyme, group IIA human secretory phospholipase A(2) (sPLA(2)-IIA), has been found to prevent collagen deposition as an important component of cardiovascular remodeling in a rat model of developing chronic hypertension. Daily treatment of young male spontaneously hypertensive rats (SHR) with an sPLA2-IIA inhibitor (KH064, 5-(4-benzyloxyphenyl)-4S-(phenyl-heptanoylamino)-pentanoic acid, 5 mg/kg/day p.o.) prevented increases in the content of perivascular,(SHR 20.6 +/- 0.9%, n = 5; SHR+KH064 14.0 +/- 1.2%, n = 5) and interstitial (SHR 7.9 +/- 0.3%, n = 6; SHR+KH064 5.4 +/- 0.7%, n = 6) collagen in the left ventricle of rat hearts, but did not affect numbers of infiltrating monocytes/macrophages, left ventricular hypertrophy (SHR 2.88 +/- 0.08, n = 12; SHR+KH064 3.09 +/- 0.08 mg/g body weight, n = 9), increased systolic blood pressure, or thoracic aortic responses. This selective antifibrotic activity suggests that sPLA2-IIA may have an important but specific role in cardiac fibrosis, and that its inhibitors could be useful in dissecting molecular pathways leading to fibrotic conditions.

Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats

Epidemiological evidence and in vitro data suggest that COX-2 is a key regulator of accelerated remodeling. Accelerated states of osteoblast and osteoclast activity are regulated by prostaglandins in vitro, but experimental evidence for specific roles of cyclooxygenase-2 (COX-2) and secretory phospholipase A(2) (sPLA(2)) in activated states of remodeling in vivo is lacking. The aim of this study was to determine the effect of specific inhibitors of sPLA(2)-IIa and COX-2 on bone remodeling activated by estrogen deficiency in adult female rats. One hundred and twenty-four adult female Wistar rats were ovariectomized (OVX) or sham-operated. Rats commenced treatment 14 days after surgery with either vehicle, a COX-2 inhibitor (DFU at 0.02 mg/kg/day and 2.0 mg/kg/day) or a sPLA(2)-group-IIa inhibitor (KH064 at 0.4 mg/kg/day and 4.0 mg/kg/day). Treatment continued daily until rats were sacrificed at 70 days or 98 days post-OVX. The right tibiae were harvested, fixed and embedded in methylmethacrylate for structural histomorphometric bone analysis at the proximal tibial metaphysis. The specific COX-2 or sPLA(2) inhibitors prevented ovariectomy-induced (OVX-induced) decreases in trabecular connectivity (P < 0.05); suppressed the acceleration of bone resorption; and maintained bone turnover at SHAM levels following OVX in the rat. The sPLA2 inhibitor significantly suppressed increases in osteoclast surface induced by OVX (P < 0.05), while the effect of COX-2 inhibition was less marked. These findings demonstrate that inhibitors of COX-2 and sPLA(2)-IIa can effectively suppress OVX-induced bone loss in the adult rat by conserving trabecular bone mass and architecture through reduced bone remodeling and decreased resorptive activity. Moreover, we report an important role of sPLA(2)-IIa in osteoclastogenesis that may be independent of the COX-2 metabolic pathway in the OVX rat in vivo. (c) 2006 Elsevier Inc. All rights reserved.