Biomolecular and Biomedical Science Research Collection

The Microbial Cell Factory

2012-03-14T00:00:00Z

The Microbial Cell Factory Murphy, Cormac D. Microorganisms have been used for decades as sources of antibiotics, vitamins and enzymes and for the production of fermented foods and chemicals. In the 21st century microorganisms will play a vital role in addressing some of the problems faced by mankind. In this article three of the current applications in which microbes have a significant role to play are highlighted: the discovery of new antibiotics, manufacture of biofuels and bioplastics, and production of fine chemicals via biotransformation.

Production of human metabolites of the anti-cancer drug flutamide via biotransformation in Cunninghamella species

2011-02-01T00:00:00Z

Production of human metabolites of the anti-cancer drug flutamide via biotransformation in Cunninghamella species Amadio, Jessica; Murphy, Cormac D. Fungi belonging to the genus Cunninghamella have enzymes similar to those employed by mammals for the detoxification of xenobiotics, thus they are useful as models of mammalian drug metabolism, and as a source for drug metabolites. We report the transformation of the anti-cancer drug flutamide in Cunninghamella sp. The most predominant phase I metabolites present in the plasma of humans, 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)aniline, were also produced in Cunninghamella cultures. Other phase I and phase II metabolites were also detected using a combination of HPLC, GC–MS and 19F-NMR.

Production of the Novel Lipopeptide Antibiotic Trifluorosurfactin via Precursor-Directed Biosynthesis.

2012-11-01T00:00:00Z

Production of the Novel Lipopeptide Antibiotic Trifluorosurfactin via Precursor-Directed Biosynthesis. O'Connor, Neil K.; Rai, Dilip K.; Clark, Benjamin R.; Murphy, Cormac D. Incorporation of fluorine into antibiotics can moderate their biological activity, lipophilicity and metabolic stability. The introduction of fluorine into an antimicrobial lipopeptide produced by Bacillus sp. CS93 via precursor-directed biosynthesis is described. The lipopeptide surfactin is synthesised non-ribosomally by various Bacillus species and is known for its biological activity. Administering 4,4,4-trifluoro-dl-valine to cultures of Bacillus sp. CS93 results in the formation of trifluorosurfactin in quantities sufficient for detection by LC–MS/MS. 19F NMR analysis of the culture supernatant revealed that the bulk of the fluorinated amino acid was transformed and thus was unavailable for incorporation into surfactin. Detection of ammonia, and MS analysis indicated that the transformation proceeds with deamination and reduction of the keto acid, yielding 4,4,4-trifluoro-2-hydroxy-3-methylbutanoic acid.

Production of anticancer polyenes through precursor-directed biosynthesis

2011-09-01T00:00:00Z

Production of anticancer polyenes through precursor-directed biosynthesis Clark, Benjamin R.; O'Connor, Stephen; Fox, Deirdre; Leroy, Jacques; Murphy, Cormac D. The biosynthesis of the pyrrolyl moiety of the fungal metabolite rumbrin originates from pyrrole-2-carboxylic acid. In an effort to produce novel derivatives with enhanced biological activity a series of substituted pyrrole-2-carboxylates were synthesised and incubated with the producing organism, Auxarthron umbrinum. Several 4-halo-pyrrole-2-carboxylic acids were incorporated into the metabolite yielding three new derivatives: 3-fluoro-, 3-chloro- and 3-bromo-isorumbrin, which were generated in milligram quantities enabling cytotoxicity assays to be conducted. The 3-chloro- and 3-bromo-isorumbrins had improved activity against HeLa cells compared with rumbrin; 3-bromoisorumbrin also showed dramatically improved activity towards a lung cancer cell line (A549).

Bacterial production of hydroxylated and amidated metabolites of flurbiprofen

2011-11-01T00:00:00Z

Bacterial production of hydroxylated and amidated metabolites of flurbiprofen Bright, Tara V.; Clark, Benjamin R.; O'Brien, Eimear; Murphy, Cormac D. Several Streptomyces and Bacillus strains were examined for their ability to transform the anti-inflammatory drug flurbiprofen 1 to the hydroxylated metabolites that are found in humans after ingestion of this compound. Of the seven Streptomyces spp. examined, all but one transformed flurbiprofen to the main mammalian metabolite 4′-hydroxyflurbiprofen 2, and the majority also produced 3′,4′-dihydroxyflurbiprofen 3. Three strains, Streptomyces griseus DSM40236 and ATCC13273, and Streptomyces subrutilis DSM40445, also elaborated 3′-methoxy, 4′-hydroxy-flurbiprofen 4. None of the Bacillus spp. examined yielded these metabolites. Examination of the extracted supernatants of Streptomyces lavenduligriseus and Streptomyces rimosus by fluorine-19 nuclear magnetic resonance (19F NMR), indicated new resonances and these new fluorometabolites were purified by HPLC and revealed to be flurbiprofenamide 5 and 7-hydroxyflurbiprofenamide 6 after MS and NMR analyses. Subsequent re-examination of the culture supernatants from Bacillus subtilis IM7, Bacillus megaterium NCIMB8291 and B. megaterium ATTC14581 showed that these strains also produced 5 and 6. Resting cell investigations suggested that the amidation reaction employed nitrogen from an as yet unidentified amino acid.

Opioid mediated activity and expression of mu and delta opioid receptors in isolated human term non-laboring myometrium

2013-01-05T00:00:00Z

Opioid mediated activity and expression of mu and delta opioid receptors in isolated human term non-laboring myometrium Fanning, Rebecca A.; McMorrow, Jason P.; Campion, Deirdre P.; Carey, Michael F.; O'Connor, J. J. The existence of opioid receptors in mammalian myometrial tissue is now widely accepted. Previously enkephalin degrading enzymes have been shown to be elevated in pregnant rat uterus and a met-enkephalin analogue has been shown to alter spontaneous contractility of rat myometrium. Here we have undertaken studies to determine the effects of met-enkephalin on in vitro human myometrial contractility and investigate the expression of opioid receptors in pregnant myometrium. Myometrial biopsies were taken from women undergoing elective caesarean delivery at term. Organ bath experiments were used to investigate the effect of the met-enkephalin analogue [d-Ala 2, d-met 5] enkephalin (DAMEA) on spontaneous contractility. A confocal immunofluorescent technique and real time PCR were used to determine the expression of protein and mRNA, respectively for two opioid receptor subtypes, mu and delta. DAMEA had a concentration dependent inhibitory effect on contractile activity (1×10−7 M–1×10−4 M; 54% reduction in contractile activity, P<0.001 at 1×10−4 M concentration). Mu and delta opioid receptor protein sub-types and their respective mRNA were identified in all tissues sampled. This is the first report of opioid receptor expression and of an opioid mediated uterorelaxant action in term human non-labouring myometrium in vitro

Targeting tumour necrosis factor-α in hypoxia and synaptic signalling

2013-01-01T00:00:00Z

Targeting tumour necrosis factor-α in hypoxia and synaptic signalling O'Connor, J. J. Tumour necrosis factor (TNF)-α is a pro-inflammatory cytokine, which is synthesised and released in the brain by astrocytes, microglia and neurons in response to numerous internal and external stimuli. It is involved in many physiological and pathophysiological processes such as gene transcription, cell proliferation, apoptosis, synaptic signalling and neuroprotection. The complex actions of TNF-α in the brain are under intense investigation. TNF-α has the ability to induce selective necrosis of some cells whilst sparing others and this has led researchers to discover multiple activated signalling cascades. In many human diseases including acute stroke and inflammation and those involving hypoxia, levels of TNF-α are increased throughout different brain regions. TNF-α signalling may also have several positive and negative effects on neuronal function including glutamatergic synaptic transmission and plasticity. Exogenous TNF-α may also exacerbate the neuronal response to hypoxia. This review will summarise the actions of TNF-α in the central nervous system on synaptic signalling and its effects during hypoxia.

Addiction as a Disease State

2012-01-01T00:00:00Z

Addiction as a Disease State Regan, Ciaran M.

The Formation of Medical Expertise

2012-01-01T00:00:00Z

The Formation of Medical Expertise Regan, Ciaran M.

Learning from interdisciplinarity

2012-01-01T00:00:00Z

Learning from interdisciplinarity Regan, Ciaran M.

The Addicted Self: A Neuroscientific Perspective

2012-11-01T00:00:00Z

The Addicted Self: A Neuroscientific Perspective Regan, Ciaran M.

Interaction of the Human Prostacyclin Receptor and the NHERF4 Family member Intestinal and Kidney Enriched PDZ Protein (IKEPP)

2012-10-01T00:00:00Z

Interaction of the Human Prostacyclin Receptor and the NHERF4 Family member Intestinal and Kidney Enriched PDZ Protein (IKEPP) Reid, Helen M.; Turner, Elizebeth C.; Mulvaney, Eamon P.; Hyland, Paula B.; McLean, Caitriona; Kinsella, B. Therese Prostacyclin and its I Prostanoid receptor, the IP, play central roles in haemostasis and in re-endothelialization in response to vascular injury. Herein, Intestinal and Kidney Enriched PDZ Protein (IKEPP) was identified as an interactant of the human (h) IP mediated through binding of PDZ domain 1 (PDZD1) and, to a lesser extent, PDZD2 of IKEPP to a carboxyl-terminal Class I ‘PDZ ligand’ within the hIP. While the interaction is constitutive, agonist-activation of the hIP leads to cAMP-dependent protein kinase (PK) A and PKC- phosphorylation of IKEPP, coinciding with its increased interaction with the hIP. Ectopic expression of IKEPP increases functional expression of the hIP, enhancing its ligand binding and agonist-induced cAMP generation. Originally thought to be restricted to renal and gastrointestinal tissues, herein, IKEPP was also found to be expressed in vascular endothelial cells where it co-localizes and complexes with the hIP. Furthermore, siRNA-disruption of IKEPP expression impaired hIP-induced endothelial cell migration and in vitro angiogenesis, revealing the functional importance of the IKEPP:IP interaction within the vascular endothelium. Identification of IKEPP as a functional interactant of the IP reveals novel mechanistic insights into the role of these proteins within the vasculature and, potentially, in other systems where they are co-expressed.

Regulation of the Human Prostacyclin Receptor Gene by the Cholesterol-responsive Sterol Response Element Binding Protein (SREBP) 1.

2012-09-11T00:00:00Z

Regulation of the Human Prostacyclin Receptor Gene by the Cholesterol-responsive Sterol Response Element Binding Protein (SREBP) 1. Turner, Elizebeth C.; Kinsella, B. Therese Prostacyclin and its prostacyclin receptor, the IP, play essential roles in regulating haemostasis and vascular tone and have also been implicated in a range cardio-protective effects, but through largely unknown mechanisms. In this study, the influence of cholesterol on human (h)IP gene expression was investigated in cultured vascular endothelial and platelet-progenitor megakaryocytic cells. Cholesterol-depletion increased hIP mRNA, hIP promoter-directed reporter gene expression and hIP-induced cAMP generation in all cell types. Furthermore, the constitutively active SREBP1a, but not SREBP2, increased hIP mRNA and promoter-directed gene expression while deletional and mutational analysis uncovered an evolutionary conserved sterol-response element (SRE), adjacent to a known functional Sp1 element, within the core hIP promoter. Moreover, chromatin immunoprecipitation assays confirmed direct cholesterol-regulated binding of SREBP1a to this hIP promoter region in vivo, while immunofluorescence microscopy corroborated that cholesterol-depletion significantly increases hIP expression levels. In conclusion, the hIP gene is directly regulated by cholesterol-depletion that occurs through binding of SREBP1a to a functional SRE within its core promoter. Mechanistically, these data establish that cholesterol can regulate hIP expression which may, at least in part, account for the combined cardio-protective actions of low serum cholesterol through its regulation of prostacyclin receptor (IP) expression within the human vasculature.

Role of Genomics and RNA-seq in Studies of Fungal Virulence

2012-09-16T00:00:00Z

Role of Genomics and RNA-seq in Studies of Fungal Virulence Riccombeni, Alessandro; Butler, Geraldine Since its introduction in the last decade, massive parallel sequencing, or “next-generation sequencing”, has revolutionized our access to genomic information, providing accurate data with increasingly higher yields and lower costs with respect to first-generation technology. Massive parallel sequencing of cDNA, or RNA-seq, is progressively replacing array-based technology as the method of choice for transcriptomics. This review describes some of the most recent applications of next-generation sequencing technology to the study of pathogenic fungi, including Candida, Aspergillus and Cryptococcus species. Several integrated approaches illustrate the power and accuracy of RNA-seq for studying the biology of human fungal pathogens. In addition, the lack of consistency in data analysis is discussed.

Biotransformation of fluorobiphenyl by Cunninghamella elegans

2010-01-01T00:00:00Z

Biotransformation of fluorobiphenyl by Cunninghamella elegans Amadio, Jessica; Murphy, Cormac D. The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by 19F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only.

Bacterial defluorination of 4-fluoroglutamic acid

2007-01-01T00:00:00Z

Bacterial defluorination of 4-fluoroglutamic acid Donnelly, Clár; Murphy, Cormac D. Fluorinated amino acids are used as enzyme inhibitors, mechanistic probes and in the production of pharmacologically active peptides. Because enantiomerically pure 4-fluoroglutamate is difficult to prepare, the selective degradation of the l-isomer is a potentially convenient method of obtaining d-4-fluoroglutamate from the racemate. In this paper, we describe our investigations on the degradation of 4-fluoroglutamate by bacteria. Fluoride ion was detected in resting-cell cultures of a number of bacteria that were incubated with racemic 4-fluoroglutamate. Analysis of the culture supernatants by chiral gas chromatography–mass spectrometry revealed that only the l-isomer was degraded. The degradation of 4-fluoroglutamate was also examined in cell-free extracts of Streptomyces cattleya and Proteus mirabilis, and it was observed that equimolar concentrations of fluoride ion and ammonia were generated. The activity was located in the soluble fraction of cell extracts, thus is not related to the l-2-amino-4-chloro-4-pentenoic acid dehydrochlorinase previously identified in membrane fractions of P. mirabilis.

Biotransformation of flurbiprofen by Cunninghamella species

2010-09-01T00:00:00Z

Biotransformation of flurbiprofen by Cunninghamella species Amadio, Jessica; Gordon, Katherine; Murphy, Cormac D. The biotransformation of the fluorinated anti-inflammatory drug flurbiprofen was investigated in Cunninghamella spp. Mono- and di-hydroxylated metabolites were detected using gas chromatography-mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy, and the major metabolite 4’-hydroxyflurbiprofen was isolated by preparative HPLC. C. elegans DSM 1908 and C. blakesleeana DSM 1906 also produced a phase II (conjugated) metabolite, which was identified as the sulfated drug via deconjugation experiments.

Class I histone deacetylase inhibition ameliorates social cognition and cell adhesion molecule plasticity deficits in a rodent model of autism spectrum disorder

2012-09-01T00:00:00Z

Class I histone deacetylase inhibition ameliorates social cognition and cell adhesion molecule plasticity deficits in a rodent model of autism spectrum disorder Foley, Andrew G.; Gannon, Shane; Rombach-Mullan, Nanette; Prendergast, Alison; Barry, Claire; Cassidy, Andrew W.; Regan, Ciaran M. In utero exposure of rodents to valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has been proposed to induce an adult phenotype with behavioural characteristics reminiscent of those observed in autism spectrum disorder (ASD). We have evaluated the face validity of this model in terms of social cognition deficits which are a major core symptom of ASD. We employed the social approach avoidance paradigm as a measure of social reciprocity, detection of biological motion that is crucial to social interactions, and spatial learning as an indicator of dorsal stream processing of social cognition and found each parameter to be significantly impaired in Wistar rats with prior in utero exposure to VPA. We found no significant change in the expression of neural cell adhesion molecule polysialylation state (NCAM PSA), a measure of construct validity, but a complete inability to increase its glycosylation state which is necessary to mount the neuroplastic response associated with effective spatial learning. Finally, in all cases, we found chronic HDAC inhibition, with either pan-specific or HDAC1-3 isoform-specific inhibitors, to significantly ameliorate deficits in both social cognition and its associated neuroplastic response. We conclude that in utero exposure to VPA provides a robust animal model for the social cognitive deficits of ASD and a potential screen for the development of novel therapeutics for this condition.

The Role of MAPK in Drug-Induced Kidney Injury

2012-01-01T00:00:00Z

The Role of MAPK in Drug-Induced Kidney Injury Cassidy, Hilary; Radford, Robert; Slyne, Jennifer; O'Connell, Sein; Slattery, Craig; Ryan, Michael P.; McMorrow, Tara This paper focuses on the role that mitogen-activated protein kinases (MAPKs) play in drug-induced kidney injury. The MAPKs, of which there are four major classes (ERK, p38, JNK, and ERK5/BMK), are signalling cascades which have been found to be broadly conserved across a wide variety of organisms. MAPKs allow effective transmission of information from the cell surface to the cytosolic or nuclear compartments. Cross talk between the MAPKs themselves and with other signalling pathways allows the cell to modulate responses to a wide variety of external stimuli. The MAPKs have been shown to play key roles in both mediating and ameliorating cellular responses to stress including xenobiotic-induced toxicity. Therefore, this paper will discuss the specific role of the MAPKs in the kidney in response to injury by a variety of xenobiotics and the potential for therapeutic intervention at the level of MAPK signalling across different types of kidney disease.

Purification and properties of fluoroacetate dehalogenase from Pseudomonas fluorescens DSM 8341

2009-01-01T00:00:00Z

Purification and properties of fluoroacetate dehalogenase from Pseudomonas fluorescens DSM 8341 Donnelly, Clár; Murphy, Cormac D. The degradation of fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases capable of hydrolyzing the stable C-F bond have been studied. The bacterium Pseudomonas fluorescens DSM 8341 was originally isolated from soil and very readily degraded fluoroacetate, thus it was thought that its dehalogenase might have some desirable properties. The enzyme was purified from cell free extracts and characterised: it is a monomer of 32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg2+, Mn2+ and Fe3+), but inhibited by others (Hg2+, Ag2+). The enzyme is specific for fluoroacetate, and the Km for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date.

Astrocytes and the regulation of cerebral cysteine/cystine redox potential: implications for cysteine neurotoxicity

2012-02-28T00:00:00Z

Astrocytes and the regulation of cerebral cysteine/cystine redox potential: implications for cysteine neurotoxicity McBean, Gethin J. The sulfur amino acid, cysteine plays an essential role in maintaining cellular redox potential and is a key constituent of the antioxidant, glutathione. Cysteine is highly reactive and readily oxidises to the disulfide form, cystine, producing oxygen radicals as a by-product. Extracellular oxidising conditions favour cystine, whereas cysteine is the dominant intracellular form of the amino acid. In the brain, astrocytes control the extracellular thiol redox potential by actively taking up cystine and exporting cysteine. Particularly, astrocytes up-regulate the cysteine/cystine cycle in response to oxidative stress, which is essential for preventing damage to neuronal function arising from loss of redox balance. Recent evidence shows that the extracellular cysteine/cystine redox state may have a significant role in a number of processes that affect synaptic activity, including signal transduction and receptor activation and may be implicated in a number of neurodegenerative diseases, for example Alzheimer’s and Parkinson’s. This review charts recent developments in understanding the role of astrocytes in neuroprotection via management of the extracellular thiol redox potential in normal brain function and provides an up-to-date account of cysteine neurotoxicity and its significance in the aetiology of neurodegenerative disease.

Precursor-directed biosynthesis of fluorinated iturin A in Bacillus spp.

2009-01-01T00:00:00Z

Precursor-directed biosynthesis of fluorinated iturin A in Bacillus spp. Moran, Stephen; Rai, Dilip K.; Clark, Benjamin R.; Murphy, Cormac D. Some iturin A-producing strains of Bacillus subtilis will elaborate the novel fluorinated analogue when incubated with 3-fluoro-L-tyrosine. The activity of iturin A is dependent on the D-tyrosine residue and the presence of fluorotyrosine may result in an improvement of the biological properties of this lipopeptide. The fluorinated iturin might also be used as a probe for studying its interaction with biological membranes.

Biosynthesis of pyrrolylpolyenes in Auxarthron umbrinum

2009-01-01T00:00:00Z

Biosynthesis of pyrrolylpolyenes in Auxarthron umbrinum Clark, Benjamin R.; Murphy, Cormac D. The biosynthesis of the pyrrolylpolyene rumbrin (1) in the fungus Auxarthron umbrinum was elucidated using feeding studies with labelled precursors. Incorporation of stable isotopes from [15N]-proline, [13C]-methionine and [13C]-acetate confirmed that these were the precursors of the pyrrole moiety, methyl groups, and backbone of rumbrin, respectively. Label-dilution experiments with pyrrole-2-carboxylate confirmed it was a direct precursor in the biosynthesis of rumbrin. Both 3- and 4-chloropyrrolecarboxylates were also accepted as precursors in polyene production.

Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea

2012-01-01T00:00:00Z

Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea Timpson, Leanne M.; Alsafadi, Diya; Mac Donnchadha, Cillin; Liddell, Susan; Sharkey, Michael A.; Paradisi, Francesca Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.

Sirolimus enhances cyclosporine a-induced cytotoxicity in human renal glomerular mesangial cells

2012-01-23T00:00:00Z

Sirolimus enhances cyclosporine a-induced cytotoxicity in human renal glomerular mesangial cells O'Connell, Séin; Slattery, Craig; Ryan, Michael P.; McMorrow, Tara End Stage Renal Disease (ESRD) is an ever increasing problem worldwide. However the mechanisms underlying disease progression are not fully elucidated. This work addressed the nephrotoxicity induced by the immunosuppressive agents’ cyclosporine A (CsA) and sirolimus (SRL). Nephrotoxicity is the major limiting factor in the long term use of CsA. SRL causes less nephrotoxicity than CsA. Therefore investigations into the differential effects of these agents may identify potential mechanisms of nephrotoxicity and possible means to prevent ESRD induced by therapeutic drugs. Using ELISA, western blotting, quantitative PCR and a reporter gene assay we detailed the differential effects of the immunosuppressive agents CsA, and SRL in human renal mesangial cells. CsA treatment increased profibrotic TGF-β1 secretion in human mesangial cells whereas SRL did not, indicating a role for TGF-β in CsA toxicity. However we observed a synergistic nephrotoxic effect when CsA and SRL were co-administered. These synergistic alterations may have been due to an increase in CTGF which was not evident when the immunosuppressive drugs were used alone. The CsA/SRL combination therapy significantly enhanced Smad signalling and altered the extracellular matrix regulator matrix metalloproteinase 9 (MMP-9). Inhibition of the ERK 1/2 pathway, attenuated these CsA/SRL induced alterations indicating a potentially significant role for this pathway.

Regulation of the human prostacyclin receptor gene in megakaryocytes : major roles for C/EBPδ and PU.1

2012-05-01T00:00:00Z

Regulation of the human prostacyclin receptor gene in megakaryocytes : major roles for C/EBPδ and PU.1 Keating, Garret L.; Turner, Elizebeth C.; Kinsella, B. Therese The prostanoid prostacyclin plays a central role in haemostasis and vascular repair. Recent studies investigating the regulation of the human prostacyclin receptor (hIP) gene identified an upstream repressor region (URR) within its regulatory promoter, herein termed the PrmIP. This study aimed to identify the main transacting factors that bind within the URR to transcriptionally repress PrmIP-directed gene expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 cell line. Of the putative cis-acting elements examined, disruption of C/EBP and PU.1 elements within the URR substantially increased PrmIP-directed gene expression. Chromatin immunoprecipitation (ChIP) confirmed that C/EBPδ and PU.1, but not C/EBPβ, bind to the URR in vivo, while ectopic expression of C/EBPδ substantially reduced hIP mRNA levels and PrmIP-directed gene expression. Phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation increased hIP mRNA and PrmIP-directed reporter gene expression and hIP-mediated cAMP generation in HEL cells. Two PMA-responsive regions, termed PRR1 and PRR2, were identified within PrmIP. Disruption of C/EBPδ and PU.1 cis-elements within the overlapping PRR1/URR and of Sp1, PU.1 and Oct-1 cis-elements within the overlapping PRR2/core PrmIP, revealed that both PRR1 and PRR2 contribute to the PMA- induction of hIP mRNA and gene expression in HEL cells. Furthermore, ChIP analysis established that induction of PrmIP-directed gene expression during megakaryocytic differentiation is largely regulated by PMA-induced dissociation of C/EBPδ and enhanced binding of PU.1 to PRR1 in addition to increased binding of Sp1, PU.1 and Oct-1 to elements within the core promoter/PRR2 in vivo. Taken together, these data provide critical insights into the transcriptional regulation of the hIP gene within the vasculature, including during megakaryocytic differentiation.

Mechanisms of calcineurin inhibitor nephrotoxicity in chronic allograft injury

2012-01-01T00:00:00Z

Mechanisms of calcineurin inhibitor nephrotoxicity in chronic allograft injury Slattery, Craig; Cassidy, Hilary; Johnston, Olwyn; Ryan, Michael P.; McMorrow, Tara The first successful transplantation of a human kidney was performed more than 50 years ago by Murray and colleagues in 1954 between identical twins. The success of this transplantation was due to the fact that no significant rejection occurs between genetically identical twins and therefore immunosuppression was not necessary in this particular case (Merrill et al., 1956). However, solid-organ transplantation could not be considered truly successful until the 1970’s after significant technical and pharmacological advances. In particular, the discovery and development of the calcineurin inhibitors (CNIs) has made allograft transplantation routinely successful with greatly reduced risk of acute rejection. In the absence of pharmacological agents to address the primary pathological mechanisms involved, renal transplantation has now been the standard management of end stage renal failure for the past four decades (Wolfe et al., 1999). Short-term renal allograft and allograft recipient survival rates have increased significantly during the last decade largely due to improved patient monitoring. However, allograft half-life beyond 1 year post-transplant remains largely unchanged. While rates of early allograft failure have significantly reduced, late renal allograft dysfunction remains a significant problem in the transplant population (de Fijter). Chronic allograft injury (CAI) is the most prevalent cause of allograft dysfunction in the first decade after transplantation. The term CAI is used to describe deterioration of renal allograft function and structure due to immunological processes (i.e. chronic rejection) and/or a range of simultaneous nonimmunological factors such as CNI-induced nephrotoxicity, hypertension and infection. This chapter will outline the pathophysiology and etiology of CAI and the role that CNI nephrotoxicity plays in this disease process. It will also review experimental studies that have identified important molecular mechanisms involved and discuss strategies utilised to minimise the development and progression of CAI.

Quantitative MRI analysis of brain volume changes due to controlled cortical impact

2010-07-26T00:00:00Z

Quantitative MRI analysis of brain volume changes due to controlled cortical impact Colgan, Niall C.; Cronin, Michelle M.; Gobbo, Olivier L.; O'Mara, S. M. (Shane M.); O'Connor, William; Gilchrist, M. D. More than 85% of reported brain traumas are classified clinically as “mild” using GCS; qualitative MRI findings are scarce and provide little correspondence to clinical symptoms. Our goal, therefore, was to establish in-vivo sequellae of traumatic brain injury following lower and higher levels of impact to the frontal lobe using quantitative MRI analysis and a mechanical model of penetrating impact injury. To investigate time-based morphological and physiological changes of living tissue requires a surrogate for the human central nervous system. The present model for TBI was a systematically varied and controlled cortical impact on deeply-anaesthetized Sprague Dawley rats designed to mimic different injury severities. Whole-brain MRI scans were performed on each rat prior to either a lower or a higher level of impact and then at hourly intervals for five hours post-impact. Both brain volume and specific anatomical structures were segmented from MR images for inter-subject comparisons post-registration. Animals subjected to lower and higher impact levels exhibited elevated intracranial pressure (ICP) in the low compensatory reserve (i.e., nearly exhausted) and terminal disturbance (i.e., exhausted) ranges, respectively. There was a statistically-significant drop in cerebrospinal fluid of 35% in the lower impacts and 65% in the higher impacts at Hr5 in comparison to the sham control. There was a corresponding increase in corpus callosum volume starting from Hr1 of 60-110% and 30-40% following the lower and higher impact levels, respectively. A statistically significant change in the abnormal tissue from Hr2 to Hr5 was observed for both impact levels, with greater significance for higher impacts. Furthermore, a statistically significant difference between the lower impacts and the sham controls occurred at Hr3. These results are statistically substantiated by a fluctuation in the physical size of the corpus callosum, a decrease in the volume of CSF, and elevated levels of atrophy in the cerebral cortex.

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

2012-02-01T00:00:00Z

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions Ceruso, Mariangela; Howe, Nicole; Malthouse, J.Paul G. The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pKa of the inhibitor carboxylate group is lowered by at least 1.5 pKa units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of carboxylate carbon of Z-[1-13C]-L-tryptophan is ≤ 3.71 Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-13C]-L-tryptophan is not bound in the S2' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.

Predicting the open conformations of protein kinases using molecular dynamics simulations

2012-01-01T00:00:00Z

Predicting the open conformations of protein kinases using molecular dynamics simulations Bjarnadottir, Una; Nielsen, Jens Erik Protein kinases (PK) control phosphorylation in eukaryotic cells, and thereby regulate metabolic pathways, cell cycle progression, apoptosis and transcription. Consequently there is significant interest in manipulating PK activity and treat diseases by using small-molecule drugs. All PK catalytic domains undergo large conformational changes as a result of substrate binding and phosphorylation. The “closed” state of a PK cataltic domain is the only state able to phosphorylate the target substrate, which makes the two other observed states (the “open” and the “intermediate” states) interesting drug targets. We investigate if MD simulations starting from the closed state of the catalytic domain of protein kinase A (C-PKA) can be used to produce realistic structures representing the intermediate and/or open conformation of C-PKA, since this would allow for drug docking calculations and drug design using MD snapshots. We perform 36 ten-nanosecond MD simulations starting from the closed conformation (PDB ID: 1ATP) of C-PKA in various liganded and phosphorylated states. The results show that MD simulations are capable of reproducing the open conformation of C-PKA with good accuracy within 1 ns of simulation as measured by Cα RMSDs and RMSDs of atoms defining the ATPbinding pocket. Importantly we are able to show that even without knowledge of the structure of the open form of C-PKA, we can identify the MD snapshots resembling the open conformation most using the open structure of a different protein kinase displaying only 23% sequence identity to C-PKA.

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1 : implications for prostate cancer.

2011-04-29T00:00:00Z

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1 : implications for prostate cancer. Turner, Elizebeth C.; Kavanagh, David J.; Mulvaney, Eamon P.; McLean, Caitriona; Wikström, Katarina; Reid, Helen M.; Kinsella, B. Therese In humans, thromboxane (TX)A2 signals through the TPalpha and TPbeta isoforms of the TXA2 receptor, or TP. Herein, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPalpha and TPbeta involving common and unique sequences within their respective carboxyl-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1640-942). While the interaction with PRK1 is constitutive, agonist-activation of TPalpha/TPbeta did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signalling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist-activation of TPalpha/TPbeta led to phosphorylation of histone H3 at Thr11 (H3Thr11), a previously recognized specific marker of androgen induced-chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in non-responsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPalpha/TPbeta in both LNCaP and PC-3 cells and targeted disruption of PRK1 impaired TPalpha/TPbeta-mediated H3Thr11 phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA2 as a potent mediator of RhoA signalling, the identification of PRK1 as a bone fide interactant of TPalpha/TPbeta, and leading to H3Thr11 phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.

Transcriptional regulation of the human prostacyclin receptor gene is dependent on Sp1, PU.1 and Oct-1 in megakaryocytes and endothelial cells

2009-02-27T00:00:00Z

Transcriptional regulation of the human prostacyclin receptor gene is dependent on Sp1, PU.1 and Oct-1 in megakaryocytes and endothelial cells Turner, Elizebeth C.; Kinsella, B. Therese Prostacyclin plays a central role in haemostasis, inflammation and nociception. However, the factors regulating expression of the prostacyclin receptor (IP) gene in humans, or in other species, have not been identified. Herein it was sought to identify the key trans-acting factors and cis-acting elements regulating IP expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 and the vascular endothelial EA.hy 926 cell lines. Using deletion and genetic reporter analyses, the essential core promoter, termed PrmIP, was localized to -1022 to -895 proximal to the transcription initiation site, while an upstream repressor region, localized to -1502 to -1271, was also identified. Bioinformatic analysis revealed evolutionary conserved Sp1, PU.1 and Oct-1 sites within the core PrmIP and disruption of those elements each led to substantial reductions in PrmIP-directed gene expression in both HEL and EA.hy 926 cells. Electrophoretic mobility shift assays (EMSAs) and supershift assays established that Sp1, PU.1 and Oct-1 can bind to elements within the core promoter in vitro while chromatin immunoprecipitiation (ChIP) assays confirmed their specific binding to chromatin in vivo. Furthermore, combination mutations of the Sp1, PU.1 and Oct-1 elements revealed that they act independently to co-regulate basal transcription of the IP gene while ectopic expression of each of the trans-acting factors led to substantial increases in PrmIP-directed gene expression and IP mRNA expression in both HEL and EA.hy 926 cells. While EMSA and antibody supershift assays established that the Ets family member Fli1, but not Ets-1, is capable of binding to the PU.1 element within PrmIP in vitro, ChIP analysis established that neither Fli1 nor Ets-1 bind to that element in vivo. Collectively, these data provide critical insights into the transcriptional regulation of the IP gene in human megakaryocytic and endothelial cells, identifying Sp1, PU.1 and Oct-1 as the critical factors involved in its basal regulation in humans.

Differential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor : independent modulation of TPalpha signaling by prostacyclin and nitric oxide

2008-08-01T00:00:00Z

Differential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor : independent modulation of TPalpha signaling by prostacyclin and nitric oxide Wikström, Katarina; Kavanagh, David J.; Reid, Helen M.; Kinsella, B. Therese In humans, thromboxane (TX) A2 signals through the TPalpha and TPbeta isoforms of the TXA2 receptor that exhibit common and distinct roles. For example, Gq/phospholipase (PL)Cbeta signaling by TPalpha is directly inhibited by the vasodilators prostacyclin and nitric oxide (NO) whereas that signaling by TPbeta is unaffected. Herein, we investigated whether TPalpha and/or TPbeta regulate G12/Rho activation and whether that signaling might be differentially regulated by prostacyclin and/or NO. Both TPalpha and TPbeta independently regulated RhoA activation and signaling in clonal cells over-expressing TPalpha or TPbeta and in primary human aortic smooth muscle cells (1o AoSMCs). While RhoA- signaling by TPalpha was directly impaired by prostacyclin and NO through protein kinase (PK)A- and PKG-dependent phosphorylation, respectively, signaling by TPbeta was not directly affected by either agent. Collectively, while TPalpha and TPbeta contribute to RhoA activation, our findings support the hypothesis that TPalpha is involved in the dynamic regulation of haemostasis and vascular tone, such as in response to prostacyclin and NO. Conversely, the role of TPbeta in such processes remains unsolved. Data herein provide essential new insights into the physiologic roles of TPalpha and TPbeta and, through studies in AoSMCs, reveal an additional mode of regulation of VSM contractile responses by TXA2.

Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoforms

1998-11-27T00:00:00Z

Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoforms Miggin, Sinead M.; Kinsella, B. Therese The human (h) TXA2 receptor (TP), a G protein-coupled receptor, exists as two isoforms, TPalpha and TPbeta, which arise by alternative mRNA splicing and differ exclusively in their carboxyl terminal cytoplasmic regions. In this study, a reverse transcriptase - polymerase chain reaction (RT-PCR) based strategy was developed to examine the expression of the TPs in tissues of physiologic relevance to TXA2. Although most of the 17 different cell / tissue types examined expressed both TP isoforms, the liver hepatoblastoma HepG2 cell line was found to exclusively express TPalpha mRNA. In most cell types, TPalpha mRNA predominated over TPbeta mRNA. Moreover, although the levels of TPalpha mRNA expression were similar in most of the cell / tissue types examined, extensive differences in the levels of TPbeta mRNA were observed. Consequently, the relative expression of TPalpha : TPbeta mRNA varied considerably due to extensive differences in TPbeta mRNA expression. Most strikingly, primary HUVEC’s were found to express: (i) low levels of TPbeta and (ii) approximately 6- fold greater levels of TPalpha than TPbeta . These data were confirmed in the spontaneously transformed HUVEC derived ECV304 cell line. Expression of TP mRNAs in the various tissue / cells correlated with protein expression, as assessed by radioligand binding using the selective TP antagonist [3H] SQ29,548.

Investigation of the role of the carboxyl terminal tails of the alpha and beta isoforms of the human thromboxane A2 receptor (TP) in mediating receptor : effector coupling

2000-04-17T00:00:00Z

Investigation of the role of the carboxyl terminal tails of the alpha and beta isoforms of the human thromboxane A2 receptor (TP) in mediating receptor : effector coupling Walsh, Marie-Therese; Foley, John F.; Kinsella, B. Therese We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A2 receptor (TP) to Galpha16 and Galpha12 members of the Gq and G12 families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP-328 cells which over-expresses a variant of TP truncated at the point of divergence of TPalpha and TPbeta we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha16 to affect increases in IP3 and mobilization of intracellular calcium ([Ca2+]i) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca2+]i mobilization in cells co-transfected with Galpha12, neither receptor generated corresponding increases in IP3 indicating that the Galpha12 mediated increases in [Ca2+]i do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca2+ channels reduced [Ca2+]i mobilization in TPalpha and TPbeta cells co-transfected with Galpha12 to approximately 40% of that mobilized in its absence whereas 3,4,5-trimethyloxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), an antagonist of intracellular Ca2+ release, had no effect on [Ca2+]i mobilization by either receptor isoform co-transfected with Galpha12. Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP-328 signaled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha11, Galpha12 or Galpha16 subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP-328 coupled to GalphaS, leading to increased cAMP, rather than to Galphai. Whereas TP-328 signaled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha co-transfection of Galphas did not augment cAMP generation by TP-328. Hence, from these studies involving the wild type TPalpha, TPbeta and TP-328, we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha11 and Galpha16 members of the Gq family or to Galpha12; it may play a role in determining GS versus Gi coupling and may act as a determinant of coupling efficiency.

The Wilms’ tumor suppressor protein WT1 acts as a key transcriptional repressor of the human thromboxane A2 receptor gene in megakaryocytes

2009-11-01T00:00:00Z

The Wilms’ tumor suppressor protein WT1 acts as a key transcriptional repressor of the human thromboxane A2 receptor gene in megakaryocytes Gannon, AnneMarie; Kinsella, B. Therese In humans, TPα and TPβ isoforms of the thromboxane A2 receptor are transcriptionally regulated by distinct promoters, designated Prm1 and Prm3. Previous investigations identified two upstream repressor regions (URR) 1 and URR2 within Prm1. Herein, it was sought to characterize Prm1, identifying the factor(s) regulating URR1 and URR2 in human erythroleukemia (HEL) 92.1.7 cells. Genetic reporter assays and 5’ deletions confirmed the presence of URR1 and URR2 but also identified a third repressor, designated RR3, within the proximal “core” promoter. Bioinformatic analysis revealed several GC elements representing putative sites for Egr1/Sp1/Wilms tumor (WT)1 within URR1, URR2 and RR3. While mutation of three GC elements within URR1 and of an adjacent GC element suggested that repressor binding occurs through a cooperative mechanism, repressors binding to the single GC elements within URR2 and RR3 act independently to regulate Prm1. While EMSAs and supershift assays demonstrated that each of the GC elements can bind Egr1 and WT1 in vitro, chromatin immunoprecipitations established that WT1 is the factor predominantly bound to each of the repressor regions in vivo. Additionally, ectopic expression of -KTS isoforms of WT1 decreased Prm1-directed gene expression and TPα mRNA expression. Collectively, these data establish WT1 as a critical repressor of Prm1, suppressing TPα expression in the platelet progenitor megakaryoblastic HEL cells.

Regulated expression of the α isoform of the human thromboxane A2 receptor during megakaryocyte differentiation : a coordinated role for WT1, Egr1 & Sp1

2009-11-20T00:00:00Z

Regulated expression of the α isoform of the human thromboxane A2 receptor during megakaryocyte differentiation : a coordinated role for WT1, Egr1 & Sp1 Gannon, AnneMarie; Turner, Elizebeth C.; Reid, Helen M.; Kinsella, B. Therese Thromboxane plays an essential role in haemostasis, regulating platelet aggregation and vessel tone. In humans, it signals through the TPalpha and TPbeta isoforms that are transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively. Herein, the consequence of megakaryocytic differentiation on Prm1-directed TPα expression was investigated. Phorbol ester (PMA) treatment substantially increased TPα mRNA and Prm1-directed gene expression in human erythroleukemia (HEL) and K562 cells. Deletional analyses localized the major responsive element(s) to the upstream -8500 and -7504 region while mutation of four WT1/Egr1/Sp1 cis-elements therein established that each contribute to the induction. Moreover, PMA increased Egr1, but not WT1 or Sp1, expression while the NAB1 co-repressor impaired PMA-induction of Egr1 and Prm1-directed gene expression. Chromatin immunoprecipitations established that WT1 is predominantly bound in vivo to the 5’ Prm1 region in non-differentiated HEL cells. In response to PMA, there was initial induction in Egr1 and associated reduction in WT1 binding to Prm1 in vivo which was displaced by Sp1 following sustained treatment. Collectively, data establish that regulated WT1 followed by sequential Egr1 and Sp1 binding to elements within Prm1 mediate repression and subsequent induction of TPα during differentiation into the megakaryocytic phenotype, shedding significant insights into factors regulating TPa expression therein.

Identification of a novel endoplasmic reticulum export motif within the eighth alpha-helical domain (alpha-H8) of the human prostacyclin receptor

2011-04-01T00:00:00Z

Identification of a novel endoplasmic reticulum export motif within the eighth alpha-helical domain (alpha-H8) of the human prostacyclin receptor Donnellan, Peter D.; Kimbembe, Cisca C.; Reid, Helen M.; Kinsella, B. Therese The human prostacyclin receptor (hIP) undergoes agonist-dependent trafficking involving a direct interaction with Rab11a GTPase. The region of interaction was localised to a 14 residue Rab11a binding domain (RBD) within the proximal carboxyl-terminal (C)-tail domain of the hIP, consisting of Val299 – Val307 within the eighth helical domain (alpha-H8) adjacent to the palmitoylated residues at Cys308 – Cys311. However, the factors determining the anterograde transport of the newly synthesised hIP from the endoplasmic reticulum (ER) to the plasma membrane (PM) have not been identified. The aim of the current study was to identify the major ER export motif(s) within the hIP initially by investigating the role of Lys residues in its maturation and processing. Through site-directed and Ala-scanning mutational studies in combination with analyses of protein expression and maturation, functional analyses of ligand binding, agonist-induced intracellular signalling and confocal image analyses, it was determined that Lys297, Arg302 and Lys304 located within alpha-H8 represent the critical determinants of a novel ER export motif of the hIP. Furthermore, while substitution of those critical residues significantly impaired maturation and processing of the hIP, replacement of the positively charged Lys with Arg residues, and vice versa, was functionally permissible. Hence, this study has identified a novel 8 residue ER export motif within the functionally important alpha-H8 of the hIP. This ER export motif, defined by ‘K/R(X)4K/R(X)K/R’, has a strict requirement for positively charged, basic Lys/Arg residues at the 1st, 6th and 8th positions and appears to be evolutionarily conserved within IP sequences from mouse to man.

Prostaglandin D2 receptor-mediated desensitization of the alpha isoform of the human thromboxane A2 receptor

2001-07-15T00:00:00Z

Prostaglandin D2 receptor-mediated desensitization of the alpha isoform of the human thromboxane A2 receptor Foley, John F.; Kelley-Hickie, Leanne P.; Kinsella, B. Therese Thromboxane (TX) A2 and prostaglandin (PG) D2 mediate opposing actions in platelets and in vascular and non-vascular smooth muscle. Here, we investigated the effects of stimulation of the PGD2 receptor (DP) on signaling by the TXA2 receptor (TP) expressed in human platelets and in human embryonic kidney (HEK) 293 cells over-expressing the individual TPalpha and TPbeta isoforms. In platelets, the selective DP agonist BW245C abolished TP-mediated mobilization of intracellular calcium ([Ca2+]i) and inhibited platelet aggregation in response to the TXA2 mimetic U46619. DP-mediated desensitization of TP signaling in platelets was prevented by pre-treatment with the cAMP-dependent PKA inhibitor, H-89, but was unaffected by the PKC inhibitor GF 109203X. In HEK 293 cells signaling by TPalpha, but not TPbeta, was subject to DP mediated desensitization in a PKA dependent, PKC independent manner. U46619-induced signaling by TP-328, a truncated variant of TP containing only those residues common to TPalpha and TPbeta, was insensitive to prior DP stimulation indicating that the carboxyl terminal tail of TPalpha contains the target site(s) for DP-mediated desensitization. Mutation of Ser329 to Ala329 within a consensus PKA site in TPalpha rendered the mutant TPalphaS329A insensitive to BW245C-mediated desensitization. Whole cell phosphorylation assays established that TPalpha, but not TPbeta or TPalphaS329A, was subject to DP-mediated phosphorylation and that TPalpha phosphorylation was blocked by the PKA inhibitor H-89. These data establish that TPalpha, but not TPbeta, is subject to DP mediated cross desensitization, which occurs through direct PKA mediated phosphorylation of TPalpha at Ser329.

Thromboxane A2 receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells

2001-05-28T00:00:00Z

Thromboxane A2 receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells Miggin, Sinead M.; Kinsella, B. Therese Both thromboxane (TX) A2 and 8-epi prostaglandin (PG) F2alpha have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA2 and 8-epiPGF2alpha mediated mitogenic signalling have not been studied in detail in human vascular SM. Thus, using the human uterine ULTR cell line as a model, we investigated TXA2 receptor (TP) mediated mitogenic signalling in cultured human vascular SM cells. Both the TP agonist U46619 and 8-epiPGF2alpha elicited time and concentration dependent activation of the extracellular signal regulated kinase (ERK)s and c-Jun N-terminal kinase (JNK)s in ULTR cells. Whereas the TP antagonist SQ29,548 abolished U46619-mediated signalling, it only partially inhibited 8-epiPGF2alpha mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF2alpha induced ERK activations were inhibited by the protein kinase (PK) C, PKA and phosphoinositide 3-kinase inhibitors GF 109203X, H-89 and wortmannin, respectively, but were unaffected by pertussis toxin. In addition, U46619 mediated ERK activation in ULTR cells involves transactivation of the EGF receptor. In humans, TXA2 signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPalpha and TPbeta, independently directed U46619 and 8-epiPGF2alpha mediated ERK and JNK activation in human embryonic kidney (HEK) 293 cells over-expressing the individual TP isoforms. However, in contrast to that which occurred in ULTR cells, SQ29,548 abolished 8-epiPGF2alpha mediated ERK and JNK activation through both TPalpha and TPbeta in HEK 293 cells providing further evidence that 8-epiPGF2alpha may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.

Estrogen increases expression of the human prostacyclin receptor within the vasculature through an ERα-dependent mechanism

2010-02-26T00:00:00Z

Estrogen increases expression of the human prostacyclin receptor within the vasculature through an ERα-dependent mechanism Turner, Elizebeth C.; Kinsella, B. Therese Prostacyclin and the prostacyclin receptor (IP) are implicated in mediating many of the atheroprotective effects of estrogen in both humans and in animal models but through unknown mechanisms. Hence, herein the influence of estrogen on IP gene expression in endothelial EA.hy926, human erythroleukemia 92.1.7 and primary human (h) aortic smooth muscle (1o hAoSM) cells was investigated. Estrogen increased hIP mRNA levels, promoter (PrmIP)-directed reporter gene expression and cicaprost-dependent cAMP generation in all cell types, effects that were abrogated by actinomycinD and the general estrogen receptor (ER)-α/ERβ antagonist ICI 182,780. Furthermore, the ERα-selective agonist 4,4’,4”-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), but not the ERβ-agonist 2,3-bis(4-Hydroxyphenly)-propionitrile, significantly increased hIP mRNA and PrmIP-directed gene expression. Deletional and mutational analysis of PrmIP uncovered an evolutionary conserved estrogen-response element (ERE) while electrophoretic mobility shift, antibody-supershift and chromatin immunoprecipitations assays confirmed the direct binding of ERα, but not ERβ, to PrmIP both in vitro and in vivo. Moreover, immunofluorescence microscopy corroborated that estrogen and PPT increased hIP expression in 1o hAoSMCs. In conclusion, the hIP gene is directly regulated by estrogen that largely occurs through an ERα-dependent transcriptional mechanism and thereby provides critical insights into the role of prostacyclin/hIP in mediating the atheroprotective effects of estrogen within the human vasculature.

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A2 receptor

2011-04-01T00:00:00Z

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A2 receptor Reid, Helen M.; Wikström, Katarina; Kavanagh, David J.; Mulvaney, Eamon P.; Kinsella, B. Therese In humans, thromboxane (TX) A2 signals through the TPα and TPβ isoforms of its G-protein coupled TXA2 receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA2 mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.

Synthetic peroxisome proliferator-activated receptor gamma agonists rosiglitazone and troglitazone suppress transcription by promoter 3 of the human thromboxane A2 receptor gene in human erythroleukemia cells

2006-04-28T00:00:00Z

Synthetic peroxisome proliferator-activated receptor gamma agonists rosiglitazone and troglitazone suppress transcription by promoter 3 of the human thromboxane A2 receptor gene in human erythroleukemia cells Coyle, Adrian T.; Kinsella, B. Therese The human thromboxane (TX)A2 receptor (TP) gene encodes two TP isoforms, TPalpha and TP beta that are regulated by distinct promoters designated promoter (Prm) 1 and Prm3, respectively. Previous studies established that 15d-delta 12,14-prostaglandin J2 (15d-PGJ2) selectively inhibits Prm3 activity and TP beta expression through a peroxisome proliferator-activated receptor (PPAR)gamma mechanism without affecting Prm1 activity or TPalpha expression in human megakaryocytic erythroleukemia (HEL) 92.1.7 cells. Herein, we investigated the effect of synthetic thiazolidinedione (TZD) PPARgamma ligands rosiglitazone and troglitazone on TP gene expression in HEL cells. Like 15d-PGJ2, both TZDs suppressed Prm3 activity, TPbeta mRNA expression and TP-mediated calcium mobilization without affecting Prm1 or TPalpha mRNA expression. However, unlike 15d-PGJ2, both TZDs mediated their PPARgamma-dependent effects through trans-repression of an activator protein-1 (AP-1) element, a site previously found to be critical for basal Prm3 activity. These data provide further evidence for the role of PPARgamma in regulating the human TP gene; they highlight further differences in TPalpha and TPbeta expression/regulation and point to essential differences between natural and synthetic PPARgamma agonists in mediating those effects.

Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK : role in endothelial cell migration and angiogenesis

2011-06-08T00:00:00Z

Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK : role in endothelial cell migration and angiogenesis Turner, Elizebeth C.; Mulvaney, Eamon P.; Reid, Helen M.; Kinsella, B. Therese Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein, the HDL scavenger receptor class B, type 1 (SR-B1) adapter protein PDZ domain-containing protein 1 (PDZK1) was identified as an interactant of the human prostacyclin receptor (hIP) involving a Class I PDZ ligand at its carboxyl-terminus and PDZ domains 1, 3 and 4 of PDZK1. While the interaction is constitutive, it may be dynamically regulated following cicaprost-activation of the hIP through a mechanism involving cAMP-dependent protein kinase (PK)A-phosphorylation of PDZK1 at Ser505. While PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface enhancing ligand binding and cicaprost-induced cAMP generation. Consistent with its role in re-endothelialization and angiogenesis, cicaprost-activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects completely abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR-B1, siRNA-targeted disruption of PDZK1 abolished cicaprost-mediated endothelial responses but did not affect VEGF-responses. Considering the essential role played by prostacyclin throughout the cardiovascular system, identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players, and potentially HDL/SR-B1, within the vascular endothelium.

Thromboxane A2 signalling in humans : a ‘tail’ of two receptors

2001-01-01T00:00:00Z

Thromboxane A2 signalling in humans : a ‘tail’ of two receptors Kinsella, B. Therese Since its discovery in 1975, we now have a wealth of knowledge relating to the biochemical, pharmacological and physiologic actions of thromboxane (TX) A2 and its related metabolites. These molecular insights have been greatly expedited by the molecular cloning and characterisation of a complementary (c) DNA for the human TXA receptor, now termed T Prostanoid or TP receptor, from a megakaryocytic / placental cDNA library in 1991 and later through the discovery of a cDNA encoding a second isoform of the human TP receptor in 1994. The requirement for two TP receptors in primates, but not in other species thus far investigated, is unclear but points to potential species-specific physiologic differences. In this review, I will describe some recent advances in the research field of TXA2/TP receptor signalling, focussing particularly on studies pertaining to the human TP receptor isoforms.

Homologous desensitization of signalling by the beta isoform of the human thromboxane A2 receptor

2006-09-01T00:00:00Z

Homologous desensitization of signalling by the beta isoform of the human thromboxane A2 receptor Kelley-Hickie, Leanne P.; Kinsella, B. Therese Thromboxane (TX) A2 is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA2 mediated through the TPbeta isoform of the human TXA2 receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.

The role of N-linked glycosylation in determining the surface expression, G protein interaction and effector coupling of the alpha isoform of the human thromboxane A2 receptor

2003-05-02T00:00:00Z

The role of N-linked glycosylation in determining the surface expression, G protein interaction and effector coupling of the alpha isoform of the human thromboxane A2 receptor Kelley-Hickie, Leanne P.; Kinsella, B. Therese In humans, thromboxane (TX) A2 signals through two TXA2 receptor (TP) isoforms, termed TPalpha and TPbeta, that diverge exclusively within their carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn4 and Asn16. Whilst it has been established that impairment of N-glycosylation of TPalpha significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPalpha on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPalpha, by assessment of its ligand binding, G-protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPalpha protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn4 or Asn16 is required and sufficient for expression of functionally active TPalpha on the PM while the fully non-glycosylated TPalphaN4,N16-Q4,Q16 is almost completely retained within the endoplasmic reticulum and remains functionally inactive, failing to associate with its coupling G protein Galphaq and, in turn, failing to mediate phospholipase Cbeta activation.

Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase

2008-12-01T00:00:00Z

Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase Wikström, Katarina; Reid, Helen M.; Hill, Maria; English, Karol A.; O'Keeffe, Martina B.; Kimbembe, Cisca C.; Kinsella, B. Therese The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val299 – Gln320) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding-domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.

Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct

2009-08-01T00:00:00Z

Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct Howe, Nicole; Rogers, Louis; Hewage, Chandralal; Malthouse, J.Paul G. The peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be ~10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 p.p.m. and 200.5 p.p.m. are observed for the glyoxal keto and aldehyde carbons of the inhibitor bound to subtilisin, showing that the glyoxal keto and aldehyde carbons are sp3 and sp2 hybridized respectively. The signal at 107.2 p.p.m. from the carbon atom attached to the hemiketal oxyanion is formed in a slow exchange process that involves the dehydration of the glyoxal aldehyde carbon. Two additional signals are observed one at 108.2 p.p.m. and the other at 90.9 p.p.m. for the glyoxal keto and aldehyde carbons respectively at pHs 6-8 demonstrating that subtilisin forms an additional tetrahedral adduct with Z-Ala-Ala-Phe-glyoxal in which both the glyoxal keto and aldehyde carbons are sp3 hybridised. For the first time we can quantify oxyanion stabilisation in subtilisin. We conclude that oxyanion stabilisation is more effective in subtilisin than in chymotrypsin. Using 1H-NMR we show that the binding of Z-Ala-Ala-Phe-glyoxal to subtilisin raises the pKa of the imidazolium ion of the active site histidine residue promoting oxyanion stabilisation. The mechanistic significance of these results are discussed.

pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor

2011-10-01T00:00:00Z

pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor Howe, Nicole; Ceruso, Mariangela; Spink, Edward; Malthouse, J.Paul G. The Stromelysin-1 catalytic domain83-247 (SCD) is stable for at least 16 hours at pHs 6.0-8.4. At pHs 5.0 and 9.0 there is exponential irreversible denaturation with half lives of 38 and 68 min respectively. At pHs 4.5 and 10.0 irreversible denaturation is biphasic. At 25°C, C-terminal truncation of stromelysin-1 decreases the stability of the stromelysin-1 catalytic domain at pH values > 8.4 and < 6.0. We describe the conversion of the carboxylate group of (βR)-β-[[[(1S)-1-[[[(1S)-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1'-biphenyl]-4-hexanoic acid (UK-370106-COOH) a potent inhibitor of the metalloprotease stromelysin-1 to a glyoxal group (UK-370106-CO13CHO). At pH 5.5 - 6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (Ki = ~1 μM). The aldehyde carbon of the glyoxal inhibitor was enriched with carbon-13 and using Carbon-13 NMR we show that the glyoxal aldehyde carbon is fully hydrated when it is in aqueous solutions (90.4 ppm) and also when it is bound to SCD (~92.0 ppm). We conclude that the hemiacetal hydroxyl groups of the glyoxal inhibitor are not ionised when the glyoxal inhibitor is bound to SCD. The free enzyme pKa values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92 ppm due to the bound UK-370106-CO13CHO inhibitor depends on pKa values of 5.8 and 7.8 respectively. No strong hydrogen bonds are present in free SCD or in SCD-inhibitor complexes. We conclude that the inhibitor glyoxal group is not directly coordinated to the catalytic zinc atom of SCD.