http://hdl.handle.net/10197/2003 2013-05-20T09:17:52Z http://hdl.handle.net/10197/4248 Principles of Electrospray Ionization Wilm, M Electrospray ionization is today the most widely used ionization technique in chemical and bio-chemical analysis. Interfaced with a mass spectrometer it allows to investigate the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which enables even the investigation of large non-covalent protein complexes. Its high ionization efficiency profoundly changed bio-molecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focusses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed which are related to this mechanism. Typical spectra of peptides, proteins and non-covalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent non-covalent complexes are described.  2011-05-19T00:00:00Z http://hdl.handle.net/10197/4247 Quantitative proteomics in biological research Wilm, Matthias Proteomics has enabled the direct investigation of biological material, at first through the analysis of individual proteins, then of lysates from cell cultures, and finally of extracts from tissues and biopsies from entire organisms. Its latest manifestation - quantitative proteomics - allows deeper insight into biological systems. This article reviews the different methods used to extract quantitative information from mass spectra. It follows the technical developments aimed toward global proteomics, the attempt to characterize every expressed protein in a cell by at least one peptide. When applications of the technology are discussed, the focus is placed on yeast biology. In particular, differential quantitative proteomics, the comparison between an experiment and its control, is very discriminating for proteins involved in the process being studied. When trying to understand biological processes on a molecular level, differential quantitative proteomics tends to give a clearer picture than global transcription analyses. As a result, MS has become an even more indispensable tool for biochemically motivated biological research. 2009-10-01T00:00:00Z http://hdl.handle.net/10197/4241 Frequency modulation atomic force microscopy in ambient environments utilizing robust feedback tuning Kilpatrick, J. I.; Gannepalli, A.; Cleveland, J. P.; Jarvis, Suzi Frequency modulation atomic force microscopy (FM-AFM) is rapidly evolving as the technique of choice in the pursuit of high resolution imaging of biological samples in ambient environments. The enhanced stability afforded by this dynamic AFM mode combined with quantitative analysis enables the study of complex biological systems, at the nanoscale, in their native physiological environment. The operational bandwidth and accuracy of constant amplitude FM-AFM in low Q environments is heavily dependent on the cantilever dynamics and the performance of the demodulation and feedback loops employed to oscillate the cantilever at its resonant frequency with a constant amplitude. Often researchers use ad hoc feedback gains or instrument default values that can result in an inability to quantify experimental data. Poor choice of gains or exceeding the operational bandwidth can result in imaging artifacts and damage to the tip and/or sample. To alleviate this situation we present here a methodology to determine feedback gains for the amplitude and frequency loops that are specific to the cantilever and its environment, which can serve as a reasonable "first guess", thus making quantitative FM-AFM in low Q environments more accessible to the nonexpert. This technique is successfully demonstrated for the low Q systems of air (Q∼40) and water (Q∼1). In addition, we present FM-AFM images of MC3T3-E1 preosteoblast cells acquired using the gains calculated by this methodology demonstrating the effectiveness of this technique. 2009-02-02T00:00:00Z http://hdl.handle.net/10197/4016 Expression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton Kochanowska, Iwona; Chaberek, Slawomir; Wojtowicz, Andrzej; Marczynski, Bartosz; Włodarski, Krzysztof; Dytko, Malgorzata; Ostrowski, Kazimierz BACKGROUND: Differences in duration of bone healing in various parts of the human skeleton are common experience for orthopaedic surgeons. The reason for these differences is not obvious and not clear.METHODS: In this paper we decided to measure by the use of real-time RT-PCR technique the level of expression of genes for some isoforms of bone morphogenetic proteins (BMPs), whose role is proven in bone formation, bone induction and bone turnover. Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. Activity of genes for BMP-2, -4 and -6 was measured by the use of fluorescent SYBR Green I.RESULTS: It was found that expression of m-RNA for BMP-2 and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. In all samples examined the expression of m-RNA for BMP-4 was higher than for BMP-2.CONCLUSION: It was shown that m-RNA for BMP-6 is not expressed in the collected samples at all. It is postulated that differences in the level of activation of genes for BMPs is one of the important factors which determine the differences in duration of bone healing of various parts of the human skeleton. 2007-12-27T00:00:00Z http://hdl.handle.net/10197/4015 Dementia in SPG4 hereditary spastic paraplegia : Clinical, genetic, and neuropathologic evidence Murphy, S.; Gorman, B.; Beetz, C.; Byrne, P.; Dytko, Malgorzata; McMonagle, P.; Kinsella, K.; Hutchinson, M. Background: Cognitive impairment and dementia has been reported in autosomal dominant hereditary spastic paraparesis (HSP) linked to the SPG4 locus. There has only been one postmortem examination described; not all accept that progressive cognitive decline is a feature of this disorder.Objective: A family with SPG4-HSP known to have a deletion of exon 17 in the spastin gene (SPG4delEx17) was cognitively assessed over a 7-year period. The index family member died and a postmortem examination was performed.Methods: Thirteen family members older than 40 years were clinically and cognitively assessed using the Cambridge Cognitive Assessment over a 7-year period. The presence of SPG4delEx17 was assessed; a neuropathologic examination of the brain of the index family member was performed.Results: Cognitive decline occurred in 6 of the 13 family members and in all 4 older than 60 years. Two genetic deletions were identified: SPG4delEx17 in 12 of the 13 family members and a deletion of SPG6 (SPG6del) in 5. Eight individuals had the SPG4delEx17 deletion only; 4 had evidence of progressive cognitive impairment. Four family members had both SPG4delEx17 and SPG6del; 2 of these had cognitive impairment. One family member with the SPG6del alone had neither HSP nor cognitive impairment. The index case with both deletions died with dementia; the brain showed widespread ubiquitin positivity within the neocortex and white matter.Conclusion: Cognitive decline and dementia is a feature of SPG4-HSP due to a deletion of exon 17 of the spastin gene. Neurology (R) 2009; 73: 378-384 2009-08-04T00:00:00Z http://hdl.handle.net/10197/3916 The Addicted Self: A Neuroscientific Perspective Regan, Ciaran M. 2012-11-01T00:00:00Z http://hdl.handle.net/10197/3891 Towards the Improved Discovery and Design of Functional Peptides: Common Features of Diverse Classes Permit Generalized Prediction of Bioactivity Mooney, Catherine; Haslam, Niall J.; Pollastri, Gianluca; Shields, Denis C. The conventional wisdom is that certain classes of bioactive peptides have specific structural features that endow their particular functions. Accordingly, predictions of bioactivity have focused on particular subgroups, such as antimicrobial peptides. We hypothesized that bioactive peptides may share more general features, and assessed this by contrasting the predictive power of existing antimicrobial predictors as well as a novel general predictor, PeptideRanker, across different classes of peptides.We observed that existing antimicrobial predictors had reasonable predictive power to identify peptides of certain other classes i.e. toxin and venom peptides. We trained two general predictors of peptide bioactivity, one focused on short peptides (4-20 amino acids) and one focused on long peptides (>20 amino acids). These general predictors had performance that was typically as good as, or better than, that of specific predictors. We noted some striking differences in the features of short peptide and long peptide predictions, in particular, high scoring short peptides favour phenylalanine. This is consistent with the hypothesis that short and long peptides have different functional constraints, perhaps reflecting the difficulty for typical short peptides in supporting independent tertiary structure.We conclude that there are general shared features of bioactive peptides across different functional classes, indicating that computational prediction may accelerate the discovery of novel bioactive peptides and aid in the improved design of existing peptides, across many functional classes. An implementation of the predictive method, PeptideRanker, may be used to identify among a set of peptides those that may be more likely to be bioactive. 2012-10-01T00:00:00Z http://hdl.handle.net/10197/3789 Profile-based short linear protein motif discovery. Haslam, Niall J.; Shields, Denis C. Background Short linear protein motifs are attracting increasing attention as functionally independent sites, typically 3-10 amino acids in length that are enriched in disordered regions of proteins. Multiple methods have recently been proposed to discover over-represented motifs within a set of proteins based on simple regular expressions. Here, we extend these approaches to profile-based methods, which provide a richer motif representation. Results The profile motif discovery method MEME performed relatively poorly for motifs in disordered regions of proteins. However, when we applied evolutionary weighting to account for redundancy amongst homologous proteins, and masked out poorly conserved regions of disordered proteins, the performance of MEME is equivalent to that of regular expression methods. However, the two approaches returned different subsets within both a benchmark dataset, and a more realistic discovery dataset. Conclusions Profile-based motif discovery methods complement regular expression based methods. Whilst profile-based methods are computationally more intensive, they are likely to discover motifs currently overlooked by regular expression methods. 2012-05-18T00:00:00Z http://hdl.handle.net/10197/3748 Predictive modelling of angiotensin converting enzyme inhibitory dipeptides Norris, Roseanne; Casey, Fergal; FitzGerald, Richard; Shields, Denis C.; Mooney, Catherine The ability of docking to predict angiotensin converting enzyme (ACE) inhibitory dipeptide sequences was assessed using AutoDock Vina. All potential dipeptides and phospho-dipeptides were docked and scored. Peptide intestinal stability was assessed using a prediction amino acid clustering model. Selected dipeptides, having AutoDock Vina scores −8.1 and predicted to be ‘stable’ intestinally, were characterised, using LIGPLOT and for ACE-inhibitory potency. Two newly identified ACE-inhibitory dipeptides, Asp-Trp and Trp-Pro, having Vina scores of −8.3 and −8.6 gave IC50 values of 258 ± 4.23 and 217 ± 15.7 μM, respectively. LIGPLOT analysis indicated no zinc interaction for these dipeptides. Phospho-dipeptides were predicted to have a good affinity for ACE. However, the experimentally determined IC50 results did not correlate since, for example, Trp-pThr and Pro-pTyr, having Vina scores of −8.5 and −8.1, respectively, displayed IC50 values of >500 μM. While docking allowed identification of new ACE inhibitory dipeptides, it may not be a fully reliable predictive tool in all cases. 2012-08-14T00:00:00Z http://hdl.handle.net/10197/3747 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. 2012-01-01T00:00:00Z http://hdl.handle.net/10197/3695 Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population Kim, Jong Ah; Åberg, Christoffer; Salvati, Anna; Dawson, Kenneth A. Nanoparticles are considered a primary vehicle for targeted therapies because they can pass biological barriers, enter and distribute in cells by energy-dependent pathways1-3. Until now, most studies have shown that nanoparticle properties, such as size4-6 and surface7,8, can affect how cells internalise nanoparticles. Here we show that the different phases of cell growth, which constitute the cell cycle, can also influence nanoparticle uptake. Although cells in different cell cycle phases internalised nanoparticles with similar rates, after 24 hours of uptake the concentration of nanoparticles in the cells is ranked according to the different cell cycle phases: G2/M > S > G0/G1. Nanoparticles were not exported from cells but the internalised nanoparticle concentration is split when the cell divides. Our results suggest that future studies on nanoparticle uptake should consider the cell cycle because in a cell population, the internalised nanoparticle dose in each cell varies as the cell cycles. 2012-01-01T00:00:00Z http://hdl.handle.net/10197/3631 High-mobility group box protein 1: a novel mediator of inflammatory-induced renal epithelial-mesenchymal transition Lynch, Julie; Nolan, Stephen; Slattery, Craig; Feighery, Ronan; Ryan, Michael P.; McMorrow, Tara Background: High mobility group box protein 1 (HMGB-1) is a chromatin binding protein that bends DNA thereby facilitating gene transcription. HMGB-1 has also been observed as an extracellular secreted protein in serum of patients with sepsis and has putative intracellular signalling effects regulating the production of interleukin-1 and tumour necrosis factor in a number of inflammatory conditions. Methods: We established a model of immune-mediated epithelial-mesenchymal transition (EMT) in human proximal tubular epithelial cells (PTECs). PTECs were cultured with conditioned medium containing supernatant from activated peripheral blood mononuclear cells (aPBMC). The model was characterised using phenotypic and transcriptomic approaches and suppression subtractive hybridisation was performed to identify differentially regulated genes. Results. Activation of PBMCs resulted in increased secretion of HMGB-1. In addition, treatment of PTECs with aPBMC-conditioned medium resulted in significant upregulation of HMGB-1 in PTECs. Direct treatment of PTECs with recombinant human HMGB-1 induced alterations in epithelial morphology consistent with EMT including reduced E-cadherin expression, increased α-SMA expression and enhanced cell migration. HMGB-1 effects were mediated at least in part by the receptor for advanced glycation end products (RAGE) and through induction of TGF-β1 secretion from PTECs. Conclusions. These results suggest that HMGB-1 is a key mediator of immune-mediated EMT of PTECs and a potentially important signalling molecule in the development of renal fibrosis. 2010-11-24T00:00:00Z http://hdl.handle.net/10197/3596 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. 2012-01-23T00:00:00Z http://hdl.handle.net/10197/3577 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. 2012-01-01T00:00:00Z http://hdl.handle.net/10197/3443 SCLpred : protein subcellular localization prediction by N-to-1 neural networks Mooney, Catherine; Wang, Yong-Hong; Pollastri, Gianluca Knowledge of the subcellular location of a protein provides valuable information about its function and possible interaction with other proteins. In the post-genomic era, fast and accurate predictors of subcellular location are required if this abundance of sequence data is to be fully exploited. We have developed a subcellular localization predictor (SCLpred), which predicts the location of a protein into four classes for animals and fungi and five classes for plants (secreted, cytoplasm, nucleus, mitochondrion and chloroplast) using machine learning models trained on large non-redundant sets of protein sequences. The algorithm powering SCLpred is a novel Neural Network (N-to-1 Neural Network, or N1-NN) we have developed, which is capable of mapping whole sequences into single properties (a functional class, in this work) without resorting to predefined transformations, but rather by adaptively compressing the sequence into a hidden feature vector. We benchmark SCLpred against other publicly available predictors using two benchmarks including a new subset of Swiss-Prot Release 2010_06. We show that SCLpred surpasses the state of the art. The N1-NN algorithm is fully general and may be applied to a host of problems of similar shape, that is, in which a whole sequence needs to be mapped into a fixed-size array of properties, and the adaptive compression it operates may shed light on the space of protein sequences. The predictive systems described in this article are publicly available as a web server at http://distill.ucd.ie/distill/. 2011-08-27T00:00:00Z http://hdl.handle.net/10197/3395 Prediction of short linear protein binding regions Mooney, Catherine; Pollastri, Gianluca; Shields, Denis C.; Haslam, Niall J. Short linear motifs in proteins (typically 3–12 residues in length) play key roles in protein–protein interactions by frequently binding specifically to peptide binding domains within interacting proteins. Their tendency to be found in disordered segments of proteins has meant that they have often been overlooked. Here we present SLiMPred (short linear motif predictor), the first general de novo method designed to computationally predict such regions in protein primary sequences independent of experimentally defined homologs and interactors. The method applies machine learning techniques to predict new motifs based on annotated instances from the Eukaryotic Linear Motif database, as well as structural, biophysical, and biochemical features derived from the protein primary sequence. We have integrated these data sources and benchmarked the predictive accuracy of the method, and found that it performs equivalently to a predictor of protein binding regions in disordered regions, in addition to having predictive power for other classes of motif sites such as polyproline II helix motifs and short linear motifs lying in ordered regions. It will be useful in predicting peptides involved in potential protein associations and will aid in the functional characterization of proteins, especially of proteins lacking experimental information on structures and interactions. We conclude that, despite the diversity of motif sequences and structures, SLiMPred is a valuable tool for prioritizing potential interaction motifs in proteins. 2012-01-06T00:00:00Z http://hdl.handle.net/10197/3230 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. 2012-01-01T00:00:00Z http://hdl.handle.net/10197/3187 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. 2011-04-29T00:00:00Z http://hdl.handle.net/10197/3186 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. 2009-02-27T00:00:00Z http://hdl.handle.net/10197/3185 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. 2008-08-01T00:00:00Z http://hdl.handle.net/10197/3184 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. 1998-11-27T00:00:00Z http://hdl.handle.net/10197/3180 SLiMSearch 2.0 : biological context for short linear motifs in proteins Davey, Norman E.; Haslam, Niall J.; Shields, Denis C.; Edwards, Richard J. Short, linear motifs (SLiMs) play a critical role in many biological processes. The SLiMSearch 2.0 (Short, Linear Motif Search) web server allows researchers to identify occurrences of a user-defined SLiM in a proteome, using conservation and protein disorder context statistics to rank occurrences. User-friendly output and visualizations of motif context allow the user to quickly gain insight into the validity of a putatively functional motif occurrence. For each motif occurrence, overlapping UniProt features and annotated SLiMs are displayed. Visualization also includes annotated multiple sequence alignments surrounding each occurrence, showing conservation and protein disorder statistics in addition to known and predicted SLiMs, protein domains and known post-translational modifications. In addition, enrichment of Gene Ontology terms and protein interaction partners are provided as indicators of possible motif function. All web server results are available for download. Users can search motifs against the human proteome or a subset thereof defined by Uniprot accession numbers or GO term. The SLiMSearch server is available at: http://bioware.ucd.ie/slimsearch2.html. 2011-05-26T00:00:00Z http://hdl.handle.net/10197/3168 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. 2000-04-17T00:00:00Z http://hdl.handle.net/10197/3167 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. 2009-11-01T00:00:00Z http://hdl.handle.net/10197/3166 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. 2009-11-20T00:00:00Z http://hdl.handle.net/10197/3165 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. 2011-04-01T00:00:00Z http://hdl.handle.net/10197/3164 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. 2001-07-15T00:00:00Z http://hdl.handle.net/10197/3163 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. 2001-05-28T00:00:00Z http://hdl.handle.net/10197/3162 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. 2010-02-26T00:00:00Z http://hdl.handle.net/10197/3161 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. 2011-04-01T00:00:00Z http://hdl.handle.net/10197/3149 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. 2006-04-28T00:00:00Z http://hdl.handle.net/10197/3148 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. 2011-06-08T00:00:00Z http://hdl.handle.net/10197/3147 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. 2001-01-01T00:00:00Z http://hdl.handle.net/10197/3146 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. 2006-09-01T00:00:00Z http://hdl.handle.net/10197/3145 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. 2003-05-02T00:00:00Z http://hdl.handle.net/10197/3144 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. 2008-12-01T00:00:00Z http://hdl.handle.net/10197/3015 Identification of β2-microglobulin as a urinary biomarker for chronic allograft nephropathy using proteomic methods Johnston, Olwyn; Cassidy, Hilary; O'Connell, Séin; O'Riordan, Aisling; Gallagher, William; Maguire, Patricia B.; Wynne, Kieran; Cagney, Gerard; Ryan, Michael P.; Conlon, Peter J.; McMorrow, Tara Chronic allograft nephropathy (CAN) remains the leading cause of renal graft loss after the first year following renal transplantation. This study aimed to identify novel urinary proteomic profiles, which could distinguish and predict CAN in susceptible individuals. Experimental Design: The study included 34 renal transplant patients with histologically proven CAN and 36 patients with normal renal transplant function. High-throughput proteomic profiles were generated from urine samples with three different ProteinChip arrays by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Following SELDI a biomarker pattern software analysis was performed which led to the identification of a novel biomarker pattern that could distinguish patients with CAN from those with normal renal function. Results: An 11.7 kDa protein identified as β2 microglobulin was the primary protein of this biomarker pattern, distinguishing CAN from control patients (ROC = 0.996). SELDI-TOF-MS comparison of purified β2 microglobulin protein and CAN urine demonstrated identical 11.7 kDa protein peaks. Significantly higher concentrations of β2 microglobulin were found in the urine of patients with CAN compared to the urine of normal renal function transplant recipients (p<0.001). Conclusions and clinical relevance: Whilst further validation in a larger more diverse patient population is required to determine if this β2 microglobulin protein biomarker will provide a potential means of diagnosing CAN by non-invasive methods in a clinical setting, this study clearly shows a capability to stratify control and disease patients. 2011-08-01T00:00:00Z http://hdl.handle.net/10197/2987 CycloPs : generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids Duffy, Fergal J.; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C.; Chubb, Anthony J. We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie. 2011-03-24T00:00:00Z http://hdl.handle.net/10197/2972 Disinfection of meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a remote non-thermal gas plasma Cotter, John J.; Maguire, Paul; Soberon, Felipe; Daniels, Stephen; O'Gara, James P.; Casey, Eoin The effective disinfection of hospital surfaces is recognised as an important factor in preventing hospital-acquired infections. The purpose of this study was to quantify the disinfection rate of a novel gas plasma system on clinically relevant biofilms. Clinical isolates of Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus (MRSA) were grown as biofilms on glass surfaces and tested in a disinfection container remote from the plasma source. The strains used in this study were known to produce substantial quantities of biofilm and average log10 counts were 9.0 and 9.1 cfu/cm2 for S. epidermidis and MRSA respectively. Counts were reduced by between 4 and 4.5 log10 after 1 h of exposure for MRSA and S. epidermidis respectively. More prolonged treatment in the case of MRSA biofilms resulted in a 5.5 log10 reduction after 90 min. Biofilm samples were also placed in medical device packaging bags and similar rates of disinfection were observed. 2011-05-01T00:00:00Z http://hdl.handle.net/10197/2942 SLiMSearch : a webserver for finding novel occurrences of short linear motifs in proteins, incorporating sequence context Davey, Norman E.; Haslam, Niall J.; Shields, Denis C.; Edwards, Richard J. Short, linear motifs (SLiMs) play a critical role in many biological processes. The SLiMSearch (Short, Linear Motif Search) webserver is a flexible tool that enables researchers to identify novel occurrences of pre- defined SLiMs in sets of proteins. Numerous masking options give the user great control over the contextual information to be included in the analyses, including evolutionary filtering and protein structural disorder. User-friendly output and visualizations of motif context allow the user to quickly gain insight into the validity of a putatively functional motif occurrence. Users can search motifs against the human proteome, or submit their own datasets of UniProt proteins, in which case motif support within the dataset is statistically assessed for over- and under-representation, accounting for evolutionary relationships between input proteins. SLiMSearch is freely available as open source Python modules and all webserver results are available for download. The SLiMSearch server is available at: http://bioware.ucd.ie/slimsearch.html. Paper presented at the 5th IAPR International Conference, PRIB 2010, Nijmegen, The Netherlands, September 22-24, 2010 2010-01-01T00:00:00Z http://hdl.handle.net/10197/2887 Identification of novel indicators of cyclosporine A nephrotoxicity in a CD-1 mouse model O'Connell, Séin; Slattery, Craig; Ryan, Michael P.; McMorrow, Tara The calcineurin inhibitor cyclosporine A (CsA) is a widely used immunosuppressive agent. However,nephrotoxicity is a serious side effect observed in patients which limits clinical use of CsA. CsA nephrotoxicity is associated with tubulointerstitial injury progressing to nephropathy. This is typically diagnosed by invasive renal biopsy and is often only detected when the disease process is well advanced. Therefore identification of novel, early indicators of CsA nephrotoxicity could be clinically advantageous. This study aimed to establish a murine model of CsA nephrotoxicity and to identify urinary proteins that may indicate the onset of CsAinduced nephropathy using 2-D gel electrophoresis. CsA nephrotoxicity was induced in CD-1 mice by daily CsA administration for 4 weeks. By week 4, elevated serum creatinine and proteinuria were observed after CsA treatment indicating significant renal dysfunction. Decreased cadherin-1, increased α-smooth muscle actin and fibroblast specific protein 1 in kidney tissue indicated disruption of normal tubular architecture. Alterations in podocin and uromodulin were also observed which may indicate damage to other segments of the nephron. Proteomic analysis of urine identified a number of differentially regulated proteins that may be involved in early CsA nephropathy including cadherin 1, superoxide dismutase and vinculin. These findings suggest novel mechanisms of CsA nephrotoxicity and identify novel potential markers of the disease. 2011-04-15T00:00:00Z http://hdl.handle.net/10197/2745 Characterisation of a modified rotating disk reactor for the cultivation of Staphylococcus epidermidis biofilm Cotter, John J.; O'Gara, James P.; Stewart, Philip S.; Pitts, Betsey; Casey, Eoin Aims:  The purpose of this study was to develop a system that would allow biofilms to be cultivated under strictly defined conditions in terms of dissolved oxygen, fluid shear and to assess whether the method was suitable for the detection of respiratory activity stratification in biofilm samples. Methods:  The system is a modified version a commercially available laboratory biofilm reactor and incorporates a number of features such as the provision of defined levels of dissolved oxygen, constant average shear, enhanced gas–liquid mass transfer, aseptic operation and the ability to remove biofilm for ex situ analysis during or after continuous cultivation. Conclusions:  The system was shown to be effective for the characterization of the effects of dissolved oxygen on a pure culture of Staphylococcus epidermidis. The versatility of the system offers the potential for cultivating pure culture biofilm in defined, controlled conditions and facilitates a range of analyses that can be performed ex situ. Significance and Impact of the Study:  The ability to provide strict regulation of environmental conditions and enhanced transfer of oxygen to the biofilm during cultivation are important, first because oxygen is known to regulate biofilm development in several micro-organisms and second because many conventional biofilm cultivation systems may not provide adequate oxygen supply to the biofilm. 2010-12-01T00:00:00Z http://hdl.handle.net/10197/2744 Oxygen-mediated regulation of biofilm development is controlled by the alternative sigma factor sigma(B) in Staphylococcus epidermidis Cotter, John J.; O'Gara, James P.; Mack, Dietrich; Casey, Eoin Using a modified rotating-disk reactor to sparge oxygen to Staphylococcus epidermidis cultures, we found that oxygen negatively regulates biofilm development by influencing the activity of {sigma}B. Under anaerobic conditions, increased {sigma}B activity activates icaADBC, which encodes enzymes responsible for polysaccharide intercellular adhesin synthesis, by repressing transcription of the negative regulator icaR. 2009-01-01T00:00:00Z http://hdl.handle.net/10197/2729 Rapid depletion of dissolved oxygen in 96 well microtitre plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration Cotter, John J.; O'Gara, James P.; Casey, Eoin Biofilm-related research using 96-well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96-well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96-well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96-well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model. 2009-08-01T00:00:00Z http://hdl.handle.net/10197/2561 SLiMFinder : a web server to find novel, significantly over-represented, short protein motifs Davey, Norman E.; Haslam, Niall J.; Shields, Denis C.; Edwards, Richard J. Short, linear motifs (SLiMs) play a critical role in many biological processes, particularly in protein–protein interactions. The Short, Linear Motif Finder (SLiMFinder) web server is a de novo motif discovery tool that identifies statistically over-represented motifs in a set of protein sequences, accounting for the evolutionary relationships between them. Motifs are returned with an intuitive P-value that greatly reduces the problem of false positives and is accessible to biologists of all disciplines. Input can be uploaded by the user or extracted directly from UniProt. Numerous masking options give the user great control over the contextual information to be included in the analyses. The SLiMFinder server combines these with user-friendly output and visualizations of motif context to allow the user to quickly gain insight into the validity of a putatively functional motif. These visualizations include alignments of motif occurrences, alignments of motifs and their homologues and a visual schematic of the top-ranked motifs. Returned motifs can also be compared with known SLiMs from the literature using CompariMotif. All results are available for download. The SLiMFinder server is available at: http://bioware.ucd.ie/slimfinder.html. 2010-07-01T00:00:00Z http://hdl.handle.net/10197/2412 Lipoxins : regulators of resolution Ryan, Aidan; Godson, Catherine Persistent inflammation underlies many of the most prevalent diseases in the developed world including atherosclerosis and diabetes. There is a growing appreciation that inflammation and its active resolution may be modulated by endogenously produced lipid mediators. Preeminent amongst these mediators are the lipoxins [LX]. The acronym lipoxin describes the provenance of these mediators: lipoxygenase interacting products . The LX are eicosanoids and display both anti-inflammatory and pro-resolving bioactions. More recently other pro-resolving lipid mediators have been described including the resolvins and neuroprotectins. In effective host defence LX biosynthesis is characterised by a switch from pro-inflammatory prostaglandin and leukotriene (LT) generation from arachidonic acid (AA) to LX production coincident with a return to tissue homeostasis ( see figure 1). Here we will provide an overview of LX pharmacokinetics, bioactions and summarise the evidence to date that indicates that LX are potential therapeutic agents for disorders involving cardiovascular and renal inflammation, leading to tissue damage and organ fibrosis. 2010-04-01T00:00:00Z