ORCID Profile
0000-0001-5548-3263
Current Organisation
Royal College of Surgeons in Ireland
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Publisher: Oxford University Press (OUP)
Date: 17-01-2018
DOI: 10.1093/CKJ/SFX148
Publisher: Public Library of Science (PLoS)
Date: 15-11-2017
Publisher: Springer Science and Business Media LLC
Date: 14-01-2007
DOI: 10.1038/NCHEMBIO854
Abstract: Short synthetic oligopeptides based on regions of human proteins that encompass functional motifs are versatile reagents for understanding protein signaling and interactions. They can either mimic or inhibit the parent protein's activity and have been used in drug development. Peptide studies typically either derive peptides from a single identified protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. Our objective was to determine whether rational bioinformatic design of oligopeptides specifically targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function. High-throughput in vitro platelet function assays of palmitylated cell-permeable oligopeptides corresponding to these regions identified many agonists and antagonists of platelet function. Many bioactive peptides were from adhesion molecules, including a specific CD226-derived inhibitor of inside-out platelet signaling. Systematic screens of this nature are highly efficient tools for discovering short signaling motifs in molecular signaling pathways.
Publisher: Elsevier BV
Date: 10-2016
DOI: 10.1111/JTH.13414
Publisher: American Chemical Society (ACS)
Date: 29-07-2006
DOI: 10.1021/CI0600760
Abstract: Sequences of human proteins are frequently prepared as synthetic oligopeptides to assess their functional ability to act as compounds modulating pathways involving the parent protein. Our objective was to analyze a set of oligopeptides, to determine if their solubility or activity correlated with features of their primary sequence, or with features of properties inferred from three-dimensional structural models derived by conformational searches. We generated a conformational database for a set of 78 oligopeptides, derived from human proteins, and correlated their 3D structures with solubility and biological assay activity (as measured by platelet activation and inhibition). Parameters of these conformers (frequency of coil, frequency of turns, the degree of packing, and the energy) did not correlate with solubility, which was instead partly predicted by two measures obtained from primary sequence analysis, that is, the hydrophobic moment and the number of charges. The platelet activity of peptides was correlated with a parameter derived from the structural modeling this was the second virial coefficient (a measure of the tendency for a structure to autoaggregate). This could be explained by an excess among the active peptides of those which had either a large number of positive charges or in some cases a large number of negative charges, with a corresponding deficit of peptides with a mixture of negative and positive charges. We subsequently determined that a panel of 523 commercially available (and biologically active) peptides shared this elevation of absolute net charge: there were significantly lower frequencies of peptides of mixed charges compared to expectations. We conclude that the design of biologically active peptides should consider favoring those with a higher absolute net charge.
Publisher: Elsevier BV
Date: 10-2006
Abstract: Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple s les simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.
Publisher: Informa UK Limited
Date: 04-08-2019
DOI: 10.1080/09537104.2019.1647527
Abstract: Soluble forms of the low-affinity immunoglobulin receptor FcγRIIa (sFcγRIIa) lacking the cytoplasmic tail have been reported in plasma however the mechanism and functional consequences are unknown. This study aimed to evaluate mechanisms of FcγRIIa release compared to GPVI release from platelets, and examine whether genetic polymorphisms at positions 27 and 131 within FcγRIIa correlate with platelet FcγRIIa stability and function. Enzyme-linked immunosorbent assays (ELISAs) were used to measure plasma sFcγRIIa and sGPVI levels. FcγRIIa genotype at positions 27 and 131 was evaluated. sFcγRIIa levels were not significantly different between non-131HH and 131HH but were significantly lower in 27W than non-27W. Treatment of platelets with aggregated immunoglobulin (Ig) G induced release of FcγRIIa and GPVI, but only sGPVI release was statistically significant, required functional FcγRIIa, and was blocked by inhibitors of signaling pathways and metalloproteinases. This indicated that sFcγRIIa was not released from platelets by metalloproteolysis. sFcγRIIa levels were not correlated with sGPVI levels in healthy in iduals however levels of sFcγRIIa and sGPVI in plasma from patients with rheumatoid arthritis (RA) were significantly elevated above levels found in healthy in iduals. Elevated level of sFcγRIIa in RA patients may reflect active immune-based arthritis and be predictive of active inflammation.
Publisher: Informa UK Limited
Date: 2008
DOI: 10.1080/09537100701817224
Abstract: Several recent findings point to an important role for redox regulation of platelet responses to collagen involving the receptor, glycoprotein (GP)VI. First, the antioxidant dietary compound, quercetin, was shown to inhibit GPVI-dependent platelet activation and signaling responses to collagen. Second, collagen increased platelet production of the oxygen radical, superoxide anion (O2-), mediated by the multi-subunit enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase. In that case, O2- was implicated in regulating not initial aggregation, but collagen-induced thrombus stabilization involving release of ADP. Third, our laboratory showed that an unpaired thiol in the GPVI cytoplasmic tail undergoes rapid oxidation to form GPVI homodimers following ligand binding, preceding GPVI signaling and ectodomain metalloproteolysis, and indicating formation of an oxidative submembranous environment in activated platelets. This review examines receptor/redox regulation in other cells, and relevance to the pathophysiological function of GPVI and other platelet receptors initiating thrombus formation in haemostasis or thrombotic diseases such as heart attack and stroke.
Publisher: Wiley
Date: 22-05-2013
DOI: 10.1111/JGH.12157
Abstract: TRIM28 is a multi-domain nuclear protein with pleotropic effects in both normal and tumor cells. In this study, TRIM28 expression in epithelial and stromal tumor microenvironment and its prognostic role in colorectal cancer were investigated. Immunohistological staining of TRIM28 was evaluated in tissue microarrays constructed from 137 colorectal cancer patients. The correlations of TRIM28 expression with clinicopathological features and p53 expression were studied. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess overall survival (OS) and recurrence-free survival (RFS). Strong epithelial TRIM28 expression was found in 42% of colorectal cancer tissues. TRIM28 expression correlated significantly with p53 expression in matched cases (P=0.0168, Spearman rank test). A high epithelial to stromal TRIM28 expression ratio was associated with shorter OS (P=0.033 log-rank test) and RFS (P=0.043 log-rank test). Multivariate analysis showed that the epithelial to stromal TRIM28 expression ratio was an independent predictor of OS (hazard ratio=2.136 95% confidence interval 1.015-4.498, P=0.046) and RFS (hazard ratio=2.100 confidence interval 1.052-4.191, P=0.035). A high TRIM28 expression ratio between stromal and epithelial compartments in colorectal cancer tissue is an independent predictor of poor prognosis. The pathophysiological role of TRIM28 in carcinogenesis may be dependent on expression levels and cell type within the tumor microenvironment.
Publisher: American Association for Cancer Research (AACR)
Date: 2015
DOI: 10.1158/1538-7445.CHTME14-B55
Abstract: Background: TRIM28 is a universal transcriptional co-repressor with pleotropic effects in both normal and tumor cells. We have previously shown that varying TRIM28 levels in epithelial cells and stromal fibroblasts have a prognostic value in colorectal cancer patients. The pathophysiological role of TRIM28 in carcinogenesis may therefore be associated with TRIM28 expression levels and cell type of expression within the tumor microenvironment. In this study we aim to dissect the molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment by isolating in idual populations of neoplastic epithelial and stromal cells and investigating their protein signaling networks. Methods: TRIM28 expression was analyzed by immunohistochemistry on FFPE tissue from 19 colorectal cancer patients. TRIM28 staining was evaluated in both epithelial and stromal compartments. IHC scoring of at least 2 units of difference in staining intensity between stromal fibroblasts and epithelial cells was defined as a high TRIM28 expression ratio and a low TRIM28 expression ratio was defined as 1 or 0 units of difference in staining intensity. Reverse-phase protein microarrays (RPMA) were constructed from laser capture micro-dissection (LCM) enriched tumor epithelium and stroma isolated from fresh-frozen tissue of the same patient cohort. The protein signaling networks were measured for 32 downstream signaling endpoints. Spearman rank analysis was used to assess the correlations between in idual protein pairs. Correlation coefficient ρ ≥ 0.75 with P ≤ 0.01 was considered significant. Results: Immunohistochemical analysis of the FFPE tissue sections identified 10 TRIM28 high ratio cases and 9 TRIM28 low ratio cases. Proteomic networks were assessed in TRIM28 high and low ratio cases in fresh-frozen tissue using RPMA. Spearman ρ rank correlation analyses for the 32 signaling proteins revealed that 184 highly correlated protein pairs exclusive to the TRIM28 high ratio group, whereas 157 protein pairs were found exclusively in the TRIM28 low ratio group. In addition 191 protein pairs were shared across both TRIM28 high and low ratio groups. The caspases 3 and 7, as well as the cell surface receptor RAGE, were prominent in the high TRIM28 ratio group proteomic network, whereas the Metalloprotease MMP9 and the GTPase Ras-GRF1 were exclusive to the low TRIM28 ratio group. Furthermore we found Survivin and JNK to be prominent in the stromal compartment of the high TRIM28 cases. Conclusions: We characterized molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment. Proteomic analysis revealed distinct protein signaling networks associated with varying TRIM28 expression levels in epithelial and stromal compartments. The study presents a novel way of deciphering molecular crosstalk between the epithelial and stromal compartments within the microenvironment. Citation Format: Seán Fitzgerald, Virginia Espina, Katherine M. Sheehan, Robert Cummins, Anthony O'Grady, Dermot Kenny, Richard O'Kennedy, Lance Liotta, Elaine W. Kay, Gregor Kijanka. Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment 2014 Feb 26-Mar 1 San Diego, CA. Philadelphia (PA): AACR Cancer Res 2015 (1 Suppl):Abstract nr B55. doi:10.1158/1538-7445.CHTME14-B55
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-07-2006
DOI: 10.1161/01.RES.0000232317.84122.0C
Abstract: Platelet activation causes conformational changes of integrin GPIIb/IIIa (α IIb β 3 ), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.
Publisher: Public Library of Science (PLoS)
Date: 13-04-2015
No related grants have been discovered for Dermot Kenny.