Elaine Sullivan

Bio: Elaine Sullivan is an academic researcher from Loughborough University. The author has contributed to research in topics: Fusion protein & Transcription factor. The author has an hindex of 7, co-authored 12 publications receiving 1499 citations.

Oscillations in NF-κB Signaling Control the Dynamics of Gene Expression

Abstract: Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.

Multi-parameter analysis of the kinetics of NF-kappa B signalling and transcription in single living cells

Abstract: Proteins of the NF-kappaB transcription factor family normally reside in the cytoplasm of cells in a complex with IkappaB inhibitor proteins. Stimulation with TNFalpha leads to proteosomal degradation of the IkappaB proteins and nuclear translocation of the NF-kappaB proteins. Expression of p65 and IkappaBalpha fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IkappaBalpha-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression, the half-time of IkappaBalpha-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IkappaBalpha and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IkappaBalpha is the true substrate for TNFalpha stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor, leptomycin B (LMB), there was nuclear accumulation of IkappaBalpha-EGFP and p65-dsRed, with IkappaBalpha-EGFP accumulating more rapidly. No NF-kappaB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IkappaBalpha-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFalpha, degradation of IkappaBalpha-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-kappaB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-kappaB-dependent transcription occur even when nuclear export of NF-kappaB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

NF-κB signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms

Abstract: NF-kappaB transcription factors are involved in the cellular response to stress, and are regulated by inhibitor (IkappaB) proteins, which prevent NF-kappaB-mediated transcription by maintaining NF-kappaB in the cytoplasm. Proteins from other pathways are also known to regulate NF-kappaB negatively, notably the glucocorticoid receptor (GR) and IL-4-responsive STAT6. Both pathways were shown to inhibit NF-kappaB-mediated transcription, by expressing either STAT6 or GR and activating the respective pathways. Using fluorescent fusion proteins, we show that GR alters the timing of activated p65 NF-kappaB nuclear occupancy by increasing the export rate of p65 and is independent of whether GR is present as a dimer or monomer. Expression of STAT6 was also shown to alter p65 nuclear occupancy but appeared to affect the import rate and hence the overall maximal level of p65 translocation. Activating STAT6 with IL-4 prior to activating NF-kappaB significantly increased this inhibition. Investigation of IkappaBa showed that activated STAT6 inhibited TNFalpha-mediated IkappaBa phosphorylation and degradation, whereas GR activation did not alter IkappaBalphakinetics. This demonstrates a clear separation of two distinct mechanisms of inhibition by STAT6 and GR upon the NF-kappaB pathway.

Network motifs: theory and experimental approaches

Abstract: Transcription regulation networks control the expression of genes. The transcription networks of well-studied microorganisms appear to be made up of a small set of recurring regulation patterns, called network motifs. The same network motifs have recently been found in diverse organisms from bacteria to humans, suggesting that they serve as basic building blocks of transcription networks. Here I review network motifs and their functions, with an emphasis on experimental studies. Network motifs in other biological networks are also mentioned, including signalling and neuronal networks.

The evolving role of natural products in drug discovery

Abstract: Natural products and their derivatives have historically been invaluable as a source of therapeutic agents. However, in the past decade, research into natural products in the pharmaceutical industry has declined, owing to issues such as the lack of compatibility of traditional natural-product extract libraries with high-throughput screening. However, as discussed in this review, recent technological advances that help to address these issues, coupled with unrealized expectations from current lead-generation strategies, have led to a renewed interest in natural products in drug discovery.

HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis

Abstract: We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-κB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein−protein and protein−DNA complexes.

Cytokines in atherosclerosis: pathogenic and regulatory pathways.

Abstract: Atherosclerosis is a chronic disease of the arterial wall where both innate and adaptive immunoinflammatory mechanisms are involved. Inflammation is central at all stages of atherosclerosis. It is implicated in the formation of early fatty streaks, when the endothelium is activated and expresses chemokines and adhesion molecules leading to monocyte/lymphocyte recruitment and infiltration into the subendothelium. It also acts at the onset of adverse clinical vascular events, when activated cells within the plaque secrete matrix proteases that degrade extracellular matrix proteins and weaken the fibrous cap, leading to rupture and thrombus formation. Cells involved in the atherosclerotic process secrete and are activated by soluble factors, known as cytokines. Important recent advances in the comprehension of the mechanisms of atherosclerosis provided evidence that the immunoinflammatory response in atherosclerosis is modulated by regulatory pathways, in which the two anti-inflammatory cytokines interleukin-10 and transforming growth factor-beta play a critical role. The purpose of this review is to bring together the current information concerning the role of cytokines in the development, progression, and complications of atherosclerosis. Specific emphasis is placed on the contribution of pro- and anti-inflammatory cytokines to pathogenic (innate and adaptive) and regulatory immunity in the context of atherosclerosis. Based on our current knowledge of the role of cytokines in atherosclerosis, we propose some novel therapeutic strategies to combat this disease. In addition, we discuss the potential of circulating cytokine levels as biomarkers of coronary artery disease.