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    ABSTRACT: Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.
    Current biology: CB 03/2014; 24(6):R215-20.
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    ABSTRACT: Little is known about how hypercholesterolemia affects Ca2+ signalling in the vasculature of ApoE−/− mice, a model of atherosclerosis. Our objectives were therefore to determine (i) if hypercholesterolemia alters Ca2+ signalling in aortic endothelial cells before overt atherosclerotic lesions occur, (ii) how Ca2+ signals are affected in older plaque-containing mice, and (iii) whether Ca2+ signalling changes were translated into contractility differences. Using confocal microscopy we found agonist-specific Ca2+ changes in endothelial cells. ATP responses were unchanged in ApoE−/− cells and methyl-β-cyclodextrin, which lowers cholesterol, was without effect. In contrast, Ca2+ signals to carbachol were significantly increased in ApoE−/− cells, an effect methyl-β-cyclodextrin reversed. Ca2+ signals were more oscillatory and store-operated Ca2+ entry decreased as mice aged and plaques formed. Despite clearly increased Ca2+ signals, aortic rings pre-contracted with phenylephrine had impaired relaxation to carbachol. This functional deficit increased with age, was not related to ROS generation, and could be partially rescued by methyl-β-cyclodextrin. In conclusion, carbachol-induced calcium signalling and handling are significantly altered in endothelial cells of ApoE−/− mice before plaque development. We speculate that reduction in store-operated Ca2+ entry may result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite the enhanced Ca2+ response.
    Cell calcium 01/2014;
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    ABSTRACT: This brief review discusses recent advances in studies of mitochondrial Ca2+ signalling and considers how the relationships between mitochondria and Ca2+ responses are shaped in secretory epithelial cells. Perhaps the more precise title of this review could have been “How to win ATP and influence Ca2+ signalling in secretory epithelium with emphasis on exocrine secretory cells and specific focus on pancreatic acinar cells”. But “brevity is a virtue” and the authors hope that many of the mechanisms discussed are general and applicable to other tissues and cell types. Amongst these mechanisms are mitochondrial regulation of Ca2+ entry and the role of mitochondria in the formation of localized Ca2+ responses. The roles of Ca2+ signalling in the physiological adjustment of bioenergetics and in mitochondrial damage are also briefly discussed.
    Cell Calcium. 01/2014;
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    ABSTRACT: In this short review we discuss how recent insights into myometrial physiology may be taken forward and translated into much needed novel therapies for problems associated with labour. We consider excitation-contraction coupling in the myometrium and how this relates to our understanding of the changes that occur to produce myometrial contractions and successful labour. We then discuss how this information has already been used in the development of drugs to either stimulate or relax the myometrium, to address the needs of women with either slow (dystocic) labours or threatened preterm labours, respectively. We next present the data showing how basic physiological findings pertaining to hypoxia and lactate production, have been taken and translated into a tool for predicting and hence better managing difficult labours. We then highlight examples of where physiological research has started to provide mechanistic insight into clinical problems associated with labour and parturition (obesity, diabetes, advanced maternal age, postdate and twin pregnancies) and suggest how these findings could be translated into new therapies for difficult labours.
    Experimental physiology 12/2013;
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    ABSTRACT: The phosphatidylinositol-3-kinase (PI3K) pathway is commonly hyperactivated in cancer. One mechanism by which this occurs is by silencing of the phosphatase and tensin homolog (PTEN), a tumor suppressor and major antagonist of the pathway, through genetic, epigenetic or posttranscriptional mechanisms. Here, we used an unbiased siRNA screen in non-small-cell lung cancer cells to identify deubiquitylases (DUBs) that have an impact on PI3K signaling by regulating the abundance of PTEN. We found that PTEN expression was induced by depleting any of three members of the Josephin family DUBs: ataxin 3 (ATXN3), ataxin 3-like (ATXN3L) and Josephin domain containing 1 (JOSD1). However, this effect is not mediated through altered PTEN protein stability. Instead, depletion of each DUB increases expression of both the PTEN transcript and its competing endogenous RNA, PTENP1. In ATXN3-depleted cells, under conditions of transcriptional inhibition, PTEN and PTENP1 mRNAs rapidly decay, suggesting that ATXN3 acts primarily by repressing their transcription. Importantly, the PTEN induction observed in response to ATXN3 siRNA is sufficient to downregulate Akt phosphorylation and hence PI3K signaling. Histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in non-small-cell lung cancer. Although PTEN exhibits a very limited response to the broad-spectrum HDACi Vorinostat (SAHA) in A549 cells, we find that combination with ATXN3 depletion enhances PTEN induction in an additive manner. Similarly, these interventions additively decrease cell viability. Thus, ATXN3 provides an autonomous, complementary therapeutic target in cancers with epigenetic downregulation of PTEN.Oncogene advance online publication, 2 December 2013; doi:10.1038/onc.2013.512.
    Oncogene 12/2013;
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    ABSTRACT: Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. Intracellular calcium ([Ca(2+)]C), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. Inhibition of OME with 4-MP converted predominantly transient [Ca(2+)]C rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation.
    Gut 10/2013;
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    ABSTRACT: Acomplex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3-clathrin binding. A dileucine motif and Aurora A-phosphorylated serine 558 on TACC3 bound to the "ankle" of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3-clathrin.
    The Journal of Cell Biology 08/2013; 202(3):463-78.
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    ABSTRACT: The microcirculation is the site of gas and nutrient exchange. Control of central or local signals acting on the myocytes, pericytes and endothelial cells within it, is essential for health. Due to technical problems of accessibility, the mechanisms controlling Ca(2+) signalling and contractility of myocytes and pericytes in different sections of microvascular networks in situ have not been investigated. We aimed to investigate Ca(2+) signalling and functional responses, in a microcirculatory network in situ. Using live confocal imaging of ureteric microvascular networks, we have studied the architecture, morphology, Ca(2+) signalling and contractility of myocytes and pericytes. Ca(2+) signals vary between distributing arcade and downstream transverse and precapillary arterioles, are modified by agonists, with sympathetic agonists being ineffective beyond transverse arterioles. In myocytes and pericytes, Ca(2+) signals arise from Ca(2+) release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca(2+) release and not via ryanodine receptors or Ca(2+) entry into the cell. The responses in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. Myocytes and pericytes are electrically coupled, transmitting Ca(2+) signals between arteriolar and venular networks dependent on gap junctions and Ca(2+) entry via L-type Ca(2+) channels. Endothelial Ca(2+) signalling inhibits intracellular Ca(2+) oscillations in myocytes and pericytes via L-arginine/nitric oxide pathway and intercellular propagating Ca(2+) signals via EDHF. Increases of Ca(2+) in pericytes and myocytes constrict all vessels except capillaries. These data reveal the structural and signalling specializations allowing blood flow to be regulated by myocytes and pericytes.
    Cell calcium 07/2013;
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    Ophthalmology 07/2013; 120(7):e50.
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    ABSTRACT: We demonstrated that increasing intracellular cAMP concentrations results in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation are mediated by PKA, whilst EPAC potentiates migration.
    Biochimica et Biophysica Acta 06/2013;
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