Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD+-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.We thank all colleagues who kindly provided antibodies and reagents; Dr. J. Donaldson (National Institutes of Health) for BFA analogs; Dr. C. P. Berrie for editorial assistance; and Drs. C. Limina, A. Tamburro, M. G. Silletta, R. Weigert, and S. Spanò (Negri Sud Institute) for performing initial experiments. We also acknowledge financial support from Italian Association for Cancer Research (AIRC) through the Grants IG4664 and IG10341 (to D.C.), IG4700 (to A.L.), and IG6074 (to A.C.); and from the Liguria Region and the Ministry of Education, University, and Research (Fund for Investments in Basic Research Project; A.D.F.). G.G. and C.V. received fellowships from AIRC (Italian Foundation for Cancer Research). Financial support from Technological Innovation Fund DM 24/09/2009, Legge 46/82-MEF, and Project FaReBio di Qualità also is acknowledgedPeer Reviewe

Endogenous ADP-ribosylation of the G Protein β Subunit Prevents the Inhibition of Type 1 Adenylyl Cyclase

Mono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein beta subunit-specific polyclonal antibody, indicating that this protein is the G protein beta subunit. The ADP-ribosylation of the beta subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited by meta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated beta subunit was also deribosylated by a cytosolic hydrolase. This ADP-ribosylation/deribosylation cycle might be an in vivo modulator of the interaction of betagamma with specific effectors. Indeed, we found that the ADP-ribosylated betagamma subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the beta subunit occurs in intact Chinese hamster ovary cells, where the NAD(+) pool was labeled with [(3)H]adenine. These results show that the ADP-ribosylation of the betagamma subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction

An Interdisciplinary Approach for the Historical and Technical Characterization of Medieval and Modern Mortars

The study concerns Italian masonries and focuses on historical, medieval and modern mortars. Within the context of the different regions under examination (Piedmont, the Po Valley area, Latium, the Umbria-Marche region, and Apulia and Sardinia) a wide variety of materials with different chemical-physical characteristics were used in masonry work, determining different structural behaviors. The project aims at improving our knowledge about historical mortars in order to further the conservation of Italian built heritage, especially in zones with seismic risk. To achieve these results, we took samples and carried out analyses to investigate the different mechanical and cohesion properties that influence the vulnerability of ruined or collapsed structures. This information has enabled advances to be made in the prevention, maintenance, protection and preservation of historical buildings. Further details will concern the history of construction techniques, with particular regard to the relationship between local resources and construction sites. Another important topic is the role of different components and additives during the preparation of the mortars and their level of hydraulicity.Lo studio riguarda le strutture murarie in Italia e si concentra sulle malte storiche, medievali e moderne. Nei contesti regionali oggetto d’indagine (Piemonte e Pianura Padana, Lazio, Umbria-Marche, Puglia e Sardegna) è stata utilizzata una grande varietà di materiali, con caratteristiche chimico-fisiche diverse, e queste differenze determinano anche comportamenti strutturali diversi. Il progetto mira a migliorare la nostra conoscenza delle malte storiche per approfondire il tema della conservazione del patrimonio edilizio italiano, soprattutto nelle zone a rischio sismico. Per ottenere questi risultati, sono stati prelevati campioni sui quali sono state effettuate analisi per indagare quanto le diverse proprietà meccaniche e di coesione incidano sulla vulnerabilità delle strutture in rovina o collassate. Grazie a queste informazioni è stato possibile contribuire alla prevenzione, manutenzione, protezione e conservazione dell'edilizia storica. Ulteriori conoscenze hanno riguardato la storia delle tecniche di costruzione, con particolare riguardo al rapporto tra risorse locali e i cantieri. Un altro importante tema è stato il ruolo dei diversi componenti e additivi durante la preparazione delle malte e il loro livello di idraulicità

SRC-dependent signalling regulates actin ruffle formation induced by glycerophosphoinositol 4-phosphate

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Glycerophosphoinositol 4-phosphate, a putative endogenous inhibitor of adenylylcyclase.

In a continuous line of rat thyroid cells transformed by the k-ras oncogene (KiKi), the expression of ras-p21 correlates with an increased activity of a phosphoinositide-specific phospholipase A2, which leads to elevated levels of glycerophosphoinositols. In this study we have characterized the biological activities of these compounds. Growth and differentiation in thyroid cells are mainly regulated by the activation of adenylylcyclase. Therefore, we have studied the effects of glycerophosphoinositols on the activity of this enzyme using a normal thyroid cell line (FRTL5). Micromolar concentrations of glycerophosphoinositol 4-phosphate (GroPIns-4-P) caused a approximately 50% inhibition of the adenylylcyclase activity in FRTL5 membranes stimulated by the GTP-binding protein activator fluoroaluminate. Similar concentrations of GroPIns-4-P were detected in KiKi cells but not in the normal FRTL5 line. Micromolar GroPIns-4-P was found to be taken up by intact FRTL5 cells and to induce nearly 50% inhibition of the thyrotropin- and cholera toxin-induced increase in cAMP levels. Similar results were also observed in other cell lines (smooth muscle, pituitary cells, and pneumocytes). GroPIns-4-P inhibited cAMP-dependent cellular functions such as iodide uptake and thymidine incorporation in FRTL5 cells when stimulated by thyrotropin and cholera toxin but not when induced by forskolin. These results are consistent with GroPIns-4-P exerting an inhibitory effect on the GTP-binding protein that stimulates adenylycyclase. We propose that GroPIns-4-P might mediate a mechanism of cross-talk between adenylylcyclase and phospholipase A2 in thyroid as well as in other cell systems

Association of the changes in cytosolic Ca2+ and iodide efflux induced by thyrotropin and by the stimulation of alpha 1-adrenergic receptors in cultured rat thyroid cells.

Abstract Thyrotropin causes a time- and concentration-dependent increase in cytosolic Ca2+ in FRTL-5 rat thyroid cells as measured by Quin2 fluorescence; the half-maximal response occurs in response to 1 X 10(-7) M thyrotropin. The effect of added thyrotropin is the same whether cells have been previously and chronically exposed to thyrotropin or whether they have been thyrotropin "starved" for several days. The thyrotropin effect on cytosolic Ca2+ has no relationship to intracellular cAMP levels with respect to dose and time course. Norepinephrine (1 X 10(-7) M) also causes increases in cytosolic Ca2+ in FRTL-5 thyroid cells. With the use of a variety of adrenergic inhibitors, norepinephrine was found to exert its effect via an alpha 1-adrenergic receptor. The exposure of FRTL-5 cells to physiological thyrotropin concentrations enhances the effect on cytosolic Ca2+ level induced by norepinephrine in vitro; the shape of the dose-response curve indicates a cooperative effect of the thyrotropin and norepinephrine. The increase in cytosolic Ca2+ seems to be derived from an intracellular pool rather than from the extracellular space. It is not prevented by nifedipine, a blocker of Ca2+ channels; it is present in cells exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid; and it is not associated with increased Ca2+ uptake into the cell. the thyrotropin- and norepinephrine-induced increase in cytosolic Ca2+ parallels the efflux of iodide and the organification of thyroglobulin in a dose-dependent manner

Molecular cloning and functional characterization of brefeldin A-ADP-ribosylated substrate. A novel protein involved in the maintenance of the Golgi structure.

Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex

Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13

60kDa Lysophospholipase, a New Sgk1 Molecular Partner Involved in the Regulation of ENaC

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling

A 14-3-3γ dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIβ to regulate post-Golgi carrier formation

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