Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

International audienceA combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase

Simvastatin promotes cardiac myocyte relaxation in association with phosphorylation of Troponin I

The number of people taking statins is set to increase across the globe due to recent changes in prescription guidelines. For example, half the US population over 40 is now eligible for these drugs, whether they have high serum cholesterol or not. With such development in policy comes a stronger need for understanding statins’ myriad of effects. Surprisingly little is known about possible direct actions of statins on cardiac myocytes, although claims of a direct myocardial toxicity have been made. Here we determine the impact of simvastatin administration (40 mg/kg/day) for 2 weeks in normocholesterolaemic rats on cardiac myocyte contractile function and identify an underlying mechanism. Under basal conditions, statin treatment increased the time to half (t0.5) relaxation without any effect on the magnitude of shortening, or the magnitude/kinetics of the [Ca2+]i transient. Enhanced myocyte lusitropy could be explained by a corresponding increase in phosphorylation of troponin I (TnI) at Ser23,24. Statin treatment increased expression of eNOS and Ser1177 phosphorylated eNOS, decreased expression of the NOS-inhibitory proteins caveolin 1 and 3, and increased (P=0.06) NO metabolites, consistent with enhanced NO production. It is well established that NO stimulates protein kinase G, one of the effectors of TnI phosphorylation at Ser23,24. Trends for parallel changes in phospho-TnI, phospho-eNOS and caveolin 1 expression were seen in atrial muscle from patients taking statins. Our data are consistent with a mechanism whereby chronic statin treatment enhances TnI phosphorylation and myocyte lusitropy through increased NO bioavailability. We see no evidence of impaired function with statin treatment; the changes we document at the level of the cardiac myocyte should facilitate diastolic filling and cardiac performance

Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx300103jTienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [14C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 non-redundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2–4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver (Methogo, R., Dansette, P. and Klarskov, K. (2007) Int. J. Mass Spectrom., 268, 284–295), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e. rat vs. human), and still another may be the method of detection of adducted proteins (i.e. Western blot vs. C-14). Knowledge of human target proteins is very limited. Of more than 350 known protein targets of reactive metabolites, only 42 are known from human and only 21 of these are known to be targets for more than one chemical. Nevertheless, the demonstration that human target proteins can be identified using isolated hepatocytes in vitro should enable the question of species differences to be addressed more fully in the future

Récit anonyme d'un voyage à Jérusalem et au Mont Sinaï

D. Régnier-BohlerInternational audienc

Le voyage d’outre-mer à la fin du xve siècle : essai de définition de l’identité pèlerine occidentale à travers le récit de Nicole Le Huen

Durant les dernières décennies du xve siècle, les pèlerinages occidentaux à Jérusalem connaissent une grande vogue auprès de groupes sociaux très divers, parmi lesquels se trouvent de nombreux clercs. Leur étude prosopographique reste difficile, car rassembler suffisamment d’informations sur leurs motivations individuelles ou collectives, n’est pas aisé. Ces motivations constituent le fond de l’identité pèlerine, qu’il n’est pas question de définir ici de façon exhaustive : il s’agira seuleme..

Jérusalem et la Terre sainte au tournant des années 1500, un enjeu politico-religieux pour l’Occident ?

L’important ouvrage du franciscain vénitien, Francesco Suriano, intitulé Il Trattato di Terra Santa e dell’Oriente di Frate Francesco Suriano , a retenu depuis longtemps l’attention des historiens par l’intérêt qu’il présente. Toutefois, les études qui se sont multipliées sur le Proche-Orient permettent de le relire à la lumière d’une problématique nouvelle sur une période charnière que l’on a pas achevé d’explorer. Période charnière pour le Proche-Orient et pour le reste du monde, puisque le..

Renan et le Second Empire

Dansette Adrien. Renan et le Second Empire. In: Bulletin de la Société des Études renaniennes, N°7, 3e trimestre 1971. pp. 11-15

Le récit de voyage à Jérusalem de Nompar de Caumont, introduction et traduction

D. Régnier-BohlerInternational audienc

Journal de voyage à Jérusalem de Louis de Rochechouart

D. Régnier-BohlerInternational audienc