Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1''-2') ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors

Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios

Influence de l'arrangement granulaire d'un sédiment polydisperses sur le seuil de mise en mouvement

La prédiction du transport d'un mélange de particules de plusieurs tailles est un point sensible d'une représentation de la dynamique sédimentaire sur de nombreux faciès en rivière ou en mer. Afin de déterminer les processus critiques qui régissent la mise en mouvement de tels mélanges et de quantifier leur contributions, des essais ont été réalisés dans un petit canal à courant. Ils ont porté sur des échantillons reconstitués, formés de sables naturels lithoclastiques bien triés ou de mélanges sableux bimodaux. L'objectif est d'observer et de quantifier les différences de comportement à l'entrainement entre un sédiment unimodal et un sédiment mélangé. Les résultats obtenus sont comparés à une sélection de formulations issues de la littérature. La série de tests a permis de mettre en évidence quelques processus particuliers du début du transport sédimentaire multiclasse dans un écoulement stationnaire. La granulométrie utilisée s'étend des sables fins aux sables très grossiers. Des paramètres du mélange tels que le rapport de taille des particules, le taux de saturation des échantillons par les grains dont la taille est la plus fine ou la porosité influent sur la modulation de la contrainte de frottement critique. Les comparaisons mesures / formules ont mis en évidence la difficulté de prédire le seuil de mise en mouvement des particules d'un mélange hétérométrique. Par conséquent, l'incertitude sur la granulométrie transportée s'accroît lorsque le frottement est proche du frottement critique d'une certaine classe de taille

The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis1. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases1, 2. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death3, 4. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family5, 6. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease

FACTORS AFFECTING MIGRATION FROM THE CROATIAN RURAL AREA

U radu se daju rezultati istraživanja migracija u seoskom području Republike Hrvatske. Cilj je istražiti čimbenike koji utječu na iseljavanje seoskog stanovništva Republike Hrvatske. Istraživanje je provedeno 2007. godine na uzorku od 914 ispitanika dobi od 24 do 45 godina u seoskom području Republike Hrvatske. Odabir naselja i ispitanika bio je slučajan. Provedeno istraživanje pokazuje da su najveće poteškoće života u hrvatskom seoskom području gospodarske naravi, manjak zaposlenja, slaba mogućnost izbora zanimanja i niža zarada u odnosu na zaposlenje u gradu. Petina ispitanika nije zadovoljna uvjetima seoskog života i namjerava se iseliti. To je zabrinjavajući pokazatelj budući da se radi o populaciji koja je u pravilu završila proces obrazovanja i većinom osnovala obitelj. Najviše mogućih iseljenika, što je bilo i za očekivati, je iz gospodarski nerazvijenih područja Republike Hrvatske. Daljnja depopulacija hrvatskog sela bila bi pogubna, a njene najveće posljedice bile bi: prevelika urbanizacija, posebice velikih gradova, daljnji neravnomjerni razvitak Republike Hrvatske te nedovoljno iskorištenje prostornog, proizvodnog i ljudskog potencijala. S obzirom na strateški cilj ulaska Republike Hrvatske u Europsku uniju, navedeno predstavlja bitno ograničenje njene uspješne prilagodbe europskoj ekonomskoj integraciji. Iseljavanje seoskog pučanstva može se spriječiti prvenstveno povećanjem zaposlenosti i dohotka te stvaranjem takve fizičke i društvene infrastrukture u seoskom području koja će bitno poboljšati životne uvjete seoskog pučanstva. Seoska područja, poglavito gospodarski nerazvijena, nemaju dovoljno vlastitih mogućnosti za ubrzanje razvoja odnosno za nužno smanjivanje razlika u kakvoći življenja prema gradskim područjima. Zbog toga je nužno da njihov razvojni proces više nego dosada potpomogne Država osmišljenim mjerama regionalnog razvoja, uz svekoliku potporu lokalne uprave i samouprave. U tome bi svoj znatan obol trebalo dati novo-osnovano Ministarstvo za regionalni razvoj.The paper presents results of the research study on migrations in rural areas of the Republic of Croatia. The aim was to determine factors influencing migrations of rural population in Croatia. The research was carried out in 2007 on 914 respondents from 25 to 45 years of age. The rural communities and respondents were selected on a random basis. The study results indicate that the major difficulties in rural life in Croatia are of economic nature: lack of employment opportunities, inadequate choice of profession and lower income in comparison with employment in urban areas. One fifth of the respondents is not satisfied with conditions of rural life and intends to leave villages. This is a very disturbing indicator, since it refers to population, which in general, has finished education and started a family. As we expect, the largest number of potential migrants comes from economically underdeveloped Croatian areas. Further depopulation of Croatian villages would have dramatic effects, and the worst consequences would be excessive urbanization, especially of large cities, further uneven development of the Republic of Croatia, and insufficient utilization of spatial, production and human resources. Since the strategic Croatian goal is to become a member of the European Union, this is a major obstacle to its successful adjustment to the European economic integration. The migration of rural population could be prevented primarily by increase in employment and income opportunities and creation of such physical and social infrastructure in rural areas that would considerably improve living conditions for rural population. The rural areas, particularly underdeveloped, have no adequate capacities for intensification of its development and diminishing differences in their quality of life compared to urban areas. Thus, the state support is increasingly required by introducing measures of regional development with complementary support of the local government. The newly founded Ministry of Regional Development is therefore inevitable in this process

ETUDE du CYTOCHROME P450 2J2 HUMAIN :<br />Recherche de substrats et d'inhibiteurs sélectifs ;<br />Détermination de la topologie de son site actif

This manuscript deals with the functional and structural study of human cytochrome P450 2J2 (CYP2J2), an enzyme mainly expressed in cardiovascular tissues, whose biological roles are not wellknown. We used terfenadone, regioselectively oxydized by CYP2J2, as a starting point for design and synthesis of several compounds, which revealed to be affine and selective inhibitors of this P450. In particular, one affine and selective inhibitor (Ki = 160 nM) and two efficient mechanism-based inactivators (kinact/Ki 3000 L/mol/s) of CYP2J2 were identified. The study of the CYP2J2-catalyzed oxidation of some of these compounds revealed a surprising regioselectivity, favoring a less reactive position, regarding the reactivity towards oxidation. The characterization of the active site and the identification of important residues for terfenadone derivatives recognition by CYP2J2 was achieved using an homology 3D model of this enzyme, and docking of several substrates in the active site. Finally, a preliminary study of the potential biological roles of CYP2J2 was performed by looking at the inhibitory effect of several compounds involved in the vascular relaxation pathways. In conclusion, this work provides the characterization of the first biochemical tools to study the biological roles of CYP2J2, the first description of the active site topology and a study on CYP2J2 biological roles.Ce manuscrit présente une étude fonctionnelle et structurale du cytochrome P450 2J2 humain (CYP2J2), enzyme exprimée dans les tissus cardiovasculaires, dont les rôles biologiques sont mal connus. En utilisant la terfénadone comme base structurale, qui est un composé oxydé régiosélectivement par le CYP2J2, plusieurs composés ont été synthétisés se sont révélés être des inhibiteurs affins et sélectifs du CYP2J2. En particulier, un inhibiteur très affin et compétitif (Ki = 160 nM) et deux substrats suicides efficaces du CYP2J2 (kinact/Ki 3000 L/mol/s) ont été mis en évidence. L'étude de l'oxydation de ces composés par le CYP2J2 a révélé une régiosélectivité surprenante, en faveur d'une position chimiquement moins réactive vis-à-vis des oxydations. La caractérisation du site actif du CYP2J2 et l'identification de résidus importants pour la reconnaissance des dérivés de terfénadone a pu être réalisée en construisant un modèle par homologie 3D de cette enzyme et par le docking de certains dérivés dans le site actif du CYP2J2. Enfin, une étude préliminaire des rôles biologiques possibles du CYP2J2 a été réalisée en étudiant les effets inhibiteur de ce P450. En conclusion, ce travail a permis de caractériser les premiers outils biochimiques d'étude des rôles biologiques du CYP2J2, de proposer une première topologie du site actif du CYP2J2, et d'affiner les rôles biologiques du CYP2J2

Rare and unusual glycosylation of peptides and proteins

International audienc

Chapter 10. Enzymatic thioglycosylation: current knowledge and challenges

International audienc

Unraveling the substrate recognition mechanism and specificity of the unusual glycosyl hydrolase family 29 BT2192 from Bacteroides thetaiotaomicronBacteroides\ thetaiotaomicron

International audienceGlycosyl hydrolase (GH) family 29 (CAZy database) consists of retaining $\alpha$-L-fucosidases. We have identified BT2192, a protein from Bacteroides thetaiotaomicron, as the first GH29 representative exhibiting both weak $\alpha$-L-fucosidase and β-d-galactosidase activities. Determination and analysis of X-ray structures of BT2192 in complex with β-d-galactoside competitive inhibitors showed a new binding mode different from that of known GH29 enzymes. Three point mutations, specific to BT2192, prevent the canonical GH29 substrate $\alpha$-L-fucose from binding efficiently to the fucosidase-like active site relative to other GH29 enzymes. β-d-Galactoside analogues bind and interact in a second pocket, which is not visible in other reported GH29 structures. Molecular simulations helped in the assessment of the flexibility of both substrates in their respective pocket. Hydrolysis of the fucosyl moiety from the putative natural substrates like 3-fucosyllactose or Lewis$^X$ antigen would be mainly due to the efficient interactions with the galactosyl moiety, in the second binding site, located more than 6–7 Å apart

Enzymatic synthesis of glycosides: from natural O- and N-glycosides to rare C- and S-glycosides

Carbohydrate related enzymes, like glycosyltransferases and glycoside hydrolases, are nowadays more easily accessible and are thought to represent powerful and greener alternatives to conventional chemical glycosylation procedures. The knowledge of their corresponding mechanisms has already allowed the development of efficient biocatalysed syntheses of complex O-glycosides. These enzymes can also now be applied to the formation of rare or unnatural glycosidic linkages