Crystallization and preliminary X-ray study of an N-terminal fragment of rat liver ribosomal P2 protein

International audienceRibosomal P proteins have been shown to be involved in the binding of elongation factors and participate in factor-dependent GTP hydrolysis. The P proteins form the pentamer (P1/P2)(2)-P0 constituting the lateral flexible stalk of the 60S ribosomal subunit. The highly soluble domain (1-65) of rat liver P2 has been overexpressed in Escherichia coli as an N-terminal poly-His-tagged protein and crystallized. To reduce nucleation and improve crystal morphology and diffraction power, the crystals were grown in a gel matrix and an oil barrier was added between the reservoir and the drop to reduce the rate of vapour diffusion. This dramatically reduced the nucleation in the drops and yielded diffraction-quality crystals. Data were collected to 2.4 A resolution at beamline ID 14-1, ESRF. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 37.7, b = 96.7, c = 135.0 A.Ribosomal P proteins have been shown to be involved in the binding of elongation factors and participate in factor-dependent GTP hydrolysis. The P proteins form the pentamer (P1/P2)(2)-P0 constituting the lateral flexible stalk of the 60S ribosomal subunit. The highly soluble domain (1-65) of rat liver P2 has been overexpressed in Escherichia coli as an N-terminal poly-His-tagged protein and crystallized. To reduce nucleation and improve crystal morphology and diffraction power, the crystals were grown in a gel matrix and an oil barrier was added between the reservoir and the drop to reduce the rate of vapour diffusion. This dramatically reduced the nucleation in the drops and yielded diffraction-quality crystals. Data were collected to 2.4 A resolution at beamline ID 14-1, ESRF. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 37.7, b = 96.7, c = 135.0 A

Etude de la specificite de substrat et de coenzyme de la glyceraldehyde-3-phosphate deshydrogenase glycolytique

SIGLECNRS T 66512 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Etude de la specificite de substrat et de coenzyme de la glyceraldehyde-3-phosphate deshydrogenase glycolytique

SIGLEINIST T 70852 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Lipémie postprandiale et lactoferrine (le Lipolysis Stimulated Receptor comme cible potentielle)

La lipémie postprandiale se caractérise par une augmentation des lipoprotéines riches en triglycérides après un repas, et joue un rôle important dans la biodisponibilité des lipides alimentaires pour les tissus périphériques. En effet, une lipémie postprandiale élevée est souvent associée à l'obésité et à une dyslipidémie, deux composantes du syndrome métabolique qui peuvent engendrer des complications médicales, incluant diabète et maladies cardiovasculaires. La lactoferrine (Lf) inhibe l'épuration hépatique des chylomicrons, conduisant à une élévation de la lipémie postprandiale par des mécanismes moléculaires non élucidés. Il est aussi établi que le Lipolysis Stimulated Receptor (LSR) contribuait à l'épuration des lipoprotéines riches en triglycérides pendant la phase postprandiale. L'objectif était de déterminer s'il existait une interaction entre la Lf et le LSR. Les études de cultures cellulaires ont montré que si la Lf n'affectait pas le taux d'expression du LSR dans des cellules Hepa 1-6 de souris, elle co-localisait avec le LSR en présence d'oléate, un composé requis pour l'activation du récepteur. Des expériences de ligand-blotting ont également montré que la Lf se fixait sur le LSR purifié et inhibait la fixation de lipoprotéines riches en triglycérides. Les domaines N et C-terminaux isolés de cette protéine, ainsi qu'un mélange de peptides obtenu après double hydrolyse de la Lf par la trypsine et la chymotrypsine, conservent cette propriété. Nous proposons que l'élévation de la lipémie postprandiale observée in vivo suite à un traitement par la Lf soit médiée par son interaction avec le LSR, inhibant ainsi l'épuration des chylomicrons et de leurs remnantsPostprandial lipemia is characterized by an increase in plasma triglyceride-rich lipoproteins after the ingestion of meal, and is important towards determining the bioavailability of dietary lipids amongst the peripheral tissues. Indeed, elevated postprandial lipemia is often observed with obesity and dyslipidemia, two disorders that can lead to health complications including diabetes and cardiovascular diseases. Lactoferrin (Lf), has been shown to inhibit hepatic chylomicron remnant removal, resulting in increased postprandial lipemia, for which the molecular mechanisms remain unclear. The lipolysis stimulated lipoprotein receptor (LSR) has been shown to contribute to the removal of triglycerides-rich lipoproteins during the postprandial phase. The aim was to determine if there was interaction between Lf and LSR. Both Lf and LSR were purified with purities upper to 95% and characterized. Cell culture studies demonstrated that while Lf does not have any significant effect on LSR protein levels in mouse Hepa1-6 cells, it co-localizes with LSR in cells, but only in the presence of oleate, which is needed to obtain LSR in its active form. Ligand blotting using purified LSR revealed that Lf binds directly to the receptor in the presence of oleate and prevents the binding of triglycerides-rich lipoproteins. Both C- and N-lobes of Lf, and a mixture of peptides derived from its tryptic and chymotryptic double hydrolysis retained the ability to bind LSR. We propose that the elevated postprandial lipemia observed upon Lf treatment in vivo is mediated by its direct interaction with LSR, thus preventing clearance of chylomicrons and their remnants through the LSR pathwayMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Caractérisation Structurale et Fonctionnelle de deux Enzymes de la Famille des Aldéhyde déshydrogénases (la Glycéraldéhyde-3-Phosphate Déshydrogénase de B. stearothermophilus et l'Erythrose-4-Phosphate Déshydrogénase d'E. coli.)

Si la GAPDH est une enzyme bien caractérisée sur le plan biochimique et structural, la contribution de ses deux sites de reconnaissance anionique lors de la catalyse reste incomprise et nécessitait la détermination des structures de l'intermédiaire thioacylenzyme et du complexe enzyme-produit. Ce manuscrit présente la mise au point des conditions de cristallisation et d'accumulation de l'intermédiaire thioacylenzyme (suivi par microspectrophotométrie) et du complexe enzyme-produit, la résolution et l'analyse des structures cristallographiques correspondantes ainsi que les implications pour le mécanisme catalytique. Les résultats obtenus mettent notamment en évidence un mouvement de "va-et-vient" du substrat au cours de la catalyse entre les deux sites de reconnaissance anionique qui est lié à l'étape d'échange du cofacteur. Bien que structuralement proche des GAPDH, l E4PDH est une enzyme pour laquelle peu de données sont disponibles. De nombreuses questions restent donc posées concernant notamment les déterminants de l'affinité pour le cofacteur, la nature du site de reconnaissance anionique, ou encore le mécanisme d'activation de la molécule d'eau nécessaire à une étape d'hydrolyse efficace. Sont présentées dans ce manuscrit trois structures cristallographiques de l'E4PDH d'E. coli, sous forme apoenzyme, en présence de phosphate ou en complexe avec un analogue de cofacteur. L'analyse de ces structures a notamment permis de caractériser l'unique site de reconnaissance anionique de l'enzyme, de proposer des hypothèses quant à la faible affinité de l'enzyme pour le cofacteur, et d'identifier un candidat possible pour l'activation de la molécule d'eau hydrolytique.Even if GAPDH is well characterized from a biochemical and structural point of view, the contribution of its two anion recognition sites to catalysis is still matter of debate. This work presents the crystallization, the strategy for the accumulation of the thioacylenzyme intermediate and for the obtaining of the enzyme-product complex, the resolution and analysis of the corresponding crystallographic structures and the implications in terms of reaction mechanism. The results mainly shed light on the existence of a flip-flop movement of the substrate between the two anion recognition sites during catalysis which is related to the cofactor exchange step. Although structurally related to GAPDHs, the E4PDH is an enzyme fairly less characterized. Many interrogations thus remain on the determinants of cofactor affinity, on the nature of the anion recognition site or on the mechanism of activation of the water molecule that is needed for an efficient hydrolysis step. This dissertation presents the resolution of three crystallographic structures of the E4PDH from E. coli, under the apoenzyme form, in the presence of phosphate or in complex with a cofactor analog. The analysis f these structures allows the characterization of the unique anion recognition site of this enzyme, to propose structural hypotheses to explain the low affinity of this enzyme for its cofactor and also to identify possible candidates for the activation of the nucleophilic water molecule.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Etude cristallographique d oxydoréductases impliquées dans la réponse au stress oxydatif chez le peuplier en vue de la compréhension de leur mécanisme catalytique

La structure de trois oxydoréductases (la glutathion peroxydase (Gpx), la thiorédoxine (Trx) et la glutarédoxine (Grx)) de Populus trichocarpa . deltoides (le peuplier) a été caractérisée par diffraction des rayons X. Les Gpxs forment un groupe d enzymes qui régulent la concentration des espèces réactives de l'oxygène (ROS) dans les cellules, et qui les protègent des effets d un stress oxydant. Contrairement à leurs homologues d origine animale, les Gpxs végétales ne dépendent pas du glutathion (GSH) mais des Trx pour leur fonctionnement. Dans cette étude, j'ai résolu les structures des formes réduite et oxydée de la Gpx5 de peuplier et montré que des changements conformationnels drastiques sont nécessaires pour passer d une forme à l autre. Les Trxs régulent diverses protéines cibles par la réduction de leur pont disulfure. Mon objectif était de comprendre le mécanisme catalytique d une nouvelle isoforme, la PtTrxh4, dont la capacité à accepter des électrons de la Grx a été récemment démontrée. Cette PtTrxh4 contient trois cystéines, la première localisée dans une extension en position N-terminale (Cys4) et deux situées dans le site actif classique (WC1GPC2) de la Trx. Les résolutions des structures de l enzyme sauvage et du mutant C4S m ont permis de proposer un mécanisme catalytique en quatre étapes en accord avec les études enzymatiques. Les Grxs sont des protéines qui utilisent des électrons du GSH en particulier pour catalyser des réactions d'échange de thiol-disulfure. Ici, je présente la structure de la PtGrxS12 (en complexe avec le GSH), la première structure de la Grx végétale de sous-classe 1 ayant un site actif de motif atypique 28WCSYS32.Three oxidoreductases (glutathione peroxidase, GPX; thioredoxin, Trx and glutaredoxin, Grx) from Populus trichocarpa . deltoides (poplar tree) were characterized using X-ray crystallography approach. GPXs are a group of enzymes that regulate the levels of oxygen species in cells, and protect them against oxidative damage. In this study, I have determined the crystal structures of the reduced and oxidized form of poplar GPX5 (PtGPX5). Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. Trxs regulate various protein partners through the thiol-disulfide(s) reduction. The aim of this study is thus to precisely describe the catalytic mechanism of a new isoform of Trx, PtTrxh4, since it has been demonstrated recently to be reduced by Grx. PtTrxh4 contains three cysteines; one localized in an N-terminal extension (Cys4) and two in the usual Trx active site (WC1GPC2). Two crystal structures of PtTrxh4 solved in this study, wild-type and C61S mutant, allow us to propose a four-step disulfide cascade catalytic mechanism in accordance with enzymatic studies. Grxs are highly conserved redox-proteins that utilize electrons from GSH particularly to catalyze thiol-disulfide exchange reactions. Here, I present the structure of glutathionylated PtGrxS12, the first structure of plant Grx of subclass 1 with an atypical 28WCSYS32 active site. Protein structures solved here shed lights to our understanding of the redox mechanism in plant and to the enzyme-substrate interactions.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Thioredoxins and related proteins in photosynthetic organisms: molecular basis for thiol dependent regulation

International audienc

Age-related changes in regiospecific expression of Lipolysis Stimulated Receptor (LSR) in mice brain

International audienceThe regulation of cholesterol, an essential brain lipid, ensures proper neuronal development and function, as demonstrated by links between perturbations of cholesterol metabolism and neurodegenerative diseases, including Alzheimer's disease. The central nervous system (CNS) acquires cholesterol via de novo synthesis, where glial cells provide cholesterol to neurons. Both lipoproteins and lipoprotein receptors are key elements in this intercellular transport, where the latter recognize, bind and endocytose cholesterol containing glia-produced lipoproteins. CNS lipoprotein receptors are like those in the periphery, among which include the ApoB, E binding lipolysis stimulated lipoprotein receptor (LSR). LSR is a multi-meric protein complex that has multiple isoforms including α and α', which are seen as a dou-blet at 68 kDa, and β at 56 kDa. While complete inactivation of murine lsr gene is embryonic lethal, studies on lsr +/-mice revealed altered brain cholesterol distribution and cognitive functions. In the present study, LSR profiling in different CNS regions revealed regiospecific expression of LSR at both RNA and protein levels. At the RNA level, the hippocampus, hypo-thalamus, cerebellum, and olfactory bulb, all showed high levels of total lsr compared to whole brain tissues, whereas at the protein level, only the hypothalamus, olfactory bulb, and retina showed the highest levels of total LSR. Interestingly, major regional changes in LSR expression were observed in aged mice which suggests changes in cholesterol homeostasis in specific structures in the aging brain. Immunocytostaining of primary cultures of mature murine neurons and glial cells isolated from different CNS regions showed that LSR is expressed in both neurons and glial cells. However, lsr RNA expression in the cerebellum was predominantly higher in glial cells, which was confirmed by the immunocytostaining profile of cerebellar neurons and glia. Based on this observation, we would propose that LSR in glial cells may play a key role in glia-neuron cross talk, particularly in the feedback control of cholesterol synthesis to avoid cholesterol overload in neurons and to maintain proper functioning of the brain throughout life

Interfacial approach to polyaromatic hydrocarbon toxicity: phosphoglyceride and cholesterol monolayer response to phenantrene, anthracene, pyrene, chrysene, and benzo[a]pyrene

International audienceInteractions of phenantrene, anthracene, pyrene, chrysene, and benzo[a]pyrene (polyaromatic hydrocarbons) with model phospholipid membranes were probed using the Langmuir technique. The lipid monolayers were prepared using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoserine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and cholesterol. Surface pressure and electrical surface potential were measured on mixed phospholipid/PAH monolayers spread on a pure water subphase. The morphology of the mixed monolayers was followed with Brewster angle microscopy. Polarization-modulation infrared reflection−absorption spectroscopy spectra obtained on DPPE/benzo[a]pyrene showed that the latter interacts with the carbonyl groups of the phospholipid. On the other hand, the activity of phospholipase A2 toward DLPC used as a probe to locate benzo[a]pyrene in the monolayers indicates that the polyaromatic hydrocarbons are not accessible to the enzyme. The results obtained show that all PAHs studied affect the properties of the pure lipid, albeit in different ways. The most notable effects, namely, film fluidization and morphology changes, were observed with benzo[a]pyrene. In contrast, the complexity of mixed lipid monolayers makes the effect of PAHs difficult to detect. It can be assumed that the differences observed between PAHs in monolayers correlate with their toxicity