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Excess brain cholesterol is strongly implicated in the pathogenesis of Alzheimer disease (AD). Here we evaluated how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of the amyloid precursor protein (APP) regulates its processing.We generated nine point mutations in the APP gene, changing the charge and/or hydrophobicity of the amino-acids which were previously shown as part of the CBS. Most mutations triggered a reduction of amyloid-β peptides Aβ40 and Aβ42 secretion from transfected HEK293T cells. Only the K to A mutation at position 28 of Aβ in the APP sequence (APPK28A) resulted in a concomitant increase in the production of shorter Aβ peptides. Mass Spectrometry (MS) confirmed the predominance of Aβx-33 and Aβx-34. The enzymatic activity of α-, β- and g-secretases remained unchanged in cells expressing the APPK28A and all other mutants. Similarly, subcellular localization of the mutants in early endosomes did not differ from the APPWT protein. A transient increase of plasma membrane cholesterol enhanced the production of Aβ40 and Aβ42 by APPWT, an effect absent in APPK28A mutant. Finally, WT but not CBS mutant Aβ derived peptides bound to cholesterol-rich exosomes. Collectively, the present data revealed a major role of juxtamembrane amino acids of the APP CBS in modulating the production of toxic Aβ species. More generally, they underpin the role of cholesterol in the pathophysiology of AD.

International audience; Rationale: Although children with primary ciliary dyskinesia (PCD) typically have low nasal nitric oxide (nNO), some children with indisputable PCD may have unexplained high nNO concentrations. Objectives: To look for relationships between nNO measures and genetic findings (and cilia motility or ultrastructure when available) in children with PCD with known genotypes. Methods: We retrospectively studied 73 children with PCD (median age, 9.5 [range, 2.1-18.2] yr). nNO was the mean value of a plateau reached while the velum was closed (nNO-VC; threshold, 77 nl ⋅ min-1) or was calculated as the average of five peaks obtained during tidal breathing (threshold, 40 nl ⋅ min-1). Ciliary beat was classified as either motile (including dyskinetic pattern) or immotile, depending on whether motility was present or absent in all cilia, or as a mixture of motile and immotile cilia. Genotypes were classified as pathogenic mutations in genes known to be associated with high nNO (mild genotype), biallelic truncating mutations in other genes (severe mutations), or putative hypomorphic pathogenic mutations (missense, single amino acid deletion, or moderate splicing mutations) in at least one allele believed to be possibly associated with residual production of a functional protein. Results: nNO was above the discriminant threshold in 16 of 73 (21.9%) children (11 nNO-VC and 5 nNO during tidal breathing). High nNO was less frequent in children with severe mutations (2 of 42) than in those with mild genotypes (7 of 10) or at least one hypomorphic mutation (7 of 21) (P

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis.

Background Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. Results Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. Conclusions The developed approach, based on parallel 13C tracer studies with GC–MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.

International audience; Leveraging systems biology approaches, we illustrate how metabolically distinct species of Clostridia protect against or worsen Clostridioides difficile infection in mice by modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survive infection with reduced disease severity, while mice colonized with the butyrate-producer, Clostridium sardiniense, succumb more rapidly. Systematic in vivo analyses revealed how each commensal alters the gut-nutrient environment to modulate the pathogen's metabolism, gene regulatory networks, and toxin production. Oral administration of P. bifermentans rescues conventional, clindamycin-treated mice from lethal C. difficile infection in a manner similar to that of monocolonized animals, thereby supporting the therapeutic potential of this commensal species. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biology approaches to define host-commensal-pathogen interactions in vivo.

Genetic studies of metabolites have identified thousands of variants, many of which are associated with downstream metabolic and obesogenic disorders. However, these studies have relied on univariate analyses, reducing power and limiting context-specific understanding. Here we aim to provide an integrated perspective of the genetic basis of metabolites by leveraging the Finnish Metabolic Syndrome In Men (METSIM) cohort, a unique genetic resource which contains metabolic measurements, mostly lipids, across distinct time points as well as information on statin usage. We increase effective sample size by an average of two-fold by applying the Covariates for Multi-phenotype Studies (CMS) approach, identifying 588 significant SNP-metabolite associations, including 228 new associations. Our analysis pinpoints a small number of master metabolic regulator genes, balancing the relative proportion of dozens of metabolite levels. We further identify associations to changes in metabolic levels across time as well as genetic interactions with statin at both the master metabolic regulator and genome-wide level., Genome-wide association studies of metabolites have revealed hundreds of genetic associations using univariate analyses. Here, the authors use a multivariate approach to perform association analyses for 158 serum metabolites, followed by fine mapping and GxE interaction tests with statin use and age.

International audience; Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27-/- cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27-/- cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.

The leucine metabolite, &beta, hydroxy-&beta, methyl butyrate (HMB), is widely used in human nutrition and animal production as a nutritional supplement. Although the HMB usage during late gestation has been demonstrated to have a positive effect on fetal development, knowledge on net absorption and metabolism of HMB and impact of HMB on branched chain amino acids (BCAAs) metabolism is lacking. To address this, we conducted a study using pigs during the perinatal period as a model organism. Eight-second parity sows were fitted with indwelling catheters in the femoral artery and in the portal, hepatic, femoral, and mesenteric veins. Eight hourly sets of blood samples were taken starting 30 min before the morning meal on day &ndash, 10 and day &ndash, 3 relative to parturition. Four control (CON) sows were fed a standard lactation diet from day &ndash, 15 and throughout the experiment, and 4 HMB sows were fed the control diet supplemented with 15 mg Ca(HMB)2/kg body weight mixed in one third of the morning meal from day &ndash, 10 until parturition. Blood gases, plasma metabolites, milk compositions, and apparent total tract digestibility of nutrients were measured. Arterial plasma concentrations of HMB (p &lt, 0.001), Cys (p &lt, 0.001), and Lys (p &lt, 0.10) were increased in HMB supplemented sows, while arterial plasma triglycerides concentration was decreased (p &lt, 0.05). The net portal recovery of Ala and Asp were increased in HMB sows (p &lt, 0.05). Sows fed HMB had increased hepatic vein flow and net hepatic fluxes of Met, Asn, and Gln (p &lt, 0.05). In contrast, the femoral extraction rates of Ala and Ser were decreased by dietary HMB supplementation (p &lt, 0.05). Dietary HMB treatment and sampling time relative to feeding had an interaction on arterial concentrations, net portal fluxes, and femoral extraction rates of BCAAs. The net portal recovery of HMB was 88%, while 14% of supplemented HMB was excreted through urine and 4% through feces. Moreover, the gastrointestinal tract metabolized 8% while the liver metabolized 12%. Finally, 26% of the daily intake of HMB was secreted via colostrum at the day of farrowing. This study demonstrated that dietary HMB supplementation increased net uptake of amino acids and increased fatty acid oxidation through improving blood flow and insulin sensitivity during the late gestation. Most importantly, oral HMB administration could maintain a stable postprandial absorption and altered metabolism in BCAAs. Net portal flux of HMB at 5.5 to 6.5 h after feeding approached zero, indicating that HMB ideally should be administrated two or three times, daily.

Protein backbone, broken and mended Small, posttranslationally modified peptides are produced by microorganisms as antimicrobial agents or to communicate with neighboring cells. Alterations to the peptide backbone can change the structure of peptides or introduce reactive chemical moieties. Morinaka et al. characterized a bacterial enzyme that excises the side chain and α-carbon of a tyrosine residue from a short peptide, leaving behind an α-ketoamide. This backbone functional group is found in some protease inhibitors and is a valuable handle for bio-orthogonal chemistry. The enzyme accepts peptide substrates with a short recognition motif, suggesting that it could be used to generate libraries of modified peptides. Science , this issue p. 779

OH (2:1 v/v), and sulfu-ric acid. In the case of chondrites, achondrites, and stony-iron meteorites, the samples were also pyrolyzed at 6008C.After the treatment, the powders did not release any traceof organic substances. The synthesis of large number of com-pounds was observed (Scheme 1): nucleobases and their an-alogues, carboxylic acids, amino acids, and low-molecular-weight derivatives (miscellanea). We dub these reactionsmeteorite-catalyzed syntheses. Given the complexity of themixtures, analysis was limited to products 1ngmL

Peptides have gained renewed interest as candidate therapeutics. However, to bring them to a broader clinical use, challenges such as the rational optimization of their pharmacological properties remain. Peptide scanning techniques offer a systematic framework to gain information on the functional role of individual amino acids of a peptide. Due to progress in mastering new chemical synthesis routes targeting amino acid backbone, they are currently diversified. Structure-activity relationship (SAR) analyses such as alanine- or enantioneric- scanning can now be supplemented by N-substitution, lactam cyclisation- or aza-amino scanning procedures addressing not only SAR considerations but also the peptide pharmacological properties.This review highlights the different scanning techniques currently available and illustrates how they can impact drug discovery.Progress in peptide scanning techniques opens new perspectives for peptide drug development. It comes with the promise of a paradigm change in peptide drug design in which peptide drugs will be closer to the parent peptides. However, scanning still remains assimilable to a trial and error strategy that could benefit from being combined with specific in silico approaches that start reaching maturity.

International audience; Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. Among 106 proteins identified, 80 were quantified in both forward/reverse SILAC experiments and quantitative proteomic analysis revealed altered levels of expression for 24 secreted proteins. These included the down-regulation of a number of extracellular matrix/cell adhesion components, and the up-regulation of secreted cyclophilins A and B, macrophage inhibition factor and phosphatidylethanolamine-binding protein 1. These changes in protein secretion were not prevented by co-incubation with the antioxidant N-acetylcysteine, suggesting that they were not triggered by cyclosporine-induced oxidative stress. The results from the present study provide important new knowledge to gain insights into the molecular mechanisms of cyclosporine-related toxicity. Some of the proteins identified here should be tested as potential biomarkers of cyclosporine nephrotoxicity in subsequent clinical studies.

International audience; Mycosporine-like compounds, comprising mycosporines and mycosporine-like amino acids (MAAs) are UV protecting secondary metabolites described in organisms such as fungi, algae, cyanobacteria or animals. Lichens however, were only poorly investigated for such constituents so far. Here, a method for the characterization of mycosporines and MAAs in purified aqueous extracts, involving HPTLC coupled to spectrophotodensitometry, HPLC-DAD-MS(n) and UPLC-HRMS analysis, is described. This optimized protocol was validated on three algae and one cyanolichen containing known MAAs and mycosporines, and then applied to 18 cyanolichen species. Analyses revealed the presence of five already described mycosporine-like compounds in the investigated species, including mycosporine serinol in Lichina and Peltigera species and mycosporine glutamicol in Degelia plumbea. Apart from that, eight unknown mycosporine-like compounds were detected and tentatively characterized on the basis of their DAD spectra and their MS(n) and HRMS data: two in the alga Porphyra dioica and six in cyanolichen species belonging to the genera Degelia, Nephroma and Stereocaulon. From Nephroma laevigatum, the mycosporine hydroxyglutamicol was preparatively isolated and identified through HRMS, 1D and 2D NMR spectroscopic data. The optimized analytical protocol allowed the characterization of mycosporine-like compounds in small amounts of material and confirmed the potential of cyanolichens as a source of mycosporine compounds. It should also be applicable to investigate lichen species with green algae photobionts for mycosporine-like compounds.

A single non-anaesthetic dose of ketamine, a non-competitive NMDA receptor (NMDAr) antagonist with hallucinogenic properties, induces cognitive impairment and psychosis, and aggravates schizophrenia symptoms in patients. In conscious rats an equivalent dose of ketamine induces key features of animal models of acute psychosis, including abnormal behaviour, hyperlocomotion, deficits in prepulse inhibition to an acoustic startle response and gating of auditory evoked potentials, and concomitantly increases the power of spontaneously occurring gamma oscillations in the neocortex. This study investigated whether NMDAr antagonist-induced aberrant gamma oscillations could be modulated by acute treatment with typical and atypical antipsychotic drugs. Adult male Wistar rats that has been implanted with extradural electrodes were placed in an arena for 30 minutes (baseline) and then subcutaneously administered either clozapine (1–5mg/kg, n=7), haloperidol (0.05 – 0.25mg/kg; n=8), LY379268 (a preclinical agonist at mGluR2/3 receptors: 0.3 – 3mg/kg; n=5) or their vehicles alone, and 30 minutes later received ketamine (5mg/kg sc). Quantitative measures of EEG gamma power and locomotor activity were assessed throughout the experiment. All three drugs significantly reduced the power of baseline EEG gamma oscillations by 30–50%, an effect most prominent after LY379268, and all inhibited ketamine-induced hyperlocomotor activity. However, only pretreatment with LY379268 attenuated trough-to-peak ketamine-induced gamma hyperactivity. These results demonstrate that typical and atypical antipsychotic drugs acutely reduce cortical gamma oscillations, an effect that may be related to their clinical efficacy.

Computational Protein Design (CPD) is a promising method for high throughput protein and ligand mutagenesis. Recently, we developed a CPD method that used a polar-hydrogen energy function for protein interactions and a Coulomb/Accessible Surface Area (CASA) model for solvent effects. We applied this method to engineer aspartyl-adenylate (AspAMP) specificity into Asparaginyl-tRNA synthetase (AsnRS), whose substrate is asparaginyl-adenylate (AsnAMP). Here, we implement a more accurate function, with an all-atom energy for protein interactions and a residue-pairwise generalized Born model for solvent effects. As a first test, we compute aminoacid affinities for several point mutants of Aspartyl-tRNA synthetase (AspRS) and Tyrosyl-tRNA synthetase and stability changes for three helical peptides and compare with experiment. As a second test, we readdress the problem of AsnRS aminoacid engineering. We compare three design criteria, which optimize the folding free-energy, the absolute AspAMP affinity, and the relative (AspAMP-AsnAMP) affinity. The sequences and conformations are improved with respect to our previous, polar-hydrogen/CASA study: For several designed complexes, the AspAMP carboxylate forms three interactions with a conserved arginine and a designed lysine, as in the active site of the AspRS:AspAMP complex. The conformations and interactions are well maintained in molecular dynamics simulations and the sequences have an inverted specificity, favoring AspAMP over AsnAMP. The method is not fully successful, since experimental measurements with the seven most promising sequences show that they do not catalyze at a detectable level the adenylation of Asp (or Asn) with ATP. This may be due to weak AspAMP binding and/or disruption of transition-state stabilization. © 2011 Wiley-Liss, Inc. 79 12 3448 3468 Cited By :12

International audience; Previous studies have shown that the mechanical properties of trabecular bone are determined by bone volume fraction (BV/TV) and microarchitecture. The purpose of this study was to explore other possible determinants of the mechanical properties of vertebral trabecular bone, namely collagen cross-link content, microdamage, and mineralization. Trabecular bone cores were collected from human L2 vertebrae (n = 49) from recently deceased donors 54-95 years of age (21 men and 27 women). Two trabecular cores were obtained from each vertebra, one for preexisting microdamage and mineralization measurements, and one for BV/TV and quasi-static compression tests. Collagen cross-link content (PYD, DPD, and PEN) was measured on surrounding trabecular bone. Advancing age was associated with impaired mechanical properties, and with increased microdamage, even after adjustment by BV/TV. BV/TV was the strongest determinant of elastic modulus and ultimate strength (r² = 0.44 and 0.55, respectively). Microdamage, mineralization parameters, and collagen cross-link content were not associated with mechanical properties. These data indicate that the compressive strength of human vertebral trabecular bone is primarily determined by the amount of trabecular bone, and notably unaffected by normal variation in other factors, such as cross-link profile, microdamage and mineralization.

International audience; Nutritional support in the intensive care setting represents a challenge but it is fortunate that its delivery and monitoring can be followed closely. Enteral feeding guidelines have shown the evidence in favor of early delivery and the efficacy of use of the gastrointestinal tract. Parenteral nutrition (PN) represents an alternative or additional approach when other routes are not succeeding (not necessarily having failed completely) or when it is not possible or would be unsafe to use other routes. The main goal of PN is to deliver a nutrient mixture closely related to requirements safely and to avoid complications. This nutritional approach has been a subject of debate over the past decades. PN carries the considerable risk of overfeeding which can be as deleterious as underfeeding. Therefore the authors will present not only the evidence available regarding the indications for PN, its implementation, the energy required, its possible complementary use with enteral nutrition, but also the relative importance of the macro- and micronutrients in the formula proposed for the critically ill patient. Data on long-term survival (expressed as 6 month survival) will also be considered a relevant outcome measure. Since there is a wide range of interpretations regarding the content of PN and great diversity in its practice, our guidance will necessarily reflect these different views. The papers available are very heterogeneous in quality and methodology (amount of calories, nutrients, proportion of nutrients, patients, etc.) and the different meta-analyses have not always taken this into account. Use of exclusive PN or complementary PN can lead to confusion, calorie targets are rarely achieved, and different nutrients continue to be used in different proportions. The present guidelines are the result of the analysis of the available literature, and acknowledging these limitations, our recommendations are intentionally largely expressed as expert opinions.

International audience; The Tet repressor (TetR) mediates the most important mechanism of bacterial resistance against tetracycline (Tc) antibiotics. In the absence of Tc, TetR is tightly bound to its operator DNA; upon binding of Tc with an associated Mg(2+) ion, it dissociates from the DNA, allowing expression of the repressed genes. Its tight control by Tc makes TetR broadly useful in genetic engineering. The Tc binding site is over 20 A from the DNA, so the binding signal must propagate a long distance. We use molecular dynamics simulations and continuum electrostatic calculations to test two models of the allosteric mechanism. We simulate the TetR:DNA complex, the Tc-bound, "induced" TetR, and the transition pathway between them. The simulations support the model inferred previously from the crystal structures and reveal new details. When [Tc:Mg](+) binds, the Mg(2+) ion makes direct and water-mediated interactions with helix 8 of one TetR monomer and helix 6 of the other monomer, and helix 6 is pulled in towards the central core of the structure. Hydrophobic interactions with helix 6 then pull helix 4 in a pendulum motion, with a maximal displacement at its N-terminus: the DNA interface. The crystal structure of an additional TetR reported here corroborates this motion. The N-terminal residue of helix 4, Lys48, is highly conserved in DNA-binding regulatory proteins of the TetR class and makes the largest contribution of any amino acid to the TetR:DNA binding free energy. Thus, the conformational changes lead to a drastic reduction in the TetR:DNA binding affinity, allowing TetR to detach itself from the DNA. Tc plays the role of a specific Mg(2+) carrier, whereas the Mg(2+) ion itself makes key interactions that trigger the allosteric transition in the TetR:Tc complex.

International audience; Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA(Leu) recognition sets and their evolution, the recognition of tRNA(Leu) by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8-A14, G18-U55 and G19-C56; and the orientation of the variable arm are critical elements for tRNA(Leu) aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.

International audience; Stereoisomers of 1-amino-2-phosphonomethylcyclopropanecarboxylic acid (APCPr), conformationally restricted analogues of L-AP4 (2-amino-4-phosphonobutyric acid), have been prepared and evaluated at recombinant group III metabotropic glutamate receptors. They activate these receptors over a broad range of potencies. The most potent isomer (1S,2R)-APCPr displays a similar pharmacological profile as that of L-AP4 (EC50 0.72, 1.95, >500, 0.34 microM at mGlu4, 6, 7, 8 receptors, respectively, and no effect at group I/II mGluRs). It was characterized on native receptors located in the basal ganglia (BG) where it induced a robust and reversible inhibition of synaptic transmission. It was tested in vivo in haloperidol-induced catalepsy, a model of Parkinsonian akinesia, by direct infusion in the globus pallidus of the BG. At a dose of 0.5 nmol/microL, catalepsy was significantly antagonized. This study reveals that (1S,2R)-APCPr is a potent group III mGluR agonist and confirms that these receptors may be considered as a therapeutic target in the Parkinson's disease.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-α (ERα) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERα in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERα remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERα ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERα monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERα ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

The molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) method was applied to the study of the protein–protein complex between a camelid single chain variable domain (cAb-Lys3) and hen egg white lysozyme (HEL), and between cAb-Lys3 and turkey egg white lysozyme (TEL). The electrostatic energy was estimated by solving the linear Poisson–Boltzmann equation. A free energy decomposition scheme was developed to determine binding energy hot spots of each complex. The calculations identified amino acids of the antibody that make important contributions to the interaction with lysozyme. They further showed the influence of small structural variations on the energetics of binding and they showed that the antibody amino acids that make up the hot spots are organized in such a way as to mimic the lysozyme substrate. Through further analysis of the results, we define the concept of “efficient amino acids,” which can provide an assessment of the binding potential of a particular hot spot interaction. This information, in turn, can be useful in the rational design of small molecules that mimic the antibody. The implications of using free energy decomposition to identify regions of a protein–protein complex that could be targeted by small molecules inhibitors are discussed. Proteins 2007. © 2007 Wiley-Liss, Inc.

The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.

The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic alpha-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChE(T) on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with Q(N), a PRAD-containing N-terminal fragment of ColQ. The formation of homo-tetramers and Q(N)-linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32-40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T(4) and T(4)-Q(N) tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with Q(N), indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the alpha-helical structure of the t peptide were found to enhance degradation. Co-expression with Q(N) generally increased secretion, mostly as T(4)-Q(N) complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChE(T) subunits and the choice between secretion and degradation.

International audience; Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. Here, we provide evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. In a genetic screen for growth modifiers, we identified slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. Our results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism.

International audience; Background: Brown algae are sessile macro-organisms of great ecological relevance in coastal ecosystems. They evolved independently from land plants and other multicellular lineages, and therefore hold several original ontogenic and metabolic features. Most brown algae grow along the coastal zone where they face frequent environmental changes, including exposure to toxic levels of heavy metals such as copper (Cu). Results: We carried out large-scale transcriptomic and metabolomic analyses to decipher the short-term acclimation of the brown algal model E. siliculosus to Cu stress, and compared these data to results known for other abiotic stressors. This comparison demonstrates that Cu induces oxidative stress in E. siliculosus as illustrated by the transcriptomic overlap between Cu and H 2 O 2 treatments. The common response to Cu and H 2 O 2 consisted in the activation of the oxylipin and the repression of inositol signaling pathways, together with the regulation of genes coding for several transcription-associated proteins. Concomitantly, Cu stress specifically activated a set of genes coding for orthologs of ABC transporters, a P 1B-type ATPase, ROS detoxification systems such as a vanadium-dependent bromoperoxidase, and induced an increase of free fatty acid contents. Finally we observed, as a common abiotic stress mechanism, the activation of autophagic processes on one hand and the repression of genes involved in nitrogen assimilation on the other hand. Conclusions: Comparisons with data from green plants indicate that some processes involved in Cu and oxidative stress response are conserved across these two distant lineages. At the same time the high number of yet uncharacterized brown alga-specific genes induced in response to copper stress underlines the potential to discover new components and molecular interactions unique to these organisms. Of particular interest for future research is the potential cross-talk between reactive oxygen species (ROS)-, myo-inositol-, and oxylipin signaling.

International audience; BACKGROUND: Machine perfusion use has been reported to promote graft outcome in case of donation after cardiac death. Our objective was to evaluate the potential for nuclear magnetic resonance (NMR) to predict graft outcome by analyzing perfusates during machine perfusion time. METHOD: We used a renal autotransplantation model mimicking deceased after cardiac death donors with pigs. Organs were subjected to 60 min of warm ischemia before the hypothermic machine preservation during 22 hr. We studied the correlation between creatinemia after transplantation and the NMR data from perfusates. RESULTS: A metabonomic analysis allowed us to highlight the evolution of several metabolites during perfusion: the concentration of lactate, choline, or amino acids such as valine, glycine, or glutamate increased with time, whereas there was a diminution of total glutathione during this period. The changes in these biomarkers were less severe in the group with the better outcome. Statistical analysis revealed a strong association between the level of those metabolites during machine perfusion and function recovery (Spearman rank ≥0.89; P

International audience; The pattern of codon usage in the amitochondriate diplomonad Giardia lamblia has been investigated. Very extensive heterogeneity was evident among a sample of 65 genes. A discrete group of genes featured unusual codon usage due to the amino acid composition of their products: these variant surface proteins (VSPs) are unusually rich in Cys and, to a lesser extent, Gly and Thr. Among the remaining 50 genes, correspondence analysis revealed a single major source of variation in synonymous codon usage. This trend was related to the extent of use of a particular subset of 21 codons which are inferred to be those which are optimal for translation; at one end of this trend were genes expected to be expressed at low levels with near random codon usage, while at the other extreme were genes expressed at high levels in which these optimal codons are used almost exclusively. These optimal codons all end in C or G so G + C content at silent sites varies enormously among genes, from values around 40%, expected to reflect the background level of the genome, up to nearly 100%. Although VSP genes are occasionally extremely highly expressed, they do not, in general, have high frequencies of optimal codons, presumably because their high expression is only intermittent. These results indicate that natural selection has been very effective in shaping codon usage in G. lamblia. These analyses focused on sequences from strains placed within G. lamblia "assemblage A"; a few sequences from other strains revealed extensive divergence at silent sites, including some divergence in the pattern of codon usage.

Poliovirus mutants (PVpi) selected during the persistent infection of human neuroblastoma cells can establish secondary persistent infections in nonneural HEp-2c cells (I. Pelletier, T. Couderc, S. Borzakian, E. Wyckoff, R. Crainic, E. Ehrenfeld, and F. Colbere-Garapin, 1991, Virology, 180, 729–737). Previous results from our laboratory have also shown that, in the genome of PVpi S11 derived from the Sabin 1 strain, the genomic region involved in this phenotype contains 11 missense mutations which map exclusively to the genes encoding the capsid proteins VP1 and VP2. We report here the identification of precise viral determinants able to confer the capacity to establish persistent infections in HEp-2c cell cultures to the lytic Sabin 1 strain. We used a strategy based on the observation that PVpi, after a few months of persistent infection in HEp-2c cells, tend to regain a more lytic phenotype in uninfected HEp-2c cell cultures. We constructed mutant viruses carrying only a few mutations potentially involved in the phenotype of persistence. Two mutations were identified, one corresponding to the substitution His>Tyr of amino acid 142 of VP2 and another corresponding to the substitution Val>Ile of amino acid 160 of VP1. Mutants carrying one or the other of the two determinants established persistent infections in HEp-2c cell cultures in about 20% of the infections. Higher frequencies were obtained with the mutant carrying both determinants (30%), and with PVpi S11 (63%), indicating that the effects of several determinants can be cumulative. The two determinants are localized on the capsid surface in a region known to be involved in the interactions between poliovirus and its cell receptor and in fact, we demonstrate here that in the case of the two persistent mutants, these interactions are modified.

International audience; The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is involved in the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop structure, which is encoded by the 5′ leader sequence found in all HIV-1 mRNAs. Herein, we report the rational design, synthesis, and in vitro evaluation of new RNA binding agents that were conceived in order to bind strongly and selectively to the stem-loop structure of TAR RNA and, thus, inhibit the Tat/TAR interaction. We have demonstrated that the conjugation of modified nucleobases, able to interact specifically with an RNA base pair, and various amino acids allows these motifs to bind the target RNA selectively and in a cooperative manner that leads to the inhibition of viral replication in HIV-infected cells.

International audience; OBJECTIVE: To compare the plasma amino acid (AA) concentrations obtained by the infusion of an AA solution (660 mOsm/L, pH 7) using the subcutaneous (SC) with that using the intravenous (i.v.) route in older patients. DESIGN: A prospective, randomized, cross-over study. SETTING: A hospital geriatric ward. PARTICIPANTS: Six patients with a mean age of 84 years. MEASUREMENTS: The infusion of the AA solution (IV or SC) lasted 6 hours. Blood was sampled at 0, 2, 4, 6, 8, 10, 14, 18, and 24 hours from the start of the infusion to determine plasma AA level by the phenyl-isothiocyanate method. RESULTS: Compared with baseline values, plasma AA concentrations increased to a significantly higher level from the second to the tenth hour and from the second to the fourteenth hour during i.v. and SC infusions, respectively. Plasma AA levels did not differ between the two routes. SC infusion was well tolerated. CONCLUSION: Assuming that nutritional sufficiency is the desired result of plasma AA infusion, we conclude the SC route is well tolerated and offers the possibility of nitrogen supplementation for older patients over short periods of time, when oral protein intake is transiently insufficient or impossible, as a way to limit, but not to treat, protein-energy malnutrition.

International audience; High-affinity IL2 receptors consist of three components, the alpha, beta, and gamma chains that are associated in a noncovalent manner. Both the beta and gamma chains belong to the cytokine receptor superfamily. Interleukin 2 (IL2) binds to high-affinity receptors on the cell surface and IL2-receptor complexes are internalized. After endocytosis, the components of this multimolecular receptor have different intracellular fates: one of the chains, alpha, recycles to the plasma membrane, while the others, beta and gamma, are routed towards late endocytic compartments and are degraded. We show here that the cytosolic domain of the beta chain contains a 10-amino acid sequence which codes for a sorting signal. When transferred to a normally recycling receptor, this sequence diverts it from recycling. The structure of a 17-amino acid segment of the beta chain including this sequence has been studied by nuclear magnetic resonance and circular dichroism spectroscopy, which revealed that the 10 amino acids corresponding to the sorting signal form an amphipathic alpha helix. This work thus describes a novel, highly structured signal, which is sufficient for sorting towards degradation compartments after endocytosis.

International audience; Protein structures are an ensemble of atoms determined experimentally mostly by X-ray crystallography or Nuclear Magnetic Resonance. Studying 3D protein structures is a key point for better understanding protein function at a molecular level. We propose a set of accurate tools, for analysing protein structures, based on the reliable method of Voronoi-Laguerre tessellations. The Voronoi Laguerre Delaunay Protein web server (VLDPws) computes the Laguerre tessellation on a whole given system first embedded in solvent. Through this fine description, VLDPws gives the following data: (i) Amino acid volumes evaluated with high precision, as confirmed by good correlations with experimental data. (ii) A novel definition of inter-residue contacts within the given protein. (iii) A measure of the residue exposure to solvent that significantly improves the standard notion of accessibility in some cases. At present, no equivalent web server is available. VLDPws provides output in two complementary forms: direct visualization of the Laguerre tessellation, mostly its polygonal molecular surfaces; files of volumes; and areas, contacts and similar data for each residue and each atom. These files are available for download for further analysis. VLDPws can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/vldp.

International audience; Defining the drug-induced neuroadaptations specifically associated with the behavioral manifestation of addiction is a daunting task. To address this issue, we used a behavioral model that differentiates rats controlling their drug use (Non-Addict-like) from rats undergoing transition to addiction (Addict-like). Dysfunctions in prefrontal cortex (PFC) synaptic circuits are thought to be responsible for the loss of control over drug taking that characterizes addicted individuals. Here, we studied the synaptic alterations in prelimbic PFC (pPFC) circuits associated with transition to addiction. We discovered that some of the changes induced by cocaine self-administration (SA), such as the impairment of the endocannabinoid-mediated long-term synaptic depression (eCB-LTD) was similarly abolished in Non-Addict- and Addict-like rats and thus unrelated to transition to addiction. In contrast, metabotropic glutamate receptor 2/3-mediated LTD (mGluR2/3-LTD) was specifically suppressed in Addict-like rats, which also show a concomitant postsynaptic plasticity expressed as a change in the relative contribution of AMPAR and NMDAR to basal glutamate-mediated synaptic transmission. Addiction-associated synaptic alterations in the pPFC were not fully developed at early stages of cocaine SA, when addiction-like behaviors are still absent, suggesting that pathological behaviors appear once the pPFC is compromised. These data identify specific synaptic impairments in the pPFC associated with addiction and support the idea that alterations of synaptic plasticity are core markers of drug dependence.

International audience; The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.

International audience; Aragonite pearl, vaterite pearl and shell nacre of the freshwater mollusc Hyriopsis cumingii (Zhejiang province, China) were chosen to analyze microstructure and organic composition in the different habits of calcium carbonate. SEM and TEM were used to reveal the microstructure and mineralogical phase. We found that tablets in vaterite exhibited more irregular texture and were packaged with more organic matrices than in aragonite forms. Then a peculiar method was introduced to extract water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM) from the three samples, and biochemical analysis of these organic matrixes involved in crystal formation and polymorph selection was carried out. High performance liquid chromatography (HPLC) confirms the hydrophobic pattern of the organic matrix intermingled with mineral, the opposite of the early mobilizable water soluble fraction. Amino acid composition confirms hydrophobic residues as major components of all the extracts, but it reveals an imbalance in acidic residues rates in WSM vs. ASM and in aragonite vs. vaterite. Electrophoresis gives evidence for signatures in proteins with a 140 kDa material specific for aragonite in WSM. Conversely all ASM extracts reveal the presence of about 55 kDa components, including a discrete band in vaterite extract.

International audience; The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.

International audience; Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

Life is made of the intimate interaction of metabolism and genetics, both built around the chemistry of the most common elements of the Universe (hydrogen, oxygen, nitrogen, and carbon). The transmissible interaction of metabolic and genetic cycles results in the hypercycles of organization and de-organization of chemical information, of living and non-living. The origin-of-life quest has long been split into several attitudes exemplified by the aphorisms “genetics-first” or “metabolism-first”. Recently, the opposition between these approaches has been solved by more unitary theoretical and experimental frames taking into account energetic, evolutionary, proto-metabolic and environmental aspects. Nevertheless, a unitary and simple chemical frame is still needed that could afford both the precursors of the synthetic pathways eventually leading to RNA and to the key components of the central metabolic cycles, possibly connected with the synthesis of fatty acids. In order to approach the problem of the origin of life it is therefore reasonable to start from the assumption that both metabolism and genetics had a common origin, shared a common chemical frame, and were embedded under physical–chemical conditions favourable for the onset of both. The singleness of such a prebiotically productive chemical process would partake of Darwinian advantages over more complex fragmentary chemical systems. The prebiotic chemistry of formamide affords in a single and simple physical–chemical frame nucleic bases, acyclonucleosides, nucleotides, biogenic carboxylic acids, sugars, amino sugars, amino acids and condensing agents. Thus, we suggest the possibility that formamide could have jointly provided the main components for the onset of both (pre)genetic and (pre)metabolic processes. As a note of caution, we discuss the fact that these observations only indicate possible solutions at the level of organic substrates, not at the systemic chemical level.

Incorporation of aza-β3-amino acids into endogenous neuropeptide from mollusks (ALSGDAFLRF-NH2) with weak antimicrobial activities allows us to design new AMPs sequences. We find that, depending on the nature of the substitution, these could result either in inactive pseudopeptides or in a drastic enhancement of the antimicrobial activity without high cytotoxicity resulted. Structural studies perform by NMR and circular dichroism on the pseudopeptides show the impact of aza-β3-amino acids on the peptide structures. We obtain the first three-dimensional structures of pseudopeptides containing aza-β3-amino acids in aqueous micellar SDS and demonstrate that hydrazino turn can be formed in aqueous solution. Overall, these results demonstrate the ability to modulate AMPs activities through structural modifications induced by the nature and the position of these amino acid analogs in the peptide sequences.

International audience; The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.

International audience; Nonsense Mediated Decay (NMD) degrades transcripts that contain a premature STOP codon resulting from mistranscription or missplicing. However NMD's surveillance of gene expression varies in efficiency both among and within human genes. Previous work has shown that the intron content of human genes is influenced by missplicing events invisible to NMD. Given the high rate of transcriptional errors in eukaryotes, we hypothesized that natural selection has promoted a dual strategy of "prevention and cure" to alleviate the problem of nonsense transcriptional errors. A prediction of this hypothesis is that NMD's inefficiency should leave a signature of "transcriptional robustness" in human gene sequences that reduces the frequency of nonsense transcriptional errors. For human genes we determined the usage of "fragile" codons, prone to mistranscription into STOP codons, relative to the usage of "robust" codons that do not generate nonsense errors. We observe that single-exon genes have evolved to become robust to mistranscription, because they show a significant tendency to avoid fragile codons relative to robust codons when compared to multi-exon genes. A similar depletion is evident in last exons of multi-exon genes. Histone genes are particularly depleted of fragile codons and thus highly robust to transcriptional errors. Finally, the protein products of single-exon genes show a strong tendency to avoid those amino acids that can only be encoded using fragile codons. Each of these observations can be attributed to NMD deficiency. Thus, in the human genome, wherever the "cure" for nonsense (i.e. NMD) is inefficient, there is increased reliance on the strategy of nonsense "prevention" (i.e. transcriptional robustness). This study shows that human genes are exposed to the deleterious influence of transcriptional errors. Moreover, it suggests that gene expression errors are an underestimated phenomenon, in molecular evolution in general and in selection for genomic robustness in particular.

Toxin synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis . Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile . SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB , encoding the toxins, and of tcdR , encoding the sigma factor required for toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets.

International audience; Protein structure analysis and prediction methods are based on non-redundant data extracted from the available protein structures, regardless of the species from which the protein originates. Hence, these datasets represent the global knowledge on protein folds, which constitutes a generic distribution of amino acid sequence-protein structure (AAS-PS) relationships. In this study, we try to elucidate whether the AAS-PS relationship could possess specificities depending on the specie. For this purpose, we have chosen three different species: Saccharomyces cerevisiae, Plasmodium falciparum and Arabidopsis thaliana. We analyzed the AAS-PS behaviors of the proteins from these three species and compared it to the "expected" distribution of a classical non-redundant databank. With the classical secondary structure description, only slight differences in amino acid preferences could be observed. With a more precise description of local protein structures (Protein Blocks), significant changes could be highlighted. S. cerevisiae's AAS-PS relationship is close to the general distribution, while striking differences are observed in the case of A. thaliana. P. falciparum is the most distant one. This study presents some interesting view-points on AAS-PS relationship. Certain species exhibit unique preferences for amino acids to be associated with protein local structural elements. Thus, AAS-PS relationships are species dependent. These results can give useful insights for improving prediction methodologies which take the species specific information into account.

International audience; Protein structure analysis and prediction methods are based on non-redundant data extracted from the available protein structures, regardless of the species from which the protein originates. Hence, these datasets represent the global knowledge on protein folds, which constitutes a generic distribution of amino acid sequence-protein structure (AAS-PS) relationships. In this study, we try to elucidate whether the AAS-PS relationship could possess specificities depending on the specie. For this purpose, we have chosen three different species: Saccharomyces cerevisiae, Plasmodium falciparum and Arabidopsis thaliana. We analyzed the AAS-PS behaviors of the proteins from these three species and compared it to the "expected" distribution of a classical non-redundant databank. With the classical secondary structure description, only slight differences in amino acid preferences could be observed. With a more precise description of local protein structures (Protein Blocks), significant changes could be highlighted. S. cerevisiae's AAS-PS relationship is close to the general distribution, while striking differences are observed in the case of A. thaliana. P. falciparum is the most distant one. This study presents some interesting view-points on AAS-PS relationship. Certain species exhibit unique preferences for amino acids to be associated with protein local structural elements. Thus, AAS-PS relationships are species dependent. These results can give useful insights for improving prediction methodologies which take the species specific information into account.

International audience; Protein structure analysis and prediction methods are based on non-redundant data extracted from the available protein structures, regardless of the species from which the protein originates. Hence, these datasets represent the global knowledge on protein folds, which constitutes a generic distribution of amino acid sequence-protein structure (AAS-PS) relationships. In this study, we try to elucidate whether the AAS-PS relationship could possess specificities depending on the specie. For this purpose, we have chosen three different species: Saccharomyces cerevisiae, Plasmodium falciparum and Arabidopsis thaliana. We analyzed the AAS-PS behaviors of the proteins from these three species and compared it to the "expected" distribution of a classical non-redundant databank. With the classical secondary structure description, only slight differences in amino acid preferences could be observed. With a more precise description of local protein structures (Protein Blocks), significant changes could be highlighted. S. cerevisiae's AAS-PS relationship is close to the general distribution, while striking differences are observed in the case of A. thaliana. P. falciparum is the most distant one. This study presents some interesting view-points on AAS-PS relationship. Certain species exhibit unique preferences for amino acids to be associated with protein local structural elements. Thus, AAS-PS relationships are species dependent. These results can give useful insights for improving prediction methodologies which take the species specific information into account.

International audience; The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein. The transcription of the lct gene was analyzed, and its promoter was mapped. DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids. Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 51-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics. The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481. Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in beta-methyllanthionine formation, and a 4th threonine residue is dehydrated. This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry.

International audience; Microiontophoretic application of selective agonists for the three major excitatory amino acid receptors, N-methyl-D-aspartate (NMDA), quisqualate and kainate, increased the discharge rate of noradrenergic locus coeruleus (LC) neurons in vivo. NMDA activation was selectively attenuated by iontophoretic application of 2-amino-5-phosphonopentanoate (AP5), an antagonist at NMDA receptors, whereas kainate- and quisqualate-evoked responses were attenuated by both NMDA and non-NMDA antagonists iontophoresis. NMDA- and quisqualate-evoked responses were significantly decreased by co-iontophoresis of serotonin (5-HT). When the NMDA receptor-mediated component of the response to kainate was blocked with AP5 iontophoresis, 5-HT increased the response of LC neurons to kainate. These results revealed that 5-HT differentially modulates the responsiveness of LC neurons to excitatory amino acids, depending on the receptor subtypes responsible for the neuronal activation.

International audience; The present experiments aimed at understanding the functional link between dopamine (DA) and glutamate (GLU) during the compensatory processes taking place after partial DA denervation. Lesion of the lateral part of substantia nigra in rats using 6-hydroxydopamine resulted in DA denervation of the lateral region of the ipsilateral caudate/putamen complex (CPc) whereas the medial CPc was spared. In vivo voltammetry revealed a large increase of extracellular dopamine (DA(ext)) in the medial CPc both ipsilateral and contralateral to the lesion. In addition, in vivo microdialysis and HPLC-ED revealed a concomitant increase of extracellular glutamate (GLU(ext)) in the ipsilateral medial CPc. Post-lesion chronic treatment with the putative neuroprotectors amantadine, memantine, and riluzole counteracted the tonic increases of DA(ext) and GLU(ext), revealing a possible role of GLU neurotransmission in the DA over-expression. Finally, acute low doses of GBR12909 had no effect on the DA(ext) in sham- operated animals, but dramatically increased DA(ext) in lesioned animals. The data suggest that a partial unilateral nigral lesion induces a bilateral increase of DA turn-over in the non-denervated striata through GLU afferences to the DA terminals.

International audience; BACKGROUND: Protein fold recognition usually relies on a statistical model of each fold; each model is constructed from an ensemble of natural sequences belonging to that fold. A complementary strategy may be to employ sequence ensembles produced by computational protein design. Designed sequences can be more diverse than natural sequences, possibly avoiding some limitations of experimental databases. METHODOLOGY/PRINCIPAL FINDINGS: WE EXPLORE THIS STRATEGY FOR FOUR SCOP FAMILIES: Small Kunitz-type inhibitors (SKIs), Interleukin-8 chemokines, PDZ domains, and large Caspase catalytic subunits, represented by 43 structures. An automated procedure is used to redesign the 43 proteins. We use the experimental backbones as fixed templates in the folded state and a molecular mechanics model to compute the interaction energies between sidechain and backbone groups. Calculations are done with the Proteins@Home volunteer computing platform. A heuristic algorithm is used to scan the sequence and conformational space, yielding 200,000-300,000 sequences per backbone template. The results confirm and generalize our earlier study of SH2 and SH3 domains. The designed sequences ressemble moderately-distant, natural homologues of the initial templates; e.g., the SUPERFAMILY, profile Hidden-Markov Model library recognizes 85% of the low-energy sequences as native-like. Conversely, Position Specific Scoring Matrices derived from the sequences can be used to detect natural homologues within the SwissProt database: 60% of known PDZ domains are detected and around 90% of known SKIs and chemokines. Energy components and inter-residue correlations are analyzed and ways to improve the method are discussed. CONCLUSIONS/SIGNIFICANCE: For some families, designed sequences can be a useful complement to experimental ones for homologue searching. However, improved tools are needed to extract more information from the designed profiles before the method can be of general use.