In silico prediction of human carboxylesterase-1 (hCES1) metabolism combining docking analyses and MD simulations.

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted in developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas little has been done to predict the hydrolytic activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES1. The study involves both docking analyses of known substrates to develop predictive models, and molecular dynamics (MD) simulations to reveal the in situ behavior of substrates and products, with particular attention being paid to the influence of their ionization state. The results emphasize some crucial properties of the hCES1 catalytic cavity, confirming that as a trend with several exceptions, hCES1 prefers substrates with relatively smaller and somewhat polar alkyl/aryl groups and larger hydrophobic acyl moieties. The docking results underline the usefulness of the hydrophobic interaction score proposed here, which allows a robust prediction of hCES1 catalysis, while the MD simulations show the different behavior of substrates and products in the enzyme cavity, suggesting in particular that basic substrates interact with the enzyme in their unprotonated form.

Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL.

Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.

trEST, trGEN and Hits: access to databases of predicted protein sequences.

High throughput genome (HTG) and expressed sequence tag (EST) sequences are currently the most abundant nucleotide sequence classes in the public database. The large volume, high degree of fragmentation and lack of gene structure annotations prevent efficient and effective searches of HTG and EST data for protein sequence homologies by standard search methods. Here, we briefly describe three newly developed resources that should make discovery of interesting genes in these sequence classes easier in the future, especially to biologists not having access to a powerful local bioinformatics environment. trEST and trGEN are regularly regenerated databases of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Hits is a web-based data retrieval and analysis system providing access to precomputed matches between protein sequences (including sequences from trEST and trGEN) and patterns and profiles from Prosite and Pfam. The three resources can be accessed via the Hits home page (http://hits. isb-sib.ch).

Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating Protein, PyAG1

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.

A theoretical model of the tridimensional structure of Bacillus thuringiensis subsp. medellin Cry 11Bb toxin deduced by homology modelling

Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp. medellin during its stationary phase; this ¶-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control. Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin. The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins. In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation. This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

An mRNA region of the canine distemper virus fusion protein gene lacking AUG codons can promote protein expression.

Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein.

Cell separation, or abscission, is a highly specialized process in plants that facilitates remodeling of their architecture and reproductive success. Because few genes are known to be essential for organ abscission, we conducted a screen for mutations that alter floral organ shedding in Arabidopsis. Nine recessive mutations that block shedding were found to disrupt the function of an ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) we have named NEVERSHED (NEV). As predicted by its homology to the yeast Age2 ARF-GAP and transcriptional profile, NEV influences other aspects of plant development, including fruit growth. Co-localization experiments carried out with NEV-specific antiserum and a set of plant endomembrane markers revealed that NEV localizes to the trans-Golgi network and endosomes in Arabidopsis root epidermal cells. Interestingly, transmission electron micrographs of abscission zone regions from wild-type and nev flowers reveal defects in the structure of the Golgi apparatus and extensive accumulation of vesicles adjacent to the cell walls. Our results suggest that NEV ARF-GAP activity at the trans-Golgi network and distinct endosomal compartments is required for the proper trafficking of cargo molecules required for cell separation.

SwissDock, a protein-small molecule docking web service based on EADock DSS.

Most life science processes involve, at the atomic scale, recognition between two molecules. The prediction of such interactions at the molecular level, by so-called docking software, is a non-trivial task. Docking programs have a wide range of applications ranging from protein engineering to drug design. This article presents SwissDock, a web server dedicated to the docking of small molecules on target proteins. It is based on the EADock DSS engine, combined with setup scripts for curating common problems and for preparing both the target protein and the ligand input files. An efficient Ajax/HTML interface was designed and implemented so that scientists can easily submit dockings and retrieve the predicted complexes. For automated docking tasks, a programmatic SOAP interface has been set up and template programs can be downloaded in Perl, Python and PHP. The web site also provides an access to a database of manually curated complexes, based on the Ligand Protein Database. A wiki and a forum are available to the community to promote interactions between users. The SwissDock web site is available online at http://www.swissdock.ch. We believe it constitutes a step toward generalizing the use of docking tools beyond the traditional molecular modeling community.

Trypanosoma cruzi ribosomal protein S4: characterization of its coding locus, analysis of transcripts, and antigenicity of the protein

Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project database reveals that our two-copy allele corresponds to a variant haplotype. However, the deduced aminoacid sequence of all the gene copies is identical. The rpS4 transcripts processing sites were determined by comparison of genomic sequences with published cDNA data. The obtained sequence data demonstrates that rpS4 genes are expressed in epimastigotes, amastigotes, and trypomastigotes. A recombinant version of rpS4 was found to be an antigenic: it was recognized by 62.5% of the individuals with positive serology for T. cruzi and by 93.3% of patients with proven chronic chagasic disease.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory.

Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates its genome in a membrane-associated replication complex. Nonstructural protein 4B (NS4B) induces the specific membrane alteration, designated as membranous web (MW), that harbours this complex. HCV NS4B is an integral membrane protein predicted to comprise four transmembrane segments in its central part. The N-terminal part comprises two amphipathic alpha-helices of which the second has the potential to traverse the membrane bilayer, likely upon oligomerisation. The C-terminal part comprises a predicted highly conserved alpha-helix, a membrane-associated amphipathic alpha-helix and two reported palmitoylation sites. NS4B interacts with other viral nonstructural proteins and has been reported to bind viral RNA. In addition, it was found to harbour an NTPase activity. Finally, NS4B has recently been found to have a role in viral assembly. Much work needs to be done with respect to further dissecting these multiple functions as well as providing a refined membrane topology and complete structure of NS4B. Progress in this direction should yield important insights into the functional architecture of the HCV replication complex and may reveal new opportunities for antiviral intervention against a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.

Molecular dissection of Arabidopsis thaliana endodermis development, structure and function

In vascular plants, the endodermis establishes a protective diffusion barrier surrounding the vasculature preventing the passive, uncontrolled entry of nutrients absorbed by the plant. It does so by means of a differentiation feature, the "Casparian Strip" (CS), a highly localized cell wall impregnation made of lignin, which seals the extracellular space. Although the existence of this differentiation feature has been intensively described, the mechanisms establishing this hallmark remain obscure. In this work I report, the developmental sequence of events that leads to a differentiated endodermis, in the plant model Arabidopsis thaliana. In addition, my descriptive approach gave important insights as to how these cells define membrane domains involved in the directional transport of nutrients. I also participated in characterizing a new transmembrane protein family, the CASPs, localized to the membrane domain underlying the CS, which we accordingly named the Casparian Strip membrane Domain (CSD). Our molecular analysis indicates that these proteins drive CS establishment. To identify more molecular factors of CS establishment, I performed a forward genetic screen. This screen led to the identification of 11 endodermis permissive mutants, which we named schengen (sgn) mutants. The causative mutations have been mapped to 5 independent loci: SGN1 to SGN5. SGN1 and SGN3 encode Receptor Like Kinases involved in the correct establishment of the CSD. A lack of those kinases leads to an incomplete CSD, which gives rise to interrupted CS barriers. Interestingly, SGN1 seems to also regulate CSD positioning to the middle of endodermal transversal walls. SGN4 encodes an NADPH oxidase involved in lignin polymerization essential for CS formation. The sgn5 mutant induces extra divisions of cortical cells strongly affecting the cell identity, but also leading to incorrect differentiation. A thorough characterization of the sgn2 mutant will follow elsewhere, yet preliminary results indicate that SGN2 encodes an Acyl-CoA N-acyltransferase. . In summary, with my work I have contributed a first set of molecular players of Casparian strip formation and initiated their characterization. Eventually, this might lead to an understanding of the molecular mechanisms of CS establishment in A.thaliana . This in turn will hopefully help to better understand nutrient uptake in higher plants and their response to environmental stresses. - Au sein des plantes vasculaires, l'endoderme représente un tissu protecteur mettant en place une barrière imperméable, empêchant n'importe quel élément de rejoindre les tissus conducteurs par simple diffusion. Cette barrière, appelée « Cadre de Caspary », correspond à une lignification de la paroi de l'endoderme et donne lieu à un cloisonnement de l'espace intercellulaire. Bien que cet élément de différenciation soit décrit en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements aboutissant à l'établissement du cadre de Caspary chez la plante modèle Arabidopsis thaliana. De plus, ce travail apporte de nouvelles connaissances expliquant comment ces cellules définissent des domaines membranaires importants pour le transport des nutriments. Nous décrivons une nouvelle famille de protéines membranaires, les CASPs (« CAparian Strip membrane domain Proteins »), localisées dans un domaine membranaire longeant le cadre de Caspary : le domaine de Caspary (CSD). L'analyse moléculaire des CASPs indique qu'elles dirigent la formation du cadre de Caspary. Par ailleurs, une approche génétique directe nous a permis d'identifier 11 mutants ayant un endoderme perméable. Nous avons nommé ces mutants Schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) qui participent à la délimitation du CSD. L'absence de ces kinases aboutit à un domaine CSD incomplet, se traduisant par un cadre de Caspary discontinu. De plus, SGN1 semble réguler le positionnement du CSD au milieu de la paroi transversale de l'endoderme. SGN4 produit une enzyme de type NADPH oxydase impliquée dans la polymérisation du cadre de Caspary. Dans le mutant sgn5, on observe une division anormale des cellules du cortex créant ainsi une nouvelle couche cellulaire incapable d'achever sa différenciation en endoderme. Quant à la mutation sgn2, bien que nous pensons qu'elle affecte une Acyl-CoA N-acyltransferase, sa caractérisation ne sera réalisée que prochainement. Au final, ce travail procure de nouveaux éléments sur l'établissement du cadre de Caspary qui pourraient être importants afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles. - De par leur immobilité, les plantes terrestres n'ont pas d'autre choix que de puiser leurs ressources dans leur environnement direct. La plante extrait du sol les nutriments qui lui sont nécessaires et les redistribue grâce à des tissus conducteurs. Afin de ne pas s'intoxiquer, il est donc essentiel de pouvoir sélectionner les éléments entrant dans la racine. Etonnement, ce n'est pas la surface des racines qui permet ce contrôle mais un tissu interne appelé endoderme. Ce dernier forme une barrière imperméable qui entoure chaque cellule et crée une jointure permettant de bloquer le passage des éléments entre les cellules. Cette structure, appelée « cadre de Caspary », oblige les éléments à entrer dans les cellules de l'endoderme et à être ainsi sélectionnés. Bien que cette structure soit décrite en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements qui aboutit à la formation du cadre de Caspary chez la plante modèle Arabidopsis thaliana. Ce travail apporte également de nouvelles connaissances expliquant comment ces cellules définissent, organisent et dirigent le transport des nutriments. Nous décrivons comment certains éléments de la cellule, les protéines CASPs (CAsparian Strip membrane domain Proteins), sont organisées un domaine particulier des membranes afin de créer une plateforme de construction longeant le cadre de Caspary : le domaine de Caspary (CSD). Afin de déterminer ce qu'il se passerait si une plante ne possédait pas de cadre de Caspary, nous avons réalisé une mutagénèse, ou approche génétique directe, et identifié 11 mutants (individu ayant un gène défectueux conduisant à la perte d'une fonction) ayant un endoderme perméable. Nous avons nommé ces mutants schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. Les gènes SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) servant à l'établissement de la plateforme de construction. L'absence de ces kinases aboutit à une base incomplète, se traduisant par un cadre de Caspary discontinu. Qui plus est, la kinase SGN1 semble réguler le positionnement de la plateforme au milieu de l'endoderme. Le gène SGN4 est par contre, impliqué dans la construction à proprement dite du cadre de Caspary. Dans le mutant sgn5, on observe une nouvelle couche de cellules ressemblant à de l'endoderme mais incapable de former correctement une barrière identique au cadre de Caspary. Quant au dernier mutant, sgn2, bien que cette étude fournisse des indices permettant de comprendre pourquoi le mutant sgn2 est défectueux, nous n'expliquerons ce cas que prochainement. En résumé, ce travail procure de nouvelles connaissances sur l'établissement du cadre de Caspary qui pourraient être importantes afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles.

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.