Caractérisation structurale et thermodynamique de la reconnaissance du substrat par le ribozyme VS de Neurospora

Les interactions ARN/ARN de type kissing-loop sont des éléments de structure tertiaire qui jouent souvent des rôles clés chez les ARN, tant au niveau fonctionnel que structural. En effet, ce type d’interaction est crucial pour plusieurs processus dépendant des ARN, notamment pour l’initiation de la traduction, la reconnaissance des ARN antisens et la dimérisation de génome rétroviral. Les interactions kissing-loop sont également importantes pour le repliement des ARN, puisqu’elles permettent d’établir des contacts à longue distance entre différents ARN ou encore entre les domaines éloignés d’un même ARN. Ce type d’interaction stabilise aussi les structures complexes des ARN fonctionnels tels que les ARNt, les riborégulateurs et les ribozymes. Comme d’autres ARN fonctionnels, le ribozyme VS de Neurospora contient une interaction kissing-loop importante. Celle-ci est impliquée dans la reconnaissance du substrat et se forme entre la tige-boucle I (stem-loop I, SLI) du substrat et la tige-boucle V (stem-loop V, SLV) du domaine catalytique. Des études biochimiques ont démontré que l’interaction kissing-loop I/V, dépendante du magnésium, implique trois paires de bases Watson-Crick (W-C). De plus, cette interaction est associée à un réarrangement de la structure du substrat, le faisant passer d’une conformation inactive dite unshifted à une conformation active dite shifted. Les travaux présentés dans cette thèse consistent en une caractérisation structurale et thermodynamique de l’interaction kissing-loop I/V du ribozyme VS, laquelle est formée de fragments d’ARN représentant les tige-boucles I et V dérivées du ribozyme VS (SLI et SLV). Cette caractérisation a été réalisée principalement par spectroscopie de résonance magnétique nucléaire (RMN) et par titrage calorimétrique isotherme (isothermal titration calorimetry, ITC) en utilisant différents complexes SLI/SLV dans lesquels l’ARN SLV est commun à tous les complexes, alors que différentes variations de l’ARN SLI ont été utilisées, soit en conformation shiftable ou preshifted. Les données d’ITC ont permis de démontrer qu’en présence d’une concentration saturante de magnésium, l’affinité d’un substrat SLI preshifted pour SLV est extrêmement élevée, rendant cette interaction plus stable que ce qui est prédit pour un duplexe d’ARN équivalent. De plus, l’étude effectuée par ITC montre que des ARN SLI preshifted présentent une meilleure affinité pour SLV que des ARN SLI shiftable, ce qui a permis de calculer le coût énergétique associé au réarrangement de structure du substrat. En plus de confirmer la formation des trois paires de bases W-C prédites à la jonction I/V, les études de RMN ont permis d’obtenir une preuve structurale directe du réarrangement structural des substrats SLI shiftable en présence de magnésium et de l’ARN SLV. La structure RMN d’un complexe SLI/SLV de grande affinité démontre que les boucles terminales de SLI et SLV forment chacune un motif U-turn, ce qui facilite l’appariement W-C intermoléculaire. Plusieurs autres interactions ont été définies à l’interface I/V, notamment des triplets de bases, ainsi que des empilements de bases. Ces interactions contribuent d’ailleurs à la création d’une structure présentant un empilement continu, c’est-à-dire qui se propage du centre de l’interaction jusqu’aux bouts des tiges de SLI et SLV. Ces études de RMN permettent donc de mieux comprendre la stabilité exceptionnelle de l’interaction kissing-loop I/V au niveau structural et mènent à l’élaboration d’un modèle cinétique de l’activation du substrat par le ribozyme VS. En considérant l’ensemble des données d’ITC et de RMN, l’étonnante stabilité de l’interaction I/V s’explique probablement par une combinaison de facteurs, dont les motifs U-turn, la présence d’un nucléotide exclu de la boucle de SLV (U700), la liaison de cations magnésium et l’empilement de bases continu à la jonction I/V.

Études structurales par résonance magnétique nucléaire du ribozyme VS de Neurospora

Le ribozyme VS de Neurospora catalyse des réactions de clivage et de ligation d’un lien phosphodiester spécifique essentielles à son cycle de réplication. Il est formé de six régions hélicales (I à VI), qui se divisent en deux domaines, soit le substrat (SLI) et le domaine catalytique (tiges II à VI). Ce dernier comprend deux jonctions à trois voies qui permettent de reconnaître le substrat en tige-boucle de façon spécifique. Ce mode de reconnaissance unique pourrait être exploité pour cibler des ARN repliés pour diverses applications. Bien que le ribozyme VS ait été caractérisé biochimiquement de façon exhaustive, aucune structure à haute résolution du ribozyme complet n’a encore été publiée, ce qui limite la compréhension des mécanismes inhérents à son fonctionnement. Précédemment, une approche de divide-and-conquer a été initiée afin d’étudier la structure des sous-domaines importants du ribozyme VS par spectroscopie de résonance magnétique nucléaire (RMN) mais doit être complétée. Dans le cadre de cette thèse, les structures de la boucle A730 et des jonctions III-IV-V et II-III-VI ont été déterminées par spectroscopie RMN hétéronucléaire. De plus, une approche de spectroscopie RMN a été développée pour la localisation des ions divalents, tandis que diverses approches de marquage isotopique ont été implémentées pour l’étude d’ARN de plus grandes tailles. Les structures RMN de la boucle A730 et des deux jonctions à trois voies révèlent que ces sous-domaines sont bien définis, qu’ils sont formés de plusieurs éléments structuraux récurrents (U-turn, S-turn, triplets de bases et empilement coaxial) et qu’ils contiennent plusieurs sites de liaison de métaux. En outre, un modèle du site actif du ribozyme VS a été construit sur la base des similarités identifiées entre les sites actifs des ribozymes VS et hairpin. Dans l’ensemble, ces études contribuent de façon significative à la compréhension de l’architecture globale du ribozyme VS. De plus, elles permettront de construire un modèle à haute résolution du ribozyme VS tout en favorisant de futures études d’ingénierie.

Sequence similarity analysis of Escherichia coli proteins: functional and evolutionary implications.

A computer analysis of 2328 protein sequences comprising about 60% of the Escherichia coli gene products was performed using methods for database screening with individual sequences and alignment blocks. A high fraction of E. coli proteins--86%--shows significant sequence similarity to other proteins in current databases; about 70% show conservation at least at the level of distantly related bacteria, and about 40% contain ancient conserved regions (ACRs) shared with eukaryotic or Archaeal proteins. For > 90% of the E. coli proteins, either functional information or sequence similarity, or both, are available. Forty-six percent of the E. coli proteins belong to 299 clusters of paralogs (intraspecies homologs) defined on the basis of pairwise similarity. Another 10% could be included in 70 superclusters using motif detection methods. The majority of the clusters contain only two to four members. In contrast, nearly 25% of all E. coli proteins belong to the four largest superclusters--namely, permeases, ATPases and GTPases with the conserved "Walker-type" motif, helix-turn-helix regulatory proteins, and NAD(FAD)-binding proteins. We conclude that bacterial protein sequences generally are highly conserved in evolution, with about 50% of all ACR-containing protein families represented among the E. coli gene products. With the current sequence databases and methods of their screening, computer analysis yields useful information on the functions and evolutionary relationships of the vast majority of genes in a bacterial genome. Sequence similarity with E. coli proteins allows the prediction of functions for a number of important eukaryotic genes, including several whose products are implicated in human diseases.

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain.

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

Le motif d’empaquetage le long du sillon: une nouvelle entité structurale récurrente dans les ARN ribosomiques

La plupart des molécules d’ARN doivent se replier en structure tertiaire complexe afin d’accomplir leurs fonctions biologiques. Cependant, les déterminants d’une chaîne de polynucléotides qui sont nécessaires à son repliement et à ses interactions avec d’autres éléments sont essentiellement inconnus. L’établissement des relations structure-fonction dans les grandes molécules d’ARN passe inévitablement par l’analyse de chaque élément de leur structure de façon individuelle et en contexte avec d’autres éléments. À l’image d’une construction d’immeuble, une structure d’ARN est composée d’unités répétitives assemblées de façon spécifique. Les motifs récurrents d’ARN sont des arrangements de nucléotides retrouvés à différents endroits d’une structure tertiaire et possèdent des conformations identiques ou très similaires. Ainsi, une des étapes nécessaires à la compréhension de la structure et de la fonction des molécules d’ARN consiste à identifier de façon systématique les motifs récurrents et d’en effectuer une analyse comparative afin d’établir la séquence consensus. L’analyse de tous les cas d’empaquetage de doubles hélices dans la structure du ribosome a permis l’identification d’un nouvel arrangement nommé motif d’empaquetage le long du sillon (AGPM) (along-groove packing motif). Ce motif est retrouvé à 14 endroits dans la structure du ribosome de même qu’entre l’ARN ribosomique 23S et les molécules d’ARN de transfert liées aux sites ribosomaux P et E. Le motif se forme par l’empaquetage de deux doubles hélices via leur sillon mineur. Le squelette sucre-phosphate d’une hélice voyage le long du sillon mineur de l’autre hélice et vice versa. Dans chacune des hélices, la région de contact comprend quatre paires de bases. L’empaquetage le plus serré est retrouvé au centre de l’arrangement où l’on retrouve souvent une paire de bases GU dans une hélice interagissant avec une paire de bases Watson-Crick (WC) dans l’autre hélice. Même si la présence des paires de bases centrales GU versus WC au centre du motif augmente sa stabilité, d’autres alternatives existent pour différents représentants du motif. L’analyse comparative de trois librairies combinatoires de gènes d’AGPM, où les paires de bases centrales ont été variées de manière complètement aléatoire, a montré que le contexte structural influence l’étendue de la variabilité des séquences de nucléotides formant les paires de bases centrales. Le fait que l’identité des paires de bases centrales puisse varier suggérait la présence d’autres déterminants responsables au maintien de l’intégrité du motif. L’analyse de tous les contacts entre les hélices a révélé qu’en dehors du centre du motif, les interactions entre les squelettes sucre-phosphate s’effectuent via trois contacts ribose-ribose. Pour chacun de ces contacts, les riboses des nucléotides qui interagissent ensemble doivent adopter des positions particulières afin d’éviter qu’ils entrent en collision. Nous montrons que la position de ces riboses est modulée par des conformations spécifiques des paires de bases auxquelles ils appartiennent. Finalement, un autre motif récurrent identifié à l’intérieur même de la structure de trois cas d’AGPM a été nommé « adenosine-wedge ». Son analyse a révélé que ce dernier est lui-même composé d’un autre arrangement, nommé motif triangle-NAG (NAG-triangle). Nous montrons que le motif « adenosine-wedge » représente un arrangement complexe d’ARN composé de quatre éléments répétitifs, c’est-à-dire des motifs AGPM, « hook-turn », « A-minor » et triangle-NAG. Ceci illustre clairement l’arrangement hiérarchique des structures d’ARN qui peut aussi être observé pour d’autres motifs d’ARN. D’un point de vue plus global, mes résultats enrichissent notre compréhension générale du rôle des différents types d’interactions tertiaires dans la formation des molécules d’ARN complexes.

Light-activated rhodopsin induces structural binding motif in G protein α subunit

A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these receptors recognize and promote nucleotide exchange on G protein α subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) α subunit C-terminal undecapeptide Gtα(340–350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gtα(340–350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an αL type. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325–346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor–G protein interface is demonstrated.

The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

Treble clef finger—a functionally diverse zinc-binding structural motif

Detection of similarity is particularly difficult for small proteins and thus connections between many of them remain unnoticed. Structure and sequence analysis of several metal-binding proteins reveals unexpected similarities in structural domains classified as different protein folds in SCOP and suggests unification of seven folds that belong to two protein classes. The common motif, termed treble clef finger in this study, forms the protein structural core and is 25–45 residues long. The treble clef motif is assembled around the central zinc ion and consists of a zinc knuckle, loop, β-hairpin and an α-helix. The knuckle and the first turn of the helix each incorporate two zinc ligands. Treble clef domains constitute the core of many structures such as ribosomal proteins L24E and S14, RING fingers, protein kinase cysteine-rich domains, nuclear receptor-like fingers, LIM domains, phosphatidylinositol-3-phosphate-binding domains and His-Me finger endonucleases. The treble clef finger is a uniquely versatile motif adaptable for various functions. This small domain with a 25 residue structural core can accommodate eight different metal-binding sites and can have many types of functions from binding of nucleic acids, proteins and small molecules, to catalysis of phosphodiester bond hydrolysis. Treble clef motifs are frequently incorporated in larger structures or occur in doublets. Present analysis suggests that the treble clef motif defines a distinct structural fold found in proteins with diverse functional properties and forms one of the major zinc finger groups.

COL18A1 is highly expressed during human adipocyte differentiation and the SNP c.1136C > T in its "frizzled" motif is associated with obesity in diabetes type 2 patients

Collagen XVIII can generate two fragments, NC11-728 containing a frizzled motif which possibly acts in Wnt signaling and Endostatin, which is cleaved from the NC1 and is a potent inhibitor of angiogenesis. Collagen XVIII and Wnt signaling have recently been associated with adipogenic differentiation and obesity in some animal models, but not in humans. In the present report, we have shown that COL18A1 expression increases during human adipogenic differentiation. We also tested if polymorphisms in the Frizzled (c.1136C>T; Thr379Met) and Endostatin (c.4349G>A; Asp1437Asn) regions contribute towards susceptibility to obesity in patients with type 2 diabetes (113 obese, BMI =30; 232 non-obese, BMI < 30) of European ancestry. No evidence of association was observed between the allele c.4349G>A and obesity, but we observed a significantly higher frequency of homozygotes c.1136TT in obese (19.5%) than in non-obese individuals (10.9%) [P = 0.02; OR = 2.0 (95%CI: 1.07-3.73)], suggesting that the allele c.1136T is associated to obesity in a recessive model. This genotype, after controlling for cholesterol, LDL cholesterol, and triglycerides, was independently associated with obesity (P = 0.048), and increases the chance of obesity in 2.8 times. Therefore, our data suggest the involvement of collagen XVIII in human adipogenesis and susceptibility to obesity.

The Last Missionary to Leave the Temple Should Turn Off the Light Sociological Remarks on the Decline of Japanese ""Immigrant"" Buddhism in Brazil

Empirical data indicate that the so-called ""Buddhism of yellow color"" that is predominantly associated with Japanese ""immigrant"" Buddhism, is constantly in decline in terms of ""explicit"" adherents. After some methodological observations, this article gives an overview of the relevant statistical data. The last part discusses possible reasons for these negative dynamics, referring to causes within Buddhist institutions, the ethnic community, and at the level of the individual.

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Distribution, expression, and motif variability of ankyrin domain genes in Wolbachia pipientis

The endosymbiotic bacterium Wolbachia pipientis infects a wide range of arthropods, in which it induces a variety of reproductive phenotypes, including cytoplasmic incompatibility (CI), parthenogenesis, male killing, and reversal of genetic sex determination. The recent sequencing and annotation of the first Wolbachia genome revealed an unusually high number of genes encoding ankyrin domain (ANK) repeats. These ANK genes are likely to be important in mediating the Wolbachia-host interaction. In this work we determined the distribution and expression of the different ANK genes found in the sequenced Wolbachia wMel genome in nine Wolbachia strains that induce different phenotypic effects in their hosts. A comparison of the ANK genes of wMel and the non-CI-inducing wAu Wolbachia strain revealed significant differences between the strains. This was reflected in sequence variability in shared genes that could result in alterations in the encoded proteins, such as motif deletions, amino acid insertions, and in some cases disruptions due to insertion of transposable elements and premature stops. In addition, one wMel ANK gene, which is part of an operon, was absent in the wAu genome. These variations are likely to affect the affinity, function, and cellular location of the predicted proteins encoded by these genes.

The c-Myb oncoprotein

The c-myb gene is the cellular homologue of the v-myb oncogenes carried by the avian leukaemia viruses AMV and E26. It encodes a transcription factor (c-Myb), as does each of the viral oncogenes, which recognises the core DNA sequence C/T-A-A-C-G/T-G via a repeated helix-turn-helix-like motif. c-myb is expressed in immature haemopoietic cells, as well as immature cells of the gastro-intestinal epithelium and is down-regulated with differentiation. Enforced expression of activated or even normal forms of Myb can transform haemopoietic cells, most often of the myeloid lineage, in vitro and in vivo. Although many genes have been identified which are likely to be regulated by c-Myb, the critical target genes involved in Myb's transforming activity are not known. Together with data showing increased c-myb expression in colonic tumours, these observations raise the possibility that c-myb may play a role in human malignant disease. (C) 1998 Elsevier Science Ltd. All rights reserved.

Turn up the HEAT

The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats, The structural properties of this solenoid protein explain many functional characteristics and account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.