Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling

<p>Abstract</p> <p>Background</p> <p>Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, <it>in vitro</it>, a residual ability to hydrolyze leukotriene A₄into the pro-inflammatory lipid mediator leukotriene B₄. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA₄hydrolase (LTA₄H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete <it>in vivo </it>functions of the enzyme.</p> <p>Results</p> <p>The rat Ap-B cDNA was expressed in <it>E. coli </it>and the purified recombinant enzyme was characterized. 18 mutants of the H³²⁵EXXHX₁₈E³⁴⁸Zn²⁺-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA₄H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA₄H and suggests that Ap-B is involved in protein-protein interactions.</p> <p>Conclusion</p> <p>Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. <it>E. coli </it>recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA₄H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing.</p

Euclid Near Infrared Spectrometer and Photometer instrument concept and first test results obtained for different breadboards models at the end of phase C

The Euclid mission objective is to understand why the expansion of the Universe is accelerating through by mapping the geometry of the dark Universe by investigating the distance-redshift relationship and tracing the evolution of cosmic structures. The Euclid project is part of ESA's Cosmic Vision program with its launch planned for 2020 (ref [1]). The NISP (Near Infrared Spectrometer and Photometer) is one of the two Euclid instruments and is operating in the near-IR spectral region (900- 2000nm) as a photometer and spectrometer. The instrument is composed of: - a cold (135K) optomechanical subsystem consisting of a Silicon carbide structure, an optical assembly (corrector and camera lens), a filter wheel mechanism, a grism wheel mechanism, a calibration unit and a thermal control system - a detection subsystem based on a mosaic of 16 HAWAII2RG cooled to 95K with their front-end readout electronic cooled to 140K, integrated on a mechanical focal plane structure made with molybdenum and aluminum. The detection subsystem is mounted on the optomechanical subsystem structure - a warm electronic subsystem (280K) composed of a data processing / detector control unit and of an instrument control unit that interfaces with the spacecraft via a 1553 bus for command and control and via Spacewire links for science data This presentation describes the architecture of the instrument at the end of the phase C (Detailed Design Review), the expected performance, the technological key challenges and preliminary test results obtained for different NISP subsystem breadboards and for the NISP Structural and Thermal model (STM)

Etude du contenu genetique d'un virus herpes du lapin cottontail (CTHV)

SIGLEINIST T 74072 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Aminopeptidase B (rôles des résidus de cystéine dans le mécanisme catalytique)

L aminopeptidase B (Ap-B ), Zn2+-métalloexopeptidase de 72 kDa, hydrolyse les acides aminés basiques présents en NH2-terminal de peptides. In vivo, deux substrats ont été identifiés : le glucagon et la cholécystokinine 9. L Ap-B possède également, in vitro, une activité résiduelle capable d hydrolyser le leucotriène A4 en leucotriène B4. Dans l attente de la détermination de la structure 3D de l Ap-B, un modèle moléculaire de cette dernière a été construit par homologie avec la structure cristallographique de la LTA4 hydrolase. L activité catalytique de l Ap-B est sensible aux réactifs des groupements thiol, ce qui suggère une implication directe ou structurale des cystéines dans le mécanisme enzymatique. L ensemble de ces résidus a été étudié par mutagenèse dirigée. 34 mutants ont été exprimés dans E. coli, purifiés et caractérisés sur le plan enzymatique et structural. Un pont disulfure, régulant la reconnaissance des substrats par l Ap-B, a été identifié par spectrométrie de masse.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Vers une meilleure compréhension de l'aminopeptidase B (Ap-B) (obtention d'outils moléculaires et expression du gène au cours du développement de la rétine de rat)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Serine protease inhibitors in ticks: an overview of their role in tick biology and tick-borne pathogen transmission

New tick and tick-borne pathogen control approaches that are both environmentally sustainable and which provide broad protection are urgently needed. Their development, however, will rely on a greater understanding of tick biology, tick-pathogen, and tick-host interactions. The recent advances in new generation technologies to study genomes, transcriptomes, and proteomes has resulted in a plethora of tick biomacromolecular studies. Among these, many enzyme inhibitors have been described, notably serine protease inhibitors (SPIs),whose importance in various tick biological processes is only just beginning to be fully appreciated. Among the multiple active substances secreted during tick feeding, SPIs have been shown to be directly involved in regulation of inflammation, blood clotting, wound healing, vasoconstriction and the modulation of host defense mechanisms. In light of these activities, several SPIs were examined and were experimentally confirmed to facilitate tick pathogen transmission. In addition, to prevent coagulation of the ingested blood meal within the tick alimentary canal, SPIs are also involved in blood digestion and nutrient extraction from the meal. The presence of SPIs in tick hemocytes and their involvement in tick innate immune defenses have also been demonstrated, as well as their implication in hemolymph coagulation and egg development. Considering the involvement of SPIs in multiple crucial aspects of tick-host-pathogen interactions, as well as in various aspects of the tick parasitic lifestyle, these molecules represent highly suitable and attractive targets for the development of effective tick control strategies. Here we review the current knowledge regarding this class of inhibitors in tick biology and tick-borne pathogen transmission, and their potential as targets for future tick control trials

Aminopeptidase B (modélisation moléculaire et étude du site actif par mutagenèse dirigée)

L aminopeptidase B (Ap-B ; EC 3.4.11.6) hydrolyse les acides aminés basiques en N-terminal de peptides et est impliquée dans la maturation d hormones et probablement de neuropeptides selon un mécanisme original récemment mis en évidence. À ce jour, un seul de ses substrats physiologiques a été identifié, le glucagon qui, de par l action successive de la NRD convertase puis de l Ap-B, est transformé en miniglucagon. Ces deux hormones participent à la régulation de l homéostasie du glucose chez les Mammifères. Une autre particularité de l Ap-B est de présenter, in vitro, une activité résiduelle de type e poxyde hydrolase, capable d hydrolyser le leucotriène A4 (LTA4) en leucotriène B4, un médiateur lipidique de l inflammation. A l inverse, son plus proche parent phylogénétique, la Leucotriène A4 Hydrolase (LTA4H ; EC 3.3.2.6) possède, en plus de son activité époxyde hydrolase, une activité aminopeptidase toutefois moins spécifique. Une analyse structurale et fonctionnelle de l Ap-B est nécessaire pour mieux comprendre les mécanismes catalytiques de l enzyme et pour parvenir à la conception d inhibiteurs spécifiques, dont l utilisation devrait nous éclairer sur l ensemble de ses fonctions in vivo. Nous avons mis au point un système d expression et de purification de l Ap-B chez E.Coli avant d entreprendre une analyse fonctionnelle par mutagenèse dirigée d un certain nombre de résidus potentiellement impliqués dans l activité de l enzyme. Ces acides aminés ont été identifiés par alignement des séquences des protéines de la famille M1 à laquelle appartiennent l Ap-B et la LTA4H. Cette étude a, par ailleurs, permis d identifier 3 sous-familles distinctes d aminopeptidases. Cette famille M1 est caractérisée par la présence d un motif de fixation du cation Zn2+ de type HEXXHX18E. Les mutations des résidus de ce motif conduisent à une perte totale de l activité de l Ap-B, confirmant ainsi le rôle essentiel de ces résidus dans le mécanisme catalytique. La famille M1 est également caractérisée par la présence d un second motif consensus (GXMEN). Nous avons également muté l ensemble des résidus de ce second motif. Si les mutations des acides aminés M, E et N conduisent à une perte de l activité, les mutations du résidu G en sérine ou proline donnent naissance à deux protéines actives. Le mutant G298S a des propriétés proches de l enzyme sauvage, tandis que le mutant G298P présente une modification de sa spécificité de substrat (clivage de R, K, P et A), de son profil d inhibition et de son activité en présence d ions Cl-. L analyse de ces mutants par dichroïsme circulaire et spectrométrie de fluorescence montre que les structures globales de ces enzymes ne semblent pas perturbées. Dix autres acides aminés ont également été mutés et leur étude fonctionnelle est en cours. Les similitudes structurales et fonctionnelles de l Ap-B et de la LTA4H dont la structure 3D est connue, nous a permis de construire un modèle moléculaire de la structure de l Ap-B. Ce modèle a été utilisé pour, d une part, essayer de comprendre le rôle du résidu Glycine du motif GXMEN et, d autre part, pour mettre en évidence un certain nombre de différences entre l Ap-B et la LTA4H, en particulier au niveau du site actif et des propriétés potentielles d interactions protéine-protéines de l Ap-B. En parallèle, nous avons mis au point un second système d expression et de purification de l Ap-B à partir d un vecteur baculovirus et de cellules d insectes. Ce système nous permet d obtenir une quantité suffisante d enzyme pour mettre au point les conditions de cristallogenèse de la protéine.Aminopeptidase B (Ap-B ; EC 3.4.11.6) cleaves basic residues at the N-terminus of peptides and participates in hormone and neuropeptide processing through an original mechanism recently described. The only known physiological substrate of Ap-B is the glucagon, which is processed into miniglucagon by NRD convertase and Ap-B. In mammals, these hormones are involved in the regulation of glucose homeostasis. One second characteristic of Ap-B is that the enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4. Inversely, LTA4 hydrolase (LTA4H ; EC 3.3.2.6), the closest homologous protein of Ap-B, possesses, besides an epoxyde hydrolase activity, an aminopeptidase activity of broader specificity. Both proteins belong to the M1 family of Zn2+-aminopeptidases. A structure-function analysis is needed for a detailed understanding of the enzymatic mechanisms of Ap-B and to aid in the design of inhibitors, which could be used to determine its whole in vivo functions. In order to carry out a functional analysis by site-directed mutagenesis, we developed an expression system and a purification procedure of Ap-B using E. Coli. Amino acid residues potentially involved in the aminopeptidase activity are identified using alignments of primary structures of proteins from the M1 family. This study allowed us to identify 3 distinct M1 sub-families. This M1 family is characterized by the presence of a HEXXHXn=18E Zn2+- binding motif. Mutations of these residues lead to a total loss of activity and confirm their essential roles in catalysis. The major part of the M1 family members (Ap-B, LTA4H, ) constitutes the main sub-family and possesses a second consensus motif (GXMEN). Mutations of the M, E or N residues also lead to a total loss of aminopeptidase activity. Surprisingly, replacement of the conserved glycine into a serine or a proline created two new active enzymes. The G298S mutant exhibits properties similar to the wild type enzyme, whereas the G298P mutant shows a modification of its substrate specificity (recognizing R, K, P and A), its inhibition profile and its behavior towards Cl-. Fluorescence spectroscopy and circular dichroïsm analyses did not show any fundamental modification in the mutant structures. Ten other residues were mutated in the Ap-B primary structure and their functional studies are in progress. The sequence and catalytic similarities shared between Ap-B and LTA4H and the determination of the X-ray structure of LTA4H led us to build a 3D structural model of Ap-B. This model was used, on one hand, to better understand the enzymatic role of the glycine residue of the GXMEN motif and, on the other hand, to highlight functional differences between Ap-B and LTA4H, particularly at the level of their active site and of their proteinprotein interaction properties. In parallel, we also developed a second system of expression and purification of Ap-B using baculovirus and insect cells. This system allows us to purify a sufficient amount of protein for crystallogenesis assays.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Suivre le chemin des nouveaux immigrés dans la ville : le cas de Châtelet de 1866 à 1900

If mobility in space is a démographie phenomenon still little known for the XIXth century, this is all the more true as concerns migration within the same town. Yet the phenomenon is important is important both in numbers and so as to be able to account for spatial segregation and social mobility within urban populations. In this paper, we have followed the movements of a thousand new immigrants coming from the country between 1867 and 1872 in the town of Châtelent and the industrial area of Charleroi. First, where do the immigrants go when they arrive in the town ? By carving up the town into districts, we have noticed that two types of neighbourhoods concentrate the most neweomers : intra muros neighbourhoods with a very dense and mobile population and extra muros neighbourhoods near the coal extraction sites. In all cases, the historical centre of the town, circled off by ancient fortifications, is abandoned. Furthermore, it appears that about half of the immigrants remains less than two years in their first lodgings in Châtelet, while one out of five stays for ten or more years. In following the migrations of these immigrants over three decades until the 1900 census, it can be ascertained that some two thirds left the town- the most often for another industrial township. Among the others remaining in the town, one out of five stayed in the same neighbourhood, even on the same street, two out of five changed neighbourhoods and the two others died. Of the 604 interior migrations of the immigrants over the three decades under observation, 23% took place on the same street, 21% were to another street of the same neighbourhood and 56% to another neighbourhood altogether. In all cases, the "peripheral" districts near the expanding coal-mines draw the most immigrants, spurred on by certain "informed" investors who hastily build row houses to absorb the town's demographic expansion.Si la mobilité spatiale reste un phénomène démographique mal connu au XIXe siècle, ceci est vrai a fortiori pour la migration à l'intérieur même de la ville. Or le phénomène est important aussi bien en nombre que pour rendre compte de la ségrégation spatiale et de la mobilité sociale au sein des populations urbaines. Dans cette contribution, nous avons suivi le cheminement d'un millier de nouveaux immigrés en provenance de la campagne entre 1867 et 1872 et ce, dans la ville de Châtelet dans le bassin industriel de Charleroi. Tout d'abord, où se dirigent les immigrés lorsqu'ils arrivent dans la ville ? Sur la base d'un découpage de la ville en quartiers, nous avons constaté que deux types de quartiers sont privilégiés : des quartiers intra muros à haute densité de population et forte mobilité et des quartiers extra muros aux abords des sièges d'exploitation de la houille. Dans tous les cas, le centre historique de cette ville ancienne cernée de fortifications est délaissé. Par ailleurs, on estime que près de la moitié des immigrés reste moins de deux années dans leur premier logement à Châtelet, alors qu'un sur cinq y demeure dix ans ou plus. En suivant le cheminement migratoire de ces immigrés pendant trois décennies jusqu'au recensement de 1900, on vérifie que près de deux tiers ont quitté la ville en émigrant le plus souvent vers une autre commune industrielle. Parmi les autres qui sont restés en ville, un sur cinq est resté dans le même quartier, voire la même rue, deux sur cinq ont changé de quartier et les deux derniers sont décédés. Au nombre des 604 migrations internes effectuées par ces immigrés pendant les trois décennies d'observation, 23 % se font dans la même rue, 21 % vers une autre rue du même quartier et 56 % vers un autre quartier. Dans tous les cas, ce sont les quartiers « périphériques » aux abords des charbonnages en forte expansion qui attirent le plus, aidés en cela par quelques investisseurs « avertis » construisant à la hâte des rangées de logements pour faire face à l'expansion démographique de la ville.Eggerickx Thierry, Foulon Michel, Poulain Michel. Suivre le chemin des nouveaux immigrés dans la ville : le cas de Châtelet de 1867 à 1900. In: Annales de démographie historique, 1999-1. Faire son chemin dans la ville. La mobilité intra-urbaine. pp. 81-105

Serine Protease Inhibitors in Ticks: An Overview of Their Role in Tick Biology and Tick-Borne Pathogen Transmission

International audienceNew tick and tick-borne pathogen control approaches that are both environmentally sustainable and which provide broad protection are urgently needed. Their development, however, will rely on a greater understanding of tick biology, tick-pathogen, and tick-host interactions. The recent advances in new generation technologies to study genomes, transcriptomes, and proteomes has resulted in a plethora of tick biomacromolecular studies. Among these, many enzyme inhibitors have been described, notably serine protease inhibitors (SPIs), whose importance in various tick biological processes is only just beginning to be fully appreciated. Among the multiple active substances secreted during tick feeding, SPIs have been shown to be directly involved in regulation of inflammation, blood clotting, wound healing, vasoconstriction and the modulation of host defense mechanisms. In light of these activities, several SPIs were examined and were experimentally confirmed to facilitate tick pathogen transmission. In addition, to prevent coagulation of the ingested blood meal within the tick alimentary canal, SPIs are also involved in blood digestion and nutrient extraction from the meal. The presence of SPIs in tick hemocytes and their involvement in tick innate immune defenses have also been demonstrated, as well as their implication in hemolymph coagulation and egg development. Considering the involvement of SPIs in multiple crucial aspects of tick-host-pathogen interactions, as well as in various aspects of the tick parasitic lifestyle, these molecules represent highly suitable and attractive targets for the development of effective tick control strategies. Here we review the current knowledge regarding this class of inhibitors in tick biology and tick-borne pathogen transmission, and their potential as targets for future tick control trials