No description available.

Les protéines de raisin thaumatin-like et chitinase sont d’un intérêt majeur, tant pour la recherche viticole de par leur implication dans les mécanismes de défense de la vigne, que pour la recherche en oenologie en raison de leur présence en quantité majoritaire dans le pool protéique d’un jus de raisin. La mise au point d’un protocole de quantification par chromatographie liquide et dosage immuno-enzymatique utilisant des gammes de protéines purifiées à partir de jus de raisin a permis de caractériser la diminution de la concentration de ces deux protéines au cours du procédé de vinification. Les propriétés tensioactives des deux protéines thaumatin-like et chitinase purifiées ont été évaluées, révélant que ni l’une ni l’autre ne pouvait à elle seule expliquer la formation et la stabilisation des bulles et de la mousse d’un Champagne. Une étude structurale de la protéine thaumatin-like VVTL1 a ensuite été menée dans l’optique de mieux comprendre les mécanismes chimiques, biologiques et physiques dans lesquels elle peut être impliquée. Une structure de VVTL1 a été modélisée par homologie et l’analyse de ses modes normaux a permis de révéler un mécanisme de type mâchoire autour d’une cavité acide, site potentiel de l’activité enzymatique de la protéine. Un feuillet beta en épingle à cheveux isolé dans la structure s’est révélé être très conservé et absolument spécifique à la superfamille des protéines thaumatin-like, ouvrant peut-être la voie vers l’élucidation complète du rôle biologique de ces protéines. Dans une seconde approche, la détermination de la structure d’un peptide sélectionné dans la séquence de VVTL1 par modélisation sous contraintes RMN a posé les bases d’une étude modèle de l’adsorption des protéines à la surface de la bentonite, argile utilisée pour la clarification des vins. / Grape proteins thaumatin-like and chitinase are of major interest, as much by the vine defense mechanisms they are involved in as by their dominance over the grape juice protein pool. Liquid chromatography and immunoassays allowed both proteins to be quantified in grape juice and Champagne, showing that their concentration decreases through winemaking. The involvement of these proteins in gas/liquid interfaces was also studied on the purified fractions from grape juice previously made for quantification standards. Results clearly indicated that neither thaumatin-like nor chitinase could alone explain bubble formation and foam stabilization in Champagne. A first study of the three-dimensional structure of the main thaumatin-like protein VVTL1 using homology molecular modeling was then achieved and normal modes analysis was performed on the VVTL1 model. It revealed a jaw-like mechanism opening and closing an acidic cleft assessed to be the enzymatic binding site. An isolated beta hairpin turned out to be highly conserved and specific to the thaumatin-like superfamilly. This domain could provide a first clue to unravel the mystery of the protein biological activity in the field of plant-pathogen interactions. A second approach was set up for the structure determination of a VVTL1 peptide using molecular modeling under NMR restraints. It could lead to a model study of protein adsorption on bentonite, a clay used for wine clarification.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 96-102). Available also in a digital version from Dissertation Abstracts.

A liquid artificial diet system, which was suitable for bioassay of added compounds, was developed for the glasshouse potato aphid, Aulacorthum solani. The diet supported normal growth and reproduction of this insect. Once established, the artificial diet bioassay system was used to test potential insecticidal activities of a variety of proteins found naturally occurring in plants. Effects on survival, development and fecundity were measured. The lectin found in snowdrop, Galanthus nivalis agglutinin (GNA) was found to significantly reduce the fecundity of A. solani, in terms of parthenogenetic nymph production, when administered in artificial diets at the 0.1% w/v level. No significant reductions in survival were found, although GNA administered in vitro did appear to slow the development of A. solani. Transgenic potato plants expressing GNA were used in a growth room trial to show that the reduction in fecundity with the in vitro trials could be reproduced in planta. Aphids feeding on the GNA-expressing potatoes had a significantly lower cumulative nymph production than those feeding on non- transformed plants. The transgenic plants had no effect on the survival of A. solani. The GNA-expressing plants were tested in a larger scale glasshouse trial and resulted in a significantly slower buildup of aphids when compared to control potatoes, thus confirming the results of the artificial diet bioassays and in planta growth room trials. Immunohistochemical studies were performed to detect the presence of GNA in the gut lumen of A. solani fed on artificial diet containing 0.1% w/v GNA; the lectin was observed to be selectively concentrated in the region of the epithelial membrane in the stomach, suggesting that binding to surface carbohydrates or glycoproteins was taking place. Binding to the gut surface has been suggested to mediate lectin toxicity in higher animals, and other insects. A synergistic effect was observed with transgenic potatoes expressing a double construct encoding GNA and bean chitinase (BCH); A. solani cumulative nymph production on these plants was significantly reduced compared to aphids feeding on control and GNA-only expressing plants. However, interestingly, BCH-only expressing plants did not significantly affect the fecundity of A. solani, although a slight reduction in nymph production was observed. On the basis of reports in the literature that suggested that chitin-binding lectins were toxic to insects, an attempt to isolate the gene encoding the chitin-binding stinging nettle lectin was made. RNA was extracted from nettle rhizomes and used to prepare a cDNA library. Successful library construction was verified. PGR methods and a primary screen of the library were used in an attempt to locate the gene.

No description available.

Le latex de Carica papaya est un milieu riche en cystéine protéinases. Celles-ci ont été régulièrement utilisées en cosmétique ou pour l’attendrissement de la viande. Mais ces protéines ont aussi un intérêt pharmaceutique. En effet, le latex est bien connu pour posséder une activité antifongique mais aussi une activité anthelminthique. Ces effets sont régulièrement attribués aux cystéine protéinases qui se trouvent en concentration importante dans le latex. Malgré ces concentrations importantes en protéinases, d’autres protéines restent actives dans ce milieu. C’est le cas de la glutamine cyclase, qui a été extraite intacte de ce milieu et cristallisée. Sa structure nous a révélé une architecture particulière en ‘’-propeller‘’ à cinq pales avec double fermeture. Cette structure lui confère sa très grande stabilité. Les industries pharmaceutiques sont aussi à la recherche de protéines très stables et résistantes aux protéinases endogènes. Nous avons donc entrepris l’étude du protéome de Carica papaya afin de mettre en évidence d’autres protéines minoritaires relativement stables pouvant conférer au latex son activité anthelminthique. Cette analyse a permis la mise en évidence de différentes protéines appartenant à diverses familles des « pathogenesis related protéins » (PR-proteins): une -1,3 glucosidase, une analogue à la barwin, une thaumatine et deux chitinases. Nous nous sommes particulièrement intéressés à ces deux dernières au cours de cette thèse. Une caractérisation de ces deux protéines a permis de montrer que celles-ci étaient bien deux protéines distinctes, identifiées comme chitinases majeure et mineure selon leur abondance dans le latex. Elles sont relativement stables et résistantes à la protéolyse. Une analyse de la séquence de la chitinase majeure a montré que celle-ci était homologue à la chitinase issue de l’orge et une analyse de sa structure révèle la présence d’une grande concentration en prolines localisées principalement dans les neuf boucles de sa structure. Cela pourrait expliquer sa grande résistance vis à vis des cystéine protéinases. La cristallisation de cette même chitinase en présence de N-acétyl-glucosamine comme additif, a conduit à une structure contenant trois molécules de GlcNac, deux dans le centre actif de notre protéine et une participant au réseau cristallin. Aucune structure de chitinase n’avait encore pu être obtenue en co-cristallisation avec un substrat. A partir des deux GlcNac observés dans le centre actif, nous avons reconstruit un complexe chitinase/(GlcNac)4. L’analyse de ce complexe a permis de mettre en évidence de nouvelles interactions entre (GlcNac)4 et les acides aminés du centre actif ainsi que de confirmer le mécanisme de la famille GH 19. Des tests préliminaires sur nématodes ont finalement confirmé l’activité anthelminthique du latex et montré que la chitinase pouvait aussi être un bon nématocide

This thesis describes the design and synthesis of small molecule derivatives and their polypeptide conjugates as high affinity binders for proteins: the D-dimer protein (D-dimer), a biomarker for diagnosis of thromboembolic diseases; human myeloperoxidase (MPO), a biomarker for cardiovascular diseases; and chitinases, potential targets for asthma therapy. The interactions between the synthetic binder molecules and those proteins were evaluated by surface plasmon resonance (SPR) biosensor analysis and fluorescence spectroscopy. Competition SPR experiments or other methods proved that the small molecule components of the binder molecules were critical for binding and specifically bound to the original binding site of small molecules. The binder molecules consisted of a 42-residue helix-loop-helix polypeptide conjugated to a small molecule via aliphatic spacers of suitable length. The small molecules could be any type of moderately binding structure. In the binder development for the D-dimer, the tetrapeptide GPRP with a dissociation constant Kd of 25 μM was used and the affinity of 4C15L8GPRP obtained was estimated to be approximately 3 nM. In the binder development for MPO, salicylhydroxamic acid (SHA) with Kd of 2 μM was used and the affinity of 4C37L34C11SHA obtained was estimated to be approximately 0.4 nM. In the binder development for chitinases, a theobromine derivative (pentoxifylline) with a Kd of 43±10 μM was used and the affinity of 4C37L34-P obtained was estimated to be considerably higher than that of pentoxifylline. The binder molecules were identified from a 16-membered pool of candidates obtained by conjugating the small molecules to each member of a set of 16 designed polypeptides. The affinities were greatly enhanced by 2-3 orders of magnitude, compared to the small molecule. The polypeptides did not bind to the proteins with measurable affinities. The discovery of these new synthetic binders for protein targets can pave the way to diagnostic tests in vivo or in vitro, independent of antibodies.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 115-132).

Hydrolytic enzymes such as chitinases and glucanases are implicated in plant defense responses against fungal pathogens. These enzymes are responsible for the breakdown of chitin and glucan, two major components of the fungal cell walls. Genes encoding these enzymes have been used to genetically engineer plants to enhance their protection against fungal pathogens. Western Australia has over 4000 endemic plant species and a largely unknown fungal biota. Given that fungi possessing chitinases and glucanases with novel activities have been isolated in other parts of the world, we propose that fungi from Western Australian soils may possess novel biochemical/enzymatic activities. The aims of this research project were to isolate chitinolytic and glucanolytic fungi from soil and to clone the genes encoding for chitinase and glucanase enzymes. To achieve these aims, fungi with activity against chitin and glucan were isolated, the activity quantified by colorimetric and inhibition assays and gene fragments with homology to known chitinase and glucanase genes were isolated and their sequences determined. Soil fungi were isolated from five locations in and around the Perth Metropolitan area of Western Australia with the use of a medium containing Rose Bengal that eliminates all actinomycetes and most bacteria and reduces the growth of fast growing mold colonies. Forty-one isolates were obtained by this method. Twenty four chitinolytic and glucanolytic fungal isolates were identified by growing them on chitin-containing media to select for those species that utilised chitin/glucan as a carbon source. These were assayed for production of exo- and endochitinolytic and glucanolytic enzymes. Enzyme activity was compared between crude and dialysed supernatants. Exochitinase activity was determined in the supernatants of 4-day old fungal cultures by the release of p-nitrophenol from p-nitrophenyl-N-acetyl-â-D glucosaminide. The supernatants were measured for endochitinase activity determined by the reduction of turbidity of suspensions of colloidal chitin. Glucanase activity was determined by release of reducing sugar (glucose) from laminarin. Supernatants from eleven of the twenty four isolates showed significant levels of enzyme activity. Eleven isolates were assayed for activity against purified cell walls of phytopathogenic fungi. Activity was determined by measuring reducing sugars in the fungal supernatants against cell wall preparations of six economically important plant pathogens. Chitinolytic activity was detected in seven isolates against cell wall preparations of Botrytis cinerea and Rhizoctonia solani, in four isolates against Fusarium solani and Sclerotinia sclerotium; in five isolates against Ascochyta faba and in six isolates against Leptosphaeria maculans. Similarly glucanolytic activity was detected in eight isolates against B. cinerea, in seven against R. solani, in two against F. solani, in three against S. sclerotium and A. faba and in one against L. maculans. The supernatants derived from the isolates were used in a bioassay to determine growth inhibition against live B. cinerea spores by measuring turbidity reduction. Growth inhibition was measured against a control (B. cinerea, grown in medium with no added supernatant). Boiled supernatant did not inhibit the growth of B. cinerea spores but there was 100% inhibition by the crude supernatant from ten of the twenty four isolates. Similarly, supernatants were used to assess growth inhibition against live mycelia cultures of F. solani and S. sclerotium. Growth inhibition of F solani ranged from 9- 59%, boiled and crude supernatants respectively whilst growth inhibition of S. sclerotium ranged from 46-75%, boiled and crude supernatants respectively. Two partial chitinase genes from the soil filamentous ungus Trichoderma asperellum,(ChiA and ChiB) and a novel glucanase gene from the filamentous fungus Aspergillus (Glu1) were cloned. ChiA, was 639 bp long, encoding 191 amino acids with identity to other chitinase genes. Two highly conserved regions, characteristic of glycosyl hydrolases from family 18, were present. ChiB, was 887 bp long and encoded a 293 amino acid sequence that was closely related to an endochitinase gene from the filamentous fungus Trichoderma asperellum. The two highly conserved regions corresponding to the substrate binding and active sites that characterise the glycosyl hydrolases from family 18, also found in ChiA, were found in this gene. Glu1 was 2844 bp long and encoded a 948 amino acid sequence that shared high identity with a â-1, 3-glucanase from the filamentous fungus Aspergillus oryzae. The sequence contained conserved regions found in glycosyl hydrolases from family 17 that encode for substrate binding, N-terminal sequences and putative asparagine linked glycosylation sites. The partial putative sequence ChiA is probably a pseudogene because it has two inframe stop codons. However, once the entire sequence of ChiB is known, both ChiB and the novel glucanase gene Glu1 could be useful contenders for engineering resistance in crop plants.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 7 uppsatser.