The presence of thiols in living systems is critical for the maintenance of cellular redox homeostasis, the maintenance of protein thiol-disulfide ratios and the protection of cells from reactive oxygen species. In addition to the well studied tripeptide glutathione (??-Glu-Cys-Gly), a number of compounds have been identified that contribute to these essential cellular roles. Many of these molecules are of great clinical interest due to their essential role in the biochemistry of a number of deadly pathogens, as well as their possible role as therapeutic agents in the treatment of a number of diseases. A series of studies were undertaken using theoretical, chemical and biochemical approaches on a selection of thiols, ergothioneine, the ovothiols and mycothiol, to further our understanding of these necessary biological components. Ergothioneine is present at significant physiological levels in humans and other mammals; however, a definitive role for this thiol has yet to be determined. It has been implicated in radical scavenging in vivo and shows promise as a therapeutic agent against disease states caused by oxidative damage. Given the clinical importance of this intracellular thiol, further investigation into the behaviour of ergothioneine appeared warranted. A high level theoretical study was performed to determine the thermodynamic driving force behind the instability of the ergothioneine disulfide, as well as the thermodynamics of the reactions of ergothioneine with a selection of biologically relevant reactive oxygen species. These results were compared to those determined for a glutathione model compound, as well as the related ovothiols. The latter are believed to act as hydrogen peroxide scavengers in vivo and are currently under review as possible therapeutics against oxidative damage. The structural differences between the ovothiols and ergothioneine dramatically affect their reactivity and this study investigates the thermodynamic driving forces behind these differences. Mycothiol is the major thiol found in the Actinomycetales bacteria, which include the causative agent of tuberculosis, and the enzymes which use mycothiol have been identified as important targets for the development of novel antimicrobials. To better understand the in vivo behaviour of mycothiol, a thorough conformational search was performed to determine what, if any, trends exist among the low energy conformers expected to be present in solution. Knowledge of the conformations preferred by mycothiol may aid in the design of substrate-based inhibitors targeted at mycothiol-dependent enzymes. In addition, the efforts towards the identification of a mycothiol-dependent glyoxalase system are described. The glyoxalase system is essential for the detoxification of methylglyoxal, a toxic by-product of glycolysis, and this system would serve as a target for the design of new therapeutics against tuberculosis and other pathogenic Actinomycetales bacteria. In addition to the study of intracellular thiols, this work details a preliminary theoretical study of the thermodynamics of the phosphorylation of proteinaceous serine residues by inositol pyrophosphates in eukaryotic cell-free extracts. It has been postulated that this observed activity may represent a novel signalling pathway in eukaryotes. This study focused on the effect of inositol pyrophosphate structure and overall charge on the thermodynamics of these reactions. This information should contribute to our understanding of this novel cellular phosphorylation process.

Les variations de la concentration cytosolique de Ca[indice supérieur 2+] contrôlent divers processus biologiques tel la contraction, la division cellulaire et la transactivation de gènes. Chez les cellules non excitables, la stimulation de certains GPCRs (G protein coupled receptor) ou récepteurs tyrosine kinase mène à l'activation d'une phospholipase C qui produit de l'inositol 1,4,5-trisphosphate (IP[indice inférieur 3]). L'IP[indice inférieur 3] active un récepteur-canal au niveau du réticulum endoplasmique (RE) et permet une relâche de Ca[indice supérieur 2+] du RE. Il existe trois isoformes distinctes d'IP[indice inférieur 3]R (IP[indice inférieur 3]R-1 à -3) différemment exprimées dans les tissus. Dans ces travaux, nous avons étudié un mode de régulation de l'IP[indice inférieur 3]R-2 par la PKC, une kinase elle-même activée lors de la réponse calcique. Nous avons aussi vérifié l'implication de chaque isoforme d'IP[indice inférieur 3]R dans l'activité transcriptionnelle des facteurs de transcription sensibles au Ca[indice supérieur 2+] NFAT et CREB.Les cellules AR4-2J expriment principalement l'IP[indice inférieur 3]R-2 (86%), ce qui en fait un bon modèle d'étude pour les mécanismes de régulation de l'IP[indice inférieur 3]R-2. Dans la première étude, nous avons vérifié si la PKC influençait l'activité calcique de l'IP[indice inférieur 3]R-2. D'abord, nous avons montré que l'IP[indice inférieur 3]R-2 est majoritairement exprimé dans les cellules AR4-2J. Nous avons montré par phosphorylation in vitro et in cellulo que l'IP[indice inférieur 3]R-2 est phosphorylé par la PKC. Nous avons montré que le traitement par la PKC réduit la réponse calcique induite par l'IP[indice inférieur 3] sur des cellules perméabilisées. Finalement, nous avons démontré que la réponse calcique induite par le CCh ou l'EGF sur des cellules entières était réduite avec l'activation de la PKC. Ces résultats indiquent que l'IP[indice inférieur 3]R-2 est une autre cible de la PKC permettant de contrôler l'intensité de la réponse calcique. Dans la deuxième étude l'objectif était de déterminer l'implication des isoformes d'IP[indice inférieur 3]R sur l'activité des facteurs de transcription sensibles au Ca[indice supérieur 2+] NFAT et CREB. Par l'approche des gènes rapporteurs de l'activité de NFAT ou CREB, nous avons montré que NFAT était activé par la voie de la calcineurine et que CREB par les voies de CamKII et de la calcineurine dans les cellules HEK 293A. Nous avons démontré que l'invalidation de l'IP[indice inférieur 3]R-2 diminuait la réponse calcique induite par le CCh et que la costimulation avec le VIP pouvait la restaurer. Nous avons montré que l'activité transcriptionnelle de NFAT était affectée, contrairement à celle de CREB, par l'invalidation de l'IP[indice inférieur 3]R-2. Finalement, nous avons montré que l'IP[indice inférieur 3]R-2, et aussi l'IP[indice inférieur 3]R-1, semblent jouer un rôle secondaire, différent de celui de transporteur de Ca[indice supérieur 2+], dans le mécanisme d'activation de NFAT. Ces résultats laissent envisager que l'IP[indice inférieur 3]R serait impliqué dans la formation d'un complexe protéique facilitant l'activation de NFAT.

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.

scyllo-Inositol is currently in phase II clinical trials as a therapeutic for Alzheimer’s disease (AD). Previous work from our lab has shown that scyllo-inositol prevents Ab1-42 fibril formation instead leading to the formation of small Ab oligomers in vitro. To further understand the molecular details of Ab-scyllo-inositol binding interactions, a library of scyllo-inositol derivatives was prepared. A sequence of protecting group transformations afforded a hydroxylamine functionalized scyllo-inositol. Subsequent oxime formation with aromatic aldehydes generated a novel class of inositol derivatives in good yield and high purity. The effects of these compounds on the Ab aggregation cascade were evaluated by a biotin-avidin Ab1-42 oligomer assay and atomic force microscopy (AFM). Preliminary plate assay data indicated that several of these derivatives increased peptide oligomerization and the corresponding AFM images showed altered fibril formation. These results suggested that this class of scyllo-inositol derivatives is active in the Ab aggregation cascade.

scyllo-Inositol is currently in phase II clinical trials as a therapeutic for Alzheimer’s disease (AD). Previous work from our lab has shown that scyllo-inositol prevents Ab1-42 fibril formation instead leading to the formation of small Ab oligomers in vitro. To further understand the molecular details of Ab-scyllo-inositol binding interactions, a library of scyllo-inositol derivatives was prepared. A sequence of protecting group transformations afforded a hydroxylamine functionalized scyllo-inositol. Subsequent oxime formation with aromatic aldehydes generated a novel class of inositol derivatives in good yield and high purity. The effects of these compounds on the Ab aggregation cascade were evaluated by a biotin-avidin Ab1-42 oligomer assay and atomic force microscopy (AFM). Preliminary plate assay data indicated that several of these derivatives increased peptide oligomerization and the corresponding AFM images showed altered fibril formation. These results suggested that this class of scyllo-inositol derivatives is active in the Ab aggregation cascade.

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.

No description available.

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

Thesis (M.S.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 50-57) and index.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.