Thesis (Ph. D.)--Cornell University, August, 2006. / Vita. Includes bibliographical references (leaves 157-170.

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 70-85).

Includes bibliographical references (p. 71-74).

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 2002. / Includes bibliographical references. Also available on the Internet.

The survival of the any living organism depends on its availability to communicate, and a breakdown of cellular signaling can have dire consequences such as uncontrolled cellular proliferations or even cell death. Environmental cues or ligands are perceived by cognate receptors, expressed primarily on the cell surface, and transmitted to the interior of the cell to elicit a response. This universal phenomenon is termed as signal transduction. During this process, second messengers such as cyclic nucleotides, cAMP and cGMP, are produced which serve to amplify the signal. Cyclic GMP is emerging as a universal second messenger, and is found in both prokaryotes and eukaryotes. It is synthesized from GTP by the action of guanylyl cyclases. Vertebrate guanylyl cyclases are of two forms, soluble and membrane-associated. Soluble guanylyl cyclases are heterodimeric enzymes which are activated by nitic oxide. Membrane-associated guanylyl cyclases on the other hand are homodimeric enzymes that act as receptors for divers polypeptide ligands. In mammals, there are seven isoforms of receptors guanylyl cyclase, GC-A through GC-G. These recptors have a highly conseved modular domin organization with an N-terminal extracellular domain, a single transmembrane domain and a C- terminal intra cellular regions. The intracellular region contains a juxtamembrane domain followed by a protein-kinase domain, a linker region and a catalytic guanylyl cyclase domain. The coordinated actions of these domains ensure fine tuned-regulations of the catalytic domain. Guanylyl cyclase-C (GC-C) is a member of the membrane-bound guanylyl cyclases. GC-C is predominantely present in the intestine, on the apical surface of epithelial cells, but has also been detected in the rat epididymis. In the intestine it serves as the guanylin, uroguanylin and lymphoguanylin which are the endogenous peptide ligands, while heat- stable entrotoxins (ST) peptides secreted by enterotoxigenic E.coil, are exogenic ligands. Activation of GC-C by these ligands results in an increase in intracellular cGMP levels, which then activates cGMP-dependent protein kinase and cross-activates protein kinase A. In turn, these activated kinases phosphorylate and active the cystic fibrosis transmembrane conductance regulator (CFTR), resulting in chloride and water secretion into the intestine lumen, thus regulating salt and water homeostasis in the intestine. ST peptide has a greater affinity for GC-C than the endogenous ligands and activation of GC-C by ST results in masiive fluid and ion efflux from the intestine cells from which manifests as Travelers’ Diarrhea. The GC-C mediated cGMP signal transduction pathway also maintains intestinal crypt-villus axis homeostatis by exerting a cytostatic effect on the epithelial cells, there by regulating their turn over. Over the years multiple mechanisms of regulation of GC-C activity has been identified including allosteric controlled by various domains in the receptor and phosphorylation-mediated regulation of guanylyl cyclase activity. The current study describes aspects of the regulation of GC-C by gycosylation, and also reports the molecular phenotypes of a naturally occurring mutation in GC-C causes sever diarrhea in affected individuals. GC-C is expressed as a differentially glycosylated protein (130k Da and 145kDa). While both forms bind with equal affinity, only one the 145 kDa form is activated by its ligands. It is this higher glycosylated form which is selectively downregulated in the process of decensitization of GC-C in colomn carcinoma cells (Caco2). Give the critical role gycosylation plays in protein folding, trafficking, receptor activity and mediating protein inter actions, its influence on GC-C was analysed.

The Gilman's cyclic AMP binding assay was used to examine the possibility of adopting this method for adenylate cyclase determinations. Cyclic AMP determinations were not invalidated by the reagents used in the adenylate cyclase reaction. Cyclic AMP measured by the binding assay was directly proportional to adenylate cyclase activity. Although variability in recovery of cyclic AMP was obtained, it could be reduced by performing triplicate assays. Thus, the cyclic AMP binding assay, with some reservations, would appear applicable for measuring adenylate cyclase activity. Adenylate cyclase in rat brain was studied by using the cyclic AMP binding method for determination of product formed. Rat brain cortex was fractionated by the method of Whittaker. The highest adenylate cyclase activity was found in the fraction containing the highest acetylcholinesterase activity, and this fraction was shown by electronmicroscopic studies to be rich in synaptosomes. A modified sucrose gradient was used for isolating satisfactory synaptosomal fractions (the layer between 1.0 M and 1.1 M sucrose). Properties of synaptosomal adenylate cyclase were examined. The enzyme was dependent on the concentrations of ATP and Mg²⁺ or Mn²⁺ ion. The enzyme was stimulated by fluoride and inhibited by calcium ion. Synaptosomal adenylate cyclase was not sensitive to catecholamines or adenosine. No hormonal stimulation was obtained in the presence of GTP. In experiments where the effects of endogenous catecholamines were reduced by the addition of α and β adrenergic blocking agents or by prior treatment of the animals with reserpine, hormonal stimulation of adenylate cyclase in particulate preparations could not be demonstrated. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Cette thèse est consacrée à la régulation de la guanylate cyclase soluble (sGC), le récepteur endogène du monoxyde d'azote (NO) chez les mammifères qui est impliqué dans la transduction du signal. L'enzyme sGC est activée par la fixation du NO sur son hème et catalyse alors la formation du cGMP à partir du GTP. Alors que la sGC est présente dans de nombreuses cellules de mammifères, le domaine hémique bactérien homologue (H-NOX) est impliqué dans la détection du NO et la régulation du métabolisme. Un objectif important est la découverte d'inhibiteurs de la sGC pour ralentir la progression tumorale.Le criblage de composés naturels d'une chimiothèque mesurant l'activité de la sGC purifiée a révélé six inhibiteurs actifs (Ki = 0.2 – 1 µM). Avec deux autres composés actifs en photothérapie (hypericin et hypocrellin) nous avons démontré que ces inhibiteurs sont des effecteurs allostériques qui ne se fixent ni sur l'hème, ni sur le site catalytique ou de fixation des activateurs, découvrant une nouvelle classe de composés pharmacologiques ciblant la voie de signalisation NO/cGMP.La transition structurale induite par l'activateur riociguat en synergie avec le CO a été étudiée par spectroscopie d'absorption résolue en temps pour démontrer des changements de coordination de l'hème. Deux états d'activation distincts de la sGC par le CO existent simultanément avec les coordiantions 6c-hème et 5c-hème en présence de l'activateur qui induit la rupture de la liaison Fe-His de l'hème, à l'instar de l'activateur naturel NO. De plus, nous montrons que l'isoliquiritigénine, commercialisée comme activateur de la sGC, et en réalité un inhibiteur de la sGC.La dynamique ds ligands CO, NO, and O2 a été mesurée sur 12 ordres de grandeur temporelle pour le type sauvage et un mutant du transporteur bactérien du NO (AXCP). La simple mutation Leu16Ala augmente l'afinité pour le CO 108 fois, celle du NO 106 fois et rend cette protéine réactive à O2. Dans le cas de CO et NO dont les affinités pour L16A-AXCP sont les plus grandes jamais mesurées, la recombinaison bimoléculaire n'est pas détectable. Des simulations de dynamique moléculaire ont démontré que le CO dissocié est contraint de rester à 4 Å du Fe2+ par Ala16, contrairement au type sauvage Leu16.La dynamique de O2 a été mesurée dans la protéine senseur Tt H-NOX par spectroscopie d'absorption transitoire et confirme l'hypothèse que Tt H-NOX n'est sans doute pas un senseur de NO stricto sensu mais un senseur redox. Les propriétés de Tt-H-NOX ne sont pas compatibles avec le rôle d'un simple transporteur de NO. / This thesis is devoted to the regulation of soluble guanylate cyclase (sGC), the endogenous nitric oxide (NO) receptor in mammals involved in signal transduction. The enzyme is activated by the binding of NO to its heme and catalyzes the formation of cGMP from GTP. While sGC is present in many mammalian cells, the homologous bacterial domain (H-NOX) is involved in NO detection and metabolism regulation. An important objective was to find sGC inhibitors to slow down tumor progression.The screening of natural compounds from a chemical library, tested on purified sGC activity, revealed six active inhibitors (Ki = 0.2 – 1 µM). Together with two agents for photodynamic therapy (hypericin and hypocrellin) we demonstrated that these inhibitors are allosteric modulators which bind neither to the heme nor to the catalytic and activator sites, revealing a new class of pharmacological compounds targetting the NO/cGMP signaling pathway.The structural transition induced in sGC by stimulator riociguat in synergy with CO was studied by transient absorption spectroscopy to demonstrate coordination changes of the heme. Two different activation states of sGC with CO 6c-heme and 5c-heme exist simultaneously in the presence of the stimulator which induces the breaking of the heme Fe-His bond, as does the sGC natural effector NO. In addition, the effect of isoliquiritigenin, which is sold as a sGC activator, was shown to be actually an inhibitor of sGC.The dynamics of the ligands CO, NO and O2 were measured over 12 orders of magnitude in time in wild type and mutant of a bacterial NO transporter (AXCP). The single mutation Leu16Ala increased 108-fold the CO affinity, ~106-fold the NO affinity and makes this protein reactive to O2. In the case of CO and NO, whose affinities for L16A-AXCP are the largest ever measured, the bimolecular rebinding was absolutely not detectable. Molecular dynamic simulations demonstrated that dissociated CO is constrained to stay within 4 Å from Fe2+ by Ala16, contrarily to wild-type Leu16.The dynamics of O2 in Tt-H-NOX proteins measured by transient absorption spectroscopy confirmed the hypothesis that Tt-H-NOX may not be a NO-sensor stricto sensu but a redox sensor. The properties of the Tt-H-NOX protein are not compatible with the role a mere NO-carrier.

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Les protéines activatrices de la guanylate cyclase (GCAPs) font partie de la famille des neuroprotéines sensibles au Ca²⁺ (NCS) et celle des protéines à EF-Hand. Il a été proposé que le mécanisme de Ca²⁺-myristoyl switch avait lieu chez toutes les protéines de la famille des NCS. Les travaux présentés dans ce mémoire permettent de déterminer si ce mécanisme est observé chez la GCAP1. En effet, des travaux de liaison membranaire à des monocouches de Langmuir effectués avec la GCAP1 ont permis d’observer ce mécanisme. De plus, l’utilisation d’un analogue du myristoyle, le 13-oxa-myristoyle, a aussi permis d’observer un Ca²⁺-myristoyl switch chez la GCAP1. Effectivement, des mesures en résonance magnétique nucléaire (RMN) ont démontré que la présence de cet analogue favorise l’extrusion du myristoyle. Finalement, différentes analyses par RMN ont été effectuées afin de déterminer si cette méthode pourrait permettre de déterminer la structure de la forme active de la GCAP1.