The aim of this thesis was to study the changes that occur in cell line H9c2 after exposure to an oxygen level reduced to 2 % for 24 hours. We monitored changes in compartmentation of chosen members of β-adrenergic signaling system. We found an increase in expression of β1AR and β2AR. Only β2AR showed change in compartmentation after hypoxia, as they relocate from membrane rafts to non-rafts fractions of membrane. AC also showed an increase of expression and was located in membrane rafts. The next aim of this work was to monitore apoptotic markers to determine whether there are activated pro-apoptotic or anti-apoptotic signals under chosen conditions of hypoxia. There was an increase in expression of both pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. We compare ratios of Bcl-2 to Bax and we found that there is a bigger increase in protein Bax expression. Another apoptotic marker, caspase 3, was tested and we also found that there was an increase in expression of caspase 3 in cells after hypoxia. Furthermore, we studied possible activation of kinase signaling pathways that may contribute to protective effects of hypoxia. Expression of Akt and ERK kinases was increased after hypoxia, but we did not confirm activation by phosphorylation of these kinases. Levels of phosphorylated Akt...

β-Adrenergic signalization plays an important role in heart, regulating cardiac frequency and contractility. It is also involved in development of hypertension and heart hypertrophy. Spontaneous hypertensive rat strain is a common model for human essential hypertension, although the origin of blood pressure abnormalities in SHR remains unknown. Dysfunction in the regulation of fatty acid translocase Cd36 was suggested as a link to development of hypertension in SHR. Transgenic strain SHR-Tg19 (also known as SHR-Cd36) was obtained, harboring a wild type of FAT/Cd36. This thesis aimed to investigate key elements of β-adrenergic signaling in the heart of SHR-Tg19 compared to their SHR controls. Expression and distribution of β1- and β2-ARs were measured using radioligand binding and Western blot analysis along with expression of selected G proteins and expression and activity of adenylyl cyclase. Our experiments showed that there were no significant changes in the Gsα and Giα subunits expressions, along with the amount of β1-AR in both left and right ventricles, according to the Western immunoblotting, but radioligand binding showed an increase in the quantity of β-ARs, particularly β2 subtype. Alongside, an increased expression of β2- ARs was observed in the right ventricle. Different...

1 Abstract Adenylate cyclase toxin (CyaA) is one of the major virulence factors of bacterium Bordetella pertussis, which is a causative agent of whooping cough. CyaA belongs to the family of RTX toxin-hemolysins. The toxin targets primarily cells expressing integrin receptor CD11b/CD18 but it can also penetrate cells lacking this receptor. CyaA acts on host cells by two independent activities. One is formation of small cation-selective channels, which can lead to colloid osmotic lysis of target cells. The second is disruption of cell signaling through the translocation of the adenylate cyclase (AC) domain to host cell cytosol, which leads to the conversion of ATP into cyclic AMP. It was recently shown that cholesterol affects endocytosis of CyaA. CyaA translocates it's AC domain after relocation of CyaA molecule to the cholesterol-rich lipid raft (Bumba et al. 2010). In this work I examined the effect of cholesterol on channel- forming activity and selectivity of ion channels created by CyaA. For measurements I used artificial membranes enriched with cholesterol. CyaA channels are voltage-dependent. The positive membrane potential on the side of toxin is rquired for incorporation of CyaA molecule into cell membrane. I tried to find out whether the value of voltage has effect on channels opening time....

Chronic postoperative pain impacts millions of individuals worldwide that undergo a variety of surgical procedures. Opioids remain the mainstay analgesics of acute and perioperative pain; however, prolonged opioid therapy may lead to life-threating adverse effects, tolerance, dependence, and addiction. Therefore, unraveling the cellular mechanisms that drive persistent pain states and opposing endogenous analgesia provided by opioid receptor signaling, may lead to novel analgesics. Evidence suggests that tissue injury leads to increased sensitization of the spinal cord nociceptive neurons which increases susceptibility to chronic pain via an N-methyl-D-aspartate (NMDA) receptor activation of calcium-sensitive adenylyl cyclase isoform 1 (AC1). This phenomenon, named latent pain sensitization (LS), is mediated by a compensatory response of endogenous inhibitory systems. In this dissertation, we test the hypothesis that surgical insult promotes prolonged activation of kappa opioid receptors (KOR) which mask LS via attenuation of pro-nociceptive AC1 signaling pathways in both male and female animals. We employed a murine model of chronic postoperative pain that promotes LS in the spinal cord and closely resembles the phenotypic features of postoperative pain in human subjects. When behavioral signs of hyperalgesia resolved, we targeted spinal opioid receptor systems and pronociceptive modulators with intrathecal delivery of selective pharmacological antagonists and assessed behavioral signs of hyperalgesia and spinal nociceptive sensitization. We propose that LS is kept in remission by a long-lasting compensatory response of tonic endogenous KOR signaling that hinders a pronociceptive LS pathway that includes not only AC1 but also two downstream targets: protein kinase A (PKA) and exchange protein activated by cAMP (Epac1/2) - in a sex-dependent manner. Our results propose new therapeutic targets for the management of persistent postoperative pain and underscore the importance of tailoring sex-specific pain management strategies.

Le récepteur endogène du NO, la guanylate cyclase (sGC) est l'objet de thèse. Cette enzyme synthétise le GMPc après fixation du NO. L'outil principal utilisé est la spectroscopie d'absorption résolue en temps picoseconde-nanoseconde. Nous avons montré que la fixation simultanée du CO et d'activateurs (YC-1, Bay 41-2272) induisent un hème 5c-CO, à l'instar du NO seul, expliquant l'activation synergique. Nous avons identifié toutes les étapes de l'interaction sGC-NO en mesurant la dynamique du NO de la picoseconde à la seconde. Cette dynamique dans la protéine entière est comparée à celle de la sous-unité beta (1-190) isolée et celle de senseurs de NO bactériens. Un mutant de la myoglobine (H93C) à été utilisé comme modèle pour l'étude de l'hème dans les états 4- et 5-coordonnés. Enfin, nous avons mesuré la variation d'absorption dans la bande III de la Mb et Hb pour mesurer le mouvement du Fer de l'hème aprés fixation du NO. Nous avons cherché un inhibiteur potentiel et un ligand endogène de la sGC.

L'autorenouvellement est une propriété fondatrice du concept de cellule souche. Cependant, malgré l'avancée des connaissances actuelles, les mécanismes moléculaires sous-jacents restent mal compris. Nous nous sommes donc intéressés à cette question, en étudiant l'équilibre entre autorenouvellement et différenciation dans des progéniteurs érythrocytaires primaires. D'une part, grâce à une étude combinant des approches pharmacologiques et de génétique fonctionnelle, nos résultats montrent que le contrôle de la synthèse cellulaire du cholestérol joue un rôle essentiel dans la régulation du basculement de l'autorenouvellement vers la différenciation. D'autre part, nous avons étudié la nature stochastique de l'expression génique au cours du passage de l'autorenouvellement vers la différenciation. En effet, contrairement au caractère déterministe initialement attribué à l'expression des gènes, les données accumulées au cours des dernières années démontrent que cette expression repose sur des processus stochastiques. Nous avons en particulier oeuvré à la conception et à la mise en place d'un dispositif permettant de suivre en temps réel l'expression génique dans des cellules individualisées, afin de pouvoir mesurer et évaluer cette stochasticité. Au final, l'ensemble de ces travaux participent à la compréhension des bases moléculaires de l'autorenouvellement et du contrôle des choix du devenir cellulaire.

During development, neurons must find their way to and make connections with their appropriate targets. Growth cones are dynamic, motile structures that are integral to the establishment of appropriate connectivity during this wiring process. As growth cones migrate through their environment, they encounter guidance cues that direct their migration to their appropriate synaptic targets. The gaseous messenger nitric oxide (NO), which diffuses across the plasma membrane to act on intracellular targets, is a signaling molecule that affects growth cone motility. However, most studies have examined the effects of NO on growth cone morphology when applied in large concentrations and to entire cells. In addition, the intracellular second messenger cascade activated by NO to bring about these changes in growth cone morphology is not well understood. Therefore, this dissertation addresses the effects that a spatially- and temporally-restricted application of physiological amounts of NO can have on individual growth cone morphology, on the second messenger pathway that is activated by this application of NO, and on the calcium cascades that result and ultimately affect growth cone morphology. Helisoma trivolvis, a pond snail, is an excellent model system for this type of research because it has a well-defined nervous system and cultured neurons form large growth cones. In the present study, local application of NO to Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number, and an increase in the intracellular calcium concentration ([Ca2+]i). In B5 neurons, the effects of NO on growth cone behavior and [Ca2+]i are mediated via sGC, protein kinase G, cyclic adenosine diphosphate ribose, and ryanodine receptor-mediated intracellular calcium release. This study demonstrates that neuronal growth cone pathfinding in vitro is affected by a single spatially- and temporally-restricted exposure to NO. Furthermore, NO acts via a second messenger cascade, resulting in a calcium increase that leads to cytoskeletal changes. These results suggest that NO may be a signal that promotes appropriate pathfinding and/or target recognition within the developing nervous system. Taken together, these data indicate that NO may be an important messenger during the development of the nervous system in vivo.

Signal transduction is a crucial event that enables cells to sense and respond to cues from their immediate environment. Guanylyl cyclase C (GC-C) is a member of the family of receptor guanylyl cyclases. GC-C is a single transmembrane protein that responds to its ligands by the production of the second messenger cGMP. The guanylin family of peptides, (including the bacterially produced heat-stable enterotoxin ST) is the ligand for GC-C, elevates intracellular cGMP levels and activates downstream pathways. GC-C regulates the cystic fibrosis transmembrane conductance regulator (CFTR) by inducing phosphorylation by protein kinase G, resulting in chloride ion and fluid efflux. GC-C also regulates cell cycle progression through cGMP-gated Ca2+ channels. These functions are seen in the intestinal epithelium, the primary site for GC-C expression. GC-C as a molecule has been studied in detail, but its functioning in the context of other signaling pathways remains unknown. The aim of the present investigation was to understand the regulation of signal transduction by GC-C and its cross-talk with other signaling pathways operating in the cell. Molecular events that commonly connect components in a signaling pathway are protein phosphorylation and protein-protein interaction. These two aspects are explored in this thesis. The possibility of tyrosine phosphorylation of GC-C has been explored earlier in our laboratory. In vitro studies indicated that the residue Tyr820 was a site for phosphorylation by the Src family of non-receptor tyrosine kinases and those studies also suggested that phosphorylated Tyr820 could bind to the SH2 domain of Src. We generated a nonphosphorylatable mutant of GC-C, GC-CY820F, and a phosphomimetic mutant GC-CY820E to study the effect of phosphorylation of Tyr820, on the functioning of GC-C. A stable cell line of HEK293:GC-CY820F cells was generated and compared with HEK293:GC-CWT. Dose response to ST in the two cell lines showed that cGMP accumulation by GC-CY820F was greater than that of GC-CWT, although the EC50 remained unchanged. The phosphomimetic GC-CY820E mutant receptor was non-responsive to ST. Further in HEK293 cells, phosphorylation of GC-CWT by constitutively active v-Src resulted in decreased ST stimulation and this effect of v-Src was reduced with GC-CY820F. Inhibition of ST stimulation brought about by v-Src required catalytically active Src, as the kinase inactive v-SrcK295R did not inhibit ST stimulation. These results were corroborated by in vitro studies by using the recombinant catalytic domain of GC-C expressed in insect cells and by phosphorylation using a purified kinase, Hck. Observations suggested that phosphorylation of Tyr820 in the catalytic domain of GC-C compromises the guanylyl cyclase activity of GC-C. T84 and Caco-2 colon carcinoma cells endogenously express GC-C. The effect of tyrosine phosphorylation of GC-C was studied by using HgCl2, a known activator of Src kinases, and by the inhibition of protein tyrosine phosphatases using pervanadate, an irreversible inhibitor. Both these ways of achieving increased tyrosine phosphorylation resulted in decreased ST-stimulated cGMP production by GC-C, as suggested from v-Src transfection studies. This decrease was reversed by using a Src kinase specific inhibitor PP2, confirming the role of Src kinases in the inhibition of GC-C activity. Interestingly, in Caco-2 cells that differentiate in culture, the effect of pervanadate on the inhibition of ST-stimulated GC-C activation was dependent on the differentiation stage. Crypt-like cells showed higher inhibition with pervanadate. As they matured into villus-like cells, the effect of pervanadate on GC-C activation was gradually lost. This effect also correlated with a decrease in the expression of Lck, suggesting that in the context of the intestine there could be differential regulation of tyrosine phosphorylation of GC-C along the crypt-villus axis. Intestinal ligated loop assays in rats demonstrated that ST-induced fluid accumulation in the intestine was abrogated on pervanadate treatment. Reduction in this fluid accumulation by pervanadate was not observed with 8-Br-cGMP, a cell permeable analogue of cGMP. This indicated that tyrosine phosphorylation of proteins is important for ST-induced fluid accumulation, and perhaps pervanadate modulates this by phosphorylation of GC-C, thereby causing a reduction in fluid accumulation. Earlier in vitro studies on Src-SH2 binding from the laboratory had suggested the possibility of activation of Src family kinases by GC-C. The activation status of Src kinases was monitored by using phosphorylation-state specific antibody, pSFK416. ST stimulation in T84 cells increased Tyr416 phosphorylation of Src kinases in a time dependent manner, indicating that Src kinases are activated downstream of GC-C. This activation of Src kinases was also seen with the endogenous ligand of GC-C, uroguanylin. Interestingly, 8-Br-cGMP a cell permeable analogue of cGMP that is known to mimic other cellular effects of GC-C, namely Cl-secretion and cell cycle progression, did not activate Src kinases, suggesting that the mechanism of Src kinase activation by GC-C could be independent of cGMP. Binding affinities of Src, Lck, Fyn and Yes SH2 domains to Tyr820 phosphorylated GCC peptide were in the nM range, indicating a high affinity of interaction. In vitro GST-SH2 pull down experiments suggested that phosphorylation of Tyr820 in full length GC-C allows interaction of GC-C to the SH2 domain of Src. These studies suggest a dual cross-talk between Src kinases and GC-C; Src phosphorylation inhibits GC-C signaling and stimulation of GC-C by its ligands activates Src kinases. Interaction of proteins containing SH2 and SH3 domains are commonly found in signaling molecules. In accordance with the observation that there are three PXXP motifs in GCC, many SH3 domains could interact with GC-C. GC-C appears to show a preference to bind the SH3 domains of Fyn, Hck, Abl tyrosine kinases, Grb2 and Crk adaptor proteins, the α-subunit of P85 PI3 kinase, PLC-γ and cortactin to various extents. The SH3 domains of spectrin and Nck did not show any detectable interaction with GC-C. In SH3 pull-down assays, the N-terminal SH3 domain of Crk, CrkSH3 (N), bound GC-C maximally, suggesting that Crk is a good candidate for interaction with GC-C. By overlay analysis, the region of GC-C that binds CrkSH3 (N) was narrowed down to the catalytic domain of GC-C containing a ‘PGLP’ motif. Mutations were generated in GC-C at this site to generate GC-CP916Q and GC-CW918R. These mutations compromised the binding of full length receptor to CrkSH3 (N). In cells, CrkII and GC-C co-transfection inhibited the ST stimulation of GC-C. A CrkII mutant, that has compromised binding through its SH3 domain, did not inhibit the activity of GC-C. CrkII from T84 cells co-immunoprecipitated with GC-C and interestingly, the phosphorylated form of CrkII did not, indicating that GC-C - Crk interaction could be regulated by the phosphorylation of Crk. In summary, this study places GC-C, in the context of tyrosine kinase signaling pathway and interaction with the adaptor protein Crk. These studies suggest that GC-C signal transduction can be altered by cross-talk with other signaling events in the cell. Reversible phosphorylation of tyrosine residues inhibits the activity of GC-C, and this is mediated by Src family kinases. Src kinases themselves are activated on stimulation of GC-C by its ligands, possibly because of SH2 domain interaction with GC-C. Association of Crk by its SH3 domain regulates GC-C functioning primarily by inhibiting ST-stimulated cGMP production. This opens up the possibility of GC-C signaling through a multimeric complex involving other binding partners of Crk, and these cross-talks involving GC-C with the two proto-oncogenes, Src and Crk, might have far reaching consequences in the regulation of cellular functions.

Natural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering. / text

Le récepteur A des peptides natriurétiques (NPRA) fait partie de la famille des guanylates cyclases membranaires. L’activation du NPRA par ses agonistes naturels, ANP et BNP, induit une production de GMPc qui est responsable de leur rôle dans l’homéostasie cardiovasculaire, l’inhibition de l’hypertrophie et de la fibrose cardiaques et la régulation de la lipolyse. Le NPRA est un homodimère non covalent composé d’un domaine extracellulaire de liaison du ligand (ECD), d’un unique domaine transmembranaire (TM), d’un domaine d’homologie aux kinases et d’un domaine guanylate cyclase. Bien que le NPRA ait un rôle physiologique important, les mécanismes moléculaires régissant son processus d’activation restent inconnus. Nous avons donc analysé les premières étapes du processus d’activation du NPRA. Nous avons d'abord étudié le rôle de la dimérisation des ECD dans l’activation du récepteur. Nous avons utilisé les techniques de liaison de radioligand, de FRET et de modélisation moléculaire, pour caractériser la liaison à l’ECD des agonistes naturels, d’un superagoniste et d’un antagoniste. L’ANP se lie à un dimère d’ECD préformé et la dimérisation spontanée est l’étape limitante du processus de liaison. De plus, comme le démontrent nos études de FRET, tous les peptides, incluant l’antagoniste, stabilisent le récepteur sous sa forme dimérique. Cependant, l’antagoniste A71915 stabilise le dimère d’ECD dans une conformation différente de celle induite par l’ANP. La dimérisation du NPRA semble donc nécessaire, mais non suffisante à l’activation du récepteur. L’état d’activation du NPRA dépend plutôt de l’orientation des sous unités dans le dimère. Nous avons ensuite étudié le mécanisme moléculaire de transduction du signal à travers la membrane. Plusieurs études ont suggéré que l’activation du NPRA implique un changement de conformation du domaine juxtamembranaire (JM). Cependant, les études de cristallographie de l’ECD soluble de NPRA n’ont pas permis de documenter la structure du JM et le changement de conformation impliqué dans la transduction du signal reste inconnu. Pour analyser ce changement de conformation, nous avons d’abord séquentiellement substitué les neuf acides aminés du JM par une cystéine. En étudiant la capacité des mutants à former des dimères covalents de façon constitutive ou induite par l’ANP, nous avons pu évaluer la proximité relative des résidus du JM, avant et après activation du NPRA. Ces résultats ont démontré la proximité élevée de certains résidus spécifiques et sont en contradiction avec les données cristallographiques. Nous avons également démontré que le domaine intracellulaire impose une contrainte conformationnelle au JM à l’état de base, qui est levée après liaison de l’ANP. En introduisant de 1 à 5 alanines dans l’hélice-α transmembranaire, nous avons montré qu’une rotation des TM de 40° induit une activation constitutive du NPRA. Le signal d’activation pourrait donc être transmis à travers la membrane par un mécanisme de rotation des TM. En utilisant nos données expérimentales, nous avons généré le premier modèle moléculaire illustrant la conformation active du NPRA, où les domaines JM et TM sont représentés. Dans son ensemble, cette étude apporte une meilleure compréhension des mécanismes moléculaires régissant les premières étapes du processus complexe d’activation du NPRA. / Natriuretic peptide receptor-A (NPRA) is a member of the particulate guanylate cyclase family. NPRA activation by natural agonists, ANP and BNP, leads to cGMP production, which is responsible for their role in cardiovascular homeostasis, cardiac hypertrophy and fibrosis inhibition and lipolysis regulation. NPRA is a non covalent dimer composed of an extracellular domain (ECD) with a ligand binding site, a single transmembrane region (TM), a kinase homology domain, and a guanylyl cyclase domain. Although NPRA plays an important physiologic role, molecular mecanisms driving its activation process are yet unknown. We thus analysed the first steps of NPRA’s activation process. First, we studied the role of ECD dimerization in receptor activation and determined the sequential steps of this dimerization process. We used radioligand binding, FRET and molecular modeling to characterize the interaction of ECD with natural agonists, a superagonist and an antagonist. ANP binds to preformed ECD dimers and spontaneous dimerization is the rate-limiting step of the ligand binding process. Furthermore, like demonstrated with fluorescence homoquenching, all the studied peptides, including A71915 antagonist, stabilize a dimeric form of the receptor. However, A71915 stabilizes the ECD dimer in a conformation distinct from those induced by ANP. Thus, ECD dimerization is necessary but not sufficient for NPRA activation. The activation state of NPRA seems to depend on the orientation of the receptor subunits within the dimer. Then, we tried to identify the molecular mechanism of signal transduction through the plasma membrane. Previous studies have shown that activation of NPRA involves a conformational change of the juxtamembrane domain (JM). However, crystallographic study of the soluble ECD of NPRA has failed to document JM structure, and the conformational change involved in transmembrane signal transduction is still unknown. To analyse this conformational change, we first sequentially substituted nine amino acids of JM by a cysteine residue. By studying the mutant’s capacity to form ANP-induced or constitutive covalent disulfide dimers, we evaluated the relative proximity of JM residues, before and after NPRA activation. These results demonstrate a high proximity of specific JM residues and are in disagreement with crystallography data. We also demonstrated that intracellular domain imposes a conformational constraint on JM at basal state, which becomes relaxed upon ANP binding. We finally confirmed, with a full-length receptor, that A71915 stabilizes NPRA in a dimeric form where JM are in a conformation distinct from the basal state. By introducing 1 to 5 alanine residues in the transmembrane α-helix, we showed that a TM rotation of 40° leads to constitutive NPRA activation. Activation signal could thus be transmitted through the membrane by a TM rotation mechanism. We finally studied the role of the TM in NPRA dimerization. By using the ToxR system, we demonstrated that the last JM residues are required to stabilize the TM dimer. Using these experimental data, we generated the first molecular model illustrating the active conformation of NPRA, where JM and TM are depicted. In summary, this study allows a better understanding of molecular mecanisms driving the first steps of NPRA’s complex activation process.