Una de las principales cuestiones en farmacología molecular de los GPCR es entender los mecanismos estructurales de las siete hélices transmembrana (TM) que se producen para estabilizar ya sea Rg o los diferentes estados R*. Para entender el mecanismo que cambia el equilibrio del conjunto a un estado activo R* se construyeron tres de los receptores de la serotonina (5-HT4, 5-HT6, y 5 HT7) sobre la base de su información más reciente de cristalografía de rayos X. Dando lugar a dos modelos de cada receptor: una inactiva y otra activa. Los modelos, mejorados y evaluados con la ayuda de datos farmacológicos y químicos se utilizaron principalmente para comprender la interacción entre un ligando y su receptor y su mecanismo de acción. Estos hallazgos estructurales pueden a su vez resultar útiles para el diseño de nuevos fármacos más eficaces y selectivos. / One of the main questions in G protein coupled receptors (GPCRs) molecular pharmacology is to understand the structural arrangements of the seven transmembrane (TM) helices that occur to stabilize either the ground state (Rg) or different active states (R*) of the receptors. In order to understand the mechanism that shift the equilibrium of the ensemble to an active R* state models of the inactive and the active state of three serotonin receptors (5-HT4, 5-HT6, and 5-HT7) were built based on the latest information from X-ray crystallography. The resulting models were mainly used to understand the interaction between a ligand and its receptor and the mechanism of action. With the help of pharmacological and chemical data these models and complexes were improved and evaluated. These findings may prove valuable for structural based drug discovery efforts and facilitate the design of more effective and selective pharmaceuticals.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 25, 2009). Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 139-155).

La petite protéine G Arf6 et son facteur d'échange EFA6 sont impliquées dans de nombreux processus cellulaires tels que le remodelage du cytosquelette d’actine, le transport vésiculaire et mise en place de la polarité épithéliale. Elles jouent également un rôle dans la voie d'endocytose dépendante de la clathrine. Ce travail de thèse nous a permis d’identifier différents mécanismes régulant l’interaction d’EFA6 avec ses différents partenaires. Nous avons pu mettre en évidence une interaction directe entre le domaine N-BAR de l’endophiline et le domaine Sec7 d’EFA6. Nous avons démontré que la courbure membranaire était un facteur régulant cette interaction. EFA6 est capable d’interagir et de recruter l’endophiline sur une membrane lipidique plane alors qu’en présence de vésicules courbées le complexe protéique ne se forme pas. Nous observons également que l’endophiline stimule l’activité d’échange nucléotidique d’EFA6 sur Arf6. Dans un second temps nous avons démontré, dans une étude menée par le Dr Cherfils, que l’activité catalytique d’EFA6 était régulée par une boucle de rétrocontrôle négatif exercée spécifiquement par la protéine Arf6-GTP. Celle-ci induit une diminution de l’activité d’échange d’EFA6 probablement grâce à sa capacité à interagir avec le domaine PH-C-terminal d’EFA6. Enfin, nous avons mis en évidence un repli intramoléculaire entre le domaine C-terminal et le domaine PH d’EFA6 qui semble contrôler l’interaction de cette extrémité C-terminale avec différents partenaires dont la β-arrestine et de façon surprenante la protéine Arf6 dans sa forme inactive. / The small G protein Arf6 and its exchange factor EFA6 control numerous cellular processes such as actin cytoskeleton remodeling, vesicular transport and apico-basal cell polarity. They are also involved in clathrin-dependent endocytosis. In this work we identify different mechanisms by which EFA6 interaction with its various partners is regulated. We have highlighted a direct interaction between the N-BAR domain of endophilin and the Sec7 domain of EFA6. We demonstrated that this interaction is regulated by the membrane curvature. EFA6 interacts and recruits endophilin on a flat lipid membrane whereas the protein complex does not occur in the presence of curved vesicules. We showed that endophilin stimulates the nucleotidic exchange activity of EFA6 on Arf6. Next we demonstrated that the catalytic activity of EFA6 is regulated by a negative feedback loop specifically mediated by the Arf6-GTP. We observed in the presence of Arf6-GTP a decrease of EFA6 catalytic activity and we showed that this effect was due to an interaction between Arf6-GTP and PH-C-terminal domain of EFA6. Finally we demonstrated an intramolecular folding between the C-terminal domain and the PH domain of EFA6 that controls the interaction of the C-terminus domain with various partners including β-arrestin and surprisingly the inactive GDP form of Arf6.

α-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. The ability of α-factor receptors to form oligomeric complexes with each other and with other proteins was investigated. Both in vivo and in vitroevidence was obtained that suggests homo-oligomerization of receptors in the plasma membrane. When the membranes from cells coexpressing two differentially-tagged receptors were solubilized with detergent and subjected to immunoprecipitation, the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Treatment of cultures with α-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind α-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. Therefore, α-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. A crosslinking and immunoprecipitation strategy was used to capture and characterize the transient complexes that contain the α-factor receptor Ste2. Tagged receptors were crosslinked to form at least three high molecular weight complexes and the complexes were immunoprecipitated with antibodies against the tag. Western blotting analysis of the precipitated material revealed the presence of β and γ subunits of the heterotrimeric G protein, Ste4 and Stel8. Similar results were obtained when the cultures had been treated with α-factor prior to analysis. A truncated receptor missing most of the cytoplasmic C-terminal tail was also active in binding Ste4. Overall, these results constitute the first biochemical evidence for a physical association between the α-factor receptor and its cognate G-protein. Endocytic signals in the C-terminal tail (residues 297-431) of the α-factor receptor were analyzed. One signaling element, SINNDAKSS, (residues 331-339) is known to be sufficient (but not necessary) for endocytosis. Internal deletions of the STE2 gene were constructed that remove sequences encoding SINNDAKSS and selected regions of the C-terminal tail. Strains containing these alelles were then assayed for endocytosis in the presence and absence of α-factor. Residues from 360 to 431 were sufficient to mediate both constitutive and ligand-mediated endocytosis of the receptor even though 63 residues including the SINNDAKSS motif had been removed. Structural features of this region that were investigated further were the highly-ubiquitinated Lys374, the neighboring Lys387, and the GPFAD motif (residues 392-396). Lys374 and Lys387 were unnecessary for the element to promote exit from the plasma membrane; however, Lys374 may play some role in intracellular trafficking. The GPFAD motif was not sufficient to promote endocytosis, since the residues 360-399 provided no detectable endocytic activity. Overall, these results suggest that a new region in the C-terminal of the α-factor receptor, redundant with the SINNDAKSS motif, is sufficient to mediate the constitutive endocytosis as well as the ligand-mediated endocytosis of the receptor.

Cdc42 is a member of the Rho family of small GTPases, which, together with its homologues RhoA and Rac1, controls a multitude of cellular functions via the actin cytoskeleton. Cdc42 exerts its effects on the cytoskeleton via effector proteins of the Wiskott-Aldrich Syndrome (WASP) family and the Transducer of Cdc42-dependent Actin assembly (TOCA) family. The WASP family and their activation by Cdc42 have been thoroughly studied in vitro and are well understood. Conversely, understanding of the TOCA family remains limited by a lack of biochemical, biophysical and structural insight. An investigation of the TOCA1-Cdc42 interaction is described here, revealing a relatively low affinity interaction with a dissociation constant in the micromolar range. This is 10-100x weaker than other Rho-effector interactions and suggests that TOCA1 must first be co-localised with Cdc42 to achieve stable binding in vivo. The solution NMR structure of the Cdc42 binding HR1 domain of TOCA1 provides the first structural data on this protein and reveals some interesting structural features that may relate to binding affinity and specificity. A structural model of the Cdc42-HR1 complex provides further insight into differential specificities and affinities of GTPase-effector interactions. NMR and actin polymerisation assays provide insight into the pathway of Cdc42/TOCA1/WASP-dependent actin assembly, suggesting unidirectional displacement of TOCA1 by N-WASP. A comparison of the Cdc42- TOCA1 model with an NMR structure of Cdc42 in complex with the GTPase binding domain of WASP reveals a possible mechanism by which an ‘effector handover’ from TOCA1 to N-WASP could take place. Small GTPases such as Cdc42 are lipid modified and membrane anchored via their C- termini in vivo, so in vitro studies using truncated, unmodified GTPases are limited in their biological interpretation. This project also aimed to develop methods to study full length and membrane-anchored GTPases in vitro. Lipid modified protein was produced, which showed a weak affinity for liposomes, and so structural studies of membrane anchored protein are within reach. Further method development is now required to achieve stable membrane anchoring of lipid modified GTPases for detailed NMR studies.

Orientador: Fabio Papes / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T08:04:04Z (GMT). No. of bitstreams: 1 Nakahara_ThiagoSeike_M.pdf: 27685441 bytes, checksum: c755af1be54c3ba9b204bed05559dd88 (MD5) Previous issue date: 2014 / Resumo: O Sistema Olfativo é um Sistema Sensorial complexo, composto por diversos subsistemas cuja integração no cérebro resulta na interação entre os animais e seus respectivos ambientes de maneira adequada. Essa adequação pode significar respostas comportamentais e fisiológicas distintas para situações diversas a que esses animais tenham sido expostos. Esse Sistema exibe compartimentos especializados na detecção de estímulos de uma mesma natureza e nesse contexto, o Sistema Olfativo Principal é responsável pela detecção de odorantes voláteis em geral e o Sistema Olfativo Acessório é responsável pela detecção de feromônios. Apesar dessa divisão formal, estudos recentes questionam essa divisão e propõem sobreposição entre a função desses subsistemas. Nesse estudo investigamos a expressão de receptores OR sendo expressos no Órgão Vomerosasal em níveis comparáveis aos receptores V2R ("endógenos"). Desses receptores, isolamos o receptor Olfr692 que possui o nível de expressão mais alto entre os OR estudados ou relatados anteriormente na literatura. As células que expressam o receptor Olfr692 foram caracterizadas molecularmente e foram feitos estudos preliminares a fim de investigar a função do receptor Olfr692 frente a possíveis funções biológicas que fossem capazes de explicar a expressão robusta de um receptor de classe OR no Órgão Vomeronasal / Abstract: The Olfactory System is a complex Sensorial System, comprised of some subsystems whose integration in the brain results in the appropriated interaction between animals and their environment, that is, proper behavioral or physiological answers to diverse situations to which these animals are exposed. This System exhibits specialized features for detection of a given kind of stimuli. The Main Olfactory System detects volatile odorants in general while the Accessory Olfactory System detects pheromones. Apart from this formal distinction, recent studies have questioned this division and propose some overlap between them. In the present study, we have investigated the expression of OR receptors in the Vomeronasal Organ whose expression level is compared to V2R Receptors (endogenous). We have isolated from these genes the Olfr692, which has the higher levels among the VNO-OR here studied and those discussed in the literature. These cells have been molecularly characterized and preliminary functional studies were also performed, searching for the possible biological functions of this Receptor, which could explain its expression in the Vomeronasal Organ / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Orientador: Fabio Papes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T21:10:44Z (GMT). No. of bitstreams: 1 Cardozo_LeonardoMinete_M.pdf: 30220846 bytes, checksum: 6793ed2dd7e959cbc81a5c912cd1c987 (MD5) Previous issue date: 2012 / Resumo: Uma propriedade fundamental do sistema nervoso em todas as espécies animais e a transformação dos estímulos sensoriais em atividade neural, levando a mudanças comportamentais e endócrinas. Dentre os sistemas sensoriais, o Sistema Olfatório destaca-se por sua complexidade molecular, capacidade de detecção de odores e modulação de comportamentos inatos. Entretanto, ainda muito pouco e conhecido sobre como este Sistema detecta, processa e interpreta as informações químicas que recebe do meio externo... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: A fundamental property of the nervous system in all animal species is the transformation of sensory stimulation into neural activity, leading to endocrine and behavioral changes. Among the sensory systems, the olfactory system stands out due to its molecular complexity, detection capacity and the modulation of innate behaviors. However, little is known about how this system detects, processes and interprets chemosignals from the environment... Note: The complete abstract is available with the full electronic document / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

GPCRs form the largest family of membrane proteins in human genome and mediate signal transmission in a wide panel of essential physiological processes, and they are thus a major source of pharmaceutical targets. Investigating GPCR interactions with their cognate ligands and their membrane environment is crucial to understand their function at a molecular level. While major breakthroughs in the determination of high resolution structures of GPCRs in inactive and active states have shed a new light on the structural basis of GPCR activation process, complementary approaches are needed to investigate its dynamic aspects in the context of a native lipid environment. Our research work falls within this scope and hinges on two main issues: on the one hand, understand which structural features of the agonist underlie the activation of S1P receptors; on the other hand determine if membrane lipids modulate the structure and the function of the vasopressin V2 receptor (V2R). First, we investigated the functional response of S1P1, S1P2, S1P4 and S1P5 receptors expressed in mammalian cells to a series of synthetic derivatives of the native ligand sphingosine-1-phosphate, of variable alkyl chain length. Our data demonstrated that the hydrophobic tail of the ligand is crucial to induce activation in S1P receptors family, and revealed subtype-specificities regarding the influence of the alkyl chain length. Our experimental results combined with molecular dynamics simulation lead us to propose an activation mechanism for S1P receptors family. In the second part of our work, we reconstituted purified V2R into systems of controlled lipid composition, mimicking the membrane bilayer. Structural and functional characterization of the receptor in different lipid environments, using infrared and fluorescence spectroscopy approaches, revealed that the lipid composition affects V2R conformation and its interaction with a specific ligand. Taken together, our research work contributes to a better understanding of GPCRs activation mechanism and its regulation by lipid environment. / Les récepteurs couplés aux protéines G (GPCRs) forment la plus grande famille de protéines membranaires du génome humain et contribuent à une kyrielle de processus physiologiques essentiels, qui leur confèrent un intérêt pharmacologique majeur. Étudier l'interaction de ces protéines avec leurs ligands et leur environnement membranaire est primordial pour appréhender leur fonctionnement à l’échelle moléculaire. Bien que de remarquables avancées dans la détermination de structures à haute résolution de GPCRs à l'état inactif et actif aient permis de comprendre certaines bases structurales du fonctionnement des récepteurs, des approches complémentaires donnant un aperçu des aspects dynamiques et dans un environnement natif sont nécessaires pour cerner pleinement leur mécanisme d'activation. Notre travail de thèse s'inscrit dans cette problématique et s'articule autour de deux sujets: d'une part, comprendre quelles caractéristiques structurales du ligand sous-tendent l'activation de la famille des récepteurs au sphingosine-1-phosphate (S1P); d'autre part, déterminer si les lipides de la membrane plasmique modulent la structure et la fonction du récepteur à la vasopressine V2. Pour répondre à notre première question, nous avons étudié la réponse fonctionnelle en système cellulaire des récepteurs S1P1, S1P2, S1P4 et S1P5 à des composés synthétiques dérivés du S1P, portant des chaînes alkyles de longueur variable. Nos données mettent en évidence que la longueur de la chaîne hydrocarbonée du ligand est un paramètre crucial dans sa capacité d'induire l'activation du récepteur et ce pour l'ensemble des sous-types étudiés. De plus, nos résultats suggèrent que le comportement vis-à-vis de la longueur de chaîne dépend du sous-type de récepteur considéré. Nos résultats expérimentaux, combinés à une approche de modélisation dynamique, ont abouti à proposer un mécanisme d'activation pour la famille des récepteurs au S1P. Dans le second volet de notre travail, nous avons reconstitué le récepteur V2 purifié dans des systèmes de composition lipidique contrôlée, mimant la bicouche membranaire. Nous avons procédé à la caractérisation structurale et fonctionnelle du récepteur inséré dans différentes types de lipides, par des méthodes spectroscopiques infrarouge et de fluorescence. Les données obtenues suggèrent que la composition lipidique affecte la conformation et la fonction du récepteur. L'ensemble de nos travaux contribue ainsi à une meilleure compréhension du mécanisme d'activation des GPCRs et de leur régulation par l'environnement lipidique. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished