Les trois proto-oncogènes RAS, soit HRAS, KRAS et NRAS (H/K/NRAS), sont les gènes les plus fréquemment mutés dans les cancers humains. Les énormes défis liés au ciblage thérapeutique des RAS soulignent la nécessité d’approfondir notre compréhension de la biologie de ces protéines et de trouver des stratégies alternatives pour traiter les cancers qu’elles induisent. Les petites GTPases RAS sont des régulateurs fondamentaux du développement et se lient à des protéines effectrices distinctes pour transmettre des signaux afin de réguler diverses voies de signalisation intracellulaires. Les effecteurs de RAS sont définis par un domaine de liaison à RAS (RBD) qui reconnaît la conformation active de RAS liée au GTP et active les voies de signalisation en aval. Par exemple, les effecteurs RAF et PI3K régulent les voies de signalisation MAPK et PI3K-AKT, respectivement, pour contrôler la prolifération, la survie et la tumorigenése. Alors que RASSF5 dirige RAS vers la voie Hippo, suppresseur de tumeur, mais cela reste moins bien compris. Il est intéressant de noter que la famille des domaines d'association à RAS (RASSF) comprend 10 effecteurs RAS supposés en aval, chacun comprenant un RBD, mais seul le RASSF5 se lie à H/K/NRAS. Les RASSF sont des suppresseurs de tumeurs connus et comptent parmi les protéines les plus fréquemment régulées à la baisse dans les cancers. La superfamille des petites GTPases RAS compte chez l’humain environ 160 protéines regroupées en cinq sous-familles : RAS, RHO, RAN, RAB et ARF. Alors que H/K/NRAS sont les mieux caractérisées et ont été au centre de la recherche sur le cancer, les fonctions cellulaires, la régulation et les protéines effectrices de nombreuses autres GTPases de la superfamille RAS restent obscures. Ma recherche doctorale visait donc à étudier le rôle des effecteurs de RASSF en cartographiant les interactions de BRAF et de quatre protéines de RASSF avec 83 GTPases appartenant aux sous-familles RAS, RHO et ARF et à utiliser ces connaissances pour démêler l'interaction complexe entre les GTPases et les effecteurs. Nous avons abordé des questions clés sur la spécificité des RBD envers les GTPases et avons révélé et validé 39 interactions RASSF-GTPase. Nous avons constaté qu'alors que BRAF démontre une spécificité restreinte pour les H/K/NRAS classiques, RASSF fait preuve de plasticité dans ses interactions avec les GTPases. RASSF5 interagit avec 10 GTPases distinctes de la sous-famille RAS (H/K/NRAS, RAP2B/2C, RRAS1/2, MRAS et RIT1/2) qui favorisent la croissance. La présence d’un complexe RASSF5-GTPase à la membrane plasmique redistribue la protéine YAP dans le cytosol et active la signalisation Hippo. Nous avons également montré que l'interaction de RASSF5 avec les kinases MST est essentielle pour l'activation de la voie Hippo médiée par le complexe RASSF5-GTPase. Nous avons également révélé que RASSF3, RASSF4 et RASSF8 lient les GTPases de la sous-famille RAS inhibitrices de croissance. RASSF8 subit une séparation de type liquide-liquide et réside avec YAP dans des gouttelettes non-membranaires. De plus, l'expression des partenaires GTPase de RASSF8 redistribue les condensats de RASSF8 et YAP de grandes structures périnucléaires. YAP et la voie Hippo entraînent une résistance aux inhibiteurs de RAS dans les cancers induits par RAS. Ainsi, nos découvertes sur l'association de RASSF5 et RASSF8 avec la voie Hippo pourraient aider à élucider les liens manquants entre les signalisations RAS et Hippo. Nous avons également identifié RASSF3 comme le premier effecteur canonique de MIRO1/2, des GTPases mitochondriales essentielles pour le fonctionnement et l'homéostasie des mitochondries. L'interaction de RASSF3 avec MIRO dans les mitochondries entraîne un effondrement du réseau mitochondrial. Pour comprendre la dynamique du réseau des GTPases, nous développons un outil de GTPase piégée inductible par la rapamycine. Ainsi, le piège qui garde la GTPase surexprimée inactive peut être libérée et la GTPase activée de manière conditionnelle en utilisant le traitement à la rapamycine. Cet outil sera utile pour élucider le rôle précis de chaque GTPase dans la régulation des effecteurs en aval in cellulo. Par conséquent, cette étude révèle la nature complexe des interactions entre GTPases et effecteurs et met en lumière l'importance biologique des protéines RASSF. / The three RAS proto-oncogenes, namely HRAS, KRAS and NRAS (H/K/NRAS) are the most frequently mutated genes in human cancers. H/K/NRAS small GTPases are fundamental regulators of development and bind distinct effector proteins to transmit signals to diverse cellular pathways. RAS effectors are defined by a RAS-binding domain (RBD) which recognizes the GTP-bound activated conformation of RAS and activates downstream signalling pathways. For example, RAF and PI3K effectors regulate the MAPK and PI3K-AKT signalling pathways, respectively, to control proliferation, survival and tumorigenesis. Whereas RASSF5 directs RAS to the tumour suppressor Hippo pathway but this remains less understood. Interestingly, the RAS Association domain family (RASSF) comprises 10 purported downstream RAS effectors, each of which comprises an RBD, but only RASSF5 binds to H/K/NRAS. RASSF are known tumour suppressors and are among the most frequently downregulated proteins in cancers. There are approximately 160 proteins in the human RAS superfamily that are clustered into five subfamilies: RAS, RHO, RAN, RAB and ARF. While H/K/NRAS are the best-characterized and have been a principal focus of cancer research, cellular functions, regulation and effectors for many other GTPases of the RAS superfamily remain recondite. My doctoral research therefore aimed to investigate the role of RASSF effectors by mapping the interactions of BRAF and four RASSF proteins with 83 GTPases belonging to the RAS, RHO and ARF subfamilies and use this knowledge to unravel the complex interplay between GTPase and effectors. I uncovered 39 RASSF–GTPase interactions and addressed key questions on RBD specificity towards GTPases. I found that while BRAF demonstrates restricted specificity for classical H/K/NRAS, RASSF shows plasticity in its interaction with GTPases. RASSF5 interacts with 10 distinct growth-promoting GTPases of the RAS subfamily (H/K/NRAS, RAP2B/2C, RRAS1/2, MRAS and RIT1/2). RASSF5–GTPase complex at the plasma membrane redistributes YAP to the cytosol and activates Hippo signalling. I also showed that RASSF5 interaction with MST hippo kinases is essential for RASSF5–GTPase complex-mediated activation of the Hippo pathway. I further revealed that RASSF3, RASSF4 and RASSF8 bind distinct growth-inhibiting RAS subfamily GTPases. RASSF8 undergoes liquid-liquid phase separation and resides in membraneless, phase-separated YAP condensates. Further, the expression of GTPase partners of RASSF8 redistributes RASSF8 and YAP condensates to large peri-nuclear structures. These findings show several GTPase–RASSF complexes play a role in Hippo signalling which may serve as potential therapeutic targets for RAS- or YAP-driven cancers. I also identified RASSF3 as the first canonical effector of MIRO1/2, mitochondrial GTPases that are essential for mitochondrial functions and homeostasis. RASSF3 interaction with MIRO at the mitochondria results in a collapse of the mitochondrial network. To understand the dynamics of the GTPase network, I am further developing a rapamycin-inducible trapped GTPase (RITG) tool, wherein a GTPase can be overexpressed while remaining occluded, and can be conditionally released or activated. This tool can be useful in elucidating the role of GTPases in the regulation of downstream effectors in cellulo. Overall, this study reveals the complex nature of GTPase–effector interactions and uncovers the biological significance of RASSF proteins.

Résumé : L’expression de surface des récepteurs couplés aux protéines G (GPCRs) est un processus hautement régulé et très important dans le maintien de l’homéostasie cellulaire. En effet, un déséquilibre dans leur niveau d’expression est souvent relié à différentes pathologies comme le cancer, le diabète, l’obésité, les maladies cardiovasculaires et les maladies neurodégénératives. C’est pourquoi la compréhension des mécanismes moléculaires influençant ce phénomène est si importante et nous permettra d’élaborer et/ou d’améliorer les médicaments ciblant la régulation de ce processus. Il est bien connu qu’un des acteurs importants dans le trafic vésiculaire des GPCRs est représenté par la famille des Rab GTPases. Effectivement plusieurs de ces dernières, soit les Rabs 1, 2, 4, 5, 6, 7, 8 et 11 pour ne nommer que les plus connues, modulent l’expression de surface des GPCRs. De plus, certaines études soulèvent la possibilité qu’un GPCR soit lui-même capable de réguler son propre trafic intracellulaire, et ce grâce à son interaction avec les Rab GTPases. Toutefois, le mécanisme emprunté par le GPCR pour atteindre cette fin reste à élucider. Dans le présent travail, je démontre que le GPCR, β2AR, module non seulement la maturation de la petite protéine G Rab11a grâce à son interaction avec la Rab GéranylGéranylTransférase (RGGT), mais influence également son activation en modulant son ubiquitination via son association avec la E3-ubiquitine ligase, HACE1. De plus, je révèle que la sous-unité alpha de la RGGT (RGGTA) accroît significativement la maturation et le transport antérograde du récepteur β2AR, ce qui souligne ainsi un nouveau rôle cellulaire pour cette protéine. L’ensemble des résultats générés appuie l’hypothèse qu’un GPCR puisse contrôler son propre routage intracellulaire, et éclaircit les mécanismes utilisés pour réguler l’activé de la Rab GTPase avec laquelle il interagit. // Abstract : Cell surface expression of G Protein-Coupled Receptors (GPCRs) is a highly regulated and very important phenomenon for keeping cellular homeostasis. In fact, dysregulation of their cell expression is related to many diseases like cancer, neurological disorders, obesity, diabetes and cardiovascular diseases. These facts illustrate how important understanding the molecular mechanisms involved in cell surface transport of those receptors is, which will help us in designing or improving drugs which actually target this pathway. Rab GTPases are proteins known for being essential regulators of GPCR vesicular trafficking. Indeed, an increasing number of studies report the implication of Rab1, 2, 4, 5, 6, 7, 8 and 11 (to cite the most frequently studied) cell surface transport of GPCRs. Moreover, some studies also put forward the possibility that a GPCR might be able to regulate its own cellular trafficking by interacting and controlling activation of Rab GTPases. However, the mechanism involved in this process remains to be clarified. In the present study, I demonstrate that the prototypic GPCR, β2AR, not only modulates prenylation/maturation of the small G protein Rab11a by interacting with Rab GeranylGeranylTransferase (RGGT), but also influences Rab11a activation by modulating its ubiquitination via its association with the E3-ubiquitin ligase, HACE1. Furthermore, I reveal that the α subunit of the RGGT (RGGTA) also promotes the maturation and anterograde transport of the receptor, which highlight a new cellular role for this protein. Altogether, those results support the hypothesis that GPCRs control their own trafficking, and shed light on some of the mechanisms that might be employed by those receptors in activation of Rab GTPases.

Le transport intracellulaire du cholestérol et sa distribution correcte au niveau des différentes membranes sont essentiels pour assurer de nombreuses fonctions cellulaires. Malgré l'importance de ce transport les mécanismes de sa régulation restent encore mal connus. L'objectif de cette thèse était de mieux caractériser les acteurs du transport intracellulaire du cholestérol. Dans ce contexte, nous nous sommes intéressés à deux acteurs de ce transport : la dynamine et les Rab GTPases. Dans la première partie de la thèse nous avons utilisé le dynasore, un inhibiteur pharmacologique de la dynamine pour étudier le rôle de la dynamine dans le contrôle du transport endolysosomal dans les cellules HeLa et les macrophages humains. Nous avons ainsi confirmé le rôle de la dynamine dans la sortie du compartiment endolysosomal et la régulation de l'homéostasie du cholestérol. Dans la deuxième partie de la thèse, nous avons étudié le rôle de Rab7 et de Rab9 dans le transport du cholestérol en utilisant la technique d'ARN interférence ainsi que l'expression de mutants dominant négatifs. Nous avons montré qu'en plus de son rôle classique dans les étapes tardives du transport du cholestérol, Rab7 contrôle les étapes précoces du transport endosomal. Enfin, nous avons évalué le rôle de Rab7 dans notre modèle de macrophages humains surchargés. Nous avons mis en évidence un effet limité de l'inactivation de Rab7 sur le contrôle de l'homéostasie du cholestérol mais à l'inverse un effet majeur pour l'efflux du cholestérol vers l'apo AI. En conclusion, notre étude a permis de mieux caractériser le transport vésiculaire du cholestérol et de démontrer son importance dans la régulation de l'homéostasie intracellulaire en cholestérol. Nos résultats permettent également d'établir le rôle critique de Rab7 dans le trafic des LDL au niveau des endosomes précoces.

A group of small GTPases consist of over twenty protein families in the super class P-loop. It has a very diverse cell functions. Small GTPases regulate the formation of vesicular follicles, cytoskeleton and nuclear transport. They participate also on cell proliferation and signaling. The aim of my work is to find important amino acids that define family and distinguish each other. I focus on families Arf, Rab, Ran, Ras and Rho. At the Rho family I am also devoted to classes Rho, Rac and Cdc42. Amino acids are identified using bioinformatic programs selected Consurf and Sca5. The objective is also to test P2RANK specialized tool developed at the Charles University in Prague that predict ligand binding sites from protein structure in different families. Founding amino acids can have a big role in the functional divergence of individual families and classes of small GTPases and can be the basis for future study example for the proliferation of cancerous cells. 1.1 Keywords Powered by TCPDF (www.tcpdf.org)

The present dissertation aims to shed light on the molecular machinery involved in the process of shell formation (thecagenesis) in Arcella (Arcellinida : Amoebozoa). Arcellinida are single-celled testate amoebae organisms, characterized by the presence of an outer shell (test or carapace); it is a monophyletic lineage of Amoebozoa, sister group to a naked amoeboid lineage. No homologous structure to shell is present in the sister group of Arcellinida, thus it is considered an evolutionary novelty. The origin and evolution of the shell in Arcellinida are currently open questions; deciphering its formation process is a key step to address these questions. During each reproductive process by budding division, these organisms build a new shell. In the span of more than a century, several authors have described the thecagenesis process on Arcellinida, primarily focusing on the genus \\textit, based on cyto-morphological evidence. Conversely, the absence of molecular data has impaired advances on describing the molecular aspects of shell formation. In this study, we designed and applied a molecular framework to identify candidate genes and develop a molecular model for the shell formation process in Arcella; we based this framework on single-cell RNA-sequencing, gene expression profiling, Gene Ontology analysis, and comparative analysis of cyto-morphological with newly generated molecular data. We identify and propose a set of 539 genes as the candidate genes for shell formation, based on expression profiling and biological process assignment. We propose a model for the the shell formation process, which describes the mechanistic aspect of this process, hypothetically based on a molecular machinery conserved in Eukaryotes. Additionally, we identified a massive expansion of the Rab GTPase family, a protein likely to be involved on the process of shell formation. In the lights of the present study, we briefly discuss possible evolutionary scenarios involved on the origin and evolution of the shell and present future perspectives; we propose the shell of Arcellinida as a prosperous model to study the origin and evolution of evolutionary novelties, as well as other evolutionary questions / A presente dissertação tem como objetivo lançar luz sobre a maquinaria molecular envolvida no processo de formação de teca (tecagênese) em \\textit (Arcellinida: Amoebozoa). Arcellinida são amebas tecadas unicelulares, caracterizadas pela presença de uma teca (carapaça ou concha) externa; é uma linhagem monofilética de Amoebozoa, grupo irmão de alguns organismos amebóides nus. Nenhuma estrutura homóloga à carapaça está presente no grupo irmão de Arcellinida, sendo considerada como uma novidade evolutiva. A origem e evolução da carapaça em Arcellinida são questões em aberto; Decifrar seu processo de formação é um passo fundamental para abordar essas questões. Durante todo processo reprodutivo, por divisão por brotamento, estes organismo constroem uma nova concha. No decorrer de mais de um século, vários autores descreveram o processo de tecagênese nestes organismos, focando principalmente no gênero \\textit, baseados em evidências cito-morfológicas. Enquanto isso, a ausência de dados moleculares impede avanços na descrição dos aspectos moleculares da formação de conchas. Neste estudo, projetamos e aplicamos uma \\textit molecular para identificar genes candidatos e desenvolver um modelo molecular para o processo de formação de teca em \\textit; Baseamos este \\textit em sequenciamento de RNA \\textit, perfil de expressão gênica, análise de \\textit{Gene Ontology} e análise comparativa de dados cito-morfológicos e moleculares. Nós identificamos e propomos um conjunto de 539 genes como genes candidatos para a formação de carapaça, com base no perfil de expressão e na atribuição de processos biológica. Propomos um modelo para o processo de formação de carapaça, que descreve o aspecto mecanicista deste processo, hipoteticamente baseado em um mecanismo molecular conservado em Eucariotos. Além disso, identificamos uma expansão maciça da família gênica das Rab GTPase, gene provavelmente envolvida no processo de formação de carapaça. À luz do presente estudo, discutimos brevemente possíveis cenários evolutivos envolvidos na origem e evolução da teca e apresentamos perspectivas futuras; propomos a teca dos Arcellinida como próspero modelo para estudar a origem e evolução das novidades evolutivas, bem como outras questões evolutivas

Septinas abrangem uma família conservada de proteínas que ligam e hidrolisam GTP e formam heterofilamentos, anéis e redes para realizar as suas funções. Apresentam três domínios estruturais: o domínio N-terminal contendo uma sequência polibásica (para ligar membranas), o domínio de ligação ao nucleotídeo (G) e o domínio C-terminal que inclui uma sequência predita de formar um coiled-coil. Em humanos, as 13 septinas são classificadas em quatro grupos (I, II, III e IV) baseadas nas sequências de aminoácidos. O único filamento caracterizado estruturalmente, até hoje, é o formado por SEPT2-SEPT6-SEPT7, mostrando que as subunidades interagem através de duas interfaces (chamadas G e NC). Os determinantes estruturais da montagem correta do filamento são pouco conhecidos, sendo o estudo limitado pela complexidade em purificar e cristalizar complexos triméricos ou tetraméricos. Uma abordagem alternativa é estudar interfaces individuais de um filamento (G e/ou NC) por separado. Assim, o presente projeto objetivou estudar, utilizando uma abordagem biofísica e estrutural, a interface G formada por SEPT5 e SEPT8 para elucidar os fatores importantes em determinar a sua especificidade. Os domínios GTPase de SEPT5 e SEPT8 foram clonadas em vetor de expressão bicistrônico pET-Duet, co-expressas e co-purificadas. Estudos de análise do estado oligomérico e homogeneidade foram conduzidos utilizando cromatografia de exclusão molecular, espalhamento dinâmico de luz e ultracentrifugação analítica, revelando um complexo dimérico e monodisperso. O complexo apresenta uma mistura aproximadamente equimolar de nucleotídeos (GTP e GDP) ligados enquanto SEPT8(G) sozinha é incapaz de ligar qualquer um dos dois. Além disto o complexo apresenta uma termoestabilidade maior que SEPT8(G), verificado por um aumento em Tm de 5°C. Com o intuito de observar os determinantes estruturais da especificidade, ensaios de cristalização foram conduzidos e assim, cristais do complexo SEPT5-SEPT8(G) que difrataram apenas a muito baixa resolução foram obtidos. Na ausência de uma estrutura cristalográfica, modelagem por homologia foi realizada para analisar as interfaces G entre diferentes combinações de septinas. Identificamos uma interação entre aminoácidos característicos (aminoácidos únicos para cada grupo de septinas) para o complexo formado entre membros do grupo III, (incluindo SEPT5) e membros do grupo II, (incluindo SEPT8). Esta interação entre Phe131 (grupo III) e Thr19 (grupo II) pode explicar a especificidade na formação de uma interface G entre septinas destes grupos durante a formação do filamento e além disso, a importância da presença do GTP ligado ao septina do grupo II. Com isto, propomos pela primeira vez uma explicação plausível da relevância da perda de atividade catalítica das septinas deste grupo, um fato inexplicado até o momento. Mutação dos resíduos identificados levou a uma mudança no seu perfil de eluição do complexo durante purificação por exclusão molecular indicando alterações na formação do complexo mutante. / Septins are a conserved family of proteins that bind and hydrolyze GTP and form heterofilaments, rings and networks in order to carry out their functions. They have three structural domains: an N-terminal domain containing a polybasic sequence (for membrane binding), a nucleotide-binding (G) domain and a C-terminal domain including a sequence predicted to form a coiled-coil. In humans, 13 septins have been classified into four groups (I, II, III and IV) based on their amino acid sequences. The only structurally characterized filament described to date is formed by SEPT2-SEPT6-SEPT7, which reveals that the subunits interact through two different interfaces (G and NC). The structural determinants of correct filament assembly are poorly known, and this is limited by the complexity of purifying and crystallizing trimeric or tetrameric complexes. An alternative approach is to study a single filament interface (G or NC) on its own. Here, we aimed to study, using biophysical and structural approaches, the G interface formed between SEPT5 and SEPT8 to elucidate the factors relevant to determining its specificity. The GTPase domain of SEPT5 and SEPT8, were cloned into the bicistronic expression vector pET-Duet, co-expressed and co-purified. Studies to determine the oligomeric state and homogeneity of the complex were conducted using size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation, revealing a monodisperse dimer for SEPT5-SEPT8(G). The complex elutes with an approximately equimolar mixture of bound nucleotides (GTP and GDP) whereas SEPT8(G) alone is shown to be unable to bind either. Furthermore, the complex has a greater thermostability than SEPT8(G), demonstrated by an increase of 5°C in Tm. In order to determine the structural determinants of specificity, crystallization trials were conducted and crystals of the SEPT5-SEPT8(G) complex were obtained, but these diffracted to only very low resolution. In the absence of a crystal structure, homology modeling was performed to analyze the potential G interfaces between different septin combinations. An interaction between characteristic amino acids (those which are unique to given septin group) was identified for the complex formed between group III septins (including SEPT5) and group II septins (including SEPT8). This interaction, between Phe131 (group II) and Thr19 (group III) may explain the specificity in the formation of a G interface between septins of these groups during filament formation and furthermore the importance of GTP bound to the group II septin. These observations allow us to propose for the first time a plausible explanation for relevance of the loss of catalytic activity by this septin group, an unexplained fact up until now. Mutation of the identified residues resulted in a change in the elution profile of the complex from the size exclusion column suggesting structural alterations in the mutants.

Septinas abrangem uma família conservada de proteínas que ligam e hidrolisam GTP e formam heterofilamentos, anéis e redes para realizar as suas funções. Apresentam três domínios estruturais: o domínio N-terminal contendo uma sequência polibásica (para ligar membranas), o domínio de ligação ao nucleotídeo (G) e o domínio C-terminal que inclui uma sequência predita de formar um coiled-coil. Em humanos, as 13 septinas são classificadas em quatro grupos (I, II, III e IV) baseadas nas sequências de aminoácidos. O único filamento caracterizado estruturalmente, até hoje, é o formado por SEPT2-SEPT6-SEPT7, mostrando que as subunidades interagem através de duas interfaces (chamadas G e NC). Os determinantes estruturais da montagem correta do filamento são pouco conhecidos, sendo o estudo limitado pela complexidade em purificar e cristalizar complexos triméricos ou tetraméricos. Uma abordagem alternativa é estudar interfaces individuais de um filamento (G e/ou NC) por separado. Assim, o presente projeto objetivou estudar, utilizando uma abordagem biofísica e estrutural, a interface G formada por SEPT5 e SEPT8 para elucidar os fatores importantes em determinar a sua especificidade. Os domínios GTPase de SEPT5 e SEPT8 foram clonadas em vetor de expressão bicistrônico pET-Duet, co-expressas e co-purificadas. Estudos de análise do estado oligomérico e homogeneidade foram conduzidos utilizando cromatografia de exclusão molecular, espalhamento dinâmico de luz e ultracentrifugação analítica, revelando um complexo dimérico e monodisperso. O complexo apresenta uma mistura aproximadamente equimolar de nucleotídeos (GTP e GDP) ligados enquanto SEPT8(G) sozinha é incapaz de ligar qualquer um dos dois. Além disto o complexo apresenta uma termoestabilidade maior que SEPT8(G), verificado por um aumento em Tm de 5°C. Com o intuito de observar os determinantes estruturais da especificidade, ensaios de cristalização foram conduzidos e assim, cristais do complexo SEPT5-SEPT8(G) que difrataram apenas a muito baixa resolução foram obtidos. Na ausência de uma estrutura cristalográfica, modelagem por homologia foi realizada para analisar as interfaces G entre diferentes combinações de septinas. Identificamos uma interação entre aminoácidos característicos (aminoácidos únicos para cada grupo de septinas) para o complexo formado entre membros do grupo III, (incluindo SEPT5) e membros do grupo II, (incluindo SEPT8). Esta interação entre Phe131 (grupo III) e Thr19 (grupo II) pode explicar a especificidade na formação de uma interface G entre septinas destes grupos durante a formação do filamento e além disso, a importância da presença do GTP ligado ao septina do grupo II. Com isto, propomos pela primeira vez uma explicação plausível da relevância da perda de atividade catalítica das septinas deste grupo, um fato inexplicado até o momento. Mutação dos resíduos identificados levou a uma mudança no seu perfil de eluição do complexo durante purificação por exclusão molecular indicando alterações na formação do complexo mutante. / Septins are a conserved family of proteins that bind and hydrolyze GTP and form heterofilaments, rings and networks in order to carry out their functions. They have three structural domains: an N-terminal domain containing a polybasic sequence (for membrane binding), a nucleotide-binding (G) domain and a C-terminal domain including a sequence predicted to form a coiled-coil. In humans, 13 septins have been classified into four groups (I, II, III and IV) based on their amino acid sequences. The only structurally characterized filament described to date is formed by SEPT2-SEPT6-SEPT7, which reveals that the subunits interact through two different interfaces (G and NC). The structural determinants of correct filament assembly are poorly known, and this is limited by the complexity of purifying and crystallizing trimeric or tetrameric complexes. An alternative approach is to study a single filament interface (G or NC) on its own. Here, we aimed to study, using biophysical and structural approaches, the G interface formed between SEPT5 and SEPT8 to elucidate the factors relevant to determining its specificity. The GTPase domain of SEPT5 and SEPT8, were cloned into the bicistronic expression vector pET-Duet, co-expressed and co-purified. Studies to determine the oligomeric state and homogeneity of the complex were conducted using size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation, revealing a monodisperse dimer for SEPT5-SEPT8(G). The complex elutes with an approximately equimolar mixture of bound nucleotides (GTP and GDP) whereas SEPT8(G) alone is shown to be unable to bind either. Furthermore, the complex has a greater thermostability than SEPT8(G), demonstrated by an increase of 5°C in Tm. In order to determine the structural determinants of specificity, crystallization trials were conducted and crystals of the SEPT5-SEPT8(G) complex were obtained, but these diffracted to only very low resolution. In the absence of a crystal structure, homology modeling was performed to analyze the potential G interfaces between different septin combinations. An interaction between characteristic amino acids (those which are unique to given septin group) was identified for the complex formed between group III septins (including SEPT5) and group II septins (including SEPT8). This interaction, between Phe131 (group II) and Thr19 (group III) may explain the specificity in the formation of a G interface between septins of these groups during filament formation and furthermore the importance of GTP bound to the group II septin. These observations allow us to propose for the first time a plausible explanation for relevance of the loss of catalytic activity by this septin group, an unexplained fact up until now. Mutation of the identified residues resulted in a change in the elution profile of the complex from the size exclusion column suggesting structural alterations in the mutants.

Current drugs used to treat Congestive Heart Failure target the renin-angiotensin and adrenergic systems. Studies showed increased mortality rates in patients treated with a combination of these medications. Angiotensin-AT1 and ?2-Adrenergic receptors were shown to form receptor heteromers. Blockade of one receptor in the complex can affect the signal transmitted by the other; suggesting that ligand-based therapy is not as selective as we might think. Modulating receptor trafficking after synthesis might prove to be a valid therapeutic strategy. Unfortunately, little is known about receptor assembly and transport from Endoplasmic Reticulum to Plasma Membrane. The objectives of this study are to identify the proteins that participate in the assembly of AT1R-?2AR heteromer and the regulators of the anterograde trafficking of G-Protein Coupled Receptors. This thesis introduces the role of important targets in those poorly understood processes. The identification of such targets could lead to developing better drugs with fewer adverse effects.

The Rho family small GTPases are key regulators of the cytoskeleton, through which they impact and control many vital cellular functions, including growth, vesicle trafficking, intercellular junctions, transepithelial transport, migration, and gene transcription. Activation of Rho GTPases is induced by Guanine Nucleotide Exchange Factors (GEFs). We have previously shown that Tumour Necrosis Factor-α (TNF), plasma membrane depolarization, and immunosuppressive drugs activate RhoA through a specific exchange factor, GEF-H1. However, the question of whether other stimuli, such as hyperosmolarity, that activate RhoA, act through GEF-H1 and whether GEF-H1 activates other RhoGTPases was not known. The overall objective of this research project has been to gain insights into the complex mechanism through which the Rho GTPases, Rac and RhoA, are regulated in tubular cells. Specifically, we wished to explore the role and pathway-specific regulation of GEF-H1 in hyperosmotic stress- and TNF-induced signalling in tubular cells. In order to accomplish our goals, we optimized and used affinity precipitation assays to detect GEF-H1 activation (RhoA(G17A) and Rac(G15A)). We found that 1) GEF-H1 is activated by hyperosmotic stress and mediates the hyperosmolarity-induced RhoA activation, as well as nuclear translocation of the Myocardin-Related Transcription Factor (MRTF); 2) TNF induces activation of both Rac and RhoA through GEF-H1, but via different mechanisms. Epidermal Growth Factor Receptor (EGFR)- and Extracellular signal Regulated Kinase (ERK)-dependent phosphorylation at the Thr678 site of GEF-H1 is a prerequisite for RhoA activation only, while both Rac and RhoA activation require GEF-H1 phosphorylation on Ser885. Interestingly, Rac is required for TNF-induced RhoA activation. Together these findings highlight a role for GEF-H1 as an osmosensitive molecule that regulates cellular reprogramming through MRTF. Importantly, we have also uncovered a novel mechanism explaining hierarchical activation of Rac and RhoA by TNF. Such a mechanism could be key in coordinating GEF function and fine-tuning Rac and RhoA activation.

Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. Steroid hormones, such as estrogen, and growth factors, which include insulin growth factor I/II (IGF-1/IGF-2) therapy has been associated with most if not all of the features of metastasis. It has been determined that IGF-1 increases cell survival of cancer cells and potentiate the effect of E2 and other ligand growth factors on breast cancer cells. However not much information is available that comprehensively expounds on the roles of insulin growth factor receptor (IGFR) and Rab GTPases may play in breast cancer. The latter, Rab GTPases, are small signaling molecules and critical in the regulation of many cellular processes including cell migration, growth via the endocytic pathway. This research involves the role of Rab GTPases, specifically Rab5 and its guanine exchange factors (GEFs), in the promotion of cancer cell migration and invasion. Two important questions abound: Are IGFR stimulation and downstream effect involved the endocytic pathway in carcinogenesis? What role does Rab5 play in cell migration and invasion of cancer cells? The hypothesis is that growth factor signaling is dependent on Rab5 activity in mediating the aggressiveness of cancer cells. The goal is to demonstrate that IGF-1 signaling is dependent on Rab5 function in breast cancer progression. Here, the results thus far, have shown that while activation of Rab5 may mediate increased cell proliferation, migration and invasion in breast cancer cells, the Rab5 GEF, RIN1 interacts with the IGFR thereby facilitating migration and invasion activities in breast cells. Furthermore, endocytosis of the IGFR in breast cancer cells seems to be caveolin dependent as the data has shown. This taken together, the data shows that IGF-1 signaling in breast cancer cells relies on IGF-1R phosphorylation, caveolae internalization and sequestration to the early endosome RIN1 function and Rab5 activation.