Le carcinome hépatocellulaire (CHC) et le cholangiocarcinome (CCA) sont 2 types de cancer primitif du foie. Le CHC est le plus fréquent, cependant l’incidence du CCA augmente partout dans le monde avec un diagnostic difficile, un mauvais pronostic et des thérapies très limitées. Ce travail avait pour objectif d'identifier des cibles pour le diagnostic et la thérapeutique du CCA. Il est basé sur l'étude de la septine 9 et des phosphoinositides (PIs). La septine 9 appartient à une famille de GTPases qui participent à l’organisation des microtubules et du cytosquelette d’actine. Les septines sont impliquées dans la cytokinèse, le trafic vésiculaire et la polarité cellulaire, elles sont aussi des partenaires importantes des PIs. Pour déterminer le rôle de la septine 9 dans le CCA nous nous sommes intéressés à son interaction avec les PIs et avec l’inhibiteur de l’inducteur et activateur de la transcription 1 (PIAS1) qui a été décrite comme une protéine pouvant agir comme une SUMO ligase pour les septines. Nous avons étudié l’expression de la septine 9 et de PIAS1 dans le CCA et le CHC. Nous avons mis en évidence un mécanisme original par lequel, la production du PtdIns5P (Phosphatidylinositol -5-phosphate) permet un recrutement de la septine 9, la stabilisation des microtubules et le transport de PIAS1 du cytoplasme vers le noyau. Il démontre un rôle important des septines en association avec les PIs dans le trafic. De plus, nous avons montré que la septine 9 est un régulateur de la signalisation de l’interféron γ qui agit au niveau de la phosphorylation de STAT1 et l’entrée de PIAS1 dans le noyau. Ce travail peut constituer une nouvelle piste pour la recherche des thérapies ciblées en immunothérapie dans le traitement de ce cancer. / Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are two types of primary liver cancer. HCC is the most frequent, however the incidence of CCA increases throughout the world with a difficult diagnosis, poor prognosis and very limited therapies. The objective of this work was to identify targets for the diagnosis and treatment of CCA. It is based on the study of septin 9 and phosphoinositides (PIs). Septin 9 belongs to a family of GTPases that participate in the organization of microtubules and the actin cytoskeleton. Septins are involved in cytokinesis, vesicular trafficking and cellular polarity and are also important partners of PIs. To determine the role of septin 9 in the CCA, we investigated its interaction with PIs and with Protein inhibitory of activated STAT1 (PIAS1), which has been described as a SUMO ligase for septins. We studied the expression of septin 9 and PIAS1 in CCA and CHC. We have demonstrated an original mechanism by which la production of PtdIns5P allows the recruitment of septin 9, the stabilization of microtubules and the transport of PIAS1 from the cytoplasm to the nucleus. It demonstrates an important role of the septins in association with the PIs in trafficking. Besides, we have shown that septin 9 is a regulator of interferon γ signaling which acts at the level of the phosphorylation of STAT1 and the entry of PIAS1 into the nucleus. This work can constitute a new avenue for the research of targeted immunotherapy for this cancer.

As proteínas pertencentes à família das septinas foram originalmente descobertas em 1971 em decorrencia de estudos genéticos em células mutantes. Essas proteínas encontradas em fungos e animais, mas não em plantas apresentam como principais características a presença de um domínio conservado de ligação aos nucleotídeos de guanina (GTP) e a formação de filamentos homo- e hetero-oligoméricos, que são estruturas altamente organizadas. Estudos filogenéticos e moleculares em humanos identificaram 14 septinas que são divididas em 4 grupos (I, II, III e IV). Estas moléculas associam-se com membranas celulares, actina, microtúbulos do citoesqueleto e estão envolvidas em inúmeros processos que ocorrem no córtex celular e requerem organização espacial, tais como: citocinese, ciclo celular, formação de barreiras de difusão, alinhamento de fuso. Alterações na expressão das septinas estão associadas a vários tipos de tumores e a doenças de Parkinson e Alzheimer. Neste trabalho, com o objetivo de obter informações estruturais e bioquímicas das septinas 7 e 9 humanas. Este projeto é parte de um esforço conjunto coordenado pelo Prof. Dr. Richard C. Garratt e conhecido informalmente como Septimoma. As construções recombinantes SEPT 7, SEPT 7G, e SEPT 9G foram expressas em Escherichia coli e as proteínas recombinantes obtidas. As análises em eletroforese SDS-Page e em gel nativo indicam que essas proteínas foram purificadas com sucesso. A atividade GTPase e o estado oligomérico na forma dimérica foram verificados. Estudos de dicroísmo circular e fluorescência determinaram que esses recombinantes são formados por uma mistura de estruturas secundárias &alfa; e β, e também que o C e o N terminais aumentam a estabilidade das proteínas. Foram obtidos cristais da SEPT 7G e, por meio da técnica de raios-X, foi determinado um modelo tridimensional da proteína com resolução de 3,4o. / Proteins belonging to the septin family were originally discovered in 1971 through genetic studies of mutant cells. These proteins found in fungi and animals, but not in plants present, as their main characteristics, a conserved guanine nucleotide-binding domain (GTP) and they also form homo and hetero-oligomeric filaments that are highly organized structures. Phylogenetic and molecular studies in humans have identified 14 septins which are divided into 4 subfamilies (groups I, II, III and IV). These molecules associate with cell membranes, actin, cytoskeleton microtubules and they are related to a number of processes that take place in the cell cortex and that require spatial organization, such as cytokinesis, cell cycle, diffusion barrier formation and spindle alignment. Alterations in the expression of septins are associated with several types of tumors and with Parkinsons and Alzheimers diseases. In this work, with the goal of obtaining structural and biochemical information of human septins 7 and 9, the recombinants SEPT 7, SEPT 7G and SEPT 9G were expressed in E. coli. Analyses both in SDS-Page electroforesis and in native gel suggest that these proteins were purified successfully for they are soluble and homogeneous. GTpase activity has been verified in all of these recombinants, which shows that these proteins are present in native form and that additional molecules are not needed for this activity. It was possible to determine through different techniques such as molecular exclusion chromatography and SAXS that all the molecules in solution are grouped as dimeric form. Circular dichroism and fluorescence spectroscopic studies have determined both that such recombinants are formed by means of a mixture of &alfa; and β secondary structures and that the C and N-terminals increase the stability of proteins. Protein stability studies under different pH and temperature conditions show that the raise of the latter produces a greater molecular aggregation. Measurements of fluorescence emissions have indicated that the SEPT 7, SEPT 7G and SEPT 9G form structures of amyloid-like filaments found in many septins. Crystal structures of SEPT 7G have been obtained and, by means of the X-ray technique, a 3-D model of the protein has been determined with a resolution of 3.4o. It has been possible to predict, with molecular modeling studies, regions formed by loops that showed low electronic density in the GTPase crystallographic model. Therefore, it has been possible to add more structural information to this domain and to form the complete polypeptide without cuts.

As proteínas pertencentes à família das septinas foram originalmente descobertas em 1971 em decorrencia de estudos genéticos em células mutantes. Essas proteínas encontradas em fungos e animais, mas não em plantas apresentam como principais características a presença de um domínio conservado de ligação aos nucleotídeos de guanina (GTP) e a formação de filamentos homo- e hetero-oligoméricos, que são estruturas altamente organizadas. Estudos filogenéticos e moleculares em humanos identificaram 14 septinas que são divididas em 4 grupos (I, II, III e IV). Estas moléculas associam-se com membranas celulares, actina, microtúbulos do citoesqueleto e estão envolvidas em inúmeros processos que ocorrem no córtex celular e requerem organização espacial, tais como: citocinese, ciclo celular, formação de barreiras de difusão, alinhamento de fuso. Alterações na expressão das septinas estão associadas a vários tipos de tumores e a doenças de Parkinson e Alzheimer. Neste trabalho, com o objetivo de obter informações estruturais e bioquímicas das septinas 7 e 9 humanas. Este projeto é parte de um esforço conjunto coordenado pelo Prof. Dr. Richard C. Garratt e conhecido informalmente como Septimoma. As construções recombinantes SEPT 7, SEPT 7G, e SEPT 9G foram expressas em Escherichia coli e as proteínas recombinantes obtidas. As análises em eletroforese SDS-Page e em gel nativo indicam que essas proteínas foram purificadas com sucesso. A atividade GTPase e o estado oligomérico na forma dimérica foram verificados. Estudos de dicroísmo circular e fluorescência determinaram que esses recombinantes são formados por uma mistura de estruturas secundárias &alfa; e β, e também que o C e o N terminais aumentam a estabilidade das proteínas. Foram obtidos cristais da SEPT 7G e, por meio da técnica de raios-X, foi determinado um modelo tridimensional da proteína com resolução de 3,4o. / Proteins belonging to the septin family were originally discovered in 1971 through genetic studies of mutant cells. These proteins found in fungi and animals, but not in plants present, as their main characteristics, a conserved guanine nucleotide-binding domain (GTP) and they also form homo and hetero-oligomeric filaments that are highly organized structures. Phylogenetic and molecular studies in humans have identified 14 septins which are divided into 4 subfamilies (groups I, II, III and IV). These molecules associate with cell membranes, actin, cytoskeleton microtubules and they are related to a number of processes that take place in the cell cortex and that require spatial organization, such as cytokinesis, cell cycle, diffusion barrier formation and spindle alignment. Alterations in the expression of septins are associated with several types of tumors and with Parkinsons and Alzheimers diseases. In this work, with the goal of obtaining structural and biochemical information of human septins 7 and 9, the recombinants SEPT 7, SEPT 7G and SEPT 9G were expressed in E. coli. Analyses both in SDS-Page electroforesis and in native gel suggest that these proteins were purified successfully for they are soluble and homogeneous. GTpase activity has been verified in all of these recombinants, which shows that these proteins are present in native form and that additional molecules are not needed for this activity. It was possible to determine through different techniques such as molecular exclusion chromatography and SAXS that all the molecules in solution are grouped as dimeric form. Circular dichroism and fluorescence spectroscopic studies have determined both that such recombinants are formed by means of a mixture of &alfa; and β secondary structures and that the C and N-terminals increase the stability of proteins. Protein stability studies under different pH and temperature conditions show that the raise of the latter produces a greater molecular aggregation. Measurements of fluorescence emissions have indicated that the SEPT 7, SEPT 7G and SEPT 9G form structures of amyloid-like filaments found in many septins. Crystal structures of SEPT 7G have been obtained and, by means of the X-ray technique, a 3-D model of the protein has been determined with a resolution of 3.4o. It has been possible to predict, with molecular modeling studies, regions formed by loops that showed low electronic density in the GTPase crystallographic model. Therefore, it has been possible to add more structural information to this domain and to form the complete polypeptide without cuts.

Les septines sont une famille de protéines GTPases qui peuvent former des structures d'ordre supérieur, comme les filaments et les anneaux, et capables de se lier avec les membranes cellulaires par leur interaction avec les phosphoinositides (PIs) via un domaine polybasique en N-terminal de leur domaine de liaison au GTP. Nous avons montré par une analyse transcriptomique réalisée en utilisant les données GSE14323 que la septine 9 est significativement surexprimée dans la cirrhose induite par le virus de l'hepatite C (VHC). Nos résultats montrent, ainsi, que la septine 9 induit l’augmentation en taille des gouttelettes lipidiques (GLs) par un mécanisme dépendant le phosphatidylinositol-5-phosphate et des microtubules. Nous avons montré, également, que cette voie de régulation des GLs est exploité par le VHC. De plus, nous avons montré que la septine 9 est impliquée dans la régulation de la morphologie de l’appareil Golgi et la mise en place de la polarité cellulaire par son interaction avec les phosphoinositides via deux domaines polybasiques. Ces résultats apportent une nouvelle compréhension du mécanisme moléculaire de l’interaction des septines avec les phosphoinositides et montrent pour la première fois l’importance de cette interaction dans des fonctions cellulaires de la septine 9. / Septins are a GTPases proteins family that can form high order structures such as filaments and rings, and able to bind cell membranes by interacting with phosphoinositides via a polybasic domain located at the N-terminal of their GTP binding domain. Here, We show by the transcriptomic analysis performed using the GSE14323 dataset that septin 9 is significantly upregulated in hepatitis C virus induced cirrhosis. Our findings show that septin 9 induce the lipid droplet growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV. In addition, we have shown that the septin 9 is involved in Golgi apparatus morphology regulation and cell polarity installation by interacting with phosphoinositides via two polybasic domains. These results provide new understanding of the molecular mechanism of septins interaction with the phosphoinositides and show its importance in septin 9 cellular functions shown for the first time.