Quality control of heteromultimeric membrane proteins includes mechanisms to ensure that only properly assembled complexes reach the cell surface. ER retrieval signals that are recognised by the COPI vesicle coat play a central role in such trafficking checkpoints. In many cases R-based ER retrieval signals are presented on one or all subunits of the heteromultimeric complexes. ATP-sensitive potassium (KATP) channels provide an example of a heteromultimeric cargo protein presenting eight R-based signals (one in each Kir6.X and SUR subunit of the 4+4 heterooctamer). KATP channels are metabolic sensors that couple cellular energy metabolism to electrical excitability by controlling the membrane potential at the plasma membrane. By this mechanism they are involved in glucose-stimulated insulin secretion, regulation of skeletal muscle excitability, cardioprotective ischemic preconditioning, neurotransmitter release and smooth muscle relaxation. In this thesis, we have established Blue Native Poly-Acrylamide Gel Electrophoresis (BN PAGE) as a method to characterize KATP channel complexes from different rodent tissues. Knock-out mice lacking individual subunits were employed as controls to probe the composition of the observed macromolecular complexes. This approach was complemented by density gradient centrifugation, subcellular fractionation and glycosylation analysis. We demonstrate the existence of distinct macromolecular complexes containing the pore-forming subunit Kir6.2 in pancreatic islets, the brain and the heart. Furthermore, we confirm assembly-dependent forward transport of channel subunits for endogenously expressed KATP channels. We report that the steady-state levels of Kir6.2 are drastically reduced in the brains of SUR1 knock-out mice, whereas we identify both fully assembled KATP channels and putative biogenetic intermediates in the hearts of SUR1 knock-out mice. Our results also suggest that SUR1, in native neuronal and cardiac KATP channels, is differentially glycosylated. Finally we have analyzed COPI-based recognition of Arg-based signals and KATP channel assembly in a mouse model (nur17) with a point mutation in the gene encoding the delta (delta)-subunit of COPI, in close vicinity to the reported binding site of Arg-based signals. We demonstrate that both the recognition of Arg-based signals and the assembly of several multimeric cargo proteins, including the KATP channel, are unaffected in the nur17 mouse. When analyzing the subunit composition of the nur17 COPI coat by two-dimensional BN SDS PAGE we discovered the presence of distinct COPI complexes in the mutant. We have also provided evidence that the association of COPI with a specific membrane compartment is compromised in the nur17 mouse. In conclusion, we have extended the study of a model COPI cargo, the KATP channel complex, to the physiologically relevant context. Furthermore, we have also provided initial insights into the molecular mechanism underlying the physiological consequences of the nur17 delta-COP mutation.

Cell polarity is a fundamental feature of eukaryotic cells. The intracellular trafficking of proteins between cellular compartments and the cytoskeleton regulates the establishment and maintenance of cell polarity. These events are largely regulated by the Ras superfamily of small GTPases. Rabs (Ras-related in brain) and Arfs (ADP-ribosylation factor), subfamilies of the Ras superfamily, play a major role in coordinating vesicle formation and in mediating vesicle association with cytoskeletal components for transport of vesicles to their correct cellular compartment or membrane domain. Although these GTPases mediate membrane trafficking, how they interact with each other and effector proteins to participate in vesicle formation and transport at the trans-Golgi network (TGN) remains poorly understood. The TGN is a major sorting station for biosynthetic cargo molecules (i.e. apical and basolateral) into distinct carriers for delivery to their correct acceptor compartment. We have analyzed the role of Rab14 at the Trans-Golgi Network (TGN), apical endosomes, and in vesicle formation at the TGN by overexpressing wild type and mutant forms of Rab14 in polarized and non-polarized cells. We localized Rab14 to a domain of the TGN distinct from that of the TGN/basolateral protein, TGN38. Overexpression of inactive Rab14 causes the TGN to expand and mislocalizes the apical membrane protein VIP/MAL to the lateral membrane. Furthermore, inactive Rab14 colocalizes with Arf1-GDP at the TGN and increases the amount of COPI at the TGN. These results suggest that Rab14 is involved in the trafficking of proteins from the TGN to apical endosomes and modulates vesicle formation at the TGN by regulating Arf1 activation and COPI localization to distinct domains of the TGN. Thus, Rab14 defines a new role for COPI-mediated vesicle formation at the TGN.

No description available.

In type 1 diabetes (T1D), an autoimmune disease, autoantibodies are preferentially directed against proteins associated with Golgi and post-Golgi secretory vesicles, including insulin secretory granules and synaptic-like microvesicles. Thus, the study of beta-cell autoantigens with yet unknown function may provide novel insight into the secretory machinery of beta-cells and led to the discovery of novel pathways. Islet cell autoantigen of 69 kDa (ICA69) is a T1D autoantigen. It is a cytosolic protein of still unknown function. An impairment in neurotransmitter release upon mutation of its homologue in C. elegans suggests, however, an involvement of ICA69 in neurosecretion. Interestingly, ICA69 contains a BAR domain, present in several proteins involved in intracellular transport. The BAR domain functions as a dimerization motif, provides a general binding interface for different types of GTPases, and is a membrane binding/bending module. Its presence in ICA69 is a further hint supporting the putative involvement of ICA69 in intracellular membrane trafficking. The first part of this thesis was concerned with the characterization of ICA69, and the elucidation of its role in membrane traffic in pancreatic beta-cells. ICA69 was shown to be enriched in the perinuclear region, where also markers of the Golgi region are found. ICA69 was shown to interact with several membrane lipids, preferentially with PI(4)P, enriched on the Golgi complex. During the course of this thesis a combination of biochemical and imaging techniques were applied to investigate the interaction between ICA69 and Rab2, a small GTPase associated with the intermediate compartment and involved in the trafficking between the ER and the Golgi complex. ICA69 was shown to co-immunoprecipitate with Rab2 from INS-1 cells extracts. GST-pull down assays demonstrated that this interaction is GTP-dependent. Furthermore, confocal microscopy indicated that ICA69 and Rab2 extensively colocalize in particulate structures throughout the cytoplasm. Immunocytochemistry and subcellular fractionation experiments suggested that Rab2 recruits ICA69 to membranes. Functional studies indicated that ICA69 over-expression in INS-1 cells has effects that resemble, and in some cases amplify those observed upon Rab2 over-expression. Specifically, it impairs the trafficking between ER and Golgi, measured through the appearance and the conversion of the pro-form of ICA512 in the mature form of the protein. Moreover, it correlates with a redistribution of the beta-COP subunit of the coatomer, participating in the early secretory pathway, between membrane-bound compartments and the cytosol and it reduces stimulated insulin secretion. The data reported in this thesis conclusively point to ICA69 as a novel Rab2 effector, and may therefore contribute to the elucidation the yet poorly understood mechanism of action of Rab2 in the secretory pathway. The second part of the thesis was devoted to the study of an ICA69 paralogue gene, called ICA69-RP. Similarly to ICA69, ICA69-RP mRNA was shown to be primarily present in tissues such as brain and pancreatic islets, showing the expression pattern of a gene preferentially expressed in neuroendocrine cells. Unlike ICA69, however, and similar to other genes associated with the secretory machinery of beta-cells, ICA69-RP appeared to be glucose regulated, as shown by a 1.55 fold increase in mRNA levels upon stimulation of the cells with 25 mM glucose for two hours.Glucose stimulation of beta-cells prompts the activation of post-transcripional mechanisms which quickly up-regulate the expression of secretory granule genes and consequently renew granule stores. The increased expression of ICA69-RP upon glucose stimulation of cells may be part of this process. Unfortunately, all attempts to elucidate the intracellular localization of endogenous ICA69-RP failed, and it was not possible to obtain significant insights about its localization by over-expressing a fusion protein between ICA69-RP and GFP. Unlike other paralogues containing the BAR domain, such as amphiphysin 1 and 2 or Rvs167p and Rvs161p, ICA69 and ICA69-RP were shown not to form heterodimers. Furthermore, ICA69-RP did not show any interaction with Rab2 or Rab1, involved in the anterograde transport between ER and Golgi. Thus, its physiological role remains to be investigated.

In type 1 diabetes (T1D), an autoimmune disease, autoantibodies are preferentially directed against proteins associated with Golgi and post-Golgi secretory vesicles, including insulin secretory granules and synaptic-like microvesicles. Thus, the study of beta-cell autoantigens with yet unknown function may provide novel insight into the secretory machinery of beta-cells and led to the discovery of novel pathways. Islet cell autoantigen of 69 kDa (ICA69) is a T1D autoantigen. It is a cytosolic protein of still unknown function. An impairment in neurotransmitter release upon mutation of its homologue in C. elegans suggests, however, an involvement of ICA69 in neurosecretion. Interestingly, ICA69 contains a BAR domain, present in several proteins involved in intracellular transport. The BAR domain functions as a dimerization motif, provides a general binding interface for different types of GTPases, and is a membrane binding/bending module. Its presence in ICA69 is a further hint supporting the putative involvement of ICA69 in intracellular membrane trafficking. The first part of this thesis was concerned with the characterization of ICA69, and the elucidation of its role in membrane traffic in pancreatic beta-cells. ICA69 was shown to be enriched in the perinuclear region, where also markers of the Golgi region are found. ICA69 was shown to interact with several membrane lipids, preferentially with PI(4)P, enriched on the Golgi complex. During the course of this thesis a combination of biochemical and imaging techniques were applied to investigate the interaction between ICA69 and Rab2, a small GTPase associated with the intermediate compartment and involved in the trafficking between the ER and the Golgi complex. ICA69 was shown to co-immunoprecipitate with Rab2 from INS-1 cells extracts. GST-pull down assays demonstrated that this interaction is GTP-dependent. Furthermore, confocal microscopy indicated that ICA69 and Rab2 extensively colocalize in particulate structures throughout the cytoplasm. Immunocytochemistry and subcellular fractionation experiments suggested that Rab2 recruits ICA69 to membranes. Functional studies indicated that ICA69 over-expression in INS-1 cells has effects that resemble, and in some cases amplify those observed upon Rab2 over-expression. Specifically, it impairs the trafficking between ER and Golgi, measured through the appearance and the conversion of the pro-form of ICA512 in the mature form of the protein. Moreover, it correlates with a redistribution of the beta-COP subunit of the coatomer, participating in the early secretory pathway, between membrane-bound compartments and the cytosol and it reduces stimulated insulin secretion. The data reported in this thesis conclusively point to ICA69 as a novel Rab2 effector, and may therefore contribute to the elucidation the yet poorly understood mechanism of action of Rab2 in the secretory pathway. The second part of the thesis was devoted to the study of an ICA69 paralogue gene, called ICA69-RP. Similarly to ICA69, ICA69-RP mRNA was shown to be primarily present in tissues such as brain and pancreatic islets, showing the expression pattern of a gene preferentially expressed in neuroendocrine cells. Unlike ICA69, however, and similar to other genes associated with the secretory machinery of beta-cells, ICA69-RP appeared to be glucose regulated, as shown by a 1.55 fold increase in mRNA levels upon stimulation of the cells with 25 mM glucose for two hours.Glucose stimulation of beta-cells prompts the activation of post-transcripional mechanisms which quickly up-regulate the expression of secretory granule genes and consequently renew granule stores. The increased expression of ICA69-RP upon glucose stimulation of cells may be part of this process. Unfortunately, all attempts to elucidate the intracellular localization of endogenous ICA69-RP failed, and it was not possible to obtain significant insights about its localization by over-expressing a fusion protein between ICA69-RP and GFP. Unlike other paralogues containing the BAR domain, such as amphiphysin 1 and 2 or Rvs167p and Rvs161p, ICA69 and ICA69-RP were shown not to form heterodimers. Furthermore, ICA69-RP did not show any interaction with Rab2 or Rab1, involved in the anterograde transport between ER and Golgi. Thus, its physiological role remains to be investigated.

La formation d'un bourgeon vésiculaire repose sur une machinerie complexe qui déforme, par une action mécanique, une membrane plane en une membrane courbée. Le manteau COPI, responsable de ce phénomène dans le Golgi, se polymérise latéralement à la surface de la membrane, sous le contrôle de la petite protéine G Arf1 activée. Suite à la formation de la vésicule, Arf1 revient à l'état inactif par hydrolyse de son GTP et se dissocie de la membrane, provoquant alors le désassemblage du manteau. Cette réaction d'hydrolyse doit être finement régulée, pour ne pas intervenir trop tôt et compromettre l'assemblage du manteau. Notre laboratoire a révélé que l'activité de la protéine ArfGAP1, responsable de la désactivation d'Arf1, est hypersensible à la courbure membranaire. Ceci permettrait à ArfGAP1 de réguler de manière spatio-temporelle l'état du manteau COPI en couplant son désassemblage à la courbure membranaire qu'il a lui-même induite.<br />Au cours de ma thèse, j'ai montré que la dépendance d'ArfGAP1 à la courbure s'explique par la présence dans cette protéine de deux motifs « ALPS », qui se replient en hélices alpha lors de leur adsorption membranaire. Ce sont des hélices amphipathiques atypiques car, si elles possèdent une face hydrophobe classique, elles ont une face polaire riche en sérine et thréonine et pauvre en résidus chargés. Puisque ces résidus hydroxylés ne peuvent interagir avec les têtes polaires des lipides, la liaison d'un motif ALPS ne repose que sur l'insertion de ses résidus hydrophobes entre les lipides, ce qui est favorisé par l'écartement lipidique induit par la courbure membranaire, mais plus difficile sur membrane plane où les lipides sont plus compactés.

Made available in DSpace on 2014-06-11T19:25:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-09Bitstream added on 2014-06-13T20:13:44Z : No. of bitstreams: 1 carignano_mlm_me_arafcl.pdf: 1146916 bytes, checksum: cfc3d10c7737c0034efa9269863206f6 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A pesquisa visa estudar a obra de Copi ( Raúl Damonte Botana 1939-1987), escritor argentino, num âmbito maior de questões, que servirão de apoio teórico- crítico possibilitando uma abordagem mais ampla. Da totalidade de sua obra (ele escreveu romances, peças teatrais e é autor também de desenhos e quadrinhos humorísticos), enfatizar-se-á o estudo da série teatral e dentro dela três peças: Cachafaz, A sombra de Wenceslao, e Eva Perón. Elas foram escolhidas por serem altamente representativas da estética controversa, humorística, paródica e irônica do autor. Primeiramente, tem-se como objetivo realizar uma investigação das formas do humor relacionando-as aos textos do autor argentino e procurando uma leitura crítica que permita repensar essas categorias à luz das particularidades da obra de Copi. Num segundo momento, situar-se-ão estas obras do autor em relação à tradição literária argentina. Elas devem ser lidas como um diálogo com a literatura e a história argentina, com os mitos e estigmas de sua cultura (tanto literários quanto históricos) que compõem ou têm composto os “grandes traços” daquilo que se supõe ser a “argentinidade”. Em relação com o primeiro objetivo, estudar-se-á teoricamente a gênesis, as especificidades e diferenças entre o humor, a comicidade, a paródia, a sátira e a ironia e formas afins, fazendo um percurso pelos principais filósofos e teóricos que abordaram estas questões. Estudar-se-á especialmente a parodia, por ser ela a forma constitutiva da escrita de Copi, mas também por ser uma das formas recorrentes da arte (tanto literária quanto pictórica) das ultimas décadas. Em relação ao segundo objetivo, ao vincular a obra de Copi a tradição literária argentina, realizar-se-á um estudo dos seguintes gêneros e movimentos da literatura rioplatense: gauchesca, sainete, grotesco, criollismo, tango. A intenção da pesquisa visa esclarecer e fornecer uma leitura... / This research aims to study the work of Copi (Raúl Damonte Botana 1939-1987), Argentinean writer, in a broader scenery of topics, that will be of theoretical and critical support, to permit a more general boarding. From the totality of his work (he wrote novels, theatrical plays, and is also the author of cartoons and humoristic comics), we will emphasize the study of his theater and within it three plays: Cachafaz, A sombra de Wenceslao, and Eva Perón. They were chosen because they are highly representative of the author’s controversial esthetics, humorous, parodic and ironic. Our first objective is to investigate about the forms of humor relating them to the texts of the Argentinean author and looking for a critical reading that allows us to represent those categories in the light of the particularities of Copi’s work. In a second time, we are going to situate the author’s texts in relation to the Argentinean literature. His work must to be read as a conversation with the Argentinean literature and history, with the myths and stigma of its culture (both literary and historical) that are part or have been part of the “great traits” of what is supposed to be the “argentinity”. In relation to our first objective, we will study the genesis, the specificity and the differences between the humor, the hilarious, the parody, the satire and the irony and related forms, beginning from the main philosophers and theoretic whom studied these themes. The parody is specially going to be analyzed, not only because it is the form that constitutes Copi’s work, but also because it is one of the recurrent forms of the art (both literary and pictorial) of the lasts decades. In relation to our second objective, when relating Copi’s work to the Argentinean literature, we are going to study the following genera and movements of Rioplatense literature: gauchesca, sainete, grotesco, criollismo, tango. The intention of this research aims to...

Les médicaments de type opioïde représentent la classe de médicaments la plus utilisée pour les douleurs modérées à sévères. C’est pour cette raison que les opioïdes et leurs récepteurs sont très étudiés et qu’il y a beaucoup de publications sur ces récepteurs. En revanche, peu d’études ont cherché à identifier les partenaires d’interactions de ces récepteurs puis à comprendre les mécanismes d’adressage à la membrane. Même si le récepteur opioïde delta (DOPr) n’est pas encore ciblé en clinique, beaucoup d’équipes s’intéressent à son rôle et à l’effet d’agonistes DOPr dans le but de trouver une nouvelle avenue thérapeutique contre la douleur. Cependant, en condition normale, les agonistes DOPr ont un faible potentiel analgésique, expliqué par le faible niveau de DOPr à la surface cellulaire des neurones. C’est pourquoi il est important de comprendre son adressage à la membrane afin de combiner les traitements aux agonistes DOPr à une thérapie qui augmenterait l’expression de surface du récepteur. De plus, on sait qu’il existe un mécanisme de régulation de l’adressage de DOPr à la surface mais il reste peu compris. Nous avons donc analysé par spectrométrie de masse le complexe protéique résultant de l’immunoprécipitation de FlagDOPr, surexprimé dans des cellules HEK293, afin d’identifier de nouveaux partenaires d’interaction. Dans cette analyse, plusieurs protéines appartenant au complexe vésiculaire COPI ont été identifiées. Nous avons montré dans l’article que DOPr interagit avec COPI via les domaines intracellulaires 2 et 3 et que ces sites d’interaction sont responsables de la rétention de DOPr. De plus, mes travaux se sont penchés sur la régulation de DOPr à la surface cellulaire, telle que l’interaction DOPr avec les protéines cdk5 et pin1, deux protéines pouvant faire partie d’un mécanisme impliqué dans la régulation du transport de DOPr à la surface cellulaire. Comprendre l’export de DOPr est important pour le développement de nouvelles thérapies contre la douleur et plusieurs théories ont été abordées dans le cadre de mes travaux de maîtrise.

EBAG9 (estrogen receptor-binding fragment-associated gene 9) hat als unabhängiger prognostischer Marker viel Aufmerksamkeit erregt, da in einigen Tumoren hohe Expressionsraten und Tumorentwicklung korrelieren. In diesen Fällen ist eine hohe EBAG9 Expression häufig mit einer schlechten klinischen Prognose verbunden. EBAG9 ist ein ubiquitär exprimiertes Golgi Protein. Aktuelle Daten demonstrieren, dass es in sekretorischen Zellen an der regulierten Exozytose und an der zytotoxischen Funktion von Lymphozyten beteiligt ist. In epithelialen Zellen führt es zur Generierung von Tumor-assoziierten O-Glykanen, welche ein Erkennungsmerkmal vieler Krebsarten sind. In dieser Arbeit wurde der pathogenetische Zusammenhang zwischen EBAG9 Expression und der Veränderung des zellulären Glykoms untersucht. Um einen tieferen Einblick in die zelluläre Funktion von EBAG9 in epithelialen Zellen zu gewinnen, wurden Zellen mit tumorähnlicher EBAG9 Expression verwendet. Innerhalb dieser Arbeit wurde demonstriert, dass EBAG9 mit anterograden COPI Vesikeln assoziiert und zwischen dem ER-Golgi intermediären Kompartiment und cis-Golgi pendelt. EBAG9 verursacht eine Verzögerung des anterograden Transportes vom ER zum Golgi und verändert die Lokalisation von Komponenten der ER Qualitätskontrolle und des Glycosylierungsapparates. Auf der anderen Seite beschleunigt die verminderte Expression von EBAG9 den Proteintransport durch den Golgi und verstärkt die Aktivität von Mannosidase II. Mechanistisch betrachtet verhindert EBAG9 die Rekrutierung von ArfGAP1 an die Membran. Dies beeinträchtigt das Auflösen der COPI Vesikelhülle und somit die Fusion von Vesikeln am cis-Golgi. Damit agiert EBAG9 in epithelialen Zellen als negativer Regulator des COPI-abhängigen ERGolgi Transportes und stellt damit ein neues phatogenetisches Prinzip dar, bei dem die Beeinflussung des intrazellulären Transportes zu der Entstehung von Tumor-assoziierten Glykanen führt. / The estrogen receptor-binding fragment-associated gene 9 (EBAG9) has received increased attention as an independent prognostic marker for disease-specific survival since in some human tumor entities high expression levels correlate with tumor progression and poor clinical prognosis. Interestingly, EBAG9 was identified as an ubiquitously expressed Golgi protein. Recent data demonstrate an involvement in regulated exocytosis in secretory cells and the cytotoxic functions of lymphocytes. However, EBAG9 is expressed in essentially all mammalian tissues, and in epithelial cells it has been identified as a modulator of tumorassociated O-linked glycan expression, a hallmark of many carcinomas. This thesis addresses the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. To gain further insights into the cellular functions of EBAG9 in epithelial cells, tumor-associated EBAG9 overexpression was mimicked in living cells. It was demonstrated that EBAG9 associates with anterograde COPI-coated carriers and shuttles between the ER-Golgi intermediate compartment and cis-Golgi stacks. EBAG9 overexpression imposes a delay in anterograde ER-to-Golgi transport and mislocalizes components of the ER quality-control and glycosylation machinery. Conversely, EBAG9 downregulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Functionally, EBAG9 impairs ArfGAP1 recruitment to membranes and consequently, interferes with the disassembly of the coat lattice at the cis-Golgi prior to fusion. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.

No description available.