No description available.

La voie TORC1 joue un rôle majeur dans le contrôle de la croissance cellulaire et de la réponse à divers stress. Le dérèglement de cette voie est constaté dans de nombreux cancers et autres maladies. Au cours de ma thèse, j’ai montré que le complexe SEA émerge comme un régulateur central des différentes activités de TORC1. Durant la carence azotée, les délétions des gènes du complexe SEA dans l’organisme modèle S.cerevisiae mènent à la délocalisation de la kinase Tor1 vers le cytoplasme, à des défauts d’autophagie et à la fragmentation de la vacuole. L’inactivation de TORC1 par le traitement avec la rapamycine ou pendant la carence azotée change le niveau d’expression des membres du complexe SEA. De plus, le complexe SEA interagit avec la mitochondrie, joue un rôle dans la réponse au stress oxydatif et peut servir de lien moléculaire entre les fonctions mitochondriales et la voie TORC1. Enfin, j’ai pu observer que le complexe SEA est impliqué dans les mécanismes de résistance à une drogue souvent utilisée en chimiothérapie, la doxorubicine. Je présente dans mes travaux la première carte d’interconnectivité des protéines composant le complexe SEA. Nos données suggèrent que le complexe SEA émerge comme une plateforme qui peut coordonner les activités structurales et enzymatiques nécessaires pour le fonctionnement efficace de la voie de signalisation TORC1. / The TORC1 pathway plays a major role in controlling cell growth and response to various stresses. Deregulation of this pathway is found in many cancers and other diseases. In my thesis, I have shown that the SEA complex emerges as a central regulator of the various activities of TORC1. During the nitrogen deficiency, deletions of SEA complex genes in the model organism S.cerevisiae lead to the relocation of Tor1 kinase to the cytoplasm, to defects in autophagy and the fragmentation of the vacuole. Inactivation of TORC1 by treatment with rapamycin or nitrogen starvation changes the level of expression of SEA complex members. Moreover, the SEA complex interacts with mitochondrion, plays a role in oxidative stress response and can serve as a molecular link between mitochondrial functions and TORC1 pathway. Finally, I observed that the SEA complex is involved in the mechanisms of resistance to a drug often used in chemotherapy, the doxorubicin. I present in my work the first interconnectivity map protein of the SEA complex component. Our data suggest that the SEA complex emerges as a platform that can coordinate structural and enzymatic activities necessary for the efficient function of the TORC1 signalling pathway.

No description available.

The laboratory strains of yeast Saccharomyces Cerevisiae form colonies which can differentiate into two main cell subpopulations. U and L cells demonstrate different morphology, metabolism and stress-resistance. It was also proved that some of metabolic pathways in U cells are a similar to ones in tumor cells. The unique metabolism is activated in U cells; the TORC1 is active in these cells together with autophagy and glycogen accumulation, which are characteristic for cells with inactivated TORC1. CORVET and HOPS complexes together with vacuolar ATPase are involved in processes related to vacuolar fusion and trafficking. Also, these complexes contribute to the regulation of TORC1 activity. Vam6p is a subunit of HOPS complex and it is also involved in regulation of TORC1 acting as GEF for Gtr1p GTPase, which activates TORC1. The aim of this study was to outline whether selected subunits of mentioned complexes affect TORC1 activity in U cells. Further aim was to confirm the effect of Vam6p on selected proteins production. These proteins were chosen based on results of proteomic analysis performed in our laboratory. In order to investigate possible effects of proteins of interest absence on colonies' morphology, strains deleted in selected genes were prepared (VPS3, VPS8, VPS33, VPS41, VPH2, VAC7 a...

[EN] The Saccharomyces cerevisiae protein kinases Sat4 (Hal4) and Hal5 are required for the plasma membrane stability of the high affinity K+ transporter Trk1 and some amino acid and glucose permeases. A transcriptomic analysis of the hal4 hal5 strain revealed that the absence of these genes causes general alterations in the metabolism of amino acids and glucose. This data is confirmed by the following approaches: activity of the Gcn2-Gcn4 pathway, uptake of methionine and leucine, activity of succinate dehydrogenase (SDH), glucose consumption and ethanol production of this mutant. In this thesis, we demonstrated that the high affinity permease Mup1 is internalized and degraded in the vacuole in the absence of potassium supplementation, like other plasma membrane permeases such as Hxt1 (glucose), Can1 (arginine), Fur4 (uracil) and Gap1 (amino acids). This destabilization of the Mup1 permease is likely to explain the reduction in the uptake of methionine in the double mutant hal4 hal5 and suggests that Hal4 and Hal5 are involved in a general mechanism of regulation of the stability of permeases in the plasma membrane. This hypothesis was corroborated by studies with inhibitors of endocytosis and mutant isoforms of the E3 ubiquitin ligase Rsp5, which is responsible for the ubiquitination and subsequent vacuolar degradation of the permeases studied. The process of Rsp5-mediated ubiquitination requires, in many cases, specific adapters for recognition of the target protein. So far, 19 Rsp5 adapter proteins have been described, among which there are 9 ARTs proteins (Arrestin-Related Trafficking adaptor). In this study, we investigated whether there is a functional connection between Hal4 and Hal5 kinases and ARTs, since this mechanism could explain the observed phenotypes. We studied whether Art1, a regulator of Mup1 and Can1 endocytosis, is involved in the internalization of these permeases in hal4 hal5 strains. Our data indicates that Art1 is not necessary for internalizing Mup1 in the hal4 hal5 strain in the absence of potassium supplementation, therefore suggesting a new role for the Hal4 and Hal5 kinases. We extended the study to include the transporter of aspartic and glutamic acid, Dip5, whose endocytosis is mainly mediated by Aly2 (Art3). The results were positive, providing support for a more general mechanism of regulation of the permeases of the plasma membrane by these kinases. It has been proposed that Npr1, a kinase that is an effector of the Target of Rapamycin Complex 1 (TORC1), controls the activity of Art1, Aly1 (Art6) and Aly2 (Art3), leading to the accumulation of some permeases in the plasma membrane. We observe lower expression levels of Npr1 in hal4 hal5 strains. We also observe a state of constitutive hyperphosphorylation, similar to WT cells under limiting potassium. Furthermore, overexpression of the Npr1 kinase partially rescues the growth defects and instability of the permeases in the plasma membrane described in the hal4 hal5 mutant. Therefore, we identify part of the pathway regulated by the Hal4 and Hal5 kinases. In eukaryotes, TOR (Target of Rapamycin) exists in two distinct multiprotein complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). We have analyzed the direct substrates of TORC1 (Sch9) and TORC2 (Ypk1) in hal4 hal5 and trk1 trk2 mutants in potassium limiting conditions, observing alterations in the phosphorylation levels of both effectors. Finally, we observed that hal4 hal5 and trk1 trk2 mutants are highly sensitive to the TORC1 inhibitor, rapamycin, and that this sensitivity is rescued by increased external potassium. We confirmed that cells treated with rapamycin had lower internal potassium levels, an effect which is dependent on TORC1 and independent of Trk1 and Trk2. Therefore, our data indicates that the Hal4 and Hal5 kinases have a more specific effect on Npr1 and that there is a reciprocal regulation between potassium and the TOR signaling pathway. / [ES] Las proteínas quinasa de Saccharomyces cerevisiae Sat4 (Hal4) y Hal5 son necesarias para la estabilidad del transportador de K+ de alta afinidad Trk1 y de algunas permeasas de aminoácidos y de glucosa. El análisis transcriptómico del mutante hal4 hal5 reveló que la ausencia de estos genes origina alteraciones generales en el metabolismo de aminoácidos y de glucosa, datos que confirmamos mediante la medida de la ruta Gcn2-Gcn4, de la toma de metionina y de leucina, de la actividad de la succinato deshidrogenasa (SDH), del consumo de glucosa y de la producción de etanol. En esta tesis, hemos demostrado que la permeasa de alta afinidad de metionina, Mup1 se degrada en la vacuola en ausencia de un suplemento de potasio en el mutante hal4 hal5, igual que otras permeasas de la membrana plasmática como Hxt1, Can1, Fur4 y Gap1. Esta desestabilización de Mup1 podría explicar el defecto en la toma de metionina observado y sugiere que Hal4 y Hal5 están implicadas en un mecanismo general de regulación de la estabilidad de las permeasas en la membrana plasmática. Esta hipótesis fue corroborada mediante estudios con inhibidores de la endocitosis y mutantes en la E3 ubiquitina ligasa Rsp5, responsable de la ubiquitinación y posterior degradación vacuolar de las permeasas estudiadas. El proceso de ubiquitinación, en muchos casos, precisa de adaptadores específicos para reconocer la proteína diana. Se han descrito 19 proteínas adaptadoras de Rsp5, entre las que se encuentran 9 proteínas ARTs (Adaptadores de tráfico relacionados con arrestina). En este trabajo, hemos investigado si existe una conexión funcional entre las quinasas Hal4 y Hal5 y los ARTs; este mecanismo podría explicar los fenotipos observados. Estudiamos si Art1, regulador de la endocitosis de Mup1 y Can1, está implicado en la internalización de estas permeases en la cepa hal4 hal5. Nuestros datos indican que Art1 no es necesario para la internalización de Mup1 y Can1 en una cepa hal4 hal5 en ausencia de un suplemento de potasio, sugiriendo un papel novedoso de las quinasas Hal4 y Hal5. Ampliamos el estudio al transportador de ácido aspártico y glutámico, Dip5, cuya endocitosis viene mediada principalmente por Aly2 (Art3). Los resultados fueron positivos apoyando un mecanismo más general de regulación de las permeasas de la membrana plasmática por parte de estas quinasas. Se ha propuesto que Npr1, una quinasa efectora del Target of Rapamycin Complex 1 (TORC1), controla la actividad de Art1, Aly1 (Art6) y Aly2 (Art3) generando la acumulación de algunas permeasas en la membrana plasmática. Observamos menores niveles de expresión de Npr1 en mutantes hal4 hal5, además de un estado de hiperfosforilación constitutivo similar al de células WT en condiciones de potasio limitante. Además, la sobreexpresión de NPR1 rescata los defectos de crecimiento observados en medios con baja disponibilidad de potasio e inestabilidad de permeasas de la membrana plasmática descritos en el mutante hal4 hal5. Por tanto, identificamos parte de la ruta regulada por las quinasas Hal4 y Hal5. En los organismos eucariotas TOR (Target of Rapamycin) existe en dos complejos multiproteicos distintos, complejo TOR1 (TORC1) y complejo TOR2 (TORC2). Hemos analizado los sustratos directos de TORC1 (Sch9) y TORC2 (Ypk1) en mutantes hal4 hal5 y trk1 trk2 y en condiciones de potasio limitante observando alteraciones en los niveles de fosforilación de ambos efectores. Finalmente, hemos observado que los mutantes hal4 hal5 y trk1 trk2 son altamente sensibles al inhibidor de TORC1, rapamicina y que esta sensibilidad se rescata con un exceso de potasio en el medio. Comprobamos que células tratadas con rapamicina presentan una disminución del potasio interno dependiente de TORC1 e independiente de Trk1 y Trk2. Por tanto, nuestros datos indican que las quinasas Hal4 y Hal5 tienen un efecto más específico sobre Npr1 y que hay una regulación reciproca entre el potasio y la r / [CA] Les proteïnes quinasa de Saccharomyces cerevisiae Sat4 (Hal4) i Hal5 són necessàries per a l'estabilitat del transportador de K+ d'alta afinitat Trk1 i d'algunes permeases d'aminoàcids i de glucosa. L'anàlisi transcriptòmic d'una soca mutant hal4 hal5 revela que l'absència d'aquests gens origina alteracions generals en el metabolisme d'aminoàcids i de glucosa, dades que confirmem mitjançant la mesura de la ruta Gcn2-Gcn4, de la presa de metionina i leucina, de l'activitat de succinat deshidrogenasa (SDH), del consum de glucosa i de la producció d'etanol d'aquest mutant. En aquesta tesi, hem demostrat que la permeasa d'alta afinitat de metionina, Mup1 es degrada en el vacúol en absència d'un suplement de potassi en el mutant hal4 hal5, igual que altres permeases de la membrana plasmàtica com Hxt1, Can1, Fur4 i Gap1. Aquesta desestabilització de Mup1 podria explicar el defecte en la presa de metionina observat i suggereix que Hal4 i Hal5 estan implicades en un mecanisme general de regulació de l'estabilitat de les permeases a la membrana plasmàtica. Aquesta hipòtesi va ser corroborada mitjançant estudis amb inhibidors de l'endocitosi i mutants en l'E3 ubiquitina ligasa Rsp5, responsable de la ubiquitinació i posterior degradació vacuolar de les permeases estudiades. El procés d'ubiquitinació, en molts casos, precisa d'adaptadors específics per reconèixer la proteïna diana. Fins al moment s'han descrit 19 proteïnes adaptadores de Rsp5, entre les quals es troben 9 proteïnes ART (Adaptadors de trànsit relacionats amb arrestina). En aquest treball hem investigat si existeix una connexió funcional entre les quinases Hal4 i Hal5 i els ARTs; aquest mecanisme podria explicar els fenotips observats. Estudiem si Art1, regulador de l'endocitosi de Mup1 i Can1, està implicat en la internalització d'aquestes permeases a la soca hal4 hal5. Les nostres dades indiquen que Art1 no és necessari per a la internalització de Mup1 i Can1 en una soca hal4 hal5 en absència d'un suplement de potassi, suggerint un paper nou de les quinases Hal4 i Hal5. Ampliem l'estudi per incloure el transportador d'àcid aspàrtic i glutàmic, Dip5, en l'endocitosi del qual intervé principalment Aly2 (Art3). Els resultats van ser positius recolzant un mecanisme més general de regulació de les permeases de la membrana plasmàtica per part d'aquestes quinases. S'ha proposat que Npr1, una quinasa que és un efector del Target of rapamycin Complex 1 (TORC1), controla l'activitat de Art1, Aly1 (Art6) i Aly2 (Art3) i genera l'acumulació d'algunes permeases a la membrana plasmàtica. Observem nivells menors d'expressió de Npr1 en mutants hal4 hal5, a més d'un estat d'hiperfosforilació constitutiu semblant al de cèl·lules WT en condicions de potassi limitant. A més, la sobreexpressió de la quinasa Npr1 rescata parcialment els defectes de creixement observats en medis amb baixa disponibilitat de potassi i la inestabilitat de permeases de la membrana plasmàtica descrits en el mutant hal4 hal5. Per tant, identifiquem part de la ruta regulada per les quinases Hal4 i Hal5. En els organismes eucariotes, TOR (Target of rapamycin) existeix en dos complexos multiproteics diferents, complex TOR1 (TORC1) i complex TOR2 (TORC2). Hem analitzat els substrats directes de TORC1 (Sch9) i TORC2 (Ypk1) en mutants hal4 hal5 i trk1 trk2 i en condicions de potassi limitant observant alteracions en els nivells de fosforilació dels dos efectors. Finalment, hem observat que els mutants hal4 hal5 i trk1 trk2 són altament sensibles a l'inhibidor de TORC1, rapamicina, i que aquesta sensibilitat es rescatada amb un excés de potassi en el medi. Vam comprovar que cèl·lules tractades amb rapamicina presenten una disminució del potassi intern dependent de TORC1 e independent de Trk1 i Trk2. Per tant, les nostres dades indiquen que les quinases Hal4 i Hal5 tenen un efecte més específic sobre Npr1 i que hi ha una regulació recíproca entre el potassi i / Primo Planta, C. (2015). Mecanismos de regulación post-traduccional de transportadores de la membrana plasmática: Papel de las quinasas Hal4 y Hal5 en el tráfico de transportadores de nutrientes e iones en el organismo modelo Saccharomyces cerevisiae [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52697

[EN] The plasma membrane H+-ATPase (Pma1) is essential for yeast growth and is activated by glucose metabolism by an unknown mechanism involving double phosphorylation of a regulatory site at the C-terminus (Ser911 Thr912). In this thesis we have investigated in Saccharomyces cerevisiae the role of two protein phosphatases, type 1 Glc7 and type 2A Sit4, and of an essential atypical protein kinase, TORC1, in the activation of Pma1 by glucose. The regulatory site of activated Pma1 can be dephosphorylated "in vitro" by recombinant Glc7 and Sit4, but inhibition "in vivo" of these phosphatases does not activate Pma1. Inhibition of Glc7 by regulated expression of a dominant-negative truncated form (the null mutant is not viable) had no effect on Pma1 activity while deletion of SIT4 gene decreased both Pma1 activity and double phosphorylation of the regulatory site. Inhibition of TORC1 protein kinase by treatment of yeast cells with the drug rapamycin or by exposure to non-permissive temperature of a temperature-sensitive mutant (tor1¿ tor2ts) inhibited Pma1 and decreased double phosphorylation of the regulatory site. We conclude that Sit4 and TORC1 are required for full activation of Pma1 by glucose while Glc7 either does not participate or is redundant with other phosphatases. / [ES] La H+-ATPasa de la membrana plasmática (Pma1) es esencial para el crecimiento de la levadura y se activa por metabolismo de glucosa por un mecanismo desconocido que lleva consigo la doble fosforilación de un sitio regulador en el extremo C-terminal (Ser911 Thr912). En la presente tesis hemos investigado en Saccharomyces cerevisiae la participación de dos proteína fosfatasas, Glc7 de tipo 1 y Sit4 de tipo 2A, y de una proteína kinasa atípica esencial, TORC1, en la activación de Pma1 por glucosa. El sitio regulador de Pma1 en su estado activo puede defosforilarse "in vitro" por Glc7 y Sit4 recombinantes pero la inhibición "in vivo" de estas fosfatasas no activa Pma1. La inhibición de Glc7 mediante la expresión regulada de una forma truncada que actúa como dominante-negativa (el mutante nulo no es viable) no tiene efecto en la actividad de Pma1 mientras que la deleción del gen SIT4 disminuye tanto la actividad de Pma1 como la doble fosforilación del sitio regulador. Inhibición de la proteína kinasa TORC1 mediante tratamiento de las células de levadura con el fármaco rapamicina o exponiéndolas a temperatura no permisiva en el caso de un mutante termosensible (tor1¿ tor2ts) resulta en inhibición de Pma1 y disminución de la doble fosforilación del sitio regulador. Estos resultados indican que Sit4 y TORC1 son necesarias para la máxima activación de Pma1 por glucosa mientras que Glc7 podría no participar o hacerlo de forma redundante con otras fosfatasas. / [CA] L'H+-ATPasa de la membrana plasmàtica (Pma1) és essencial per al creixement dels llevats i s'activa gràcies al metabolisme de glucosa per un mecanisme desconegut que porta associat la doble fosforilació d'una regió reguladora a l'extrem C-terminal (Ser911 Thr912). En aquesta tesi hem investigat en Saccharomyces cerevisiae la participació de dos proteïnes fosfatases, Glc7 de tipus 1 i Sit4 de tipus 2A, i d'una proteïna quinasa essencial atípica, TORC1, en l'activació de Pma1 per glucosa. La regió reguladora de Pma1, en seu estat activat, pot desfosforar-se "in vitro" per Glc7 i Sit4 recombinants, però la inhibició "in vivo" d'aquestes fosfatases no activa Pma1. La inhibició de Glc7 mitjançant l'expressió regulada d'una forma truncada que actua com a dominant-negativa (el mutant nul no és viable) no té cap efecte en l'activitat de Pma1 mentre que la deleció del gen SIT4 disminueix tant l'activitat de Pma1 com la doble fosforilació de la regió reguladora. La inhibició de la proteïna quinasa TORC mitjançant un tractament de cèl·lules de llevat amb el fàrmac rapamicina o la seua exposició a temperatures no permissives en el cas d'un mutant termosensible (tor1¿ tor2ts) resulta en la inhibició de Pma1 i la disminució de la doble fosforilació de la regió reguladora. Aquests resultats indiquen que Sit4 i TORC1 són necessàries per a l'activació màxima de Pma1 per glucosa, mentre que Glc7 podria no participar o fer-ho d'una forma redundant amb altres fosfatases. / Mahmoud Ali Ibrahim Hamouda, S. (2015). Protein kinases and phosphatases regulating the yeast proton pump [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/54131

Saccharomyces cerevisiae are unicellular organisms that are highly adaptable to acute changes in nutrient availability. The two main signaling pathways that allow S. cerevisiae to sense and respond to changes in glucose availability in the environment are the conserved cAMP/PKA and AMPK/Snf1 kinase-dependent pathways. The conserved TORC1 pathway is primarily responsible for allowing cells to respond to the availability of nitrogen. Studies have shown that S. cerevisiae, but not mammalian and plant cells, regulate nuclear tRNA trafficking in response to nutrient stress. Here, we show that the yeast species of the Saccharomyces genus, but not Schizosaccharomyces pombe and Kluyveromyces lactis specifically regulate nuclear tRNA export in response to nutrient stress, providing further evidence that regulation of nuclear tRNA export in response to nutrient availability is not evolutionarily conserved. We also established that amino acid and nitrogen starvation affects nuclear export of a subset of tRNAs in S. cerevisiae. Inhibition of TORC1 signaling by rapamycin treatment, which simulates nitrogen starvation, also affects nuclear export of the same subset of tRNAs, suggesting that the TORC1 signaling pathway plays a role in regulating nuclear export of the tRNAs in response to nitrogen level. Regulation of nuclear export of these tRNAs by nitrogen deprivation is most likely due to an effect on the function of the nuclear tRNA export receptors, as overexpression of the tRNA export receptor, Los1p, restores export of the tRNAs during nitrogen starvation. These findings suggest that the TORC1 signaling pathway may, in part, regulate nuclear export of the tRNAs by affecting the function of the tRNA export receptors. In contrast to amino acid and nitrogen starvation, glucose depletion affects nuclear export of all tRNA species in S. cerevisiae. Evidence obtained suggests that nuclear retention of tRNA in cells deprived of glucose is due to a block in nuclear re-import of the nuclear tRNA export receptors. Retention of the receptors in the cytoplasm is not caused by activation of Snf1p, but by the inactivation of PKA during glucose deprivation. Furthermore, regulation of nuclear re-import of the receptors is not due to phosphorylation of the tRNA export receptors by PKA. However, PKA phosphorylates known components of the tRNA export machinery. A model that is consistent with the data is that PKA and an unknown mechanism regulate the activity of these components or an unidentified protein(s) to control nuclear re-import of the receptors in response to glucose availability.

Ataxin-2 is an RNA-binding protein that is involved in many crucial cellular processes such as R-loop regulation, mRNA stability, TOR signaling regulation, and stress granule formation. Ataxin-2 is highly conserved, found in organisms ranging from Saccharomyces cerevisiae to Caenorhabditis elegans and Homo sapiens. Recently, ataxin-2 has been linked to the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal disease that causes loss of motor neurons. In addition to ataxin-2 interacting with known ALS risk factor proteins, research into the relationship between ataxin-2 and ALS shows that polyglutamine expansions in ataxin-2 are gain-of-function mutations that lead to overactivity of ataxin-2 and probable neurodegeneration. In fact, targeting ataxin-2 using gene silencing techniques dramatically slows the progression of ALS in both mice and man.The Grose laboratory has characterized a serine-threonine kinase, PAS kinase as upstream kinase and putative activator of ataxin-2. We hypothesize that knockdown of PAS kinase could, therefore, have similar effects to directly downregulating ataxin-2 and its cellular functions. Characterization of Ataxin-2 has revealed that its gain or loss of function lead to distinct cellular phenotypes. One study concluded that lowering ataxin-2 levels reduced the size and number of stress granules in mammalian cells, which was observed through microscopy. Another study found that activation and overexpression of ataxin-2 lead to reduced mTOR levels because of its sequestration to stress granules. Lastly, preliminary data obtained by the Grose laboratory suggests that yeast deficient in Pbp1 (the yeast homologue of ataxin-2) have altered cell cycles.This project describes the cellular readouts used to determine if PAS kinase downregulation confers the same cellular phenotypes as ataxin-2 downregulation. First, we found that PAS kinase does influence ataxin-2 abundance in mammalian cells. Using yeast as a model, we found that Pbp1 influences the cell cycle through its binding partners, causing a reduction in the percentage of cells in the G2 phase compared to the G1 phase. PAS kinase conferred an opposite change, most likely due to the activity of other PAS kinase substrates. Additionally, we found that Pbp1 deficiency is synthetically lethal when in conjunction with deficiency of any one of its cell cycle-related binding partners. The cellular changes cause by Pbp1 deficiency highlight not only the importance of ataxin-2 in the cell, but also the importance of understanding the effects of downregulation of ataxin-2.

Tesis por compendio / La exposición crónica a sustancias de abuso lleva a cambios adaptativos en el cerebro que implican alteraciones en la expresión génica. Se ha propuesto que los factores de transcripción CREB y deltaFosB serían dianas moleculares para la regulación de la plasticidad, la cual lleva a la adicción.En este trabajo hemos estudiado los cambios en la activación de CREB, en PVN y NTS, y las quinasas que mediarían su activación durante la dependencia y síndrome de abstinencia a morfina, así como la respuesta del eje HHA durante dicho síndrome. También se investigó la posibilidad de que la activación de CREB y su coactivador transcripcional TORC1 dependan de la activación de receptores adrenérgicos. Además se evaluaron las posibles modificaciones en la expresión de FosB/deltaFosB en diferentes áreas cerebrales implicadas en la adicción, así como los cambios neuroendocrinos/neuroquímicos responsables de las alteraciones metabólicas observadas durante el tratamiento crónico con morfina. / Chronic exposure to opioids and other abused drugs results in adaptive changes in the brain involving alterations in gene expression. It is proposed that the transcription factors CREB and deltaFosB be molecular targets for the regulation of plasticity, which leads to addiction.In this work we studied changes in activation of the cAMP-response element binding protein (CREB) in PVN and NTS and the kinases that may mediate this activation during dependence and morphine withdrawal and the HPA axis response after naloxone-induced morphine withdrawal. We also investigated the possibility that the activation of CREB and the transcriptional coactivator of CREB, TORC1, arises from the activation of adrenergic receptors. We also evaluated the possible modifications in FosB/deltaFosB expression in several brain areas involved in addiction and neuroendocrine/neurochemical changes that are responsible for the metabolic alterations seen during chronic morphine treatment.

Nitrogen Catabolite Repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae reduces the expression of genes encoding components involved in the utilization of poor nitrogen sources when rich ones are available. Expression of NCR-sensitive genes is controlled by the negative regulator Ure2 and four DNA-binding GATA-like transcription factors: two activators (Gln3 and Gat1) and two repressors (Dal80 and Gzf3). In the presence of preferred nitrogen sources, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner, whereas upon growth under non-preferred nitrogen conditions, the GATA activators relocate to the nucleus and mediate the transcription of NCR-sensitive genes. Even though the Target of Rapamycin Complex 1 (TORC1) as well as several phosphatases are involved in regulating Gln3 and Gat1 subcellular localization, a detailed mechanistic understanding of the NCR process is still lacking. <p>In the first part of this work, we have shown that class C and D VPS (vacuolar protein sorting) components, involved in Golgi-to-vacuole vesicular trafficking, are required for intact Gat1 and Gln3 nuclear localization in response to TORC1-inhibiting rapamycin treatment or upon shifting cells from rich to poor nitrogen conditions. The requirements of Vps proteins for Gln3 function are media-specific: a requirement after rapamycin treatment was observed in minimal but not in rich medium. Moreover, we have seen that a significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. These observations support the view that GATA factor regulation in response to nitrogen signals seems to occur at intracellular compartments.<p>In a second step, we confirmed an important role for the anabolic glutamate dehydrogenase (Gdh1) within NCR, through the control of Gat1 function. However, since we observed a strong correlation between the anabolic activity of Gdh1 and its NCR regulatory capacity, we do not exclude that an alteration of Gdh1-substrates or any other metabolite could be responsible for the phenotype exhibited by gdh1 mutants. We also showed that there is no simple and direct link between the intracellular levels of glutamine/glutamate (reported in the literature as signals for NCR), TORC1 activity and NCR. In conclusion, the mechanisms regulating the perception of the quality of the nitrogen sources are still not fully understood. <p>Several screens for multi-copy suppression of mutated phenotypes were conducted during this work and led to the identification of several elements (URE2, BAP2, STP2, GZF3 and KDX1) that could interfere with NCR-sensitive gene expression. Among these, the gene encoding the Kdx1 kinase was identified in two independent screens. <p>In the last part of this work, we uncovered a role for leucine in NCR signaling. We showed that the addition of leucine in the culture medium could impair Gat1-dependent expression of certain NCR genes, while leucine starvation had no effect at this level. The repressive effect of leucine appeared to involve elements of the SPS signaling pathway which is required for the induction of genes encoding amino acid transporters in response to extracellular amino acids. The mechanism(s) by which leucine regulates Gat1 function is still not fully clear and requires further investigation:La levure Saccharomyces cerevisiae adapte l’expression de ses gènes selon la disponibilité en azote dans son environnement au moyen d’un contrôle majeur appelé répression catabolique azotée (NCR, pour « nitrogen catabolite repression ». L’expression des gènes NCR est contrôlée par un régulateur négatif de type prion (Ure2) et quatre facteurs de transcription de type GATA :deux activateurs, Gat1 et Gln3 et deux répresseurs, Dal80 et Gzf3. Bien que le complexe TORC1 et les phosphatases qu’il régule soient impliquées dans la régulation NCR, le mécanisme précis par lequel la NCR se produit est loin d’être compris.<p>Dans la première partie de ce travail, nous avons montré que les composants VPS (vacuolar protein sorting) de classe C et D, impliqués dans le trafic vésiculaire entre le Golgi et la vacuole, sont requis pour que Gat1 et Gln3 rejoignent le noyau en réponse à un traitement par la rapamycine, un inhibiteur de TORC1. En accord avec cette observation, nous avons montré que Gat1, comme Gln3, est associé aux membranes intracellulaires légères. <p>Dans un second temps, nous avons confirmé un rôle important pour la glutamate déshydrogénase anabolique (Gdh1) au sein de la NCR, par l’intermédiaire du contrôle de la fonction de Gat1. Cependant, étant donné qu’il semble exister une forte corrélation entre l’activité anabolique de Gdh1 et sa capacité à réguler la NCR, nous n’excluons pas qu’une altération des substrats de Gdh1 ou de tout autre métabolite pourrait être responsable du phénotype observé du mutant gdh1. Nous avons également montré qu’il n’existait pas de lien simple et direct entre niveaux intracellulaires de glutamine/glutamate, activité de TORC1 et signalisation NCR. En conclusion, les mécanismes conditionnant la perception de la qualité de l’aliment azoté sont encore méconnus à ce jour. <p>Plusieurs cribles de suppression multicopie ont été menés pendant ce travail et ont conduit à l’identification de plusieurs éléments pouvant éventuellement intervenir dans la voie de signalisation NCR. Parmi ceux-ci, le gène codant pour la kinase KDX1 a été identifié à deux reprises. Nous avons caractérisé en détail le rôle qu’elle joue dans la régulation des gènes NCR.<p>Dans la dernière partie de ce travail, nous avons montré que l’addition de leucine dans le milieu de culture pouvait affecter l’expression Gat1-dépendante de certains gènes NCR, alors que par ailleurs une carence en leucine est sans effet à ce niveau. Cet effet de répression par la leucine semble nécessiter des éléments de la voie de signalisation SPS, requise pour l’induction, en réponse aux acides aminés extracellulaires, de gènes codant pour des transporteurs d’acides aminés. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished