Indiana University-Purdue University Indianapolis (IUPUI) / The unfolded protein response (UPR) is important for cell adaptation to accumulation of unfolded proteins in the endoplasmic reticulum (ER). A central UPR sensor of ER stress is PKR- like ER Kinase (PERK), which phosphorylates eIF2 to reduce global translation and help mitigate ER stress. While this is a survival mechanism that serves to save the cell from catastrophic events during ER stress, PERK can also be activated in cancer cells due to genetic changes and exposure to stresses inherent in the tumor micro-environment. Published reports have indicated that PERK is activated in cancer cells in response to hypoxia, nutrient deprivation, matrix detachment, and increased protein load by oncogene activation to facilitate cell survival. The UPR features PERK and another ER stress sensory protein, IRE1α, which also regulates the dynamic assembly of the actin cytoskeleton; loss of either PERK or IRE1α functions decrease cell migration activity. We hypothesized that PERK is required for anchorage-independent survival of the cancer cell line HCT116 and that PERK is essential for cell migration. Consistent with these premises, inhibition of PERK using pharmacological inhibitors GSK2656157 and LY-4 in suspended cells showed reduced growth. Furthermore, PERK-deficient cells showed reduced migration in transwell migration assays as compared to their wild type counterpart. These results suggest that PERK facilitates anchorage-independent growth of cancer cells and cell migration.

Made available in DSpace on 2015-03-26T13:36:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1875030 bytes, checksum: 2c2c1497d43bb10ad7e10f009f941c57 (MD5) Previous issue date: 2006-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Geminiviruses constitute a large group of plant virus whose genome is packed as single-stranded DNA circles in a small, twinned isometric particle and is converted to double- stranded forms in nuclei of differentiated plant cells. Members of the genus Begomovirus, such as Cabbage leaf curl virus (CaLCuV), possess two genomic components, DNA-A and DNA-B. The DNA-A has the potential to code for five gene products (AV1, AC1, AC2, AC3, AC4) and is involved in replication, transcriptional activation of viral genes and encapsidation of the viral genome. The DNA-B encodes two movement proteins, the movement protein MP (BC1) and the nuclear shuttle protein NSP (BV1), both required for systemic infection. NSP shuttles the viral DNA between the nucleus and the cytoplasm and then acts cooperatively with MP to move the viral DNA cell-to-cell across the wall. The localization of NSP and its proposed role in cell-to-cell movement of the viral DNA predict that interactions with host factors may occur in both the cytoplasm and the nucleus. In fact, NSP has been demonstrated to interact with a plasma membrane receptor protein, designated NIK, and a nuclear acetyltransferase. A proline-rich extensin-like receptor protein kinase (PERK) was found to interact specifically with NSP of CaLCuV and of tomato-infecting geminiviruses, through yeast two-hybrid screening. The PERK-like protein, which we designated NsAK (NSP-associated kinase), is structurally organized into a proline-rich N-terminal domain followed by a transmembrane segment and a C-terminal serine/threonine kinase domain. The viral protein interacted stably with defective versions of the NsAK kinase domain but not with the potentially active enzyme in an in vitro binding assay. In vitro translated NsAK enhanced the phosphorylation level of NSP, indicating that NSP functions as substrate for NsAK. These results demonstrated that NsAK is an authentic serine/threonine kinase and suggested a functional link for the NSP-NsAK complex formation. This interpretation was corroborated by in vivo infectivity assays showing that loss of NsAK function delays the onset of CaLCuV infection and attenuates symptom development. Our data implicate NsAK as a positive contributor to geminivirus infection and suggest it may regulate NSP function. / Geminivírus constitui um grande grupo de vírus de planta, cujo genoma é empacotado na forma de DNA circular fita simples em partículas icosaédricas geminadas e é convertido em uma forma fita dupla no núcleo de células diferenciadas de plantas. A maioria dos membros do gênero Begomovirus, como o Cabbage leaf curl vírus (CaLCuV), possuem dois componentes genômicos, DNA-A e DNA-B. O DNA-A apresenta o potencial para codificar cinco produtos gênicos (AV1, AC1, AC2, AC3, AC4) e está envolvido na replicação, ativação transcricional de genes virais e encapsidação do genoma viral. O DNA-B codifica duas proteínas de movimento, Movement Protein MP (BC1) e Nuclear Shuttle Protein NSP (BV1), ambas requeridas para o estabelecimento de uma infecção sistêmica. NSP transporta o DNA viral entre o núcleo e o citoplasma e então atua cooperativamente com MP para transportar o DNA viral de célula-a-célula através da parede celular. A localização de NSP e seu papel proposto no movimento célula- a-célula do DNA viral predizem que podem ocorrer interações com fatores do hospedeiro tanto no citoplasma quanto no núcleo. De fato, foi demonstrado que NSP interage com uma proteína receptora da membrana plasmática, designada NIK, e uma acetiltransferase nuclear. Através de ensaio de duplo híbrido, foi identificada, uma PERK quinase ( proline-rich extensin-like receptor protein kinase ) de Arabidopsis thaliana que interage especificamente com NSP de CaLCuV e, também de geminivírus que infectam tomate, a qual foi designada NsAK ( NSP-associated kinase ) e é estruturalmente organizada em um domínio N-terminal rico em prolina seguido de um segmento transmembrana e de um domínio C-terminal de serina/treonina quinase. A proteína viral interagiu estavelmente com versões defectivas do domínio de quinase de NsAK, mas não com a enzima potencialmente ativa em um ensaio de ligação in vitro. NsAK traduzida in vitro aumentou o nível de fosforilação de NSP, indicando que NSP atua como substrato de NsAK. Estes resultados demonstram que NsAK é uma autêntica serina/treonina quinase e sugerem elo funcional para a formação do complexo NSP-NsAK. Esta interpretação foi corroborada por ensaios de infectividade in vivo, demonstrando que a perda de função de NsAK reduz a eficiência da infecção por CaLCuV e atenua o desenvolvimento dos sintomas. Estes dados implicam NsAK como um contribuidor positivo para a infecção por geminivírus e sugerem que NsAK pode regular a função de NSP.

No description available.

Le réticulum endoplasmique (RE) est un organite assurant de nombreuses fonctionscellulaires telles que la conformation et des modifications post-traductionnelles des protéines ou lemaintien de l’homéostasie calcique. Cet organite est donc un site crucial pour réguler le maintien del’homéostasie cellulaire et tissulaire des organismes multicellulaires. Des altérations de ses fonctionsconduisent à l’accumulation de protéines mal-conformées qui sont observées dans de nombreusespathologies humaines telles que des cancers ou des maladies inflammatoires chroniques. Ce stressdéclenche une réponse adaptative connue sous le nom de réponse aux protéines mal-conformées(UPR) qui permet à la cellule de supprimer ses sources et conséquences. Néanmoins, l’intensité et lachronicité du stress peuvent entrainer une modification de l’UPR qui conduit alors à l’élimination dela cellule par apoptose. A ce jour, les processus moléculaires qui permettent à l’UPR d’induirel’apoptose restent flous. De plus, l’implication de l'UPR dans la régulation de processuscompensatoires n'a jamais été étudiée. Mes travaux de thèse apportent une meilleurecompréhension de ces mécanismes à travers l’étude comparative de différents modèles de stresschronique du RE, qui dépendent d’une dérégulation de l’homéostasie protéique et/ou calcique. Ilssoulignent également le rôle essentiel de la branche PERK/ATF4 de l’UPR dans l’induction de deuxvoies parallèles et indépendantes. D’une part, PERK promeut une apoptose dépendante des caspasesvia une répression de l'expression de diap1, et d‘autre part, elle induit un retard de développement àtravers une induction de l’expression de dilp8 dépendante de la voie JNK. Mes données suggèrentégalement une spécificité tissulaire des signalisations déclenchées en réponse à un stress chroniquedu RE. / The endoplasmic reticulum (ER) is an organelle which ensures various cellular functionssuch as protein maturation and folding or calcium homeostasis maintenance. That is why ER is acrucial site of cell and tissue homeostasis regulation in multicellular organisms. Disruption of ERfunctions leads to misfolded-protein accumulation and is observed in a great number of devastatinghuman diseases. This ER stress triggers an adaptive response named Unfolded Protein Response(UPR) in order to attempt to resolve its sources and consequences. Nevertheless, the intensity andchronicity of ER stress can change this response and lead to the apoptosis of stressed cells. To thisdate, the molecular processes that regulate UPR-induced apoptosis remain unclear. Furthermore, theUPR contribution in the modulation of compensatory mechanisms in response to ER stress has neverbeen studied. This work contributes to a better understanding of these processes through acomparative study of various chronic ER stresses, which depend on the disruption of proteostasis orcalcium homeostasis. During my thesis, I have established the essential role of the PERK/ATF4 branchof the UPR in the induction of two parallel and independent pathways. One promotes apoptosisthrough the down-regulation of the diap1 gene while the other interferes with the induction of adevelopmental delay though a JNK signaling-dependent dilp8 expression. My results also suggest thatchronic ER stress response is tissue specific.

Thesis (Ph.D.)--Indiana University, 2010. / Title from screen (viewed on July 21, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Ronald C. Wek, Howard J. Edenberg, Peter J. Roach, Martin Bard. Includes vitae. Includes bibliographical references (leaves 125-132).

京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第13322号 / 論理博第1569号 / 新制||理||1663(附属図書館) / 京都大学大学院理学研究科 / (主査)教授 森 和俊, 教授 川口 真也, 教授 平野 丈夫 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Two important nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), participate in the control of yeast growth and metabolism in response to changes in nutrient availability. Starvation for amino acids activates the GAAC through Gcn2p phosphorylation of the translation initiation factor eIF2 and preferential translation of GCN4, a transcription activator. TOR senses nitrogen availability and regulates transcription factors, such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome during amino acid starvation and rapamycin treatment. We found that the GAAC is a major effector of the TOR pathway, with Gcn4p and Gln3p each inducing a similar number of genes during rapamycin treatment. While Gcn4p activates a common core of 57 genes, the GAAC directs significant variations in the transcriptome during different stresses. In addition to inducing amino acid biosynthetic genes, Gcn4p activates genes required for assimilation of secondary nitrogen sources, such as -amino-butyric acid (GABA). Gcn2p activation upon shifting to secondary nitrogen sources is suggested to occur by means of a dual mechanism. First, Gcn2p is induced by the release of TOR repression through a mechanism involving Sit4p protein phosphatase. Second, this eIF2 kinase is activated by select uncharged tRNAs, which were shown to accumulate during the shift to GABA medium. This study highlights the mechanisms by which the GAAC and TOR pathways are integrated to recognize changing nitrogen availability and direct the transcriptome for optimal growth adaptation.

Les leucémies aiguës myéloïdes (LAM) représentent un groupe d’hémopathies malignes agressives, de pronostic sombre en dépit des traitements intensifs actuellement proposés. Malgré une grande hétérogénéité clinique et moléculaire, les cellules de LAM sont caractérisées par l’activation de voies de signalisation essentielles à leur prolifération et leur survie, comme par exemple celle du complexe mTORC1 (mammalian target of rapamycin complex 1). Cependant, l’utilisation clinique d’inhibiteurs tels que la rapamycine ou des inhibiteurs catalytiques s’est avérée décevante, ce qui suggère qu’il n’y a pas d’addiction oncogénique à mTORC1 dans les LAM. Au cours de ce travail, nous avons démontré que l’activation de mTORC1 est au contraire une condition nécessaire à l’induction de la mort cellulaire en réponse à l’activation d’AMPK (AMP-activated protein kinase), établissant une relation de létalité synthétique entre ces deux voies. Pour cela, nous avons utilisé un nouveau composé activateur spécifique d’AMPK, le GSK621. En invalidant la sous-unité catalytique AMPKα1 par ARN interférence ou par le système CRISPR/Cas9, nous avons démontré que les effets antileucémiques de ce composé sont bien dépendants de l’activation d’AMPK. Nous avons observé que ce composé favorise l’autophagie, et que ce processus est impliqué dans la mort des cellules leucémiques puisque l’inhibition des protéines ATG5 ou ATG7 a un effet protecteur sur les cellules leucémiques. Les effets antileucémiques du composé GSK621 ont été confirmés sur des cellules primaires, ainsi que sur un panel de vingt lignées de LAM, et dans un modèle murin de xénogreffe. De façon intéressante, l’activation d’AMPK pourrait également compromettre la survie des cellules souches leucémiques, comme en atteste l’atténuation du potentiel clonogénique en méthylcellulose de cellules murines transformées par MLL-ENL ou FLT3-ITD. Nous avons observé que le composé GSK 621 n’avait pas de toxicité envers les progéniteurs hématopoïétiques normaux, ouvrant ainsi une fenêtre thérapeutique intéressante. Comme l’activation d’AMPK conduit dans de nombreux modèles cellulaires à l’inhibition de mTORC1, et comme l’activation de mTORC1 est observée dans les cellules de LAM mais pas dans les progéniteurs hématopoïétiques normaux, nous avons proposé l’hypothèse que le niveau d’activation de mTORC1 déterminait les effets de l’activateur d’AMPK. Pour cela, nous avons inhibé mTORC1 dans les cellules leucémiques d’une part, et activé mTORC1 dans les progéniteurs normaux d’autre part. De façon inattendue, mTORC1 échappe au contrôle d’AMPK dans les LAM, et nous avons observé que l’activation de mTORC1 est une condition nécessaire et suffisante pour que le composé GSK621 entraîne la mort des cellules. Le substrat moléculaire de cette létalité synthétique est le facteur de transcription proapoptotique ATF4, dont la transcription est favorisée par mTORC1, et la traduction par AMPK via la phosphorylation d’eIF2A. Ces travaux proposent donc que malgré l’absence d’addiction oncogénique, l’activation de mTORC1 dans les LAM représente une opportunité thérapeutique originale via une relation de létalité synthétique avec l’activation d’AMPK. Ils constituent un rationnel au développement clinique d’activateurs d’AMPK dans les LAM, voire dans d’autres cancers ayant une activation constitutive de mTORC1. / Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis despite intensive treatments. Virtually all recurrent molecular alterations in AML functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as in clinical trials, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR/Cas9 and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, and blocking autophagy via shRNA-mediated knockdown of ATG5 or ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation between AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1, sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. The same synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. We further observed that the level of ATF4 protein is under a transcriptionnal control by mTORC1 and a translational control by AMPK (through eIF2A), and explains the synthetic lethal relationship between AMPK and mTORC1. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials.

La fibrose hépatique est la conséquence de toutes les maladies chroniques du foie et se caractérise par un dépôt excessif de matrice extracellulaire synthétisée par les myofibroblastes. Les myofibroblastes portaux (MFP), l'une des sous populations de myofibroblastes, jouent un rôle majeur dans la progression de la fibrose et sont pro-angiogéniques. Des études ont montré un rôle important du stress du réticulum endoplasmique (RE) dans la fibrose du foie. Nos objectifs étaient de déterminer si un stress du RE survient dans les MFP lors de la fibrose et affecte les fonctions de ces cellules, et d'étudier l'effet du TUDCA, une molécule chaperonne utilisée en clinique dans les maladies biliaires, sur le stress du RE. Le phénotype de MFP activés in vivo, isolés à partir de foie de rats fibreux après cholestase, a été comparé à celui de MFP contrôles que nous avons préalablement bien caractérisés. Nos résultats montrent que les MFP activés in vivo subissent un stress du RE se traduisant par l'activation de la voie PERK. Ce stress du RE n'a pas d'effet sur la différenciation myofibroblastique, diminue les capacités de prolifération et de migration des MFP mais augmente leur pouvoir angiogénique. En revanche, le TUDCA n'a aucun effet sur les paramètres étudiés. Les MFP subissent donc un stress du RE lors de leur activation myofibroblastique qui stimule leur propriété pro-angiogénique et pourrait ainsi favoriser la progression de la fibrose. Cependant le stress du RE inhibe également leurs fonctions de prolifération et de migration ce qui pourrait induire une boucle de contrôle négative limitant leur expansion. / Hepatic fibrosis is the consequence of all chronic liver diseases and is characterized by an abnormal extra cellular matrix deposition by myofibroblasts. Portal myofibroblasts (PMF), a subpopulation of hepatic myofibroblasts, play a major role in fibrosis progression and angiogenesis. Accumulating evidences indicate an important role of endoplasmic reticulum (ER) stress in hepatic fibrosis. The aims of this study were to determine whether an ER stress occured in PMF during fibrosis and affected the functions of these cells, and to study the effect of the molecular chaperone TUDCA used in biliary diseases, on ER stress. The phenotype of in vivo activated-PMF obtained from rat fibrotic liver after cholestasis was compared with the phenotype of control PMF that we previously characterized. Our results showed that in vivo activated-PMF underwent ER stress with PERK pathway activation. This ER stress had no effect on myofibroblastic differentiation but reduced PMF proliferation and migration and increased PMF angiogenesis capacity. TUDCA had no effect on these parameters. In conclusion, PMF display ER stress during their activation. ER stress stimulates their pro-angiogenic proprieties and thereby may promote fibrosis progression. However, ER stress also inhibits their proliferation and migration functions, and thereby could provide a negative control loop to restrict their expansion.

No description available.