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The role of the peptidyl prolyl isomerase Rrd1 in the transcriptional stress responsePoschmann, Jeremie 08 1900 (has links)
La régulation de la transcription est un processus complexe qui a évolué pendant des millions
d’années permettant ainsi aux cellules de s’adapter aux changements environnementaux. Notre
laboratoire étudie le rôle de la rapamycine, un agent immunosuppresseur et anticancéreux, qui
mime la carence nutritionelle. Afin de comprendre les mécanismes impliqués dans la réponse a
la rapamycine, nous recherchons des mutants de la levure Saccaromyces cerevisiae qui ont un
phenotype altérée envers cette drogue. Nous avons identifié le gène RRD1, qui encode une
peptidyl prolyl isomérase et dont la mutation rend les levures très résistantes à la rapamycine et il
semble que se soit associé à une réponse transcriptionelle alterée. Mon projet de recherche de
doctorat est d’identifier le rôle de Rrd1 dans la réponse à la rapamycine. Tout d’abord nous
avons trouvé que Rrd1 interagit avec l’ARN polymérase II (RNAPII), plus spécifiquement avec
son domaine C-terminal. En réponse à la rapamycine, Rrd1 induit un changement dans la
conformation du domaine C-terminal in vivo permettant la régulation de l’association de RNAPII
avec certains gènes. Des analyses in vitro ont également montré que cette action est directe et
probablement liée à l’activité isomérase de Rrd1 suggérant un rôle pour Rrd1 dans la régulation
de la transcription. Nous avons utilisé la technologie de ChIP sur micropuce pour localiser Rrd1
sur la majorité des gènes transcrits par RNAPII et montre que Rrd1 agit en tant que facteur
d’élongation de RNAPII. Pour finir, des résultats suggèrent que Rrd1 n’est pas seulement
impliqué dans la réponse à la rapamycine mais aussi à differents stress environnementaux, nous
permettant ainsi d’établir que Rrd1 est un facteur d’élongation de la transcription requis pour la
régulation de la transcription via RNAPII en réponse au stress. / Transcriptional regulation is a complex process that has evolved over millions of years of
evolution. Cells have to sense environmental conditions and adapt to them by altering their
transcription. Herein, we study the role of rapamycin, an immunosuppressant and anticancer
molecule that mimics cellular starvation. To understand how the action of rapamycin is
mediated, we analyzed gene deletion mutants in the yeast Saccharomyces cerevisiae that have an
altered response to this drug. Deletion of RRD1, a gene encoding a peptidyl prolyl isomerase,
causes strong resistance to rapamycin and this was associated with a role of Rrd1 in the
transcriptional response towards rapamycin. The main focus of my PhD was therefore to unravel
the role of Rrd1 in response to rapamycin. First, we discovered that Rrd1 interacts with RNA
polymerase II (RNAPII), more specifically with its C-terminal domain and we showed that in
response to rapamycin, Rrd1 alters the structure of this C-terminal domain. This phenomenon
was confirmed to be directly mediated by Rrd1 in vitro, presumably through its peptidyl prolyl
isomerase activity. Further, we demonstrated that Rrd1 is capable of altering the occupancy of
RNAPII on genes in vivo and in vitro. With the use of ChIP on chip technology, we show that
Rrd1 is actually a transcription elongation factor that is associated with RNAPII on actively
transcribed genes. In addition, we demonstrate that Rrd1 is indeed required to regulate the
expression of a large subset of genes in response to rapamycin. This data let us propose a novel
mechanism by which Rrd1 regulates RNAPII during transcription elongation. Finally, we
provide evidence that Rrd1 is not only required for an efficient response towards rapamycin but
to a larger variety of environmental stress conditions, thus establishing Rrd1 as a transcriptional
elongation factor required to fine tune the transcriptional stress response of RNAPII.
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V-ATPase regulation of Hypoxia Inducible transcription FactorsMiles, Anna Louise January 2018 (has links)
Metazoans have evolved conserved mechanisms to promote cell survival under low oxygen tensions by initiating a transcriptional cascade centered on the action of Hypoxia Inducible transcription Factors (HIFs). In aerobic conditions, HIFs are inactivated by ubiquitin-proteasome-mediated degradation of their a subunit, which is dependent on prolyl hydroxylation by 2-oxoglutarate (2-OG) and Fe(II)-dependent prolyl hydroxylases (PHDs). In hypoxia, HIF-$\alpha$ is no longer hydroxylated and is therefore stabilised, activating a global transcriptional response to ensure cell survival. Interestingly, HIFs can also be activated in aerobic conditions, however the mechanisms of this oxygen-independent regulation are poorly understood. Here, I have explored the role of the vacuolar H+-ATPase (V-ATPase), the major proton pump for acidifying intracellular vesicles and facilitating lysosomal degradation, in regulating HIF-$\alpha$ turnover. Unbiased forward genetic screens in near-haploid human cells identified that disruption of the V-ATPase leads to activation of HIFs in aerobic conditions. Rather than preventing the lysosomal degradation of HIF-$\alpha$, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-$\alpha$ isoforms by altering the intracellular iron pool and preventing HIF-$\alpha$ prolyl hydroxylation. In parallel, I characterised two putative mammalian V-ATPase assembly proteins, TMEM199 and CCDC115, identified by the forward genetic screen and subsequent mass spectrometry analysis. I confirmed that both TMEM199 and CCDC115 are required for V-ATPase function, and established assays to determine how TMEM199 and CCDC115 associate with components of the core V-ATPase complex. Lastly, to measure how V-ATPase activity leads to changes in the labile iron pool, I developed an endogenous iron reporter using CRISPR-Cas9 knock-in technology. This approach confirmed that iron homeostasis is impaired during V-ATPase inhibition, and demonstrated that exogenous ferric iron can restore the labile iron pool in a transferrin-independent manner. Together my studies highlight a crucial link between V-ATPase activity, iron homeostasis, and the hypoxic response pathway.
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The role of the peptidyl prolyl isomerase Rrd1 in the transcriptional stress responsePoschmann, Jeremie 08 1900 (has links)
La régulation de la transcription est un processus complexe qui a évolué pendant des millions
d’années permettant ainsi aux cellules de s’adapter aux changements environnementaux. Notre
laboratoire étudie le rôle de la rapamycine, un agent immunosuppresseur et anticancéreux, qui
mime la carence nutritionelle. Afin de comprendre les mécanismes impliqués dans la réponse a
la rapamycine, nous recherchons des mutants de la levure Saccaromyces cerevisiae qui ont un
phenotype altérée envers cette drogue. Nous avons identifié le gène RRD1, qui encode une
peptidyl prolyl isomérase et dont la mutation rend les levures très résistantes à la rapamycine et il
semble que se soit associé à une réponse transcriptionelle alterée. Mon projet de recherche de
doctorat est d’identifier le rôle de Rrd1 dans la réponse à la rapamycine. Tout d’abord nous
avons trouvé que Rrd1 interagit avec l’ARN polymérase II (RNAPII), plus spécifiquement avec
son domaine C-terminal. En réponse à la rapamycine, Rrd1 induit un changement dans la
conformation du domaine C-terminal in vivo permettant la régulation de l’association de RNAPII
avec certains gènes. Des analyses in vitro ont également montré que cette action est directe et
probablement liée à l’activité isomérase de Rrd1 suggérant un rôle pour Rrd1 dans la régulation
de la transcription. Nous avons utilisé la technologie de ChIP sur micropuce pour localiser Rrd1
sur la majorité des gènes transcrits par RNAPII et montre que Rrd1 agit en tant que facteur
d’élongation de RNAPII. Pour finir, des résultats suggèrent que Rrd1 n’est pas seulement
impliqué dans la réponse à la rapamycine mais aussi à differents stress environnementaux, nous
permettant ainsi d’établir que Rrd1 est un facteur d’élongation de la transcription requis pour la
régulation de la transcription via RNAPII en réponse au stress. / Transcriptional regulation is a complex process that has evolved over millions of years of
evolution. Cells have to sense environmental conditions and adapt to them by altering their
transcription. Herein, we study the role of rapamycin, an immunosuppressant and anticancer
molecule that mimics cellular starvation. To understand how the action of rapamycin is
mediated, we analyzed gene deletion mutants in the yeast Saccharomyces cerevisiae that have an
altered response to this drug. Deletion of RRD1, a gene encoding a peptidyl prolyl isomerase,
causes strong resistance to rapamycin and this was associated with a role of Rrd1 in the
transcriptional response towards rapamycin. The main focus of my PhD was therefore to unravel
the role of Rrd1 in response to rapamycin. First, we discovered that Rrd1 interacts with RNA
polymerase II (RNAPII), more specifically with its C-terminal domain and we showed that in
response to rapamycin, Rrd1 alters the structure of this C-terminal domain. This phenomenon
was confirmed to be directly mediated by Rrd1 in vitro, presumably through its peptidyl prolyl
isomerase activity. Further, we demonstrated that Rrd1 is capable of altering the occupancy of
RNAPII on genes in vivo and in vitro. With the use of ChIP on chip technology, we show that
Rrd1 is actually a transcription elongation factor that is associated with RNAPII on actively
transcribed genes. In addition, we demonstrate that Rrd1 is indeed required to regulate the
expression of a large subset of genes in response to rapamycin. This data let us propose a novel
mechanism by which Rrd1 regulates RNAPII during transcription elongation. Finally, we
provide evidence that Rrd1 is not only required for an efficient response towards rapamycin but
to a larger variety of environmental stress conditions, thus establishing Rrd1 as a transcriptional
elongation factor required to fine tune the transcriptional stress response of RNAPII.
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Estudo fitoquímico e avaliação das atividades antimicrobiana, de inibição enzimática e antitumoral de Erythrina crista-galli nativa do RS / Phytochemical study and evaluation of antimicrobial, enzymatic inhibition and antitumor activities Erythrina crista-galli native from RSÁvila, Janaína Medeiros de 05 September 2013 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The phytochemical study of the crude extract hexane, methanol and fractions (acid ether, basic ether and basic acetate) from the stem bark of E. crista-galli (Fabaceae) resulted in the isolation of four compounds: The phytosterol stigmasterol (70), the triterpene lupeol (71) and the alkaloids erysotrine (1) and epierythratidine (50), usual in the genus Erythrina. The structures of the isolated metabolites were elucidated by 1H and 13C NMR uni and bidimensional, and compared with standard sample and data available in the literature. The extracts, fractions and isolated compounds were tested for their antimicrobial and antitumor front cancer cells HT29 (colorectal) activities, as well as regarding the capacity of inhibition of enzymes prolyl oligopeptidase, dipeptidil peptidase-VI and acetylcholinesterase. The crude methanolic extract, all fractions and individual compounds showed high antimicrobial activity mainly against Gram-positive and Gram-negative bacteria. The results were satisfactory in POP inhibition assays, when the crude methanolic extract and its fractions, mainly acid ether fraction, showed great inhibitor potential against this enzyme. For DPP-IV only the crude hexane extract was active. The in vitro antitumoral activity of the crude methanolic extract, basic fractions and the isolate alkaloids was investigated at different concentrations against the human colon cancer cell line HT-29 (PicoGreen dsDNA assay). The results suggest that the anti-proliferative effect of E. crista-galli extract on HT-29 cancer cells may be attributed, at least in part, to the presence of the erythrinian alkaloids 1 and 50. / O estudo fitoquímico do extrato bruto hexânico, metanólico e frações (éter ácida, éter básica e acetato básica) das cascas do caule de E. crista-galli (Fabaceae) resultou no isolamento de quatro compostos: o fitoesterol estigmasterol (70) e o triterpeno lupeol (71) além dos alcaloides erisotrina (1) e epieritratidina (50) usuais do gênero Erythrina. As estruturas dos metabólitos isolados foram elucidadas através de RMN 1H e 13C, uni e bidimensionais, além de comparação com amostra padrão quando existente e dados disponíveis na literatura. Os extratos, as frações e os compostos isolados foram testados quanto à sua atividade antimicrobiana, antitumoral frente a células cancerígenas HT29 (colorretal) e de inibição das enzimas POP, DPP-IV e AChE. Dentre as amostras testadas, o extrato bruto metanólico (EBM), suas frações (FEA, FEB e FAB) e compostos isolados apresentaram grande potencial antimicrobiano principalmente frente as bactérias Gram-positivas e Gram-negativas. Os resultados obtidos nos ensaios enzimáticos foram satisfatórios para a enzima POP, onde o EBM e suas frações, principalmente a fração éterea ácida (FEA), demonstraram grande potencial inibidor desta enzima. Para a DPP-IV apenas o extrato bruto hexânico (EBH) mostrou-se ativo. O efeito antitumoral da planta em questão também foi investigado e os resultados obtidos indicam que o EBM, a combinação das frações FEB e FAB e o alcaloide epieritratidina (50) possuem um grande efeito antiproliferativo frente às células do colorretal (HT29) após 72 horas de exposição.
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