Utilisation de la levure en tant que modèle et outil : de la compréhension de la cytokinèse à l'étude de sélectivité d'une molécule à potentiel thérapeutique

The yeast Saccharomyces cerevisiae can be used as a model but also as a tool for research in Biology. During my PhD, I have worked on two different research projects: I have used yeast as a model system to study cytokinesis, but also as a tool to study the selectivity of roscovitine, a small-molecule drug candidate currently in phase IIb of clinical trials against different cancers. Cytokinesis is the last step of cell cycle division. Contrary to mitosis, this step is rarely studied and is still poorly understood. Results obtained previously in our laboratory had established that the yeast protein Hof1 must be degraded in an ubiquitin-proteasome dependant way for cells to be physically split at the end of cell cycle. Nevertheless, the biological role of Hof1 was still unknown. The work presented here highlights the essential role of this protein in membrane recycling, and more specifically in exchanges between endosomes and the Golgi apparatus. These results establish for the first time in yeast, a link between membrane recycling and cytokinesis. Among protein kinases, the CDKs (Cyclin dependant kinases) are the main regulators of several essential biological mechanisms such as the cell cycle. Abnormalities of their regulation are involved in a number of pathologies, and particularly cancers. Pharmacological inhibitors of CDKs thus constitute potential therapeutic molecules. To fully understand the mechanism of action of these drugs and to optimize them so as to bring them towards clinical use, it is important to know their in vivo selectivity. The purpose of this study was to identify targets of roscovitine, a purine that have been previously discovered in our laboratory. To this end, we applied the method of affinity chromatography on immobilized molecules to the yeast proteome. Many targets have been identified and validated. Moreover, this study opens the way for the identification of cellular drug targets of therapeutic molecules by genetic methods.La levure Saccharomyces cerevisiae peut être employée en tant que modèle mais aussi en tant qu’outil pour la recherche en biologie. C’est sur cette base que, lors de ce travail, deux axes de recherches ont été développés : la levure a servi de modèle pour une étude fondamentale de la cytokinèse, mais elle a aussi été un outil pour l’étude de la sélectivité d’un composé de bas poids moléculaire, la roscovitine. La cytokinèse est la dernière étape du cycle de division cellulaire. A l’inverse de la mitose, cette phase fait l’objet de peu d’études et reste encore mal connue. Des résultats obtenus au laboratoire montraient que la protéine de levure Hof1 doit être dégradée de manière ubiquitine-protéasome dépendante pour que les cellules puissent se séparer physiquement à l’issu du cycle cellulaire. Malgré cela, le rôle biologique de la protéine Hof1 était encore inconnu. Le travail présenté ici met en avant le rôle essentiel de cette protéine dans le recyclage membranaire, et plus particulièrement dans les échanges entre les endosomes et l’appareil de Golgi. Ces données permettent pour la première fois, chez la levure, de faire un lien entre ce processus biologique et la cytokinèse. Parmi les protéine kinases, les CDKs (Cyclin dependant kinases) sont les principaux régulateurs de plusieurs mécanismes biologiques cruciaux dont le cycle cellulaire. Des anomalies de leur régulation sont impliquées dans de nombreuses pathologies, en particulier les cancers. Les inhibiteurs pharmacologiques des protéine kinases, et plus particulièrement des CDKs constituent donc une famille de produits thérapeutiques potentiels. Afin de comprendre de façon exhaustive le mode d’action de ces drogues et de les optimiser de façon à les amener vers une utilisation clinique, il est important d’en connaître la sélectivité in vivo. Ainsi, le but de cette étude est d’identifier les cibles de la roscovitine, une purine isolée au laboratoire et actuellement en phase IIb de tests cliniques pour différents cancers. Afin d’aborder cette question, nous avons choisi la technique de la chromatographie d’affinité sur inhibiteurs immobilisés appliquée à la levure. Plusieurs cibles ont été identifiées et validées. De plus, ce travail apporte les premières bases expérimentales d’un vaste projet de caractérisation par voie génétique chez la levure de cibles cellulaires de drogues à haut potentiel thérapeutique

Utilisation de la levure en tant que modèle et outil (de la compréhension de la cytokinèse à l'étude de sélectivité d'une molécule à potentiel thérapeutique)

La levure Saccharomyces cerevisiae peut être employée en tant que modèle mais aussi en tant qu outil pour la recherche en biologie. C est sur cette base que, lors de ce travail, deux axes de recherches ont été développés: la levure a servi de modèle pour une étude fondamentale de la cytokinèse, mais elle a aussi été un outil pour l étude de la sélectivité d un composé de bas poids moléculaire, la roscovitine. La cytokinèse est la dernière étape du cycle de division cellulaire. A l inverse de la mitose, cette phase fait l objet de peu d études et reste encore mal connue. Des résultats obtenus au laboratoire montraient que la protéine de levure Hof1 doit être dégradée de manière ubiquitine-protéasome dépendante pour que les cellules puissent se séparer physiquement à l issu du cycle cellulaire. Malgré cela, le rôle biologique de la protéine Hof1 était encore inconnu. Le travail présenté ici met en avant le rôle essentiel de cette protéine dans le recyclage membranaire, et plus particulièrement dans les échanges entre les endosomes et l appareil de Golgi. Ces données permettent pour la première fois, chez la levure, de faire un lien entre ce processus biologique et la cytokinèse. Parmi les protéine kinases, les CDKs (Cyclin dependant kinases) sont les principaux régulateurs de plusieurs mécanismes biologiques cruciaux dont le cycle cellulaire. Des anomalies de leur régulation sont impliquées dans de nombreuses pathologies, en particulier les cancers. Les inhibiteurs pharmacologiques des protéine kinases, et plus particulièrement des CDKs constituent donc une famille de produits thérapeutiques potentiels. Afin de comprendre de façon exhaustive le mode d action de ces drogues et de les optimiser de façon à les amener vers une utilisation clinique, il est important d en connaître la sélectivité in vivo. Ainsi, le but de cette étude est d identifier les cibles de la roscovitine, une purine isolée au laboratoire et actuellement en phase IIb de tests cliniques pour différents cancers. Afin d aborder cette question, nous avons choisi la technique de la chromatographie d affinité sur inhibiteurs immobilisés appliquée à la levure. Plusieurs cibles ont été identifiées et validées. De plus, ce travail apporte les premières bases expérimentales d un vaste projet de caractérisation par voie génétique chez la levure de cibles cellulaires de drogues à haut potentiel thérapeutique.The yeast S. cerevisiae can be used as a model but also as a tool for research in Biology. During my PhD, I have worked on two different research projects: I have used yeast as a model system to study cytokinesis, but also as a tool to study the selectivity of roscovitine, a small-molecule drug candidate currently in phase IIb of clinical trials against different cancers. Cytokinesis is the last step of cell cycle division. Contrary to mitosis, this step is rarely studied and is still poorly understood. Results obtained previously in our laboratory had established that the yeast protein Hof1 must be degraded in an ubiquitin-proteasome dependant way for cells to be physically split at the end of cell cycle. Nevertheless, the biological role of Hof1 was still unknown. The work presented here highlights the essential role of this protein in membrane recycling, and more specifically in exchanges between endosomes and the Golgi apparatus. These results establish for the first time in yeast, a link between membrane recycling and cytokinesis. Among protein kinases, the CDKs (Cyclin dependant kinases) are the main regulators of several essential biological mechanisms such as the cell cycle. Abnormalities of their regulation are involved in a number of pathologies, and particularly cancers. Pharmacological inhibitors of CDKs thus constitute potential therapeutic molecules. To fully understand the mechanism of action of these drugs and to optimize them so as to bring them towards clinical use, it is important to know their in vivo selectivity. The purpose of this study was to identify targets of roscovitine, a purine that have been previously discovered in our laboratory. To this end, we applied the method of affinity chromatography on immobilized molecules to the yeast proteome. Many targets have been identified and validated. Moreover, this study opens the way for the identification of cellular drug targets of therapeutic molecules by genetic methods.RENNES1-BU Sciences Philo (352382102) / SudocROSCOFF-Observ.Océanol. (292393008) / SudocSudocFranceF

Identification of potential cellular targets of aloisine A by affinity chromatography.

International audienceAffinity chromatography was used to identify potential cellular targets of aloisine A (7-n-butyl-6-(4'-hydroxyphenyl)-5H-pyrrolo[2,3b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine

Plankton Planet: A frugal, cooperative measure of aquatic life at the planetary scale

International audienceIn every liter of seawater there are between 10 and 100 billion life forms, mostly invisible, called marine plankton or marine microbiome, which form the largest and most dynamic ecosystem on our planet, at the heart of global ecological and economic processes. While physical and chemical parameters of planktonic ecosystems are fairly well measured and modeled at the planetary scale, biological data are still scarce due to the extreme cost and relative inflexibility of the classical vessels and instruments used to explore marine biodiversity. Here we introduce ‘Plankton Planet’, an initiative whose goal is to engage the curiosity and creativity of researchers, makers, and mariners to ( i ) co-develop a new generation of cost-effective (frugal) universal scientific instrumentation to measure the genetic and morphological diversity of marine microbiomes in context, ( ii ) organize their systematic deployment through coastal or open ocean communities of sea-users/farers, to generate uniform plankton data across global and long-term spatio-temporal scales, and ( iii ) setup tools to flow the data without embargo into public and explorable databases. As proof-of-concept, we show how 20 crews of sailors were able to sample plankton biomass from the world surface ocean in a single year, generating the first seatizen-based, planetary dataset of marine plankton biodiversity based on DNA barcodes. The quality of this dataset is comparable to that generated by Tara Oceans and is not biased by the multiplication of samplers. The data unveil significant genetic novelty and can be used to explore the taxonomic and ecological diversity of plankton at both regional and global scales. This pilot project paves the way for construction of a miniaturized, modular, evolvable, affordable and open-source citizen field-platform that will allow systematic assessment of the eco/morpho/genetic variation of aquatic ecosystems and microbiomes across the dimensions of the Earth system

Plankton Planet : ‘seatizen’ oceanography to assess open ocean life at the planetary scale

Abstract In every liter of seawater there are between 10 and 100 billion life forms, mostly invisible, called plankton, which form the largest and most dynamic ecosystem on our planet, at the heart of global ecological and economic processes. While physical and chemical parameters of planktonic ecosystems are fairly well measured and modelled at the planetary scale, but biological data are still scarce due to the extreme cost and relative inflexibility of the classical vessels and instruments used to explore marine biodiversity. Here we introduce ‘ Plankton Planet ’, an initiative whose goal is to merge the creativity of researchers, makers, and mariners to ( i ) develop frugal scientific instrumentation and protocols to assess the genetic and morphological diversity of plankton life, and ( ii ) organize their systematic deployment through fleets of volunteer sailors, fishermen, or cargo-ships to generate comparable and open-access plankton data across global and long-term spatio-temporal scales. As proof-of-concept, we show how 20 crews of sailors (“planktonauts”) were abl to sample plankton biomass from the world surface ocean in a single year, generating the first citizen-based, planetary dataset of plankton biodiversity based on DNA barcodes. The quality of this dataset is comparable to that generated by Tara Oceans and is not biased by the multiplication of samplers. This dataset has unveiled significant genetic novelty and can be used to explore the taxonomic and ecological diversity of plankton at both regional and global scales. This pilot project paves the way for construction of a miniaturized, modular, evolvable, affordable and open-source citizen field-platform that will allow systematic assessment of the eco/morpho/genetic variation of aquatic ecosystems across the dimensions of the Earth system