The budding yeast S. cerevisiae is widely used as a model organism to study biological processes that are conserved among eukaryotes. Di fferent genomic approaches have been applied successfully to interrogate the mode of action of small molecules and their combinations. In this thesis, these technologies were applied to di fferent sets of chemical compounds in the context of two collaborative projects. In addition to insight into the mode of action of these molecules, novel approaches for analysis of chemical-genetic pro files to integrate GO annotation, genetic interactions and protein complex data have been developed. The fi rst project was motivated by a pressing need to design novel therapeutic strategies to combat infections caused by opportunistic fungal pathogens. Systematic screens of 1180 FDA approved drugs identifi ed 148 small molecules that exhibit synergy in combination with uconcazole, a widely used anti-fungal drug (Wright lab, McMaster University, Canada). Genome-wide chemical-genetic profiles for 6 of these drugs revealed two di fferent modes of action of synergy. Five of the compounds a ffected membrane integrity; these chemical-genetic interactions were supported by microscopy analysis and sorbitol rescue assays. The sixth compound targets a distinct membrane-associated pathway, sphingolipid biosynthesis. These results not only give insight into the mechanism of the synergistic interactions, they also provide starting points for the prediction of synergistic anti-fungal combinations with potential clinical applications. The second project characterised compounds that aff ected melanocytes in a chemical screen in zebra fish (Patton lab, Edinburgh). Chemical-genetic screens in S.cerevisiae enabled us to show that melanocyte pigmentation reducing compounds do so by interfering with copper metabolism. Further, we found that defects in intracellular AP1 and AP3 trafficking pathways cause sensitivity to low copper conditions. Surprisingly, we observed that the widely-used MAP-kinase inhibitor U0126 a ffects copper metabolism. A nitrofuran compound was found to speci fically promote melanocyte cell death in zebrafi sh. This enabled us to study off -target eff ects of these compounds that are used to treat trypanosome infections. Nifurtimox is a nitrofuran prodrug that is activated by pathogen-specifi c nitroreductases. Using yeast and zebra fish we were able to show that nitrofurans are also bioactivated by host-specifi c aldehyde dehydrogenases suggesting that a combination therapy with an aldehyde dehydrogenase inhibitor might reduce side e ffects associated with nifurtimox.

Hematopoietic stem and progenitor cells (HSPCs) repopulate the blood system upon transplantation. A large-scale genetic approach to understand the factors that participate in successful engraftment has not been undertaken. In this thesis, I present the development of a novel live imaging-based competitive marrow repopulation assay in adult zebrafish, which allows fast and quantitative measurement of HSPC engraftment capability. Using this assay, a transplantation-based chemical screen was performed, which led to the discovery of 10 compounds that can enhance the marrow engraftment capability in zebrafish. Among them, the arachidonic acid-derived epoxyeicosatrienoic acids (EET), had conserved effects on both short- and long-term bone marrow engraftment in mice. Genetic analysis in zebrafish embryos demonstrated that EET acts through a \(G\alpha12/13\)-mediated receptor, which activates PI3K and induces transcription factors of the AP-1 family. This PI3K/AP-1 pathway directly induced the transcription of HSC marker, runx1, in embryos. The activation of PI3K by EET promoted HSPC migration and interactions with niche cells. Our studies define a role for EETs in the development of blood stem cells during embryogenesis, and in engraftment in adult vertebrates. The other compounds discovered in the screen implicate additional novel signaling pathways involved in the HSPC engraftment process, which require further investigation. In summary, this thesis elucidated an important role of bioactive lipids in regulating HSC engraftment in adults and during embryo development. Systematically mapping out the regulatory network will tremendously benefit both the basic understanding of stem cell biology and the clinical manipulation to generate better stem cells for transplantation.

Bacterial toxins have co-opted host cell machinery in order to enter cells and exert their deleterious effects. Anthrax toxin is composed of the receptor binding protein protective antigen (PA), and the enzymatic subunits lethal factor (LF) and edema factor (EF), which form the binary toxin complexes lethal toxin, LeTx (PA + LF), and edema toxin, EdTx (PA + EF). PA binds to receptors on the surface of host cells and shuttles LF and EF into cells through the endocytic pathway. Upon endosome acidification, PA oligomers insert into the endosomal membrane and form functional pores that deliver LF and EF into the cytoplasm. Translocation of the N-terminal domain of LF, \(LF_N\), through PA pores formed in lipid bilayers in vitro does not require host machinery. However, translocation of the related fusion protein \(LF_N\)-DTA across the membrane of toxin-loaded endosomes in vitro requires the addition of cytosolic translocation factors that include the COPI coatamer complex. We performed high-throughput small molecule and RNAi screens to identify host factors required for LF translocation, using LeTx-induced cell death as a phenotype. We describe the characterization of small molecule inhibitors of LeTx-induced cell death that inhibit toxin entry. Further, we describe the role of the endosomal chaperone GRP78 and the cytoplasmic CCT chaperonin in toxin translocation. RNAi knockdown of GRP78 and CCT subunits inhibited LeTx and EdTx delivered through the endocytic pathway. CCT knockdown additionally inhibited translocation of LF through PA pores formed directly in the plasma membrane, while GRP78 had no effect. Furthermore, we show that the role of GRP78 in toxin translocation is specific to translocation from the early endosome. Together with biochemical data, we propose that GRP78 facilitates translocation by unfolding LF and EF at near-neutral pH. In addition, we show that in CCT-knockdown cells, lethal levels of toxin reach the endosome, suggesting that CCT has a role in translocation and/or refolding of LF and EF. These studies highlight previously unidentified strategies used by anthrax toxin to hijack host cellular machinery in order to gain access to the cytosol.

This work presents the investigation of fungal AdoMet-dependent methyltransferases. The first part of the dissertation focuses on two distinct methyltransferases with previously unknown functions in the budding yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. To characterize these enzymes I used a combinatorial approach that exploits contemporary high-throughput techniques available in yeast (chemical genetics, expression, lipid profiling and genetic interaction analysis) combined with rigorous biological follow-up. First, I showed that S. cerevisiae CRG1 (ScCRG1) is a small molecule methyltransferase that methylates cytotoxic drug cantharidin and is important for maintaining lipid homeostasis and actin cytoskeleton integrity in response to small-molecule cantharidin in the baker’s yeast. Similarly to ScCRG1, orf19.633 in the human fungal pathogen C. albicans (CaCRG1) methylates cantharidin and is important for GlcCer biosynthesis. I also demonstrated that CaCrg1 is a ceramide- and PIP-binding methyltransferase involved in Candida’s morphogenesis, membrane trafficking and fungal virulence. Together, the analysis of two genes in yeast illuminated the important roles of the novel small molecule methyltransferases in coupling drug response to lipid biosynthesis and fungal virulence. In the second part of my dissertation, I present the systematic characterization of the genetic architecture of the yeast methyltransferome by examining fitness of double-deletion methyltransferase mutants in standard and under environmental stress conditions. This analysis allowed me to describe specific properties of the methyltransferome network and to uncover functional relationships among methyltransferases inspiring multiple hypotheses and expanding the current knowledge of this family of enzymes.

This work presents the investigation of fungal AdoMet-dependent methyltransferases. The first part of the dissertation focuses on two distinct methyltransferases with previously unknown functions in the budding yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. To characterize these enzymes I used a combinatorial approach that exploits contemporary high-throughput techniques available in yeast (chemical genetics, expression, lipid profiling and genetic interaction analysis) combined with rigorous biological follow-up. First, I showed that S. cerevisiae CRG1 (ScCRG1) is a small molecule methyltransferase that methylates cytotoxic drug cantharidin and is important for maintaining lipid homeostasis and actin cytoskeleton integrity in response to small-molecule cantharidin in the baker’s yeast. Similarly to ScCRG1, orf19.633 in the human fungal pathogen C. albicans (CaCRG1) methylates cantharidin and is important for GlcCer biosynthesis. I also demonstrated that CaCrg1 is a ceramide- and PIP-binding methyltransferase involved in Candida’s morphogenesis, membrane trafficking and fungal virulence. Together, the analysis of two genes in yeast illuminated the important roles of the novel small molecule methyltransferases in coupling drug response to lipid biosynthesis and fungal virulence. In the second part of my dissertation, I present the systematic characterization of the genetic architecture of the yeast methyltransferome by examining fitness of double-deletion methyltransferase mutants in standard and under environmental stress conditions. This analysis allowed me to describe specific properties of the methyltransferome network and to uncover functional relationships among methyltransferases inspiring multiple hypotheses and expanding the current knowledge of this family of enzymes.

<p>Ribosome biogenesis is a major metabolic expense of bacteria and a promising target for antibacterial drug discovery. <em>Trans-</em>acting proteins, called ribosome biogenesis factors, aid this complex and cooperative process. EngA (YfgK, Der) is a widely distributed bacterial GTPase that is shown here to be important for normal ribosome biogenesis. EngA is an attractive antibacterial target because it is essential for viability in bacteria but is absent in humans.</p> <p>The GTPase activity and cellular function of EngA was investigated in <em>Escherichia coli</em>. Depletion of EngA caused accumulation of 30S and 50S ribosomal subunits at the expense of 70S ribosomes, showing for the first time that EngA is important for normal ribosome biogenesis. Mutation of either of the tandem GTPase domains of EngA led to abnormal ribosome profiles, cell death and loss of GTPase activity, revealing that the two GTPase domains act cooperatively to carry out an essential function. EngA bound the 50S subunit of the ribosome in cells and <em>in vitro</em>. Depletion of EngA resulted in sensitization to aminoglycoside antibiotics, which bind at the aminoacyl-tRNA binding site of ribosomes. To search for an inhibitor of ribosome biogenesis, a high-throughput screen of the GTPase activity of EngA was developed. A specific inhibitor was not identified, however, this robust screen can be extended to other compound libraries. Thus, we showed that the GTPase domains of EngA have a cooperative function in ribosome biogenesis, probably in maturation of the 50S subunit, and that EngA is an amenable target for further inhibitor screens.</p> / Doctor of Philosophy (PhD)

JLP (JNK associated Leucine zipper protein) is a scaffolding protein that has been shown to interact with and activate the JNK/p38MAPK pathway. Its interaction with various signaling proteins is associated with coordinated regulation of cellular processes such as endocytosis, motility, neurite outgrowth, cell proliferation and apoptosis. Here, we undertook a mass spectrometric approach to identify novel interaction partners of JLP and identified the mitotic Ser/Thr kinase, Polo like Kinase 1 (PLK1) and the Fox transcription factor, Forkhead box protein K1 (FoxK1), as proteins that interact with and form a ternary complex with JLP during mitosis. Domain mapping studies showed that the N-terminal domain of JLP interacts with the polo-box domain (PBD) of PLK1 in a phosphorylation-dependent manner. Our results indicate that, JLP is phospho-primed on Thr351, which is recognized by the PBD of PLK1 and leads to phosphorylation of JLP at additional sites. Moreover, treatment of cells with the PLK1 inhibitor BI2536 affects this interaction, demonstrating the importance of PLK1 kinase activity in this process. Because JLP is a scaffolding protein that recruits proteins to mediate specific cell signaling events, the interaction of JLP with PLK1 likely results in the recruitment of other proteins to this complex. To test this hypothesis, we carried out SILAC labeling of proteins in mitotic cells in the presence or absence of BI2536. Through mass-spectrometry, we identified the FoxK1 transcription factor as a PLK1-dependent JLP-interacting protein. Furthermore, we show that JLP, PLK1 and FoxK1 form a ternary complex that is present only during mitosis. Knockdown of PLK1 and not JLP affected the interaction between JLP and FoxK1, indicating that the formation of the ternary complex is PLK1-dependent. FoxK1 is a known transcriptional repressor of the cyclin dependent kinase inhibitor, p21/WAF1. Knockdown of JLP in U2OS cells resulted in increased FoxK1 protein levels and a reduction of p21 expression. Moreover, immunofluorescence studies in asynchronous cells showed that FoxK1 is excluded from the nucleus during mitosis and that a fraction of FoxK1 localizes to the midbody region during cytokinesis. Analysis of FoxK1 protein in cells exiting S-phase suggests that FoxK1 is post-translationally modified during mitosis. In this study we characterized the ternary complex formed between JLP, PLK1 and FoxK1 during mitosis. Based on our observations, we propose that formation of the JLP/PLK1/FoxK1 ternary complex regulates the stability and/or transcriptional activity of FoxK1. / Molecular Biology and Genetics

La fitohormona ácido abscísico (ABA) juega un papel crucial en el control de la respuesta a estrés y en la regulación del crecimiento y desarrollo de la planta. La unión del ABA a los receptores intracelulares PYR/PYL/RCAR conlleva la inhibición de las PP2Cs del clado A tales como ABI1 o HAB1, causando la activación de la ruta de señalización del ABA. Para obtener más información en la señalización del ABA nos hemos centrado en la caracterización de miembros de estas dos familias proteicas. Hemos generado una versión mutada de HAB1 que contiene una mutación en el Trp-385, residuo clave para la interacción con los receptores y con la molécula de ABA. Como resultado, hab1W385A se mostró refractaria a la inhibición por los receptores PYR/PYL/RCAR. Así, en ensayos de actividad quinasa in vitro encontramos que hab1W385A era capaz de desfosforilar a OST1 incluso en presencia de ABA y de los receptores. hab1W385A y hab1G246D pueden ser clasificadas como mutaciones dominantes hipermórficas. Mientras que hab1G246D posee una actividad fosfatasa reducida, el nuevo alelo dominante muestra una actividad idéntica al genotipo salvaje. Líneas transgénicas de Arabidopsis sobreexpresando hab1W385A mostraron una fuerte insensibilidad al ABA. También hemos analizado el papel de las PP2Cs del clado A pertenecientes a la rama representada por PP2CA. La generación de un mutante doble pp2ca-1hai1-1, que muestra mayor sensibilidad a la hormona en comparación con el genotipo salvaje y con los mutantes sencillos, reveló que HAI1 es un regulador negativo de la ruta de señalización del ABA. El análisis de la localización subcelular mostró que tanto HAI1 como PP2CA se localizan en el núcleo, aunque también están presentes en el citosol y en la fracción microsomal. Tres miembros de la rama de PP2CA i.e.: PP2CA, AHG1 y HAI1, mostraron una inhibición selectiva por los receptores PYR/PYL/RCAR. Estos resultados sugieren que estos receptores pueden discriminar entre miembros del clado A de las PP2Cs. pyl8 es el único mutante sencillo que muestra sensibilidad reducida al ABA en ensayos de crecimiento de raíz. Análisis usando el gen reportero GUS mostraron que PYL8 estaba presente en la estela, en la epidermis de la raíz y en la caliptra, y la cuantificación de la actividad beta-glucuronidasa en raíz mostró que PYL8 es uno de los receptores con mayor nivel de expresión. La caliptra juega un papel crucial en la respuesta hidrotrópica. El estudio de esta respuesta en mutantes múltiples de las PP2Cs y de los PYR/PYL/RCAR reforzó la idea de que el ABA regula este proceso. Así, mientras el mutante séxtuple pyr/pyl112458 presentó una curvatura menor al aplicársele un gradiente de humedad, el mutante cuádruple de las PP2Cs (Qabi2-2) mostró una curvatura más pronunciada en estas condiciones, evitando las zonas con menor potencial hídrico. Finalmente, en la última parte de este trabajo se utilizaron abordajes genético-químicos para aumentar la resistencia a la sequía. Hemos llevado a cabo un rastreo con compuestos químicos para aislar nuevos agonistas del ABA. Basado en datos estructurales de los receptores, se seleccionaron 500 compuestos que fueron ensayados en Arabidopsis. De estos, el compuesto 2C06 inhibió el crecimiento de raíz en plantas salvajes más que en mutantes pyr/pyl/rcar insensibles a ABA y produjo resultados prometedores in vitro al inhibir a las PP2Cs e interaccionar con éstas en ensayos de doble híbrido. / Antoni Alandes, R. (2013). Molecular and genetic analyses of the PP2C-ABA receptor interaction in the abscisic acid signaling pathway [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/29756 / Premios Extraordinarios de tesis doctorales

La Phostine (PSN ; PHOSphate STarvation response INductor) est une petite molécule organique de synthèse, identifiée dans une chimiothèque par son effet inducteur de l’expression du gène PHT1;4 codant un transporteur de phosphate chez Arabidopsis thaliana. Au cours de cette thèse, j’ai caractérisé l’effet de la PSN chez Arabidopsis et montré que cette drogue induit plusieurs réponses typiques de la carence en phosphate : expression de gènes régulés localement ou à longue distance, accumulation d’anthocyanes et d’amidon, activité des phosphatases acides, inhibition de la croissance des racines. De plus, la PSN provoque une accumulation de phosphate soluble dans les racines.L’étude d’analogues structuraux de la PSN m’a permis d’identifier un motif commun (#10) nécessaire à son activité biologique. Il apparaît que la PSN et ses analogues actifs sont instables à pH acide, ce qui libère le motif #10. Nous montrons que le motif #10 pénètre dans la plante essentiellement par les racines et qu’il ne circule pas des racines vers les feuilles, et faiblement des feuilles vers les racines. L’accumulation de #10 dans les racines expliquerait l’ensemble des effets observés de la PSN et de ses analogues actifs. Nous avons isolé un mutant d’Arabidopsis dont la croissance racinaire résiste à la PSN. Chez ce mutant, la PSN ne provoque plus l’accumulation d’anthocyanes et d’amidon dans les feuilles, ni celle de phosphate dans les racines. En absence de PSN, et sur un milieu de culture partiellement appauvri en phosphate, les feuilles et les racines de ce mutant accumulent plus de phosphate que celles du type sauvage. Chez les plantes, l’homéostasie du phosphate est soumise à une régulation complexe, la caractérisation approfondie de ce mutant apportera de nouveaux éléments pour sa compréhension. / The Phostin (PSN ; PHOSphate STarvation response INductor) is a small organic synthetic molecule identified in a chemical library by its induction effect on PHT1;4 expression, a gene encoding a phosphate transporter in Arabidopsis thaliana. During my PhD, I characterized the effects of PSN in Arabidopsis and shown that this drug induces several phosphate starvation responses: expression of locally or systemically regulated genes, anthocyanin and starch accumulation, acid phosphatase activity, primary root growth inhibition. Moreover, PSN induces inorganic soluble phosphate accumulation in roots.Thanks to PSN’s structural analogues, we identified a common motif (motif #10) necessary to its biological activity. PSN and its analogues are unstable in acidic conditions, leading to the release of #10 motif. We have shown that #10 motif enters the plant mostly by the roots and do not circulate from roots to shoots, and weakly from shoots to roots. The accumulation of #10 motif in the roots explains all the PSN and analogues observed effects. We characterized an Arabidopsis mutant resistant to PSN for its root growth. In this mutant, PSN does not induce anthocyanin or starch accumulation in leaves, neither phosphate in roots. When growing without PSN, on partially phosphate depleted medium, the mutant presents higher phosphate content than the wild type. Phosphate homeostasis is a very complex process in plant, the deep characterization of this mutant will bring new elements for the comprehension of this pathway.

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