Chez Saccharomyces cerevisiae, les souches mutantes pour Rrd1, une protéine qui possède une activité de peptidyl prolyl cis/trans isomérase, montrent une résistance marquée à la rapamycine et sont sensibles au 4-nitroquinoline 1-oxide, un agent causant des dommages à l’ADN. PTPA, l’homologue de Rrd1 chez les mammifères, est reconnu en tant qu’activateur de protéine phosphatase 2A. Notre laboratoire a précédemment démontré que la surexpression de PTPA mène à l’apoptose de façon indépendante des protéines phosphatase 2A. La fonction moléculaire de Rrd1/PTPA était encore largement inconnue au départ de mon projet de doctorat. Mes recherches ont d’abord montré que Rrd1 est associé à la chromatine ainsi qu’à l’ARN polymérase II. L’analyse in vitro et in vivo par dichroïsme circulaire a révélé que Rrd1 est responsable de changements au niveau de la structure du domaine C-terminal de la grande sous-unité de l’ARN polymérase II, Rpb1, en réponse à la rapamycine et au 4-nitroquinoline 1-oxide. Nous avons également démontré que Rrd1 est requis pour modifier l’occupation de l’ARN polymérase II sur des gènes répondant à un traitement à la rapamycine. Finalement, nous avons montré que suite à un traitement avec la rapamycine, Rrd1 médie la dégradation de l’ARN polymérase II et que ce mécanisme est indépendant de l’ubiquitine. La dernière partie de mon projet était d’acquérir une meilleure connaissance de la fonction de PTPA, l’homologue de Rrd1 chez les mammifères. Nos résultats montrent que le «knockdown» de PTPA n’affecte pas la sensibilité des cellules à différentes drogues telles que la rapamycine, le 4-nitroquinoline 1-oxide ou le peroxyde d’hydrogène (H2O2). Nous avons également tenté d’identifier des partenaires protéiques pour PTPA grâce à la méthode TAP, mais nous ne sommes pas parvenus à identifier de partenaires stables. Nous avons démontré que la surexpression de la protéine PTPA catalytiquement inactive n’induisait pas l’apoptose indiquant que l’activité de PTPA est requise pour produire cet effet. Finalement, nous avons tenté d’étudier PTPA dans un modèle de souris. Dans un premier lieu, nous avons déterminé que PTPA était exprimé surtout au niveau des tissus suivants : la moelle osseuse, le thymus et le cerveau. Nous avons également généré avec succès plusieurs souris chimères dans le but de créer une souris «knockout» pour PTPA, mais l’allèle mutante ne s’est pas transférée au niveau des cellules germinales. Mes résultats ainsi que ceux obtenus par mon laboratoire sur la levure suggèrent un rôle général pour Rrd1 au niveau de la régulation des gènes. La question demeure toujours toutefois à savoir si PTPA peut effectuer un rôle similaire chez les mammifères et une vision différente pour déterminer la fonction de cette protéine sera requise pour adresser adéquatement cette question dans le futur. / In Saccharomyces cerevisiae, mutants devoid of Rrd1, a protein possessing in vitro peptidyl prolyl cis/trans isomerase activity, display striking resistance to rapamycin and show sensitivity to the DNA damaging agent 4-nitroquinoline 1-oxide. PTPA, the mammalian homolog of Rrd1, has been shown to activate protein phosphatase 2A. Our laboratory previously found that overexpression of PTPA leads to apoptosis independently of PP2A. At the outset of my thesis work, the molecular function of Rrd1/PTPA was largely unknown. My work has shown that Rrd1 is associated with the chromatin and interacts with RNA polymerase II. In vitro and in vivo analysis with circular dichroism revealed that Rrd1 mediates structural changes of the C-terminal domain of the large subunit of RNA pol II, Rpb1, in response to rapamycin and 4-nitroquinoline 1-oxide. Consistent with this, we demonstrated that Rrd1 is required to alter RNA pol II occupancy on rapamycin responsive genes. We also showed that upon rapamycin exposure Rrd1 mediates the degradation of RNA polymerase II and that this mechanism is ubiquitin-independent. Another part of my work was to gain insight into the function of PTPA, the mammalian counterpart of Rrd1. PTPA knockdown did not affect sensitivity to rapamycin, 4-nitroquinoline 1-oxide or H2O2. We also attempted to find protein interaction partners for PTPA using tandem affinity purification, but no stable partners for PTPA were found. We also demonstrated that overexpression of a catalytically inactive PTPA mutant did not induce apoptosis, indicating that PTPA activity is required to produce this effect. Finally, we attempted to study PTPA in a mouse model. We first determined that PTPA was expressed in a tissue-specific manner and was most abundant in bone marrow, thymus and brain. We pursued creation of a knockout mouse and successfully generated chimeras, but the mutated allele was not transmitted to the germline. My data and other data from our laboratory regarding the yeast work suggest a general role for Rrd1 in regulation of gene transcription. Whether PTPA has a similar function in mammalian cells remains unknown, and a different vision of what the protein does in mammalian cells will be required to adequately address this question in the future.

Selective ‘super-specific’ inhibitors of Protein Phosphatase 1 (PP1) are not available. Several natural product toxins possessing marginal selectivity between PP1 and the closely related Protein Serine/Threonine Phosphatase (PSTP), Protein Phosphatase 2A (PP2A) have been used to study the role of PP1 and PP2A in cellular signaling processes, such as the cyclic peptide inhibitors (microcystins and nodularins); terpenoid (cantharidin); polyketides (okadaic acid, calyculin, and tautomycin). The organic molecule tautomycetin is a natural product which has the highest selectivity for PP1 compared to the closely related PSTP PP2A, albeit slightly so (about 39 times more selective). Calyculin A is equally selective to PP1 and PP2A. On the other hand, okadaic acid is about 100 times more selective towards PP2A compared to PP1. Specific protein inhibitors are not suitable for cell-based assay due to low, intrinsic cellular permeability of proteins. A si-RNA mediated knockdown approach though feasible, is not ‘fast-acting’. The knockdown often lasts for an extended time period and cannot be modulated (turned on or off) as desired. Also, analysis of knockdown data is complex as the system can regulate itself in complex ways, making any effort to interpret the data liable to misinterpretation. The ultimate goal of this project is to develop a cell-permeable, potent, and selective inhibitor for PP1 (which does not target the related protein phosphatases PP2A, PP2B and PP5) whose activity inside cells can be modulated as desired so that spatiotemporal control over the activity of PP1 can be achieved. Development of such an inhibitor can be used as a chemical tool to study the cellular signaling of PP1 and not by the related PSTP PP2A. To address the problem of a lack of inhibitor targeting Protein Phosphatase 1 selectively over the closely related PSTP, PP2A; design of a peptide based inhibitor has been envisioned which targets the acidic groove and hydrophobic groove of Protein Phosphatase 1 in addition to targeting the active site (triple approach combination). The parent peptide (V6.2.10) of this study has been designed using a co-crystal structure of rat PP1cγ complexed with mouse inhibitor-2 (PDB ID: 2O8A). The parent peptide V6.2.10 has an IC50 value of 4.2 µM, which has been confirmed in the present study. A combination of single site mutations has been made using N-terminus arginine scanning, C-terminus arginine scanning, active site mutations, cyclohexylalanine scanning, and miscellaneous site-specific mutations. A hydrophobic pocket present in Protein Phosphatase 1 has been probed using ortho and meta fluorophenyalanine residue to increase potency and metabolic stability of the peptide. The rationale for such mutations was based upon a combination of approaches: mutagenesis in PyMOL, calculation of binding energies in FoldX, suitability of parent residues to be mutated, and how important are parent and substituent residues for cellular permeability and metabolic stability. Several peptides were identified from single-site mutations which had lower (improved) IC50 compared to the parent peptide of the study, V6.2.10. Several double mutations combining potent single-mutant peptides identified from this study has lower (improved) IC50 values than either of the single mutant peptides. #30 (combination of #15 and #4.2) has an IC50 value of about 334 nM and #36 (combination of #15 and 4-Fluoro Phenylalanine at the F5 position) has an IC50 value of 531 nM. #30 is the optimized peptide inhibitor from this study which is currently being utilized for crystallization trails in the laboratory. Far UV Circular dichroism study of #4.2 peptide shows mostly random coil conformation along with contributions from other secondary structures. Moreover, #4.2 is capable of adopting an alpha helical conformation in the presence of the well-known helix inducer chemical trifluoroethanol. Purification of PP1α protein using affinity chromatography has been optimized in order to increase the yield of pure protein phosphatase 1. Attempts to express and purify PP1α protein in BL21 (DE3) bacterial cells gave low yield. Thus, expression and purification of PP1α protein derived from human genomic sequence has been attempted in BL21 (RIL) codon-optimized cells which resulted in increased production of pure protein.

Pendant le bris de l'enveloppe nucléaire, la kinase dépendante des cyclines liée à la cycline B (CDK1-cycline B) et d'autres kinases phosphorylent des protéines nucléaires conduisant au désassemblage des complexes de protéines de l'enveloppe nucléaire. Les protéines nucléaires phosphorylées sont ensuite déphosphorylées par un groupe de phosphatases en sortie mitotique. La protéine phosphatase 2A en complexe avec la sous-unité régulatrice B55 (PP2A-B55) est connue pour être la principale phosphatase à déphosphoryler les protéines critiques à la fin de la mitose. Cependant, les substrats nucléaires déphosphorylés par PP2A-B55 à la sortie mitotique sont peu connus. En utilisant des cellules de drosophile en culture, nous avons démontré que PP2A-B55 est nécessaire pour le recrutement de protéines de l'envelope nucléaire telles que BAF, la protéine de lamina nucléaire Lamin B et la nucléoporine Nup107. De plus, nous avons trouvé que les œufs de femelles des drosophiles hétérozygotes pour une mutation dans les gènes codant pour la Lamine B et Tws (B55 chez la drosophile) n’éclosent pas. Ces œufs présentent divers défauts au stade de la méiose et des divisions nucléaires de l’embryon syncytial. De plus, des tests in vitro et d'autres analyses biochimiques indiquent que PP2A-Tws se lie et déphosphoryle BAF. J'ai d'autres résultats qui suggèrent un rôle de la protéine Ankle2 dans la régulation du recrutement de BAF pour réassembler les noyaux à la sortie mitotique. Mes résultats suggèrent également que Ankle2 en complexe avec PP2A est responsable de la bonne progression mitotique. Mes résultats mettent en évidence l'utilité de la drosophile comme système modèle dans l'étude de différents aspects du cycle cellulaire. Ils démontrent également / During nuclear envelope breakdown, the cyclin-dependent kinase 1 bound to Cyclin B (CDK1-Cyclin B) and other kinases phosphorylate a number of nuclear proteins leading to the disassembly of nuclear envelope protein complexes. Phosphorylated nuclear proteins are then dephosphorylated by a group of phosphatases at mitotic exit. The protein phosphatase 2A in complex with the regulatory subunit B55 (PP2A-B55) is known to be the major phosphatase to dephosphorylate critical proteins at the end of mitosis. However, little was known about the nuclear substrates dephosphorylated by PP2A-B55 at mitotic exit. Using Drosophila cells in culture, we demonstrated that PP2A-B55 is required for the recruitment of nuclear envelope proteins such as BAF, the nuclear lamina protein Lamin B, and the nucleoporin Nup107. Also, eggs from Drosophila females heterozygous for a mutation in genes coding for Lamin B and Tws (B55 in Drosophila), didn’t hatch. These eggs showed various defects during the nuclear division stage and meiosis. Moreover, in vitro assays and other biochemical analyses indicate that PP2A-B55 binds and dephosphorylates BAF. I have other results that suggest a role of the protein Ankle2 in regulating BAF recruitment to reassembling nuclei at mitotic exit. My results also suggest that Ankle2 in complex with PP2A is responsible for the proper mitotic progression. Our results highlight the importance of Drosophila in investigating different aspects of the cell cycle. It also demonstrates a role of PP2A in the nuclear envelope reformation at the end of mitosis.

<p>Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (<b>PDAC</b>) and are considered the initiating event during the development of pancreatic intraepithelial neoplasia (<b>PanIN</b>) precursor lesions. While it is well established that KRAS mutations can drive the initiation of pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (<b>PP2A</b>) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both Cancerous Inhibitor of PP2A (<b>CIP2A</b>), an endogenous inhibitor of PP2A activity, and the PP2A target, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56a, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation <i>in vivo</i>, knockout of B56a promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (<b>ADM</b>) and the formation of PanIN lesions. The process of ADM was attenuated <i>ex vivo</i> in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (<b>SMAP</b>s). Together, the results of this study suggest that suppression of PP2A-B56a through KRAS signaling can promote Myc-driven initiation of pancreatic tumorigenesis.</p>

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Studies have shown that the alternative splicing of caspase 9 and the phospho-status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and de novo ceramide via the action of protein phosphatase-1 (PP1). Two RNA splice variants are derived from the caspase 9 gene, pro-apoptotic caspase 9a and anti-apoptotic caspase 9b, via alternative splicing by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. In this study, the link between SR proteins and the alternative splicing of caspase 9 was established. Sequence analysis of the exon 3, 4, 5, and 6 cassette of the caspase 9 gene identified five possible high affinity sequences for interaction with the SR protein, SRp30a, a well-established regulator of exon inclusion/exclusion. Replacement mutagenesis identified purine-rich sequences between exons 4 and 5 and wthin exon 6 as important for binding SRp30a and required for expression of the caspase 9a splice variant. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-acting proteins and SRp30a with these sequences. Furthermore, SDS-PAGE analysis of cross-linked RNA trans-acting factors with these possible RNA cis-elements revealed the specific binding of an approximate 66, 56, 45, and 38 kDa protein/protein complex to these sequences. A previous application of RNAi technology to downregulate SRp30a in A549 lung adenocarcinoma cells induced an approximately 75% decrease in SRp30a expression and induced a dramatic change in the ratio of caspase 9a/caspase 9b. Therefore, these studies have identified SRp30a as a major regulator of the alternative splicing of caspase 9 directly linking de novo ceramide generation, PP1, and SRp30a as the signal transduction pathway regulating the expression of caspase 9.

Saxitoxins were extracted from a bloom of toxic Anabaena circinalis and used to spike treated water from Hope Valley Reservoir (HVTW) and Milli-Q water. The waters were treated with ozone using the batch method and saxitoxin levels were measured in the samples using HPLC. The results for oxidation of saxitoxins in Milli-Q water versus HVTW show that despite the presence of natural organic matter(NOM) and the production of vastly different ozone residuals, there was a similar removal of all saxitoxins in both waters. The results show that high concentrations of saxitoxins were present in solution after ozonation with doses and contact times typically used in water treatment. Relating the toxin destruction to ozone residual showed that even with a residual ozone concentration of 0.8 mg/L after 10 minutes contact in HVTW, over 60% of the initial saxitoxin content was still present in the samples. The presence of an ozone residual in the water could not be related to saxitoxin destruction and it appeared that saxitoxin removal occurred more rapidly when ozone was consumed rather than stabilised in solution. The results indicate that the mechanism for toxin removal is probably based on the reaction with a hydroxyl radical species as the oxidant rather than molecular ozone. The results obtained during these experiments indicate that ozone is not an effective oxidant for this class of compound. A range of ozone doses were applied to two different treated reservoir waters that had been spiked with microcystins LA (mLA) and LR (mLR). At the ozone dose where a residual was first measured in the sample after 5 minutes exposure time, no microcystins were detected by HPLC in either water. The removal of mLA and mLR was identical in all samples. The absence of mLA and mLR by HPLC was supported by a loss of toxicity using a highly sensitive and specific bioassay (PP2A) and by in vivo studies in mice. In both waters microcystins were removed with an ozone dose typical of that used in drinking water treatment. The results indicate that conventional ozone treatment was effective in removing hepatotoxicity at microcystin levels greater than those likely to be found in drinking water. Two waters were sampled from reservoirs in South Australia. One was collected directly from Happy Valley Reservoir (HVRW) and the other from Myponga Reservoir after treatment but before chlorination (MFCW). They were spiked with mLA and mLR and chlorinated to measure toxin removal and chlorine consumption using the CT concept. In MFCW at pH 7 there was a better removal of both mLA and mLR than in HVRW at pH 8.1. There was also a lesser effect from water temperature upon toxin removal in MFCW. Microcystin LA was less easily removed than mLR at both temperatures in both waters. For HVRW, at the higher pH, this required an initial dose of 7 mg/L of chlorine which corresponded to a CT of around 70 min.mg/L. If the water temperature was reduced to 6??C then under these conditions there would still be 40% of the initial concentration of mLA and mLR present in this water. At this temperature a final chlorine residual of 3.5 mg/L after 30 minutes, requiring a chlorine dose of 8mg/L and corresponding to a CT of about 95 min.mg/L, was required to reduce microcystin levels below the WHO guidelines. This implies that in colder climates the application of chlorine for microcystin removal may require elevated chlorine doses and CT values. Arrhenius activation energies were calculated for mLA and mLR in both waters, revealing different Ea values for both toxins. Due to the complexity of the reactions and the possible effects of pH in solution, this system was considered too complicated to be described by the Arrhenius equation. NOM was collected from Myponga Reservoir in South Australia using magnetic ion exchange (MIEX??) resin. The collected NOM was desorbed and separated into fractions of different molecular weight and character using ultrafiltration and mixed resin ion exchange. At approximately 5 mg/L dissolved organic carbon (DOC) the measured apparent second order rate constant (kapp) for mLA and mLR removal was fairly similar in both the high molecular weight fraction (designated F3), and the intermediate high molecular weight fraction (designated F2). The low molecular weight fraction (designated EN) had slightly higher kapp values as would be expected due to the less reactive nature of the NOM in this fraction. This meant more chlorine was available to react with microcystins in this fraction. Fractions F3 and F2 produced similar kapp values to those from the parent water source following treatment, indicating the similar reactivity of these NOM fractions at comparable DOC levels. Increasing the DOC concentration in the F2 fraction increased kapp for both mLA and mLR due to the additional chlorine needed to react with the additional NOM present. The results showed that pH, temperature and DOC concentration have a higher impact upon chlorination rates, and the efficiency of toxin removal, than NOM character alone. In general it is assumed that chlorine will be more effective at removing toxins in water with a low SUVA and low specific colour as these indicate less 'reactive' NOM in the water. The results of this study show that toxin removal was more effective in the EN fraction as indicated by the higher kapp. This fraction also had the lowest SUVA and lowest specific colour which supports the generally held view in water treatment. Relating the toxin removal to chlorine residual in these reconstituted fractionated NOM samples, indicated that a residual of around 1.5 mg/L after 30 minutes contact was generally adequate to remove all toxins in water with a DOC level of around 5 mg/L. This is consistent with the results obtained in real waters, where at 20??C a chlorine residual of 2 mg/L was found to be sufficient for removal of both mLA and mLR.

La progression mitotique est principalement régulée par la phosphorylation et la déphosphorylation des protéines. La kinase dépendante des cyclines liée à la cycline B (Cdk1 - cycline B) et d'autres kinases phosphorylent une myriade de protéines pour promouvoir l’entrée à la mitose. Ces phosphorylations sont réversées par les phosphatases lors de la sortie mitotique. La protéine phosphatase 2A avec sa sous-unité régulatrice B55 (PP2A-B55) est une des principales phosphatases neutralisant les phosphorylations par Cdk1. Dans ce projet, nous avons vu que Ankle2 participe à la sortie de la mitose. En utilisant D. melanogaster comme modèle puissant, nous avons observé que Ankle2 est important pour le recrutement des protéines BAF et Lamin associées à l'enveloppe nucléaire (NE) à la télophase, assurant la formation d'un seul noyau. In vivo, nos résultats indiquent que Ankle2 est crucial pour le développement de l'embryon de drosophile, car les embryons ARNi Ankle2 sont arrêtés lors de la première mitose. Pour amorcer l’étude des mécanismes moléculaires par lesquels Ankle2 remplit ces foncions, nous avons identifié ses partenaires d’interactions. Nous avons constaté que Ankle2 est associé à une forme active de PP2A, suggérant Ankle2 comme une sous-unité régulatrice potentielle de PP2A. De plus, Ankle2 forme un complexe avec la cycline B et les Cdks mitotiques, et nos résultats génétiques suggèrent que les deux protéines peuvent avoir des rôles opposés. Nous avons également découvert que Ankle2 interagit avec la protéine du réticulum endoplasmique (ER) Vap33 à travers son motif FFAT. Dans ce projet, nous avons constaté que Ankle2 est une protéine associée au ER chez la drosophile qui est cruciale pour la complétion de la mitose, et que pourrait réguler l'activité des kinases et phosphatases mitotiques. Cette étude servira de base pour déchiffrer les mécanismes moléculaires précis par lesquels Ankle2 favorise la sortie de la mitose. / Protein phosphorylation and dephosphorylation is one of the mechanisms that regulates mitotic progression. Cyclin dependent kinase 1 bound to cyclin B (Cdk1 – cyclin B) and other kinases phosphorylate a myriad of proteins to promote early mitotic events. These phosphorylations are reversed by phosphatases during mitotic exit. The Protein Phosphatase 2A with its regulatory subunit B55 (PP2A-B55) is the major phosphatase counteracting Cdk1 phosphorylations. In this project, we have found that Ankle2 participates in mitotic exit. Using D. melanogaster as a model, we have found that Ankle2 is important for the Nuclear Envelope (NE)-associated proteins BAF and Lamin recruitment at telophase, ensuring the formation of a single nucleus. In vivo, we have found that Ankle2 is crucial for Drosophila embryo development, as RNAi Ankle2 embryos are arrested in the first mitosis. To study the molecular mechanisms by which Ankle2 promotes mitotic exit, we identified its interacting partners. We found that Ankle2 is associated with an active form of PP2A, suggesting Ankle2 as a potential regulatory subunit of PP2A. Moreover, Ankle2 engages in a complex with cyclin B and mitotic Cdks, and our genetic results suggest that Ankle2 and mitotic cyclin – Cdk complex may have opposite roles. We have also found that Ankle2 interacts with the Endoplasmic-Reticulum (ER) protein Vap33 through its FFAT motif. In this project, we have found that Ankle2 is an ER-associated protein in Drosophila that is crucial for completion of mitosis, probably regulating the activity of mitotic kinases and phosphatases. This study will serve as a basis to decipher the precise molecular mechanisms by which Ankle2 promotes mitotic exit.

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