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.

No description available.

Les mécanismes moléculaires contrôlant les rythmes circadiens sont conservés parmi les organismes des différents règnes (plantes, animaux et champignons). Ils se composent de boucles de rétroaction où un complexe d’activation transcriptionnelle, l’hétérodimère CLK/CYC chez la drosophile, entraîne l'expression des répresseurs de son activité, les gènes et protéines PER et TIM chez la mouche. De manière importante, la période de l'oscillateur dépend en grande partie par des mécanismes post-transcriptionnels qui régulent l’accumulation et l'activité des composantes positifs et négatifs de la boucle. Bien que de nombreux partenaires d'interaction modifiant les composants d'horloge de base ont déjà pu être isolés, le schéma reste encore incomplet. Dans le cadre de la recherche de nouveaux composants de cette horloge, nous avons réalisé un crible comportemental basé sur l'expression ciblée de transgènes ARNi dirigés contre la moitié du génome de Drosophila melanogaster. Cinquante-quatre nouveaux gènes putatifs ont pu être identifiés. Au cours de ce travail, j'ai étudié le rôle de deux d’entre eux, sélectionnés pour les forts défauts comportementaux de l'expression de leur transgène ARNi. Le gène CG12082 de la drosophile est l’orthologue de l’Ubiquitin-specific protéase 5 (USP5) chez l’homme. La dérégulation d’Usp5 retarde les oscillations de la protéine PER dans les neurones d'horloge et allonge la période d'activité locomotrice des mouches. Chez les mouches ARNi Usp5, des formes à haut poids moléculaire des protéines PER et TIM s'accumulent pendant le matin, alors qu’elles sont normalement dégradées chez les contrôles. On a pu montrer que Usp5 participe directement à la dégradation de la protéine PER, indépendamment de TIM. En accord avec le rôle décrit pour l’orthologue humaine, Usp5 serait susceptible de contrôler la dégradation des protéines par son activité de démontage des chaînes libres de polyubiquitine présents dans la cellule, qui peuvent entrer en compétition avec les protéines ubiquitinylées pour la reconnaissance au niveau du protéasome, bloquant leur dégradation. La majorité des travaux ont porté sur un gène isolé au cours de notre crible, Strip, dont les fonctions étaient encore inconnues. Strip interagit avec Cka, une nouvelle sous-unité régulatrice de l’enzyme phosphatase PP2A. La dérégulation à la fois de Strip et/ou de Cka amène à des phénotypes comportementaux de période longue. D’un point de vue moléculaire, des formes hyper-phosphorylées de la protéine CLK s’accumulent dans la matinée quand Cka et/ou Strip sont perturbées. La dérégulation des activités générales de PP2A produit également une hyper-phosphorylation de CLK le matin, indiquant que, grâce à Cka/Strip, les complexes PP2A contrôlent la déphosphorylation de CLK à la fin du cycle. Il est connu que les formes hyper-phosphorylés de CLK sont transcriptionnellement inactives. En effet, la transcription des gènes tim et vrille, cibles de CLK, est fortement réduite dans les mouches ARNi Cka. En plus de PP2A/Cka, des complexes PP2A contenant une autre sous-unité régulatrice, Wdb, ont été montré pour déstabiliser CLK en culture des cellules (Kim et Edery, 2006). Nous montrons que la dérégulation de Wdb affecte la stabilité du CLK également dans la mouche adulte, sans toutefois induire aucun effet apparent sur sa phosphorylation. En conclusion, deux complexes PP2A différents agissent sur la protéine CLK : le complexe PP2A/Cka/Strip contrôle la déphosphorylation de CLK et sa réactivation, tandis que PP2A/Wdb affecte la stabilité de CLK indépendamment ou après PP2A/Cka. Ces résultats enrichissent l’étude de la régulation post-traductionnelle de la protéine CLK, qui était largement mal connue.Pour conclure, cette étude a permis de décrire deux nouveaux composants de la boucle moléculaire qui contrôle les rythmes circadiens chez la mouche du vinaigre, Drosophila melanogaster. / The molecular mechanism underlying circadian rhythms is conserved among organisms and consists of feedback loops where a transcriptional activating complex (the CLOCK (CLK)/CYCLE (CYC) heterodimer in Drosophila) drives the expression of the repressors of its activity (the period (per) and timeless (tim) genes and proteins in Drosophila). Importantly, the pace of the oscillator largely depends on post-transcriptional mechanisms that regulate the accumulation and activity of both the positive and negative components of the loop. A number of interacting partners that modify core clock components have already been isolated, but more are expected. Looking for new clock components, we set up a behavioral screen based on targeted expression of RNAi transgenes directed to half of the Drosophila genome. 54 putative new clock genes have been identified. Among them, some were independently reported to function within the fruit fly molecular clock, thus validating the screen. In this work, I investigated the circadian role of additional “positive” genes, selected for the strong behavioral defect induced by the expression of the corresponding RNAi. The CG12082 gene codes for the fruit fly ortholog of the human Ubiquitin-specific protease 5 (USP5). Downregulation of USP5 in clock cells lengthens the period of locomotor activity of flies as well as PER protein oscillations in clock neurons. High molecular weight forms of PER and TIM proteins accumulate during the morning after USP5 knockdown, while these forms are degraded in controls. In addition, TIM is not stabilized in the absence of PER, while PER still accumulate in the absence of TIM. Therefore, USP5 directly participates in the degradation of the PER protein and, later, of the TIM protein at the end of the cycle. Being a deubiquitinylase enzyme, USP5 may directly deubiquitinate PER. However, accordingly to the role described for the human ortholog, USP5 likely controls protein degradation through the disassembling of the unanchored polyubiquitin chains present in the cell that could compete with ubiquitinated-PER for proteasome recognition and subsequent breakdown.The majority of the work has focused on an unknown gene isolated in the screen, that, accordingly to the human homolog, we named STRIP. We show that STRIP interacts with Connector of Kinase to AP-1 (CKA), a novel regulatory subunit for the PP2A phosphatase holoenzyme, both in insect S2 cells and in fly head extracts. Downregulation of both STRIP and/or CKA causes long-period behavioral phenotypes and high molecular weight forms of the CLK protein to accumulate in the morning. Perturbation of general PP2A activities also produces hyper-phosphorylated CLK in the morning indicating that, through CKA/STRIP, PP2A complexes controls CLK dephosphorylation at the end of the cycle. Hyper-phosphorylated CLK forms are transcriptionally inactive. Accordingly, transcription of the tim and vrille (vri) CLK targets is strongly reduced in Cka-RNAi fly head extracts. PP2A complexes containing the Widerborst (WDB) regulatory subunits were already shown to affect CLK stability in insect S2 cells (Kim and Edery, 2006). We show that WDB downregulation also affects the stability of CLK in fly head extracts, but has no apparent effects on CLK phosphorylation. Therefore, we could describe two different PP2A complexes acting on the CLK protein: PP2A/CKA/STRIP complex controls CLK dephosphorylation and reactivation, while PP2A/WDB affects CLK stability independently or after PP2A/CKA functions. Moreover, STRIP, but not CKA, downregulation affects the stability of PER, indicating that STRIP possesses some functions unrelated to CKA. In conclusion, this work has allowed the isolation of new components of the Drosophila molecular clock. In particular, we give evidence for a double role for the PP2A phosphatase in modulating the activity and stability of the CLK protein, the regulation of which is not well understood yet.