Pancreatic cancer is one of the deadliest cancers with a five-year survival rate of 6%. Pancreatic cancer is resistant to conventional chemotherapy and is usually diagnosed at late stages. Current treatment options have minimal effects in extending patients' lives beyond 10 months. One significant limitation in developing treatments to combat pancreatic cancer is its immunosuppressive microenvironment. Pancreatic cancer secretes factors that activate immunosuppressive cells, such as regulatory T cells (Tregs). These Tregs suppress effector CD4+ and CD8+ T cell anti-tumor immune responses. Therefore, novel treatment options to reduce Treg-mediated immune suppression and increase the numbers and functions of CD4+ and CD8+ T cells are paramount to enhance anti-tumor immunity in pancreatic cancer tumor-bearing (TB) hosts. The alternatively spliced transcription factor Ikaros is essential for lymphocyte development and is considered a tumor suppressor in T cells. Ikaros' protein stability and function are regulated by its phosphorylation and dephosphorylation by protein kinase CK2 and phosphatase 1 (PP1), respectively. Mutations and functional inactivation of Ikaros have mainly been investigated in T cell leukemias and lymphomas. In this dissertation, we investigated the role of Ikaros in regulating T cell homeostasis in murine pancreatic cancer. In this study, we report that Ikaros proteins are degraded by the ubiquitin-proteasome pathway in response to factors produced by murine pancreatic cancer cells. Our results further suggest that an increase in CK2 activity leads to Ikaros' degradation and disrupts its localization to pericentromeric heterochromatin in our murine pancreatic TB model. This loss of Ikaros expression is accompanied by an imbalance in T cell homeostasis. More specifically, we observe a significant decrease in effector CD4+ and CD8+ T cells but an increase in Treg percentages in TB and spontaneous pancreatic cancer models. T-cell specific defects in Ikaros protein expression were also observed in TB CD3+ T cells. Apigenin, a natural plant flavonoid and CK2 inhibitor, restored expression of some Ikaros isoforms in our TB model. Apigenin also displayed immunological benefits evident by enhanced anti-tumor immunity in TB mice. These data provide mechanistic and functional evidence that pharmacological inhibition of CK2 can regulate Ikaros expression and identifies the possible involvement of Ikaros in regulating T cell immune responses in murine pancreatic cancer.

Indiana University-Purdue University Indianapolis (IUPUI) / Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.

No description available.

No description available.

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.

No description available.

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.

Le paludisme, première endémie parasitaire mondiale ayant engendré près d’un demi-million de morts en 2017 (d’après l’OMS), est due à une infection par un parasite du genre Plasmodium. Cet apicomplexe infecte, au cours de son cycle de vie, un hôte définitif, un moustique femelle du genre Anopheles, et un hôte intermédiaire homéotherme (l’Homme pour au moins 6 espèces). Chez ce dernier, après une phase de développement hépatique, le parasite envahit puis lyse les érythrocytes. L’accroissement exponentiel de la parasitémie engendre les symptômes du paludisme et permet la production de formes sexuées (gamétocytes) qui seront transmises au vecteur arthropode, permettant ainsi la complétion du cycle de vie du parasite.Plasmodium a co-évolué avec ses hôtes et mis en place divers modes de régulation de l’expression de ses gènes. La phosphorylation est l’une des modifications post-traductionnelles majeures et rapides qu’il utilise pour répondre aux changements environnementaux auxquels il est confronté au cours de son cycle de vie. Nombre de ses kinases et phosphatases jouent un rôle essentiel dans l’invasion de cellules hôtes, la croissance et la division cellulaires, ainsi que la motilité de certains stades. En revanche, le rôle des cinq pseudokinases de Plasmodium dans son développement n’a jusqu’ici pas été exploré.Durant ma thèse, j’ai caractérisé l’unique pseudo-Tyrosine Kinase-like (pTKL) de Plasmodium et étudié son rôle au cours du cycle intra-érythrocytaire du parasite.L’annotation de la pTKL de P. falciparum (PfpTKL) m’a permis d’identifier différents domaines et motifs, et notamment un domaine SAM (Sterile Alpha Motif), deux motifs RVxF (connus pour leur capacité d’interaction avec la Protéine Phosphatase de type 1, PP1) et un pseudo-domaine kinase appartenant à la famille des Tyrosine Kinases-like (TKL). Nous avons montré que ce pseudo-domaine kinase est capable de lier l’ATP de manière cation-indépendante, mais est dépourvu d’activité enzymatique. Des études d’interaction in vitro couplées à l’utilisation de modèles hétérologues (Levure, ovocytes de Xénope) m’ont permis d’identifier deux protéines parasitaires partenaires de PfpTKL : le domaine SAM de PfpTKL interagit directement avec la pseudo-protéase PfSERA5 (SErine Repeat Antigen 5), alors que les deux régions de la protéine contenant les motifs RVxF de PfpTKL interagissent avec PfPP1c (phosphatase majeure de Plasmodium). De façon intéressante, le deuxième motif RVxF est directement impliqué dans l’interaction avec PP1c et serait capable de moduler l’activité de cette dernière de manière allostérique.La localisation de la pTKL de P. berghei (PbpTKL) a ensuite été étudiée par immunofluorescence et confirmée par des expériences de fractionnement cellulaire. Nous avons ainsi observé que PbpTKL est exportée dans l’érythrocyte infecté au stade trophozoïte, puis retenue dans le parasite et la vacuole parasitophore au stade schizonte. L’étude de l’interactome de PbpTKL par IP/MS au stade trophozoïte a montré que PbpTKL s’associe à diverses protéines impliquées dans l’organisation du cytosquelette de l’érythrocyte, ainsi que dans l’érythropoïèse et l’homéostasie cellulaire. Ces observations suggèrent que pTKL joue un rôle, direct ou via ses partenaires, à l’interface entre le parasite et sa cellule hôte.Enfin, afin d’approcher la fonction de pTKL chez le parasite, nous avons généré différentes lignées génétiquement modifiées. L’étude phénotypique des souches de P. berghei KO et iKD pour pTKL a montré qu’elle était dispensable pour la complétion du cycle intra-érythrocytaire, l’expression des gamétocytes ainsi que l’activation des gamétocytes mâles. Ces données suggèrent que pTKL est dispensable pour ces stades de développement ou que l’expression de gènes redondants compense son absence. Quoi qu’il en soit, il est important de poursuivre les recherches sur le rôle de cette protéine aux autres stades de développement du parasite, notamment du zygote aux stades hépatiques. / Malaria is the first endemic parasitic disease in the world with nearly half million deaths in 2017 according to the WHO. This disease is the result of an infection by an agent belonging to the Plasmodium genus. This apicomplexan parasite infects two hosts over its complex life cycle: a definitive one – a mosquito belonging to the Anopheles genus – and a homoeothermic intermediate host. At least six Plasmodium species can infect humans. In its intermediate host, Plasmodium first replicates in hepatocytes before releasing erythrocyte-infectious stages in the bloodstream. Once there, parasites invade and replicate within erythrocytes, before lysing them to release other infectious stages. This triggers an exponential rise in the parasitemia, as well as malaria symptoms. Sexual stages, called gametocytes, are produced over this intra-erythrocytic cycle to be transmitted to the arthropod vector, thus allowing the completion of the parasite life cycle.Plasmodium co-evolved with its hosts and set up diverse gene expression regulation pathways accordingly. Phosphorylation is one of the major and fastest post-translational modifications used by the parasite to respond to environmental changes. Many of its kinases and phosphatases play key roles in host cell invasion, cellular growth and division, as well as motility of specific developmental stages. However, the role of the five pseudo-kinases expressed by Plasmodium has not been explored yet.During my PhD project, I have performed the characterization of the unique Plasmodium pseudo-Tyrosine Kinase-like (pTKL) and explored its role over the parasite intra-erythrocytic cycle.P. falciparum pTKL (PfpTKL) in silico annotation allowed the delineation of the protein domains. Notably, a SAM (Sterile Alpha Motif) domain, two RVxF motifs (known for their binding potential with the major protein phosphatase type 1, PP1) and a pseudo-kinase domain belonging to Tyrosine Kinase-like (TKL) family were found. This pseudo-kinase domain was found to be able to bind ATP in a cation-independent way although devoid of kinase activity. Two parasite protein partners of PfpTKL have been identified using in vitro protein-protein interaction studies together with heterologous models (yeast, Xenopus ovocytes). First, PfSERA5 (SErine Repeat Antigen 5) specifically and strongly interacts with PfpTKL SAM domain and second, PfPP1c binds the two RVxF-containing regions of PfpTKL. Interestingly, the second RVxF motif, which is located within the pseudo-kinase domain, directly binds PfPP1c and seems to be involved in the allosteric regulation of the phosphatase activity. The subcellular localization of P. berghei pTKL (PbpTKL) was studied by IFA as well as sequential lysis of erythrocytes followed by immunoprecipitation assays. PbpTKL was shown to be exported to the host cell cytosol at the trophozoite stage, but retained in the parasitophorous vacuole and the parasite cytosol at the schizont stage. Furthermore, our interactome analysis conducted at the trophozoite stage by IP/MS showed that PbpTKL binds many host cell proteins involved in erythrocyte cytoskeleton organization, as well as erythropoiesis and cell homeostasis. These data suggest that pTKL plays a role at the parasite/host interface, either directly or via its protein partners.Finally, in an attempt to understand the role of pTKL for the parasite development, we generated genetically modified P. berghei strains. The phenotypic study of PbpTKL KO and iKD strains did not show any difference between the defective parasites and the parental wild type ones during the intra-erythrocytic cycle, gametocyte expression and male gametocyte activation. These data suggest the dispensability of pTKL or the expression of redundant gene(s) with similar functions in these parasite stages. Whatever the explanation, it is still important to follow up this investigation in other parasite stages, from zygotes to hepatic stages.

Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.