Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xviii, 158 p. : ill. (some col.). Advisor: Young C. Lin, Veterinary Biosciences Graduate Program. Includes bibliographical references (p. 148-158).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 82-90). Also available in electronic version.

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.

Trypanosomiases and leishmaniases are amongst the world’s most neglected infectious diseases. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are the primary human pathogens of the trypanosomatidae family and the causative agents of African sleeping sickness, Chagas’ disease and cutaneous leishmaniasis, respectively. The molecular mechanism controlling each parasite’s life cycle and virulence is poorly understood however protein phosphatases are expected to play a critical role. This study presents the biochemical characterisation data of two groups of phosphatases from the trypanosomatids: the LRR-DSP and LM phosphatases. The LRR-DSPs are a group of twelve proteins, characterised by a unique domain architecture: a leucine-rich repeat (LRR) domain together with a dual- specificity phosphatase (DSP) catalytic domain. In this study, the recombinant expression of a representative LRR-DSP orthologue from T. brucei (TbLRR-DSP), proved to be highly problematic in E. coli. Most full-length and catalytic domain constructs were expressed at very low levels or as insoluble proteins. Soluble protein was obtained by denaturation, treatment with detergents, non-denaturing extraction from inclusion bodies and fusion to solubility-enhancing proteins. However, no method yielded protein with catalytic activity or detectable secondary structure. Soluble expression of TbLRR-DSP was achieved using baculovirus-infected insect cells, but the protein co-purified with endogenous chaperones and exhibited no catalytic activity thus implying a lack of correct folding. In the second part of this study, two phosphatases specific to Leishmania major, LM1 and LM2, were characterised and structural studies were initiated. LM2 was shown to readily hydrolyse phospho-tyrosine substrates in vitro, but not phosphoinositides like its homologue, LM1. Both proteins therefore have a differentiated catalytic profile and are likely to have different functions in vivo. Purification protocols for both proteins were established and crystallisation screenings set up. Preliminary hits were obtained for LM2 and a mutagenesis strategy was developed to improve chances of obtaining diffraction quality crystals. Recombinant LM1 samples exhibited heterogeneity and therefore will require additional engineering to improve chances of crystallization. Promising pilot NMR data was also obtained for both phosphatases. In conclusion, this study demonstrates that the recombinant expression of multi- domain trypanosomatid proteins (like the LRR-DSPs) can be highly problematic and may pose a challenge for their biochemical characterisation and functional elucidation. Future work into trypanosomatid phosphatases, however challenging, will improve our understanding of their cell biology and potentially identify therapeutic targets.

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No / Acid phosphatases hydrolyse phosphomonoesters at acidic pH in a variety of physiological contexts. The recently defined class C family of acid phosphatases includes the 32 kDa LppC lipoprotein of Streptococcus equisimilis. To define further the distribution of acid phosphatases in the genus Streptococcus we have examined the equine pathogens Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Whole cell assays indicated that these organisms possess two acid phosphatases with activity optima at pH 5.0 and pH 6.0-6.5 and that only the former of these was, like LppC, resistant to EDTA. Western blotting with a polyclonal anti-LppC antiserum revealed the presence of a cross-reactive 32 kDa protein in both organisms. The cross-reactive protein in S. equi was shown to be a surface accessible lipoprotein as its processing was inhibited by the antibiotic globomycin and it was released from whole cells by treatment with trypsin. The presence of DNA sequences homologous to the S. equisimilis lppC gene were confirmed by PCR. These data strongly suggest that Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus produce a lipoprotein acid phosphatase homologous to LppC of S. equisimilis.

Le paludisme est la première endémie parasitaire mondiale causée par le protozoaire Plasmodium. Cette parasitose est responsable de 219 millions de cas et 660 000 décès par an. La prévalence et la mortalité élevées sont liées notamment à la résistance des parasites aux traitements existants, ce qui rend primordial le développement de nouvelles thérapeutiques. Pour ce faire, une meilleure connaissance de la biologie fondamentale du parasite est nécessaire. Dans ce contexte l’un des axes de recherche concerne la régulation du cycle cellulaire chez Plasmodium et notamment les mécanismes de phosphorylation/déphosphorylation qui sont essentiels.Parmi les nombreux acteurs des mécanismes de phosphorylation, la sérine/thréonine protéine phosphatase de type 2A (PP2A) est, avec PP1, l’une des phosphatases majeures. Cette phosphatase est impliquée dans de nombreux processus cellulaires notamment la mitose, la méiose ou encore l’apoptose. Elle est composée d’une sous-unité catalytique (PP2Ac), d’une sous-unité d’aide à l’agencement spatial (A) et d’une sous-unité régulatrice (B). Il existe quatre familles de sous-unités régulatrices contenant chacune plusieurs membres qui permettent de réguler la localisation, la spécificité et l’activité de PP2A. Il existe également des protéines régulatrices indépendantes, notamment les inhibiteurs 1 et 2, la protéine α4 et le PhosphoTyrosyl Phosphatase Activator (PTPA). Chez Plasmodium falciparum, la protéine phosphatase de type 2A ou PfPP2A a été identifiée et semble essentielle pour le développement asexué du parasite. Cependant, peu de choses sont connues sur sa régulation chez le parasite. En effet, seul l’inhibiteur 2 de PP2A a été décrit et caractérisé. Au cours de cette thèse, nous avons effectué par des études in silico un recensement des régulateurs putatifs de PfPP2A. Ces études nous ont permis d’identifier la sous-unité A et une unique sous-unité B. Parmi les régulateurs spécifiques, outre l’inhibiteur 2 déjà caractérisé, l’analyse du génome du parasite montre qu’il contient un orthologue de l’inhibiteur 1, d’α4 et de PTPA. Le projet de cette thèse s’articule autour de la caractérisation moléculaire et fonctionnelle de l’un de ces régulateurs : PfPTPA.La caractérisation moléculaire de PfPTPA a permis de montrer dans ce travail la conservation de cette protéine au cours de l’évolution. L’analyse de sa séquence a révélé que cinq des six motifs de fixation à la PP2A identifiés chez l’homme sont conservés. Par des études in vitro et in vivo dans un modèle hétérologue, nous avons pu confirmer le rôle d’activateur de PfPTPA vis-à-vis de la PP2A. Par une approche de mutation unique d’acides aminés, nous avons identifié trois résidus impliqués dans l’interaction et l’activité de PfPTPA notamment le résidu G292 qui est essentiel pour l’interaction PfPTPA/PfPP2A. Nous avons ensuite montré par des études de génétique inverse que PfPP2A et PfPTPA, qui sont présents dans le même compartiment cellulaire au cours du cycle érythrocytaire, sont essentielles pour la complétion du cycle intra-érythrocytaire du parasite. De plus, PfPTPA semble impliqué dans le cycle cellulaire chez le xénope.En parallèle, l’analyse de la séquence de PfPTPA, a révélé la présence, spécifique au parasite, d’un motif de fixation à la PP1 (motif RVxF). L’identification de ce motif, nous a incités à étudier la relation entre PfPTPA et PfPP1. Nous avons ainsi pu montrer que PfPTPA était capable de se lier à PfPP1 même si elle est incapable de réguler son activité.L’ensemble de ce travail de thèse a permis de caractériser chez Plasmodium falciparum un activateur de la protéine phosphatase de type 2A et de montrer sa spécificité par rapport à la protéine humaine. Nos résultats, et notamment l’implication de PfPTPA dans la régulation du cycle cellulaire, font de ce régulateur une cible thérapeutique potentielle. / Malaria is the most deadly parasitic disease in the world caused by the Apicomplexa protozoan Plasmodium falciparum. This parasite is responsible for 219 million cases and 660 000 deaths per year and the drug resistance increases the prevalence and the morbidity. The emergence of multi-drug resistance requires the development of new therapeutics. Hence, a better understanding of parasitic fundamental biology is necessary. In this context, one research axis is the cell cycle regulation of Plasmodium, notably phosphorylation/dephosphorylation mechanisms which are essential for the parasite.Among the actors of the reversible phosphorylation, the serine/threonine phosphatase type 2A (PP2A) in eukaryote is, with PP1, one of the major phosphatases. It is involved in several cell processes like mitosis, meiosis or apoptosis. PP2A is composed of a catalytic subunit (PP2Ac), a scaffold subunit (A) and a regulatory subunit (B). There are four regulatory subunit families which regulate location, specificity and activity of PP2A. Furthermore, several independent regulatory proteins including inhibitor 1 and 2, the α4 protein or the phosphotyrosyl phosphatase activator (PTPA) were identified.In Plasmodium falciparum, the protein phosphatase type 2A named PfPP2A has been characterized and seems to be essential for the parasite asexual development as shown by the inhibition of parasitic growth after treatment with natural toxins inhibiting phosphatases. However, its regulation is still poorly understood in Plasmodium. Indeed, only the PP2A inhibitor 2 is characterized in P. falciparum and in P. berghei (a rodent specific Plasmodium species). Using an in silico study, we have identified a putative scaffold subunit and only one B subunit. Among the regulatory proteins, we have identified orthologs of the inhibitor 1, α4 and PTPA. The purpose of this thesis is to study PfPTPA both of the molecular and functional levels.The molecular characterization of PfPTPA showed the evolutionary conservation of this protein. The PfPTPA sequence analysis revealed that five out of six amino acids involved in interaction with PP2A in human, are conserved in P. falciparum. In vitro binding and functional studies revealed that PfPTPA binds to and activates PfPP2A. Mutation studies showed that three residues (V283, G292 and M296) of PfPTPA are indispensable for the interaction and that G292 residue is essential for its activity. Localization studies indicated that PfPTPA and PfPP2A are localized in the same cellular compartment throughout the erythrocytic cycle of P. falciparum, suggesting a possible interaction of both proteins in vivo. In Plasmodium falciparum, genetic studies likely suggested the essentiality of PfPTPA for the completion of intraerythrocytic parasite lifecycle. Functionnal studies, using Xenopus oocyte, showed that PfPTPA blocked the G2/M transition. Further analysis of PfPTPA sequence revealed that PfPTPA, unlike its human counterpart, possess one of the most canonical binding motif to PP1 (RVxF motif). The identification of this RVxF motif led us to study the role PfPTPA on PfPP1. Thus, we have shown that PfPTPA interacts with PfPP1 but was unable to regulate PfPP1 activity in vitro. This work allowed characterizing the PfPTPA, an activator of protein phosphatase type 2A in Plasmodium falciparum and to show some specificities when compared to its human ortholog. Our data which suggest that this regulator could be involved in cell cycle regulation, together with its essentiality for the growth of P. falciparum strongly support the idea to explore it as potential drug target.

Placental alkaline phosphatase (PLAP) is a membrane bound enzyme normally synthesized by the syncytiotrophoblasts in the human placenta. Recent studies have indicated that trace amounts of placental-like alkaline phosphatases also are present in several normal organs like testes and endocervix. PLAP and PLAP-1ike enzymes are furthermore synthesized by some tumors and can be detected in sera of approximately 12 % of patients with any type of cancer, more often in patients with genital tumors. This synthesis has been considered to be ectopic.PLAP is known to be electrophoretically highly polymorphic. Both poly- and monoclonal antibodies were used to study this enzyme. One of the monoclonal antibodies was able to discriminate between different phenotypes of PLAP and thus immunochemical approaches to elucidate enzyme polymorphism were established.To evaluate the potential clinical use of PLAP as a tumor marker serum levels of the enzyme were measured by a radioimmunoassay in 100 patients with the testicular tumor seminoma. Elevated levels of PLAP were found in 43 % of the patients with primary tumors and in 75 % of the patients with recurrent or metastatic disease. After successful treatment of seminoma the PLAP levels decreased. This indicates that measuring PLAP give useful information during follow up of treatment of seminomas.The content of PLAP-like enzymes in seminoma tumors was determined in 13 typical seminomas. The levels, of enzyme found in the tumor tissue ranged from 870-13 404 ng/g wet weight, which should be compared to around 100 ng/g in normal testes. Analysis using monoclonal antibodies and enzyme inhibitors showed the PLAP-like enzymes present in seminomas to be similar to the enzymes in normal testes. This suggests that the increased expression of PLAP-like enzymes in seminomas results from an enhanced eutopic expression of enzymes found in normal testis.A sensitive catalytic assay was used to quantify enzyme levels in sera from women with malignant gynaecological tumors. In the group of patients with cervical carcinoma 68 % had values exceeding the normal limit. For patients with ovarian cancer and carcinoma of the breast the percentages were 35 and 23 respectively.Monoclonal and polyclonal antibodies against PLAP were evaluated for tumor immunolocalization of human PLAP-producing tumors in nude mice.The antibodies were labeled with "125j and injected into mice with tumors. The distribution of 25j_an-ti-PLAP in various tissues showed that the labeled antibodies were enriched in the tumors, with a mean concentration ratio of 7. This indicates that there is a potential use of PLAP in localizing tumors in humans. / <p>S. 1-37: sammanfattning, s. 39-88: 5 uppsatser</p> / digitalisering@umu