A new procedure has been developed to concentrate the phosphofructokinase from muscle of Ascaris suum with minimum loss of activity. By utilizing this method, 50 ml fraction was concentrated to a final volume of 3 ml in about 1.5 h without loss in enzyme activity. The concentrated enzyme had a specific activity of 64 units per mg. Ascaris muscle-cuticle was incubated in 50 1M solutions of either acetylcholine, serotonin, y-aminobutyric acid, levamisole, or saline alone. Phosphate analysis of the isolated phosphofructokinase from each incubation revealed that the enzyme contained the following moles of phosphate per subunit: 2.9 (acetylcholine), 2.2 (serotonin), 2.0 (y-aminobutyric acid), 1.5 (levamisole), and 3.4 (salne alone). The present study did not establish a direct correlation between degree of phosphorylation and phosphofructokinase activity. Phosphofructokinase from muscle of Ascaris suum appears to contain several phosphorylation sites, and one of these sites is required to be phosphorylated in order for the enzyme to exhibit maximum activity under physiological conditions.

Advances in the antiretroviral therapy (ART) have dramatically reduced the death rate from human immunodeficiency virus type 1 (HIV-1) induced acquired immune deficiency syndrome (AIDS). However, it is still necessary to develop anti-HIV-1 new drugs. In this study, two projects were conducted and may contribute to the new drug development. The first project is focused on characterizing the anti-HIV activity of a kinase inhibitor Kenpaullone (Ken). We found a cyclin dependent kinase (CDK) and glycogen synthase kinase-3β (GSK-3β) inhibitor named Ken can significantly inhibit HIV-1 replication. Mechanistic analysis by RT-PCR revealed that Ken inhibited HIV-1 replication by disrupting transcription possibly through CDK-dependent pathways. The second project is focused on understanding the association between HIV-1 integrase (IN) and dynein components. Our investigation indicated that HIV-1 IN is associated with DIC1 and DYNLT1. Further investigation this IN/dynein component association may help to reveal new anti-HIV targets. / May 2016

Um Änderungen in seiner Umwelt wahrnehmen zu können, benötigt S. aureus unterschiedliche Signaltransduktionssysteme. In dieser Arbeit wurde erstmals die Eukaryoten-ähnliche Serin/Threonin-Proteinkinase (STPK) PknB umfassend charakterisiert. Die posttranslationale Proteinmodifikation mittels Phosphorylierung spielt sowohl in Eukaryoten als auch in Prokaryoten eine wichtige Rolle. Man glaubte lange, dass die Phosphorylierung von Serin-, Threonin- und Tyrosinresten ein nur auf Eukaryoten beschränkter Regulationsmechanismus ist. Dagegen wurde die Phosphorylierung an Histidin- und Aspartatresten durch die Zweikomponenten-Systeme allein den Prokaryoten zugeordnet. Die Genomanalysen der letzten Jahre identifizierten jedoch STPKs und Serin/Threonin-Proteinphosphatasen (STPP) in nahezu allen prokaryotischen Genomen. Auch S. aureus codiert für eine STPK, die eine hohe Homologie zu den beschriebenen STPKs aufweist. In dieser Arbeit wurden mittels Microarray-Analyse einer ΔpknB-Mutante im Stamm 8325 erste Hinweise zur Funktion von PknB als Regulator der Zellwandsynthese sowie zentraler Stoffwechselwege gewonnen. Es wurden mittels Phosphopreoteom-Analysen in vivo-Substrate identifiziert und weiterhin die Kinase biochemisch charakterisiert. / S. aureus needs effective signal transduction systems to be able to sense a changing environment. In this work we characterize for the first time the regulatory ser/thr protein kinase PknB. The posttranslational protein modification via phosphorylation plays an important role in eukaryotes as well as in prokaryotes. The phosphorylation of proteins on serine, threonine, and tyrosine residues was originally thought to be a mechanism of signal sensing and translation only restricted to eukaryotes. In contrast, prokaryotes were thought to achieve signal transduction exclusively via the phosphorylation of histidine and aspartate residues by using two-component systems. However, recent bacterial genome sequencing identified STPKs and STPPs in almost all bacterial genomes. S. aureus also encodes a STPK, which shows high similarity to the described STPKs. In this study the putative function of PknB as a regulator of cell wall-synthesis as well as central metabolic pathways was analysed by a competitive microarray approach. Furthermore, a phosphoproteome analysis was used to identify in vivo substrates and a biochemical characterization was performed.

Die c-Jun-N-terminale Kinase (JNK), ein Mitglied der Familie der MAP-Kinasen (Mi-togen Activated Protein Kinases), wirkt als signalübertragender Effektor, der den klei-nen GTPasen der Rho–Familie Rac und Cdc42 nachgeschaltet ist. Rho-GTPasen spielen eine Schlüsselrolle in der Regulation von zellulären Aktinstrukturen und steuern Prozesse in der Zelle, die Änderungen der Aktinstruktur erfordern, wie z.B. Änderungen der Zellmorphologie, Zellmigration, Wachstum und Differenzierung. Genetische Studien an der Fruchtfliege Drosophila melanogaster konnten eine Rolle des Drosophila-JNK-Homologs DJNK(basket) in der Regulation von Zellbewegungen und Zellmorphologieänderungen während der Drosophila-Embryogenese zeigen. Inhibierung der Funktion von DJNK auf allen Stufen der DJNK-Signaltransduktions-kaskade führt zum sogenannten dorsal closure-Phänotyp der Embryonen mit fehlender Zellstreckung und fehlender Migration dorsaler Epithelzellen. Der molekulare Mechanismus, mit dessen Hilfe Rho-GTPasen Aktinstrukturen regu-lieren und wie JNK Einfluss auf Zellmorphologie und Zellbewegung nimmt, ist bisher nicht bekannt. Die Identifizierung neuer, mit JNK interagierender Proteine könnte zum besseren Verständnis der Funktion und Regulation von JNK führen. In dieser Arbeit wurde ein Yeast-Two-Hybrid-Screen mit dem Drosophila-Homolog DJNK/basket durchgeführt, der zur Entdeckung des Drosophila-Proteins p150-Spir als Interaktionspartner von DJNK führte. Der C-terminus des p150-Spir-Proteins enthält eine JNK-Interaktionsdomäne, ein DEJL-Motiv (Docking Site for Erk and JNK, LxL) und wird von aktivierten JNK-Proteinkinasen phosphoryliert. p150-Spir ist ein Multi-Domänen-Protein, das in seiner aminoterminalen Hälfte eine Aufeinanderfolge von vier WH2-Domänen (Wiskott Aldrich Homology Domain 2) enthält. WH2-Domänen binden monomeres Aktin, Proteine mit WH2 Domänen, wie z.B. WASP oder WAVE sind Aktinreorganisatoren. Die transiente Überexpression von p150-Spir in NIH3T3-Mausfibroblasten führt ebenfalls zu einer Aktinreorganisation. Eine weitere Domäne in p150-Spir ist eine modifizierte FYVE-Zinkfinger-Struktur (mFYVE) im zentralen Bereich des Proteins, die für die subzelluläre Lokalisation von p150-Spir von Bedeutung ist. Mutationen, welche die Zinkfingerstruktur zerstören, führen bei Überexpression in NIH3T3-Zellen zu einer zytoplasmatischen Lokalisation der mutierten p150-Spir-Proteine, während Wildtyp-p150-Spir perinukleär akkumuliert. Spir-Proteine sind evolutionär hoch konserviert. Es konnten Spir-ähnliche Sequenzen auf den humanen Chromosomen 16 und 18, in der Maus und in der Seescheide Ciona savignyi gefunden werden. Der höchste Grad an Konservierung besteht im Bereich der funktionellen Proteindomänen. Ein in allen Spir-Proteinen ent-haltenes, als Spir-Box bezeichnetes hoch konserviertes Sequenzmotiv befindet sich unmittelbar vor dem mFYVE-Zinkfinger. Die Spir-Box zeigt Strukturverwandschaft zur Rab-GTPase-Bindungsregion in Rabphilin 3A, einem Protein, das ebenfalls eine FYVE-Domäne besitzt. Rab-GTPasen sind wie FYVE-Domänenproteine in die Regulation zellulärer Vesikeltransportprozesse involviert. Das Vorhandensein beider Do-mänen in p150-Spir deutet auf eine Rolle des Proteins in zellulären Transportprozes-sen hin. Ein denkbares Modell wäre, daß p150-Spir unter der Kontrolle von JNK-Signalen zelluläre Aktinstrukturen reguliert, die für Transportprozessse in der Zelle von Bedeutung sind; p150-Spir fungiert damit möglicherweise als direktes Bindeglied zwischen MAPK-Signaltransduktionskaskaden und dem Aktinzytoskelett. / Summary c-Jun-N-terminal kinases (JNKs) are members of the MAPK family (mitogen activated protein kinases) and act as downstream effectors of Rho family-GTPases, Rac and Cdc42. Rho family GTPases are involved in the regulation of cellular actin structures and control cellular processes which require remodelling of the actin skeleton, such as morphological changes, migration, growth and differentiation. A role for the Drosophila JNK-homolog DJNK/basket in the regulation of cell move-ment and cell shape changes during Drosophila embryogenesis arises from its func-tion in the process of dorsal closure. Inhibition of the DJNK-cascade results in a mu-tant phenotyp, where the dorsal elongation and migration of the epithelial cells fails. However, a direct link between JNK signaling and actin reorganization has not yet been established. A Yeast-Two-Hybrid-Screen using DJNK as a bait led to the discovery of the new Drosophila protein p150-Spir. p150-Spir is a multi-domain protein with a stretch of acidic amino acids, a cluster of 4 WH2-domains (Wiskott Aldrich Homology Domain 2), a Spir-Box and a modified FYVE zinc-finger motif (mFYVE). In addition, the C-terminus of p150-Spir harbors a docking site for ERK and JNK, LXL (DEJL-motif) and is phosphorylated by JNK in vitro and in vivo. When coexpressed with p150-Spir in NIH3T3 cells, JNK translocates to and colocalizes with p150-Spir at discrete spots around the nucleus. In its N-terminal part p150-Spir possesses 4 WH2-Domains. WH2-domains bind monomeric actin and WH2-family proteins, such as WASP and WAVE are involved in actin reorganization. We can show that in NIH3T3 mouse fibroblasts, p150-Spir co-localizes with F-actin and its overexpression induces clustering of filamentous actin around the nucleus. A modified FYVE zinc-finger structure (mFYVE) is located in the central region of the protein. FYVE-fingers mediate cell membrane localization of proteins. Disruption of the p150-Spir mFYVE-structure by deletion mutagenesis or cysteine/serine substitu-tions shows that the mFYVE-domain determines the subcellular localisation of p150-Spir. In contrast to the perinuclear distribution of the wild type p150-Spir, the mutated protein exhibits a uniform cytoplasmic distribution. Spir-family proteins are highly conserved among different species. Comparison of Drosophila p150-Spir sequences to EST data bases identified similar sequences in human (on chromosomes 16 and 18), mouse and the ascidian Ciona savignyi. A con-served sequence motif found in all Spir proteins - called Spir-Box - is located in the N-terminus, next to the mFYVE domain. Close inspection of the Spir-Box sequence revealed homology to the GTPase binding region of Rabphilin 3A, a FYVE domain containing protein which binds the small GTPase Rab3a. Rab-GTPases are involved in the regulation of cellular vesicle trafficking processes. The presence of a mFYVE domain in p150-Spir protein and a Spir-Box - a possible Rab-GTPase binding motif - suggests a potential function of Spir proteins in vesicel transport. In a possible model Spir initiates remodelling processes of the actin cytoskeleton, necessary for cellular transport processes under the control of JNK signals and thereby provides a direct link between MAPK-signaling and the actin cytoskeleton.

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Class I phosphoinositide 3-kinases (PI3K) and mammalian target of rapamycin complexes 1/2 (mTORC1/2) are enzymes that play important roles in elementary biology and disease. As a consequence, numerous small-molecule inhibitors of their catalytic activity have been developed and these have shown clinical utility in certain cancers. Unfortunately, acquired resistance to these therapies is a common phenomenon and often occurs relatively quickly following treatment. Our understanding of how resistance develops is hampered by the difficulty of measuring the circuitry and plasticity of the signalling networks that these and other kinases signal within. Advances in mass spectrometric technologies have rendered the routine quantitative interrogation of the phosphoproteome (the set of phosphorylated proteins expressed in a particular biological system at a specific time) more tractable than ever before. The aim of this project therefore, was to improve upon existing mass spectrometry (MS)-based phosphoproteomics methods, and to utilise these to contribute to our understanding of kinase signalling networks and examine their plasticity in models of acquired resistance to PI3K and mTORC1/2-targeted therapies. Novel approaches for the enrichment of phosphopeptides from complex biological matrices (and their analysis by MS) were designed, tested and optimised. These methods were then used to systematically characterise a kinase signalling network comprising the Akt/PI3K/mTOR and MEK/ERK signalling axes in MCF7 breast cancer cells. The biological relevance of this network was confirmed through the assessment of its dynamics upon EGF and IGF-1 stimulation. Finally, the plasticity of this network following chronic treatment with targeted PI3K and mTORC1/2 inhibitors (GDC-0941 and KU-0063794) was examined in cell-line models of acquired resistance to these two compounds. This revealed that these cells each remodelled this network in a different manner, thus indicating that the initial conditions of the system were not the sole determinant of how resistance was acquired.

Over the last twenty years, fragment-based drug discovery (FBDD) has emerged as a highly successful way to provide lead compounds for subsequent optimisation into drug candidates. Initial hits usually exhibit lower potency than those identified by more traditional techniques, such as High-Throughput Screening (HTS), but the optimisation phase of FBDD is highly efficient, thus providing superior lead-like compounds. The recent application of FBDD in a variety of protein kinase campaigns has successfully led to the identification of novel binding sites and highly efficient chemical ligands. This demonstrates the utility of the FBDD strategy against well-established kinase targets, where selectivity is otherwise challenging due to significant conservation of the ATP-binding site. Protein kinase CK2 is a ubiquitously expressed and constitutively active regulator of cell growth, proliferation and apoptosis. Elevated levels of CK2 protein and activity have historically been involved in human cancer, including lung, cervical and head and neck cancer types, and its overexpression is associated with worse prognosis. A number of CK2 inhibitors are currently available displaying activity against multiple cancers in vitro and in the clinic, however the majority of these candidates target the ATP-binding site and thus display poor selectivity in kinase panel assays. Here we explore the application of FBDD towards the development of potent and selective inhibitors of the catalytic α-subunit of CK2. This project exploits a novel, conserved binding site, named the αD pocket, for the generation of allosteric inhibitor molecules. Following structure-based optimisation of a potent inhibitor series, and characterisation of a previously unreported binding mode, a fragment linking strategy between the lead αD-site fragment and a low-affinity pseudosubstrate peptide is investigated. This work validates the utility of FBDD towards the discovery of new binding modes, presents a first in class CK2α allosteric inhibitor series and provides the first X-ray crystal structure of protein kinase CK2 in complex with a ligand binding in the substrate-binding channel.

Many diseases correlate with abnormal glucose metabolism in cells and organisms. For instance, the human M2 isoform of the glycolytic enzyme pyruvate kinase (M2PYK) plays an important role in metabolic reprogramming of tumour cells whereby aerobic glycolysis or the ‘Warburg effect’ supports cell proliferation by accumulating necessary biomass. By contrast, gluconeogenesis may play an important role, as observed in certain types of trypanosomatid parasites (e.g. the amastigote form of Leishmania major) where anabolism is essential for infectious properties. Hence, these glucose metabolising enzymes are important potential drug targets for cancer and trypanosomiasis. However, many aspects of their regulatory mechanisms are still poorly understood. This thesis describes biochemical and structural studies on M2PYK and on L. major fructose-1,6-bisphosphatase (LmFBPase), providing insights into allosteric mechanisms and structure-based drug design for both enzymes. Human PYKs and LmFBPase were expressed and purified from Escherichia coli, and their kinetics were fully characterised. It was shown that certain amino acids regulate the activity of M2PYK allosterically, but in opposite ways, with some being inhibitors and others activators. X-ray crystallographic structures and biophysical data of M2PYK complexes with alanine, phenylalanine, serine or tryptophan reveal an R-/T-state oscillating model of M2PYK involving a 11° rotation of each subunit. In addition, M2PYK was demonstrated to be a redox-sensitive enzyme. Reducing reagents were shown to help maintain the tetramer and prevent its dissociation, and thereby to activate M2PYK, whereas oxidation and nitrosylation reagents functioned in the opposite sense. Nitrosylation assays showed that the main nitrosylated residue is Cys326 of M2PYK, which is located on the tetramer interface. Dynamics and modulator effects of PYKs were further studied by hydrogen–deuterium exchange by mass spectrometry. These observations highlight the important effects of amino acids on M2PYK regulation. M1PYK by contrast, was demonstrated to be a constitutively fully active pyruvate kinase, with minor effects from modulators. The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is a potential drug target against leishmaniasis. Here we present biochemical and structural studies for LmFBPase, by characterising its activity in a metal-dependent reaction, as well as its inhibition by AMP. The crystal structure of LmFBPase is a homotetramer, composed of monomers with alternating α/β/α/β/α ‘club sandwich’ topologies. In comparison with previously revealed LmFBPase structures, the AMP-complexed structure shows a rotated form of the tetramer. Comparisons of the structures reveal an ‘unlock-androtate’ allosteric mechanism in which AMP binding causes a series of structural changes culminating in an incomplete and non-productive active site. The structure of the effector site of LmFBPase shows a different conformation from human FBPases, thereby offering a potential specific target for Leishmania.

C. elegans is as an extremely powerful model for the study of innate immunity. MAP kinase signaling pathways in C. elegans are involved in the response of C. elegans to infection by pathogenic bacteria. The yeast S. cerevisiae can infect C. elegans, producing pathogenic effects. In this project, we tested whether several MAP kinase pathways are important for C. elegans¡¯ resistance to yeast infection. We tested members of several MAP kinase pathways including tir-1, nsy-1, sek-1 and pmk-1 in the p38 pathway, mek-1, jnk-1 and kgb-1 in JNK pathway and mek-2 and mpk-1 in the ERK pathway. We used survival assays to compare the responses of mutants of components of these pathways to the control responses of wild-type C. elegans. In the survival assay, we found that mutants in all three MAP kinase pathways showed a decreased survival relative to wild type; therefore all three pathways are important for innate immunity against the yeast pathogen. With respect to the p38 pathway, mutations affected survival but not the deformed anal region (Dar) phenotype, a putative defensive response induced by yeast in wild-type C. elegans. This indicates that for the p38 pathway, survival depends on some other immune response besides Dar. Finally, we hypothesize that cross talk occurs between p38 and JNK MAPK pathways in the C. elegans immune responses.

Glioblastoma is the most malignant form of brain cancer. Due to its aggressive nature, extensive research has been performed, but little progress has been made in identifying effective treatment options. Glycogen synthase kinase-3 (GSK-3) is a ubiquitous, multifaceted protein kinase. Previous studies have shown that small molecule inhibitors of GSK-3 block the migration of glioblastoma cells and may prevent spread of tumor in the brain. However, these studies were performed using non-selective GSK-3 inhibitors (LiCl and an indirubin derivative, BIO); thus, it was unclear whether GSK-3 was the most important target. In this study, we used recently generated highly selective GSK-3 inhibitors (CHIR99021, AZD1080, and AZD2858, as well as BIO) to investigate these questions. These were applied to four glioblastoma cell lines: G30, G9, U251, and U1242, in three migration assays: transwell, spheroid, and wound healing (scratch) assay to further assess the suitability of GSK-3 as a target in glioblastoma. We also utilized the ATP Luciferase reporter assay for cell viability to assess the influence of our panel of drugs on cell migration versus viability. In addition, the TOPFlash Luciferase reporter assay was performed as an indicator of the level of GSK-3 inhibition. The TOPFlash assay showed that all GSK-3 inhibitors were able to increase luciferase levels. This indicates that GSK-3 was inhibited in our cells after drug treatment. The transwell assays showed us that the GSK-3 inhibitors were able to block migration significantly in all cell lines tested in a dose-dependent manner. The effectiveness of GSK-3 inhibition in the three-dimensional collagen spheroid assays was cell line-dependent, with the non-selective GSK-3 inhibitor BIO showing the most potent effects. Cell migration was not blocked by any of the three selective GSK-3 inhibitors in the wound healing scratch assay. Thus we have found that the three distinct highly selective inhibitors of GSK-3 block glioblastoma cell migration, but only work consistently in the transwell assay. Therefore, we conclude that GSK-3 might be important in the contraction and morphological changes necessary for glioblastoma cells to migrate through the 8 micron pores in the transwell. Further investigation into this observation is necessary. Though results were variable between assays, we conclude that the inhibition of GSK-3 is a promising potential therapeutic strategy for glioblastoma treatment.