This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning. Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions. Samples were evaluated by Western blot analysis with monoclonal antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were quantified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cells with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells, the particulate fraction contained about 30% of PKC epsilon, 5-10% of PKC alpha and 60-70% of PKC zeta. Preconditioning with adenosine (100 μM) or 10 min ischemia had no significant effect on these percentages. Furthermore, the relative amounts of the PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a post-incubation in oxygenated buffer or during a final ischemic incubation. PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon. The distribution of PKC zeta between fractions was not affected by any drug, The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the final ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro.

Oncolytic viruses (OVs) have made tremendous strides as a viable cancer therapeutic in recent years; however, variable infectivity rates have since limited clinical efficacy. Residual type 1 interferon (IFN-1) responses are integral to the tumour’s innate antiviral defense and confer resistance to OVs. To combat this, small molecules with viral sensitizing ability can be used in combination to transiently knockdown IFN-1 responses, allowing OVs to gain a foothold for increased infectivity and therapeutic efficacy. Accordingly, we hypothesize that some chemical or genetic manipulations of cellular processes can indirectly antagonize antiviral IFN-1 responses and modulate pro-inflammatory pathways to potentiate oncolytic virotherapy. In this thesis, we identify several avenues to modify cell signalling events to increase OV therapeutic efficacy through IFN-1 inhibition. Firstly, with respect to the demonstrated OV-enhancing effects of vanadium, a pan-phosphatase (PP) inhibitor, we elucidate that its IFN-1 suppressing activity involves activation of the epidermal growth factor receptor (EGFR) pathway via STAT1/2 and NF-κB. Pharmacological inhibition of EGFR abrogated vanadium’s viral sensitizing ability in vivo. Secondly, using high-throughput screening methodology, we identify protein phosphatases that inherently regulate the IFN-1 response as targets for oncolytic vesicular stomatitis virus (VSV∆51) potentiation. Indeed, cloning interfering RNA against one of these PP targets, acid phosphatase 2 (ACP2), into the VSV∆51 platform demonstrated superior infectivity and cancer cell cytotoxicity compared to the non-targeting VSV∆51 control. Thirdly, we characterize pevonedistat, a first in-class neddylation activating enzyme inhibitor, to potentiate OV therapeutic efficacy across several in vitro and in vivo contexts. We demonstrate pevonedistat’s ability to inhibit IFN-1 signalling and pro-inflammatory cytokine production using both neddylation independent and dependent mechanisms. Taken altogether, we dissect multiple signaling mechanisms by which the IFN-1 response can be modulated for the purposes of improving OV therapeutic efficacy. This knowledge can subsequently be directly translated into designing optimized OV strategies for clinical testing.

[EN] The plasma membrane H+-ATPase (Pma1) is essential for yeast growth and is activated by glucose metabolism by an unknown mechanism involving double phosphorylation of a regulatory site at the C-terminus (Ser911 Thr912). In this thesis we have investigated in Saccharomyces cerevisiae the role of two protein phosphatases, type 1 Glc7 and type 2A Sit4, and of an essential atypical protein kinase, TORC1, in the activation of Pma1 by glucose. The regulatory site of activated Pma1 can be dephosphorylated "in vitro" by recombinant Glc7 and Sit4, but inhibition "in vivo" of these phosphatases does not activate Pma1. Inhibition of Glc7 by regulated expression of a dominant-negative truncated form (the null mutant is not viable) had no effect on Pma1 activity while deletion of SIT4 gene decreased both Pma1 activity and double phosphorylation of the regulatory site. Inhibition of TORC1 protein kinase by treatment of yeast cells with the drug rapamycin or by exposure to non-permissive temperature of a temperature-sensitive mutant (tor1¿ tor2ts) inhibited Pma1 and decreased double phosphorylation of the regulatory site. We conclude that Sit4 and TORC1 are required for full activation of Pma1 by glucose while Glc7 either does not participate or is redundant with other phosphatases. / [ES] La H+-ATPasa de la membrana plasmática (Pma1) es esencial para el crecimiento de la levadura y se activa por metabolismo de glucosa por un mecanismo desconocido que lleva consigo la doble fosforilación de un sitio regulador en el extremo C-terminal (Ser911 Thr912). En la presente tesis hemos investigado en Saccharomyces cerevisiae la participación de dos proteína fosfatasas, Glc7 de tipo 1 y Sit4 de tipo 2A, y de una proteína kinasa atípica esencial, TORC1, en la activación de Pma1 por glucosa. El sitio regulador de Pma1 en su estado activo puede defosforilarse "in vitro" por Glc7 y Sit4 recombinantes pero la inhibición "in vivo" de estas fosfatasas no activa Pma1. La inhibición de Glc7 mediante la expresión regulada de una forma truncada que actúa como dominante-negativa (el mutante nulo no es viable) no tiene efecto en la actividad de Pma1 mientras que la deleción del gen SIT4 disminuye tanto la actividad de Pma1 como la doble fosforilación del sitio regulador. Inhibición de la proteína kinasa TORC1 mediante tratamiento de las células de levadura con el fármaco rapamicina o exponiéndolas a temperatura no permisiva en el caso de un mutante termosensible (tor1¿ tor2ts) resulta en inhibición de Pma1 y disminución de la doble fosforilación del sitio regulador. Estos resultados indican que Sit4 y TORC1 son necesarias para la máxima activación de Pma1 por glucosa mientras que Glc7 podría no participar o hacerlo de forma redundante con otras fosfatasas. / [CA] L'H+-ATPasa de la membrana plasmàtica (Pma1) és essencial per al creixement dels llevats i s'activa gràcies al metabolisme de glucosa per un mecanisme desconegut que porta associat la doble fosforilació d'una regió reguladora a l'extrem C-terminal (Ser911 Thr912). En aquesta tesi hem investigat en Saccharomyces cerevisiae la participació de dos proteïnes fosfatases, Glc7 de tipus 1 i Sit4 de tipus 2A, i d'una proteïna quinasa essencial atípica, TORC1, en l'activació de Pma1 per glucosa. La regió reguladora de Pma1, en seu estat activat, pot desfosforar-se "in vitro" per Glc7 i Sit4 recombinants, però la inhibició "in vivo" d'aquestes fosfatases no activa Pma1. La inhibició de Glc7 mitjançant l'expressió regulada d'una forma truncada que actua com a dominant-negativa (el mutant nul no és viable) no té cap efecte en l'activitat de Pma1 mentre que la deleció del gen SIT4 disminueix tant l'activitat de Pma1 com la doble fosforilació de la regió reguladora. La inhibició de la proteïna quinasa TORC mitjançant un tractament de cèl·lules de llevat amb el fàrmac rapamicina o la seua exposició a temperatures no permissives en el cas d'un mutant termosensible (tor1¿ tor2ts) resulta en la inhibició de Pma1 i la disminució de la doble fosforilació de la regió reguladora. Aquests resultats indiquen que Sit4 i TORC1 són necessàries per a l'activació màxima de Pma1 per glucosa, mentre que Glc7 podria no participar o fer-ho d'una forma redundant amb altres fosfatases. / Mahmoud Ali Ibrahim Hamouda, S. (2015). Protein kinases and phosphatases regulating the yeast proton pump [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/54131

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1996, härtill 6 uppsatser</p> / digitalisering@umu

Infectious and neurodegenerative diseases continue to be a major concern worldwide. In spite of the great advances in drug therapy for treating various infectious and neurodegenerative diseases, there is still an urgent need for new and improved drugs due to increasing drug resistance among pathogens, emergence of new pathogens, ease of transmission of infections, ineffective available treatments, toxicity associated with current standard of care, aging populations and the lack of better alternative treatment options. The first part of this manuscript (chapters 1 - 5) describes the synthesis of novel agents active against Leishmania donovani. According to the World Health Organization (WHO), a significant number of deaths worldwide can be attributed to infectious diseases – particularly neglected tropical diseases (NTDs), one of which is leishmaniasis - a complex and clinically diverse disease transmitted through the bite of an infected female phlebotomine sand-fly. The pathogen that causes leishmaniasis develops through a complex life cycle via different morphological changes. Its clinical presentations range from the less severe (cutaneous) to lethal/fatal (visceral) forms depending upon the level of systemic involvement, infecting species and the endemic environment. Treatments (and vaccines) must be species-specific to be particularly effective since sensitivity to commonly used drugs is largely species-specific. Heat shock protein 90 (Hsp 90) has been shown to promote the differentiation of the protozoan parasite that causes leishmaniasis from the promastigote stage to the amastigote pathogenic stages. To this end a series of compounds were prepared based on known Hsp 90 inhibitors, SNX2112 and XL888. The synthetic approach allows the probing of a hydrophobic pocket and rapid access to a collection of anti-leishmanial compounds. The most active compound, was found to be more than twice as active as the climivally used drug, miltefosine, in an infected J774 macrophage at IC50 = 0.65 µM. The second part of this manuscript (chapters 6 - 9) describes the synthesis novel anti-Alzheimer’s agents. Alzheimer’s disease is a progressive neurodegenerative disease believed to be caused by tau hyperphosphorylation and plaque aggregation in the brain. It is known to affect about 44 million people worldwide and it is marked as the 6th leading cause of death in the United States. Slingshot homology-1 (SSH1) proteins, important protein phosphatases, are promising targets for the discovery of a new generation of small molecule inhibitors as treatment for Alzheimer’s disease, since SSH1 is known to contribute to both tau hyperphosphorylation and plaque aggregation in the brain. Through structure and activity relationships (SAR) studies, two (2) series of compounds were synthesized, thiazoles and pyridones, bearing a carboxylic acid or phosphonic acid functionality as inhibitors of SSH1 enzymes. In the preliminary screening efforts against SSH1 phosphatase activity, the thiazole series were found to be more potent at inhibiting the phosphatase activity than the pyridone series. Among the active thiazole series, eight (8) analogs exhibited significant inhibitory activity over the initial hit compound, observed via phosphatase inhibition curves (using a pNPP phosphatase assay). Further investigations into the molecular target (SSH1) are currently underway.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Ischaemic heart disease is a major contributor to global morbidity and mortality rates. Manoeuvres such as ischaemic preconditioning confer cardioprotection against ischaemia/reperfusion (I/R) injury by activating several intracellular signalling pathways. These pathways have been defined solely in terms of the kinases involved, despite the realization in recent years that protein phosphatase activity also contributes significantly to the attributes of the propagated signal. Protein phosphatase 2A (PP2A) is a heteromultimeric enzyme involved in an array of phosphatase reactions. We hypothesized that PP2A is an important participant in the myocardial response to I/R by regulating intracellular signalling. This project aimed to (i) characterize PP2A during myocardial I/R; (ii) determine the importance of its contribution to the cellular response to I/R; and (iii) investigate its role in the signalling pathways mediated by PKB/Akt, GSK-3β, ERK p42/p44 and p38 MAPK. Two models were used to characterize PP2A during I/R: (i) H9c2 cells exposed to simulated ischaemia (SI) buffer in conjunction with hypoxia (0.5% O2) for a maximum of 2 hours, followed by reoxygenation in standard growth medium for up to 30 minutes; and (ii) isolated working rat hearts exposed to a maximum of 20 minutes global ischaemia and 10 minutes reperfusion. In both models samples were collected at several time points during I/R for Western blotting analysis. PP2A-C (the catalytic subunit) accumulated in the nucleus during early ischaemia, but later redistributed to the cytosol. At the end of ischaemia there was an elevation of PP2A-C relative to PP2A-A in the unfractionated whole cell preparation concomitant with an increase in the inhibitory phosphorylation of PP2A-C. The impact of PP2A activity was evaluated by either inhibiting PP2A using okadaic acid (OA, 10 nM) or activating it by administering FTY720 (1 μM) in an isolated working rat heart model exposed to either 35 minutes of regional ischaemia (RI) with infarct size (IFS) as primary end-point, or 20 minutes global ischaemia (GI) with functional recovery as end-point. The results showed that the pre-ischaemic administration of OA or FTY720 reduced or exacerbated IFS respectively, indicating that PP2A activation during I/R favours cell death. OA and FTY720 were also employed to assess the contribution of PP2A to intracellular signalling in an isolated working rat heart exposed to I/R. Samples were collected at several timepoints and analyzed using Western Blotting. Pre-ischaemic administration of OA enhanced the phosphorylation of PKB/Akt, ERK p42/p44 and GSK-3β at the onset of reperfusion, while FTY720 given before ischaemia reduced the phosphorylation of GSK-3β, p38 MAPK and PKB/Akt at the end of ischaemia and onset of reperfusion. In summary, PP2A is part of an early nuclear-based response to ischaemia, while long-term ischaemia induces an increase in PP2A-C. A portion of this PP2A-C is stored in an inactive form, while an active portion acts as a regulator of the pro-survival signalling components PKB/Akt, GSK- 3β and ERK p42/p44 at the end of ischaemia and the onset of reperfusion. PP2A is therefore an important component of the myocardial response to I/R by regulating pro-survival signalling. / AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die belangrikste komponente wat bydra tot globale morbiditeit en mortaliteit. Ingrepe soos iskemiese prekondisionering aktiveer veelvoudige intrasellulêre seintransduksiepaaie om kardiobeskerming teen iskemie/herperfusie (I/H)-besering te ontlok. Die kinases betrokke in hierdie seintransduksiepaaie is reeds deeglik nagevors, terwyl die potensiële belang van die proteïenfosfatases in seintransduksie tot onlangs misken is. Ons hipotese was dat Proteïenfosfatase 2A (PP2A), wat in ‘n wye verskeidenheid fosfatase reaksies betrokke is, ‘n belangrike rolspeler in die miokardiale reaksie op I/H-besering is, deur deelname aan die regulering van intrasellulêre seintransduksie. Hierdie projek het ten doel gehad om (i) PP2A te karakteriseer tydens miokardiale I/H; (ii) die belang van PP2A in die sellulêre reaksie op I/H-besering te bepaal; en (iii) PP2A se rol in die seintransduksiepaaie, gemedieer deur PKB/Akt, GSK-3β, ERK p42/p44 en p38 MAPK, te evalueer. Twee modelle is aangewend om PP2A tydens I/H te karakteriseer: (i) H9c2-selle blootgestel aan ‘n simuleerde iskemiebuffer tesame met hipoksie (0.5% O2) vir ‘n maksimum van 2 uur gevolg deur heroksiginasie in standaardgroeimedium vir verskillende tydsperiodes tot ‘n maksimum van 30 minute; en (ii) geïsoleerde, werkende rotharte blootgestel aan ‘n maksimum van 20 minute globale iskemie en 10 minute herperfusie. In beide modelle is monsters op verskillende tye versamel vir Western-kladanalise. Tydens vroeë iskemie het PP2A-C in die kern toegeneem, waarna dit met verloop van tyd na die sitosol herversprei het. Teen die einde van iskemie was daar ‘n toename in die vlakke van PP2A-C relatief tot PP2A-A in ongefraksioneerde weefselhomogenate, tesame met ‘n toename in die inhibitoriese fosforilering van PP2A-C. Die belang van PP2A-aktiwiteit is ondersoek deur die effek te bepaal van die inhibisie of aktivering daarvan op infarktgrootte (IFS) en funksionele herstel in ‘n geïsoleerde werkende rothartmodel, blootgestel aan onderskeidelik 35 minute streeksiskemie (RI) of 20 minute globale iskemie. Preiskemiese toediening van die PP2A-inhibitor okadaïensuur (OA, 10 nM), of aktiveerder FTY720 (1 μm) het infarktgrootte respektiewelik beperk of vergroot. PP2A-aktivering tydens I/H is dus nadelig. OA en FTY720 is ook aangewend om die bydrae van PP2A tot I/H-verwante, intrasellulêre seintransduksie in die geïsoleerde, werkende rothart te bepaal. Monsters is op verskeie tydintervalle versamel en ontleed deur gebruik te maak van die Western-kladtegniek. Preiskemiese toediening van OA het die fosforilering van PKB/Akt, ERK p42/p44 en GSK-3β by die aanvang van herperfusie bevoordeel, terwyl pre-iskemiese toediening van FTY720, die fosforilering van GSK-3β, p38 MAPK en PKB/Akt aan die einde van iskemie en die begin van herperfusie verminder het. Ter opsomming: PP2A is deel van ‘n vroeë gelokaliseerde kerngebaseerde reaksie op iskemie, terwyl langdurige iskemie ‘n toename in PP2A-C relatief tot PP2A-A induseer. ‘n Deel van hierdie PP2A-C is onaktief, terwyl die res funksioneer in die regulering van die seintransduksiekomponente PKB/Akt, GSK-3β en ERK p42/p44 wat oorlewing fasiliteer met die aanvang van herperfusie. PP2A is dus ‘n belangrike komponent in die miokardiale reaksie op I/H deurdat dit tot die beheer van seintransduksiepaaie bydra.

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