Le p300/CBP acétyltransférase est un co-activateur transcriptionnel très important qui est impliqué dans la régulation d'un grand nombre de processus biologiques, comme la transcription d'ADN, le développement et l'immunité innée. Jusqu'à présent, le rôle de p300/CBP dans la régulation de l'expression des gènes a été largement étudiée, mais les mécanismes qui régulent son activité enzymatique sont encore peu connus. Des études ont montré que le dysfonctionnement de p300/CBP est associé à plusieurs formes de cancer et de maladies neurodégénératives. Dés lors, chaque progrès concernant les mécanismes de régulation de p300/CBP est devenu primordial pour le développement de nouvelles thérapies. Le 'noyau' de p300/CBP contient deux domaines pour la reconnaissance des modifications post-traductionnelles (MPTs), un bromodomaine et un PHD finger (le module BP), adjacent à un domaine HAT (ou domaine histone acétyltransférase). Plusieurs enzymes, modifiant la chromatine, contiennent des domaines de reconnaissance des MPTs. Fréquemment des groupements particuliers de ces domaines sont très conservés et liés, au sein de la même protéine ou du même complexe protéique, suggérant qu'ils réalisent des fonctions coordonnées. Ces domaines adjacents peuvent agir en concertation dans la reconnaissance simultanée de différents MPTs ou peuvent exercer des fonctions différentes de celles qui sont effectuées par ces deux domaines particuliers, tels que les fonctions de régulation enzymatique. Plusieurs études suggèrent que les cycles acétylation/désacétylation dans la boucle d'auto-inhibition, à l'intérieur du domaine HAT, jouent un rôle important dans la régulation de l'activité enzymatique de p300/CBP. La proximité du module BP et du domaine HAT suggère que la spécificité de liaison, appartenant au module BP, peut être intrinsèquement liée à la régulation de l'activité du domaine HAT. L'objectif de ma thèse est de déterminer le rôle du module BP dans la régulation de l'activité du domaine HAT. Je propose que le module BP soit impliqué dans la régulation de p300/CBP de deux façons. La première consiste à établir un lien avec le domaine HAT qui stabilise la conformation auto-inhibée de l'enzyme. La deuxième exige que le module BP joue un rôle dans le choix des substrats de p300/CBP. J'ai été en mesure de montrer que BP peut se lier au domaine HAT et à la chromatine modifiée et qu'il peut reconnaître les modifications effectuées par p300/CBP lui-même. Les données obtenues indiquent que le module BP peut être impliqué dans la régulation de l'activité de p300/CBP et dans son ciblage à la chromatine. / The p300/CBP acetyltransferase is an important transcriptional co-activator which is involved in regulating a wide range of biological processes, such as DNA transcription, development and innate immunity. To date, the role of p300/CBP in gene regulation has been extensively described but little is known about the mechanisms which regulate its activity. Since misregulation of p300/CBP has been associated to the development of several forms of cancers and neurodegenerative diseases, studies directed to decipher the mechanisms of regulation of p300/CBP are of great importance for the development of new therapies. The p300/CBP 'core' contains two post-translational modifications (PTMs) recognition motifs, a bromodomain and a PHD domain (the bromo-PHD module, BP), in close proximity to a histone acetyltransferase domain (HAT). Many chromatin modifying enzymes contain recognition modules for PTMs. Frequently particular groupings of such modules are conserved and linked within the same protein or the same multisubunit complex, suggesting that they perform concerted functions. These linked modules may act combinatorially to allow recognition of multiple PTMs or display new functions that are not possessed by the single modules, such as regulatory properties. Accumulating evidence suggests that acetylation/deacetylation in a conserved autoinhibitory loop of the p300/CBP HAT domain plays an important role in regulation of HAT activity. The close apposition of the BP module and the HAT domain suggests that BP substrate recognition is intrinsically linked to regulation of HAT activity. During my thesis work, I have investigated the role of BP in HAT regulation. I propose that the BP module is involved in p300/CBP regulation by binding to the HAT domain and stabilizing the autoinhibited conformation of the enzyme. I have also investigated substrate specificity of the BP module towards modified chromatin. I could show that the BP module binds histone modifications including those that are p300/CBP dependent. Altogether, the data suggests that the BP module is involved in regulating p300/CBP HAT activity and in targeting of chromatin.

Abstract In the cells, the last step of the beta-oxidation cycle, aiming at the degradation of fatty acids, is catalyzed by the enzyme named thiolase. It shortens the acyl chain of the acyl-CoA by two carbons. The reaction is reversible, it can proceed for both directions. Thiolases are divided into two categories, synthetic and degradative ones. These two classes of thiolases differ not only by their biological function, but also by their substrate specificity. Degradative thiolases accept substrates with various lengths but synthetic thiolases only accept short chain-acyl-CoAs as a substrate. In humans, at least six isozymes of thiolases are found. The mitochondrial biosynthetic thiolase, T2, differs from other thiolases by getting activated by potassium. In addition, it accepts branched acyl-CoA, namely 2-methyl-acetoacetyl-CoA, as a substrate. This molecule is an important reaction intermediate in the degradation of the amino acid isoleucine. Many human patients have been diagnosed to have a mutation in the gene of T2, and they are treated with a special diet. The results of this theses show that potassium ion rigidifies the groups of the T2 protein involved in the substrate binding. The presence of potassium increases the reaction rate and it also raises the affinity towards some of the substrates. The enzyme mechanistic studies with bacterial thiolase revealed that the oxyanion hole 1, formed by a water molecule and histidine side chain, is important for the synthetic reaction, not so much for the degradative direction. Binding studies showed that both the terminal sulfur of the substrate and the sulfur of the catalytic cysteine are important for the right positioning of the substrate. The electrostatics of the active site also have a significant role in the catalysis. These studies give a good basis for future studies aiming at drug development against this enzyme in pathogenic species.

Die Acetyltransferase TIP60/KAT5 steuert die circadiane Uhr in Drosophila Endogene Uhren steuern verhaltensbezogene und physiologische Prozesse in einer rhythmischen Weise, die eine Periodizität von ~24 h aufweist. Drosophila zeigt einen robusten Rhythmus von Aktivitäts- /Ruhephasen, der von im Gehirn befindlichen zirkadianen Schrittmacherneuronen gesteuert wird. Angeborene Rhythmen der Genexpression werden durch positive und negative transkriptionelle/translationale Rückkopplungsschleifen, die die circadiane Uhr antreiben, generiert. Obwohl die Regulierung der Rückkopplung von Uhrengenen relativ gut verstanden ist, sind die Faktoren, die die Aktivatoren stimulieren, einen neuen circadianen Zyklus zu beginnen, noch unbekannt. Posttranslationale Modifikationen spezifischer Proteine durch Acetylierung erleichtert die Gentranskription. TIP60/KAT5 ist ein Mitglied der hochkonservierten MYST-Familie von Acetyltransferasen und ist an einer Vielzahl zellulärer Prozesse beteiligt, wie beispielsweise Zellwachstum und DNA-Reparatur, undzwar indem es verschiedene Zielproteine acetyliert. Das Hauptziel dieser Arbeit ist es, die Mechanismen, durch die Lysin-Acetyltransferase TIP60/KAT5 die circadiane Uhr von Drosophila steuert, zu untersuchen. Das binäre UAS/GAL4 System wurde eingesetzt, um einen gezielten knock down (durch RNA-Interferenz) oder eine Überexpression (eine Mutation in der für die Katalyse essentiellen Aminosäure, wodurch eine dominant negative Funktion verursacht wird) von TIP60/KAT5 in zirkadianen Schrittmacherneuronen zu erreichen. Das temperatursensitive GAL80 (TARGET) System wurde verwendet, um die normale Entwicklung der Schrittmacherneuronen bis ins Erwachsenenstadium zu ermöglichen. Unter Verwendung von nicht-invasiven lokomotorischen Aktivitätstests konnte gezeigt werden, dass TIP60 in Schrittmacherneuronen für den robusten zirkadianen Rhythmus in Abwesenheit von Zeitgebern im adulten Stadium erforderlich ist. In Übereinstimmung mit den beobachteten Veränderungen des circadianen Bewegungsverhaltens, war die rhythmische Expression von wichtigen clock-controlled genes wie Period und Timeless sowie Genen der Hilfsschleife Vrille und Clockwork orange gedämpft. Im Gegensatz dazu konnte gezeigt werden, dass die Tip60 mRNA unabhängig von der Uhr reguliert wird. Das zirkadiane Bewegungsverhalten wurde genutzt, um zu beurteilen, ob Untereinheiten des TIP60 (Nu4A) multimeren Komplexes auch an der Regulation des Rhythmus beteiligt sind. Um die Zielproteine von TIP60 in der circadianen Uhr zu bestimmen, wurde ein genetischer Interaktions-Test unter Verwendung des Phänotyps der circadianen Lokomotoraktivität durchgeführt und es wurde eine starke Interaktion mit dem zirkadianen Co-Aktivator CYCLE und dem Repressor PERIOD in trans-heterozygoten Kreuzungen gefunden. Aufgrund des Fehlens eines spezifischen Antikörpers, um die Funktion von TIP60 in vivo in adulter Drosophila zu untersuchen, wurde ein transgener Stamm (UAS dTIP60-HA) mit einem Hämagglutinin-Epitop-Tag erzeugt. Dieser transgene Stamm wurde genutzt, um TIP60-HA unter Verwendung eines ubiquitären Promotors (tub GAL4) überzuexprimieren. Ein Antikörper gegen das Epitop (Anti-HA) wurde verwendet, um einen Chromatin-Immunopräzipitations-Assay (ChIP) in diesen transgenen Fliegen durchzuführen. Es zeigte sich, dass TIP60 rhythmisch in der regulatorischen Region von clock-controlledgenes lokalisiert ist, was eine rhythmische Belegung von DNA-Elementen, ähnlich dem CLOCK/CYCLE Komplex, nahelegt. Co-Immunpräzipitation wurde mit transgenen Fliegen durchgeführt, um eine Protein-Protein-Wechselwirkung zwischen TIP60 und CYCLE nachzuweisen. Die Ergebnisse dieser Studie legen deutlich eine Beteiligung von TIP60/KAT5 als Co-Aktivator an der präzisen Steuerung der circadianen Uhr in Drosophila nahe. Deutsche Übersetzung: Dr. Inga Urban

Cholinergic enzymes and muscarinic receptors in nuclei of rat medulla oblongata were examined after unilateral vagotomy to determine their association with efferent vagal neurons. Vagotomy caused an ipsilateral depletion of acetylcholinesterase from the dorsal motor nucleus of the vagus (DNV) and the nucleus ambiguus (NA). Choline acetyltransferase activity was reduced in ipsilateral DNV, nucleus tractus solitarius and rostral NA. Muscarinic receptor localization by autoradiography with [3H]quinuclidinyl benzilate (QNB) revealed marked intranuclear variations in receptor density. Vagotomy had no effect on the QNB binding pattern. Loss of cholinergic enzymes is a consistent response of motor and preganglionic autonomic neurons to axotomy. Depletion of muscarinic receptors is an additional component of axon reaction in brain stem motoneurons. Accordingly, previous studies have shown a decrease in neurotransmitter-related proteins after axotomy of motoneurons. In the present study, cholinergic enzymes were depleted from axotomized vagal neurons but receptors were not. It is concluded that muscarinic receptors in the DNV and NA are not associated with vagal efferent neurons.

Trimethyltin (TMT) destroys specific subfields of the hippocampus in the rat. TMT also increases choline acetyltransferase (ChAT) activity in CA1 of Ammon's horn and the outer molecular layer of the dentate gyrus. This observation suggests that axonal sprouting occurs in the cholinergic septohippocampal system in response to TMT. However, neither does-response nor time course data are available for the effects of TMT on this enzyme. The effects of three dose levels of TMT on ChAT activity in CA1 and the dentate gyrus were determined in Experiment 1 and ChAT activity in these two areas was measured at six time points following exposure to TMT in Experiment 2. Only the highest dose of TMT (6 mg/kg) significantly increased ChAT activity. ChAT activity in the dentate gyrus increased significantly by 3 d after administration and continued to increase until 21 d after exposure. A significant increase was not observed in CA1 until 7 d after exposure to TMT. Asymptotic levels were still reached at d 21. These results indicate a steep dose-response curve for TMT-induced changes in ChAT activity in the hippocampal formation and that this marker of cholinergic activity is more sensitive to perturbation by TMT in the dentate gyrus than Ammon's horn.

Polyamines are ubiquitously present in all living cells. The abnormal metabolism of polyamines that is associated with certain types of cancers has been the focus of several investigations. Enzymes that are involved in the transacetylation of polyamines have been studied extensively, so as to develop inhibitors of these enzymes which may be used as drugs in cancer therapy. Based on indirect evidence, the nuclear spermidine acetyltransferase has been thought to be a critical enzyme that is associated with genetic derepression leading to cancerous growth. In the present study a novel, rapid, sensitive and highly reproducible radio chemical procedure has been developed for assaying spermidine (polyamine) acetylation. The study contains data showing range of linearity of the procedure, percent product recovery, as well as low interference from the unreacted acetyl coenzyme A. Rat liver nuclear spermidine acetyltransferase has been purified using the biochemical procedures annmonium sulfate precipitation, DEAE chromatography, Hydroxyapatite chromatography, Diaminobutyl agarose chromatography and Polyacrylamide P-300 gel filtration. The enzyme obtained at the end of such procedures was found to be essentially homogeneous as seen on native gel electrophoresis. The purified enzyme has been shown to have an isoelectric point of 5.2. Bicine and Hepes were found to be more suitable as buffering species for good enzyme activity. The enzymatic reaction velocity was found to increase with temperature upto 36$\sp\circ$C and was found to increase linearly up to four minutes under non limiting conditions in the presence of 20% glycerol. Using the purified enzyme it has also been established that of the three nuclear polyamines, spermidine is the preferred substrate. The apparent Km for acetyl Co A with spermidine as substrate was found to be about 5 mM. The purified enzyme does acetylate histones. All the substrate analogs containing aminobutylamino group are acetylated by the enzyme.

Indiana University-Purdue University Indianapolis (IUPUI) / Infection with the single-celled parasite Toxoplasma gondii (phylum Apicomplexa) is usually benign in normal healthy individuals, but can cause congenital birth defects, ocular disease, and also life-threatening infection in immunocompromised patients. Acute infection caused by tachyzoites is controlled by a healthy immune response, but the parasite differentiates into a latent cyst form (bradyzoite) leading to permanent infection and chronic disease. Current therapies are effective only against tachyzoites, are highly toxic to the patient, and do not eradicate the encysted bradyzoites, thus highlighting the need for novel therapeutics. Inhibitors of histone deacetylases have been shown to reduce parasite viability in vitro demonstrating that chromatin remodeling enzymes, key mediators in epigenetic regulation, might serve as potential drug targets. Furthermore, epigenetic regulation has been shown to contribute to gene expression and differentiation in Toxoplasma. This dissertation focused on investigating the physiological role of a Toxoplasma GCN5-family histone acetyltransferase (HAT), termed TgGCN5-B. It was hypothesized that TgGCN5-B is an essential HAT that resides within a unique, multi-subunit complex in the parasite nucleus. Studies of TgGCN5-B have revealed that this HAT possesses a unique nuclear localization signal (311RPAENKKRGR320) that is both necessary and sufficient to translocate the protein to the parasite nucleus. Although no other protein motifs have been identified in the N-terminal extension of TgGCN5-B, it is likely that this extension plays a role in protein-protein interactions. All GCN5 homologues function within large multi-subunit complexes, many being conserved among species, but bioinformatic analysis of the Toxoplasma genome revealed a lack of many of these conserved components. Biochemical studies identified several potential TgGCN5-B associating proteins, including several novel apicomplexan transcription factors. Preliminary evidence suggested that TgGCN5-B was essential for tachyzoites; therefore, a dominant-negative approach was utilized to examine the role of TgGCN5-B in the physiology of Toxoplasma. When catalytically inactive TgGCN5-B protein was over-expressed in the parasites, there was a significant decrease in tachyzoite growth and viability, with initial observations suggesting defects in nuclear division and daughter cell budding. These results demonstrate that TgGCN5-B is important for tachyzoite development and indicate that therapeutic targeting of this HAT could be a novel approach to treat toxoplasmosis.

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No description available.

This thesis describes the design, synthesis, analysis, mechanistic evaluation and optimisation of small molecule probes for the specific detection of proteins, focusing on the target protein human arylamine <i>N</i>-acetyltransferase type 1 (HUMAN(NAT1)) and its murine homologue, mouse arylamine <i>N</i>-acetyltransferase type 2 (MOUSE(NAT2)). The HUMAN(NAT1) gene is reported to be one of the most highly overexpressed genes in estrogen-receptor-positive (ER+) breast tumours, leading to its potential use as both a novel diagnostic biomarker and a novel therapeutic target for this disease. <strong>Chapter 1</strong> reviews the literature on optical methods for the specific detection of a protein target, exploring strategies both based on biosensors and on chemical probes, before introducing the arylamine <i>N</i>-acetyltransferases as a family of enzymes. In <strong>Chapter 2</strong>, a family of naphthoquinone inhibitors of HUMAN(NAT1) are introduced, which undergo a colour change from red to blue upon binding specifically to the enzyme. The mechanism of this colour change, a proton transfer-mediated process, is discussed via the synthesis, pharmacological and colorimetric evaluation of close analogues of the hit compound lacking a key acidic sulfonamide-N<i>H</i> proton. During these studies, it was found that direct <i>O</i>-methylation of a sulfonamide is possible under certain conditions; such a reaction has not previously been reported. Furthermore, upon heating in polar solvents the <i>O</i>-methylated sulfonamide was observed to undergo rearrangement, and the mechanism of this process is investigated via NMR and kinetic studies. In <strong>Chapter 3</strong>, the design, synthesis and evaluation of HUMAN(NAT1) inhibitors with improved pharmacological and colorimetric profiles over the initial hit are described. From this optimisation, structure-activity relationships and an in silico model of interactions between the inhibitors and enzyme are evaluated. Testing of these compounds in cellular environments, however, exposes some limitations of this approach, notably the lack of sensitivity of the probes when dosed at low concentrations in cellular samples. In order to overcome this limitation, in <strong>Chapter 4</strong> fluorescent analogues of the hit compound are designed and synthesised. Initial compounds developed in this series possess promising properties, but each compound generated suffers from either a low fluorescent intensity, lack of a <i>p</i>H-dependent switch in fluorescence or a low fluorescence excitation wavelength, which overlaps with those of tryptophan or tyrosine residues in proteins. Insights into the mechanism of molecular fluorescence and application of some simple quantum mechanical principles, however, lead to the design of a species which possesses all the required properties. The fluorescent emission intensity of this probe correlates linearly with [MOUSE(NAT2)] in E. coli cell extracts, and can quantify as little as 0.64% MOUSE(NAT2) in the samples; furthermore, the probe is capable of unambiguously detecting HUMAN(NAT1) within a cell extract from the ER+ breast cancer cell line ZR-75-1; future work on this probe may therefore enable its clinical use in improved early diagnosis of breast tumours. This study also represents, to the best of our knowledge, the first ever example of a small molecule, non-covalent probe capable of quantifying the concentration of a target protein in cellular extracts. In <strong>Chapter 5</strong>, the series of naphthoquinone probes is further optimised in order to study the roles of HUMAN(NAT1) in a cellular environment. Firstly, structure-activity relationships are utilised to design inhibitors with improved physical properties such as aqueous solubility and cell membrane permeability, in order to test the effect of HUMAN(NAT1) inhibitors in tumour cell models, which could have implications for the future use of a HUMAN(NAT1) inhibitor as a therapeutic agent in oncology. Secondly, the effect of the cofactor folic acid on the function and activity of HUMAN(NAT1) is explored. Finally, in <strong>Chapter 6</strong>, the conclusions of this study are outlined and a hypothesis as to how the concepts developed in this thesis might be applied to alternative, more ubiquitous biological targets is discussed, paving the way for future investigations.