Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...

The ability of proteins to bind other molecules in response to various stimuli in their microenvironment serves as a platform for extensive regulatory networks coordinating downstream cell actions. The correct function of these signaling pathways depends mostly on noncovalent interactions often affecting the structure of proteins and protein complexes. Understanding the molecular mechanism of a protein function in cell signaling therefore often depends on our knowledge of a three-dimensional structure. In this doctoral thesis, I present the work that led to the understanding of several protein-protein and protein-ligand interactions implicated in cell signaling at the molecular level. I applied nuclear magnetic resonance spectroscopy, small angle X-ray scattering and other biophysical methods to determine the molecular basis of inhibition of four signaling proteins: Calcium/Calmodulin (Ca2+ /CaM)-dependent protein kinase kinase 2 (CaMKK2); protease Caspase-2; Forkhead transcription factor FOXO3, and Apoptosis signal-regulating protein kinase 1 (ASK1). In particular, I investigated the distinct roles of 14-3-3 and Ca2+ /CaM in the regulation of CaMKK2 activity. I also studied in detail the mechanism how 14-3-3 interferes with the caspase-2 oligomerization and its nuclear localization as well as...

Gegenstand dieser Arbeit sind systematische Untersuchungen zu den Anwendungsmöglichkeiten der am Institut für Physikalische Chemie entwickelten IC-Kalorimeter im Bereich flüssiger Reaktionssysteme, mit Schwerpunkt auf enzymkatalysierten Reaktionen. Dazu kamen IC-Durchfluss- und IC-Batch-Kalorimeter zum Einsatz, mit denen durch Zusammenführung zweier Lösungen initiierte Mischungs- und Reaktionsprozesse untersucht werden können. Anhand einer Vielzahl untersuchter Reaktionssysteme wird unter stofflichen und methodischen Gesichtspunkten aufgezeigt, unter welchen Bedingungen ein Einsatz der IC-Kalorimeter sinnvoll erscheint. Außerdem werden für die miniaturisierten kalorimetrischen Anordnungen spezifische Aspekte diskutiert; u. a. Probleme bei der elektrischen Kalibrierung und die Nachweisgrenzen bzgl. der bei einem zu detektierenden Prozess mindestens zu generierenden Wärme bzw. Wärmeleistung. Beide IC-Kalorimeter konnten durch konstruktive Veränderungen gegenüber vorhandenen Anordnungen in ihrer Leistungsfähigkeit verbessert werden.

Influenza is an infectious disease caused by the influenza virus. This virus causes a severe viral infection that spreads easily from person to person in yearly pandemics. Vaccination is the most effective way to prevent the infection, however, due to the high rate in mutations of the virus, the vaccine needs to be often reformulated. Another option to combat influenza is based on administration of antiviral drugs. Clinical studies of isolated influenza strains ("avian flu" H5N1, 2004; "swine flu" H1N1, 2009) revealed resistance towards known influenza neuraminidase inhibitors (zanamivir, oseltamivir). The resistance is caused by structural changes close to the enzymatic site. This calls for the development of new neuraminidase inhibitors as well for development of inhibitors targeting different influenza enzymes. This Thesis is focused on design and synthesis of new inhibitors of influenza neuraminidase and RNA-dependent RNA polymerase, namely PA subunit and the assembly of PA-PB1 heterodimer enzymes (Scheme 1). Influenza neuraminidase inhibitors were prepared by C-5 derivatization of oseltamivir followed by subsequent extension of its structure with binders of 150-cavity. Binding potencies of new oseltamivir derivatives against two influenza strains were determined. The next part contributed to...

<p> I viskosprocessens inledande merceriseringssteg behandlas cellulosa (dissolvingmassa) med natriumhydroxid (NaOH) varvid cellulosan omvandlas till alkalicellulosa. Därpå följer en sulfidering med koldisulfid (CS₂) som omvandlar alkalicellulosa till natriumxantogenat. Xantatet löses i en alkalisk lösning och en trögflytande vätska, viskos, bildas. Vid tillverkning av spinnviskos är sista steget i processen en surgörning där koldisulfiden spjälkas av och cellulosan återbildas i form av en tråd. Vid tillverkning av högviskös viskos (Freudenberg HP) sker regenereringen i basisk miljö men vid förhöjd temperatur (100°C), och återbildningen av cellulosa ger då en cellulosabaserad bädd. Genom att öka dissolvingmassans reaktivitet skulle förbrukningen av koldisulfid i sulfideringssteget kunna minskas. Med en ökad reaktivitet menas att fler hydroxylgrupper på cellulosan blir tillgängliga för vidare reaktioner med natriumhydroxid och koldisulfid. Detta skulle kunna möjliggöras med en enzymatisk förbehandling av massan.</p><p>Det första delmålet i projektet var att producera en viskos med hög viskositet i laboratorieskala. Projektets andra mål var att undersöka effekterna av en enzymatisk behandling, med enzymet Carezyme^®, på massans viskositet och reaktivitet.</p><p>En studie gjordes för att se hur olika tider i viskosprocessens andra steg, pressteget, påverkade cellulosahalten och luthalten för alkalicellulosa. Resultaten tydde på att en längre presstid gav en högre cellulosahalt upp till en viss tidpunkt. Vid 195 sekunder avklingade kurvan och effekten av en längre presstid minskade. Resultatet visade också på att mängden lut i alkalicellulosaprovet var relativt konstant och att luthalten inte påverkas nämnvärt av pressningen.</p><p>Vidare genomfördes försök kring viskosprocessens sulfideringssteg. Det fanns under projektets gång stora svårigheter i att uppnå samma höga nivå på gammatalet vid produktion av viskos i laborativ skala (52-58 %) som vid produktion i fabriksskala (68-70 %). Gammatalet är ett mått på hur väl koldisulfid har reagerat med cellulosa under sulfideringen. I ett försök att öka gammatalet satsades en större mängd koldisulfid, med förhoppningen att kunna kompensera för den relativt stora andel koldisulfid som befann sig i gasfas under reaktionen och som därmed inte var aktiv under sulfideringen. Den ökade mängden koldisulfid resulterade dock inte i en ökning av gammatalet. I ett annat försök tillsattes en svag natriumhydroxidlösning direkt till sulfideringskärlet vid avslutad reaktion, utan att någon effekt på gammatalet kunde påvisas. Det undersöktes även om ett ökat förhållande mellan luthalt och cellulosahalt i alkalicellulosan kunde ge någon positiv effekt på gammatalet. Denna förändring gav dock inget ökat gammatal.   </p><p>Dissolvingmassaprover behandlades med enzymet Carezyme^® för att kunna studera dess inverkan på massans reaktivitet och viskositet.  Resultaten visade på en tydlig nedgång i viskositet med högre koncentrationer av enzym. Reaktiviteten på den enzymbehandlade massan ökade i jämförelse med den obehandlade massan. Då viskos producerades med en enzymbehandlad massa kunde ingen effekt av enzymbehandlingen noteras, med avseende på gammatalet.</p><p> </p> / <p>In the first step of the conventional viscose process, called mercerization, cellulose (dissolving pulp) is treated with sodium hydroxide (NaOH) in which the cellulose is converted to alkali cellulose. Alkali cellulose is then treated with carbon disulphide (CS₂) to be converted into a sodium xanthate.  This xanthate is dissolved in an alkali solution and a viscous liquid, called viscose, is formed. The last step in the process is an acidification where the carbon disulphide is splinted off and the cellulose is regenerated in the shape of threads. When producing high viscosity viscose (Freudenberg HP) the regeneration takes place in an alkaline environment and the re-formation of cellulose gives a cellulosic based bed. By increasing the reactivity of the dissolving pulp the amount of carbon disulphide could be reduced, compared at the same degree of substitution. An increase in reactivity means that a larger amount of hydroxyl groups on the cellulose molecule are available to react with sodium hydroxide and carbon disulphide. This could be enabled by an enzymatic pretreatment of the pulp prior to the mercerization step.</p><p>The first aim of this project was to produce a high viscosity viscose in a laboratory scale, comparable to the viscose quality that is produced by Freudenberg HP. The second aim of the project was to investigate the effects of an enzymatic treatment (with the enzyme named Carezyme^®) on the viscosity and reactivity of the dissolving pulp. </p><p>A study was made to examine the influence of the time in the pressing step (after the mercerization) on the cellulose and sodium hydroxide content in the alkali cellulose. The results indicated a linear correlation between the cellulose content and the pressing time up to 195 seconds. At this point the correlation declined and the effects of a longer pressing time decreased. The results also showed that the amount of lye in the alkali cellulose sample was nearly constant and therefore not effected by the pressing time.</p><p>Tests were also carried out concerning the sulphidation step in the process. During the whole project there were difficulties in reaching the same gamma value of the viscose in a laboratory scale (52-58 %) compared to large-scale production (68 - 70%). The gamma value is a measurement of the degree of substitution for carbon disulphide on the cellulose backbone. In one attempt to enhance the gamma value the carbon disulphide charge was increased. The expectation was to compensate for the relatively high amounts of inactive carbon disulphide expected to be found in the gaseous phase in the reactor. However, this did not result in a higher gamma value. In another experiment a weak solution of sodium hydroxide was added directly to the sulphidation vessel after the reaction was completed, but no change in the gamma value was obtained. It was also investigated if an increased relation between the cellulose- and sodium hydroxide content in the alkali cellulose could affect the gamma value positively.  Unfortunately, this modification did not give an increased gamma value.</p><p>Samples of dissolving pulp were treated with the enzyme Carezyme^® to see its impact on viscosity and reactivity of the pulp. The results showed a distinct loss in viscosity with an increased enzyme concentration. The reactivity of the pulp increased compared to the untreated pulp. No effects of the enzymatic treatment could be seen on the final viscose when it was produced from an enzyme treated pulp.</p>

Zugl.: Berlin, Techn. Univ., Diss., 2008

Zugl.: Hohenheim, Univ., Diss., 2008

Context: Beta-site alpha-amyloid protein cleaving enzyme1 (BACE1) plays a key role in the pathogenesis of Alzheimer’s disease. Additional to its moderate expression in the brain, high levels of BACE1 mRNA were found in the pancreas. Murine Bace1 has been immunohistochemicaly detected at the apical pole of acinar cells within the exocrine pancreas of mice and Bace1 activity was observed in pancreatic juice. In vitro experiments revealed enteropeptidase as a putative substrate for Bace1 suggesting a role in acute pancreatitis.

Regioselektive Halogenasen sind Enzyme, die für die Biosynthese verschiedener halogenierter Metaboliten verantwortlich sind. Der Stamm Streptomyces rugosporus produziert die bioaktive halogenierte Verbindung Pyrroindomycin B. In dieser Arbeit wurde ein Gen einer Tryptophan-5-Halogenase aus dem Pyrroindomycinproduzenten Streptomyces rugosporus isoliert. Durch gezielte Mutation des Gens konnte die Beteiligung der Halogenase an der Biosynthese von Pyrroindomycin B nachgewiesen werden. Das Tryptophan-5-Halogenase-Gen wurde heterolog exprimiert. In einem spezifischen Enzymtest konnte die Aktivität der Tryptophan-5-Halogenase gezeigt werden. Tryptophan wurde im Enzymtest durch dieses Enzym monobromiert und monochloriert. Das Protein konnte über Nickelaffinitäts-Chromatographie gereinigt werden. / Regioselectively acting halogenases are enzymes which are responsible for the biosynthesis of a large variety of halogenated secondary metabolites. In many cases the biological activity of these metabolites is influenced by the halogen substituents. Isolation and characterization of the genes of specific halogenases and understanding the biochemistry of these enzymes are prerequisites for genetic engineering aiming at the production of novel halogenated compounds by combinatorial biosynthesis. In the work presented here a halogenase with novel regioselectivity was isolated. It is one of only three specific halogenases for which the enzymatic activity and the involvement in the biosynthesis of a halogenated metabolite could be shown. The availability of this specific tryptophan 5-halogenase is expected to open up novel possibilities for combinatorial biosynthesis and the enzymatic production of halogenated compounds.

Agar is a marine heteropolysaccharide with repeating units consisting of 3,6-α-anhydro-L-galactopyranose and D-galactopyranose linked by α-(1,3) and β-(1,4) linkages. It has been promoted as a prospective replacement for petroleum-based feedstocks and other applications. Enzymatic biotransformation of agar generates high specific products: It is also more environmentally friendly than chemical hydrolysis. In particular, agarolytic bacteria and their agarases are preferred for the processing of agar into sugar derivatives. Agar-producing macroalgae are one of Indonesia's national commodities. However, agar-based products and technology are rarely developed in Indonesia. This research is aimed to explore the potential of an Indonesian marine bacterium and its agarases as bioagents for agar bioprocessing. The research objectives are to identify the novelty of the isolate among known agarolytic bacteria using microbiology and molecular biology approaches, to elucidate the agarolytic system of the bacterium using in silico genome analysis, to express and characterize the recombinant agarases, and to elucidate their potential for producing agar-derived saccharides from Indonesian natural agar. Microbulbifer elongatus PORT2 is a gram-negative marine bacterium that had been isolated from Batu Karas seawater, Pangandaran, West Java Indonesia. PORT2 shows potential as biocatalysts for agar saccharides conversion by showing remarkable agar liquefaction. The annotation of the draft genome identifies six putative β-agarases consist of three GH50, two GH86, and one GH16 in M. elongatus PORT2. Those agarases are clustered at two different contigs. Besides agarases, other genes for D-galactose and 3,6 anhydro-L galactose metabolism, sugar transports and regulatory system are found in the vicinity of the agarases clusters. Despite the ability to utilize agar as a sole carbon sole, PORT2 lacks any putative α-agarase GH117 or GH96. Both are responsible for the cleavage of α-glycosidic bonds in agar. Indeed, several hypothetical proteins are in the neighborhood of the agarase gene clusters in M. elongatus PORT2. They probably could have a function as the alternative machinery or pathway for agar monomerization that needs clarification in future research work. Four recombinant β-agarases from PORT2; AgaA50, AgaB50, AgaC50, and AgaF16A have been successfully overexpressed in E.coli and characterized. The AgaA50 and AgaC50 exhibit metal-dependent activity. They perform exo-agarolytic modes and generates neoagarobiose (NA2). The AgaB50 can act as endo-and exo-β-agarase without any additional activator and produces neoagarohexaose (NA6), neoagarotetraose (NA4), and NA2. AgaF16 produces NA6 and NA4. The enzyme shows pure endo-catalytic action which thiol agents positively affect its activity. The synergetic reaction of AgaF16A and AgaA50 converts Indonesian Gelidium agar into NA2 and Gracilaria agar into modified NA2. The modified NA2 from Gracilaria agar could promise new potential bioactivity that is different from agarose-derived NA2 due to the presence of additional side chains on the saccharide backbone. The NA6, NA4, and NA2 products from agarose have shown potential pharmaceutical applications such as immunomodulator, anti-tumor, antioxidant, anti-diabetic, and moisturizer. Despite being isolated from a mesophilic marine bacterium, the recombinant agarases from M. elongatus PORT2 are active at 50 °C and pH between 6.5 to 8. They maintain more than 75% of their activities even after 1 h preincubation at 50 °C, except for AgaC50. Their thermostability gives advantages for the effective biocatalytic conversion of agar because the substrate is more accessible at mild pH and the temperature above the sol-gel condition (> 40 °C).:Contents 1. Introduction 1 1.1. Motivation and Scientific Goals 1 1.2. Literature Review 3 2. Materials and Methods 12 2.1. Materials 12 2.2. Methods 13 3. Agarolytic Bacterium Microbulbifer elongatus PORT2 22 3.1. Results 22 3.2. Discussion 28 4. Genome Profiling for In Silico Elucidation of the Agarolytic System 32 4.1. Results 32 4.2. Discussion 41 5. Recombinant Agarases from Microbulbifer elongatus PORT2 44 5.1. Results 44 5.2. Discussion 71 6. Conclusions and Outlooks 78 References 81 Appendices 97 Acknowledgements 110