Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Archiv

The thesis is addressing different aspects of the chemistry of silicate related nitridic compounds. One main focus was the investigation of the influence of substitution (cations, O/N, Si/Al, halides) on the material properties of the compounds. For this concern known compounds (nitridosilicates, sions, sialons) as well as a new class of compounds (oxonitridosilicate halides) were investigated. Very important for these investigations was the detailed characterisation of the synthesised compounds. In this context the establishment of an especially optimised microprobe technique has to be highlighted. The new technique allows the quantitative analyses of light elements as O, N, Cl and Si, Al, Br beside elements like Sr, Ba and the lanthanides. In addition a lot of different analytical tools were used to understand the crystal chemistry of the investigated systems, e.g. single-crystal x-ray analyses, powder neutron diffraction, XAS, EDX and spectroscopic methods, some of them also performed under high pressure. The second main focus of the work was the synthesis and characterisation of compounds with new crystal structures in the area of oxonitridosilicates, sions and sialons.

In Escherichia coli, the cylindrical chaperonin GroEL and its cofactor GroES promote the folding of a fraction of newly synthesized polypeptide chains by acting as an Anfinsen cage. GroEL recognizes substrate proteins with its apical domains of the tetradecameric structure. Exposed hydrophobic side chains in non-native proteins interact with GroEL and bound substrates are subsequently encapsulated under the GroES lid, where they can fold in a protected environment. Despite the detailed knowledge about structural and mechanistic features of GroEL and GroES, little is known about its genuine in vivo substrate proteins. Here, the nearly complete set of GroEL interacting proteins in vivo was identified and quantified by an approach using affinity chromatography for the isolation of GroEL/GroES/substrate complexes and subsequent analysis by mass spectrometric methods. GroEL substrate proteins were analyzed with respect to their fold types and functional classes, revealing a preference for proteins which fold into the versatile TIM barrel fold to interact with GroEL. Further in vivo and in vitro experiments with individual proteins identified as GroEL substrates verified the data obtained by the proteomic approach and allowed conclusions on the usage of the other main chaperone system in E. coli: DnaK/DnaJ/GrpE. Taken together, the results culminated in the classification of GroEL interacting proteins according to their dependence on chaperones for folding. Class I proteins are largely independent of chaperones but their folding yield can be increased by chaperone interaction. Class II proteins do not refold efficiently in the absence of chaperones in vitro, but can utilize either the DnaK or the GroEL/GroES systems for folding. Class III substrates are fully dependent on GroEL. DnaK can bind class III proteins and thus prevent their aggregation, but folding is achieved only upon transfer to GroEL.

Die Triomzell-Vakzinierung ist ein neuer, überaus potenter Ansatz in der Immuntherapie maligner Lymphome. Aus A20-Lymphomzellen der Maus wurde durch Fusion mit der Ratten-Hybridomzelllinie 2.4G2 die Triomzelllinie BiV hergestellt. Die entstandenen Triomzellen enthalten potenziell alle Antigene der parentalen Lymphomzellen und zusätzlich die anti-Fcγ-Rezeptor-Spezifität des Hybridoms. Die Präsentation der Tumorantigene wird somit durch Redirektion gegen internalisierende Rezeptoren auf Antigen-präsentierenden Zellen (APZ) ermöglicht. Durch Triom-Immunisierung konnte nicht nur ein Langzeit-Tumorschutz in Mäusen vermittelt werden, sondern es wurden sogar etablierte Tumoren abgestoßen. Der Tumorschutz wird dabei über CD4+ und CD8+ T-Zellen vermittelt, die humorale Immunantwort spielt nur eine untergeordnete Rolle. Mehrfach in vitro restimulierte und adoptiv transferierte T-Zellen aus immunisierten Mäusen waren im Gegensatz zu identisch behandelten T-Zellen aus naiven Mäusen tumorprotektiv. Das war insofern erstaunlich, als beide T-Zellpopulationen in vitro den gleichen A20-spezifischen Aktivierungszustand zeigten. Ein eingeschränktes T-Zell-Rezeptor-(TZR-) Repertoire von in vitro restimulierten T-Zellen aus immunisierten Mäusen korrelierte mit dem Tumorschutz. Restimulierte T-Zellen aus naiven Mäusen zeigten hingegen keine Einschränkung im TZR-Repertoire. Durch die Immunisierung der Mäuse erfolgt offensichtlich eine In-vivo-Aktivierung, die sich durch In-vitro-Stimulationen nicht nachstellen lässt.

Introduction: Factors and other stimuli that lead to the release of basic fibroblast growth factor (bFGF) from endothelial cells may be essential for physiological processes such as development and angiogenesis. The release mechanisms are somewhat obscure and it has previously been shown that in the case of shear stress induced bFGF release cell matrix interaction is critically mediating that bFGF release (Gloe et al., 2002). Considering the potential role of proteolytically modified extra-cellular matrix components in the induction of cellular signaling cascades, the aim of the present study was to investigate whether elastase activity contributes to the release of bFGF from endothelial cells. Methods and results: Treatment of porcine aortic endothelial cells with elastase led to a release of bFGF in a concentration-dependent manner. This release was strictly regulated and could be reduced by inhibition of integrin v3. Moreover, bFGF was translocated towards the cell membrane after elastase treatment as well as shear stress exposure, in close proximity to HSP27. Furthermore, elastase treatment led to a p38 MAP Kinase dependent HSP27 phosphorylation and this phospho-HSP27 could be shown to co-precipitate with bFGF. Conclusion: We conclude that elastolytic activities activated by shear stress are involved in the active release of bFGF from endothelial cells and that phosphorylation of HSP27 is prerequisite for this release mechanism. The results may reflect the critical role of proteases in the initial process of angiogenesis induction.

Gene therapy is a very promising approach to treat or to prevent diseases. However, progress in this field is hindered by lack of suitable vectors. Current research focuses on the development of novel nonviral biodegradable gene carriers with improved gene transfer activity and low toxicity. In the course of this thesis, a library of degradable DNA compacting domains based on oligomerized polyamines was synthesized and analyzed. Degradation of the originated polymers was either based on site-specific reductive cleavage of disulfide bonds or on time-dependent ester/amide hydrolysis. DNA binding activity, polyplex stability, transfection efficiency, toxicity, and hemocompatibility studies were performed in order to identify promising candidates. Some of the novel gene carriers, especially the degradable oligoethylenimine (OEI) derivatives were successfully applied for in vitro transfection and could easily compete with the current ‘golden standard’ linear polyethylenimine with an average Mw of 22 kDa (PEI22lin). Furthermore, screening results revealed critical structure activity relationships which were very helpful for improving the polymer design. According to transfection and biocompatibility results, efficiency and toxicity correlated to some degree. Polymers with an overall high charge density and a high molecular weight like OEI-HD-1 provided polyplex stability and formed small uniform particles. On the other hand these polymers tended to induce erythrocyte aggregation and exhibited a pronounced cytotoxicity when applied at high concentrations. Polycation with a lower molecular weight (~ 10 kDa) like e.g. OEI-IP-1 were essentially nontoxic, but had to be applied at high concentrations in order to achieve efficient gene transfer. Intrinsic membrane activity of certain polymers could damage cellular membranes but may also trigger endosomal release and therefore boost transfection activity. Crosslinking of OEI 800 with 1,6-hexanedioldiacrylate resulted in highly efficient degradable polycations. Different reaction temperatures during OEI-HD-1 synthesis had a strong impact on molecular weight and the ester/amide ratio. Despite structural differences, both OEI-HD-1 (synthesized at 60°C) and lt-OEI-HD-1 (synthesized at 20°C) possessed equal gene transfer activity as the ‘golden standard’ PEI22lin when applied at their optimal polymer/DNA-ratio (w/w). It was important to note that lt-OEI-HD-1, the LMW-derivative which is predominantly based on ester linkages, was significantly less toxic than its HMW amide-linked counterpart. OEI-HD displayed a very promising basis for the development of further powerful gene carriers. A two-step synthesis protocol was established in order to generate OEI-HD cores bearing excessive linker which could be subsequently modified with various functionalities like spermine. OEI-HD-Sper pseudo-dendrimers were characterized by a pronounced intrinsic membrane activity and possess high transfection efficiency. Since current nonviral vectors are still very inefficient as compared to their viral competitors, natural viruses present an ideal example educating us how to further optimize polycationic gene carriers in terms of specific cell-targeting and improved endosomal release. Modification of polyplexes towards a “smart” virus-like system was achieved in the following way. Degradable DNA compacting domains (OEI-HD-1) were utilized for complex formation. Furthermore, epidermal growth factor (EGF) was incorporated as targeting ligand into OEI-HD-1 polyplexes and thus allowed cell-specific cellular uptake via the EGF receptor (EGFR). Gene transfer potential of EGFR-targeted degradable polyplexes was further improved by applying technologies which promoted the endosomal release of endocytosed particles. Photochemical intracellular release (PCI) is based on accumulation of amphiphilic photosensitizers (PS) in endosomal membranes. Illumination of PS pre-treated transfected cells results in activation of the PS and subsequent light-induced rupture of endocytic vesicles. Combination of biological (EGFR) and physical (PCI) targeting greatly enhanced reporter gene delivery mediated by OEI-HD-1 polyplexes. Finally, the incorporation of membrane active melittin derivatives into EGF/OEI-HD-1 polyplexes was the first example of a biodegradable synthetic virus for gene delivery.

Prions are unconventional pathogens that cause transmissible spongiform encephalopathies (TSEs). According to the "protein only" hypothesis, prions consist of an infectious protein that is capable of converting a normal host protein termed PrPc into a protease resistant form termed PrPSc. PrPSc is poorly degraded by the host and accumulates in the CNS. Normal biological functions of PrPc and mechanisms involved in neurodegeneration remain obscure. During the past two decades, considerable efforts have been made to elucidate prion diseases and in particular to identify PrP interactors for a better understanding in prion biology. A major break-through was the identification of the 37 kDa laminin receptor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), as the cell surface receptor for the cellular prion protein. We investigated the role of LRP/LR in the propagation of PrPSc in chronically infected cells by different approaches. Three strategies resulted in downregulation or blocking of LRP and prevented PrPSc accumulation in different scrapie infected neuronal cell lines (i) transfection with an antisense LRP RNA expression plasmid (ii) transfection with small interfering (siRNAs) specific for the LRP mRNA and (iii) incubation with the polyclonal anti-LRP antibody, W3. We observed that the treatment with W3 abolished PrPSc deposition and reduced PrPc levels after one week of incubation. PrPSc did not reappear in cells being cultured for 14 additional days without therapeutic antibody treatment. Taken together, these results indicate that LRP is not only required for PrPc metabolism under non-pathological conditions but also has a pivotal role in prion propagation in a cell culture model. LRP/LR appears then to be a promising potential target for the development of therapeutics for the treatment of prion disease. Due to these encouraging cell culture data, we decided to select single chain antibodies (scFv) encompassing a suitable format for therapy. ScFvs are composed of variable parts of heavy and light chains of an immunoglobulin that are connected by a peptidic linker. The antibodies were screened on recombinant GST::LRP employing a phage display strategy. Two scFvs termed N3 and S18 were screened and selected by ELISA. Both antibodies were further characterized by western blotting and FACS analysis: both N3 and S18 specifically recognized mouseLRP and humanLRP overexpressed in mammalian cells under denaturating conditions (western blot) and under native conditions at the cell surface (FACS). Epitope mapping revealed that as expected both scFvs are directed against the extracellular part of LRP: S18 and N3 recognized amino acid residues 225-233 and 273-278, respectively. The ability of N3 and S18 to interfere with LRP/PrP interaction was tested by pull-down assays. In contrast to the control scFv C9 directed against the pre-S1 coat-protein of hepatitis B virus, both anti-LRP scFvs were able to block the specific LRP/PrP binding. In order to investigate a potential curing effect of scFv S18 in vivo, this scFv was tested in a scrapie mouse model by passive immunization. The application of S18 by intra-peritoneal injection was able to reduce PrPSc deposition in the spleen in comparison to mice injected with PBS or C9. However the survival times of S18 treated animals was not increased. Anti-LRP scFv S18 seems to contribute to block prion propagation in the periphery but it is likely that this effect was not enough strong to have an impact on the CNS invasion. Thus, we hypothesized that a strategy targeting directly the brain should be more effective. In this context, an approach based on the expression of single chain antibodies as secretory molecules in the brain via an adeno-associated virus (AAV) vector was initiated. To assure secretion of the scFv expressed in mammalian cells, a signal sequence was fused to the scFvs. Tranfection experiments demonstrated that neuronal cells were able to express and secrete high quantities of both scFvs. Furthermore, the generated scFvs were still functional as shown by western blotting. To find the appropriate AAV serotype for scFv expression, neuronal cells were transduced with varying serotypes carrying a GFP. AAV serotype 2 was chosen due to (i) its good transduction performance in two neuronal cell lines and (ii) the possibility of its purification by affinity chromatography. The sequences encoding for the scFvs N3, S18 and C9 have been cloned in an AAV-based vector. The AAV system was also able to drive high expression of scFvs into the supernatant by transfection or transduction. rAAV-scFv particles were produced and purifed for further stereotaxic injections into mice. Although the investigation of this therapeutic strategy is still in progress in a murine scrapie model, we already proved that a single injection of rAAV led to the expression of scFvs into the brain of mice 30 days post injection. This study represents the first gene therapeutic approach for the treatment of prion diseases.

In dieser Dissertation wurden Kobalt-katalysierte Aryl-Aryl-Kreuzkupplungen zwischen funktionalisierten magnesium- oder kupferorganischen Reagenzien und Arylbromiden, -chloriden, -fluoriden und -tosylaten durchgeführt. Außerdem wurden neuartige Ein-Topf-Synthesen zur Darstellung bororganischer Verbindungen und anschließender Palladium-katalysierter Suzuki-Kreuzkupplungen untersucht. Abschließend wurden funktionalisierte Aryl- und Heteroaryldisulfide durch Reaktion zinkorganischer Reagenzien mit Schwefelmonochlorid synthetisiert.

In der vorliegenden Dissertation wurde die Stabilitätsproblematik therapeutischer Proteine am Beispiel zweier Entwicklungssubstanzen, der Immunzytokine huKS-IL 2 und hu14.18-IL 2, erörtert. Bei beiden Molekülen handelt es sich um neuartige Fusionsproteine, in denen eine variable Antikörper-Komponente mit jeweils zwei Interleukin-2 Resten verknüpft ist. Ziel der Untersuchungen war es, das Abbauverhalten der Substanzen näher zu charakterisieren und destabilisierende Einflussfaktoren zu identifizieren. Im Fokus der Arbeit stand die Frage, inwiefern sich das Stabilitätsverhalten der Fusionsproteine gegenüber den einzelnen Proteinkomponenten verändert und welche Auswirkungen eine Variation der Molekülbestandteile auf die Stabilität der Konstrukte hat. Hierzu wurden den Stabilitätsstudien von huKS-IL 2 und hu14.18-IL 2 Untersuchungen mit den monoklonalen Antikörpern huKS und hu14.18 bzw. mit Interleukin-2 gegenübergestellt. Ein weiteres Ziel dieser Arbeit war es, eine lagerstabile flüssige oder gefriergetrocknete Formulierung für huKS-IL 2 und hu14.18-IL 2 zu entwickeln, wobei im Rahmen der Rezepturfindung neben etablierten Excipienten auch einige neuartige Hilfsstoffe erprobt wurden. Der letzte Teil dieser Dissertation befasste sich schließlich mit der Frage, inwiefern für huKS-IL 2 und hu14.18-IL 2 Stabilitätsvorhersagen aufgrund beschleunigter Haltbarkeitsstudien getroffen werden können. Hierbei wurde die Anwendbarkeit der klassischen isothermen Methode sowie einer nonisothermen Methode getestet.

Halophilic archaea inhabit hypersaline environments and share common physiological features such as acidic protein machineries in order to adapt to high internal salt concentrations as well as electron transport chains for oxidative respiration. Surprisingly, nutritional demands were found to differ considerably amongst haloarchaeal species, though, and in this project several complete genomes of halophilic archaea were analysed to predict their metabolic capabilities. Comparative analysis of gene equipments showed that haloarchaea adopted several strategies to utilize abundant cell material available in brines such as the acquisition of catabolic enzymes, secretion of hydrolytic enzymes, and elimination of biosynthesis gene clusters. For example, metabolic genes of the well-studied Halobacterium salinarum were found to be consistent with the known degradation of glycerol and amino acids. Further, the complex requirement of H. salinarum for various amino acids and vitamins in comparison with other halophiles was explained by the lack of several genes and gene clusters, e.g. for the biosynthesis of methionine, lysine, and thiamine. Nitrogen metabolism varied also among halophilic archaea, and the haloalkaliphile Natronomonas pharaonis was predicted to apply several modes of N-assimilation to cope with severe ammonium deficiencies in its highly alkaline habitat. This species was experimentally shown to possess a functional respiratory chain, but comparative analysis with several archaea suggests a yet unknown complex III analogue in N. pharaonis. Respiratory chains of halophilic and other respiratory archaea were found to share similar genes for pre-quinone electron transfer steps but show great diversity in post-quinone electron transfer steps indicating adaptation to changing environmental conditions in extreme habitats. Finally, secretomes of halophilic and non-halophilic archaea were predicted proposing that haloarchaea secretion proteins are predominantly exported via the twin-arginine pathway and commonly exhibit a lipobox motif for N-terminal lipid anchoring. In N. pharaonis, lipoboxcontaining proteins were most frequent suggesting that lipid anchoring might prevent protein extraction under alkaline conditions. By contrast, non-halophilic archaea seem to prefer the general secretion pathway for protein translocation and to retain only few secretion proteins by N-terminal lipid anchors. Membrane attachment was preferentially observed for interacting components of ABC transporters and respiratory chains and might further occur via postulated C-terminal anchors in archaea. Within this project, the complete genome of the newly sequenced N. pharaonis was analysed with focus on curation of automatically generated data in order to retrieve reliable gene prediction and protein function assignment results as a basis for additional studies. Through the development of a post-processing routine and expert validation as well as by integration of proteomics data, a highly reliable gene set was created for N. pharaonis which was subsequently used to assess various microbial gene finders. This showed that all automatic gene tools predicted a rather correct gene set for the GC-rich N. pharaonis genome but produced insufficient results in respect to their start codon assignments. Available proteomics results for N. pharaonis and H. salinarum were further analysed for posttranslational modifications, and N-terminal peptides of haloarchaeal proteins were found to be commonly processed by N-terminal methionine cleavage and to some extent further modified by N-acetylation. For general function assignment of predicted N. pharaonis proteins and for enzyme assignment in H. salinarum, similarity-based searches, genecontext methods such as neighbourhood analysis but also manual curation were applied in order to reduce the number of hypothetical proteins and to avoid cross-species transfer of misassigned functions. This permitted to reliably reconstruct the metabolism of H. salinarum and N. pharaonis. Generated metabolic data were stored in a newly developed metabolic database that also integrates experimental data retrieved from the literature. The pathway data can be assessed as coloured KEGG maps and were combined with data resulting from transcriptomics and proteomics techniques. In future, expert-curated reaction entries of the created metabolic database will be a valuable source for the design of metabolic experiments and will deliver a reliable input for metabolic models of halophilic archaea.

Small ubiquitin-related modifier (SUMO) is a protein that is attached to lysine residues in a variety of target proteins. Sumoylation of proteins can alter their intracellular localisation, stability, activity and interaction with other proteins. The pathway of sumoylation is analogous to that of ubiquitination. The reaction is ATP dependent and requires the E1-activating enzyme (Aos1/Uba2), the E2-conjugation enzyme (Ubc9) and for most target proteins SUMO E3 ligases. The aim of this study was to characterise the SUMO E1 enzyme, a heterodimer consisting of the subunits Aos1 and Uba2. On one hand I characterised an Uba2 splice variant, which lacks one exon encoding 50 amino acids. Using RT-PCR I could determine the tissue specific distribution of the Uba2 splice variant. I furthermore showed that Uba2 variant protein is still able to form an active E1 enzyme complex with Aos1. I could demonstrate that variant Aos1/Uba2 complex is fully active in RanGAP1 sumoylation with SUMO1 or SUMO2. This finding was surprising in light of the missing amino acids, and will have implications for the understanding of E1 function. A large part of my work was dedicated to the identification and characterization of a novel SUMO substrate called ELKS. According to literature, ELKS proteins have been linked to intracellular membrane traffic and NFB signaling pathways. I identified ELKS in membrane fractions as a binding partner for the Aos1 subunit of the SUMO E1 enzyme and confirmed in vivo interaction with ELKS antibodies that I generated. Because recombinant proteins did not interact directly, I searched for potential bridging factors. Neither SUMO nor Ubc9 or Rab6 (one ELKS partner) mediated interaction between ELKS and Aos1. Performing a large scale immunoprecipitation and analysis by mass spectrometry, I could find several candidates, including nucleoporin RanBP2, a SUMO E3 ligase. This suggested that ELKS may be a target for sumoylation. Indeed, I could show that ELKS was SUMO-modified in vivo and in vitro. Moreover, RanBP2 enhanced ELKS sumoylation. By mass spectrometry I identified two SUMO acceptor sites in ELKS. Mutation of these two residues had no effect on ELKS localisation, but strongly inhibited ELKS induced NFB activation. In conclusion, work described in this thesis implicates sumoylation as an important mechanism for ELKS function in NFkB signaling.

The molecular structures of all silver halide monomers, Ag2X, AgX, AgX2 and AgX3, (X = F, Cl, Br, I), have been calculated at the B3LYP, MP2 and CCSD(T) levels of theory by using quasirelativistic pseudopotentials for all atoms except fluorine and chlorine. All silver monohalides are stable molecules, while the relative stabilities of the subhalides, dihalides and trihalides considerably decrease toward the larger halogens. The ground-state structure of all Ag2X silver subhalides has C2v symmetry, and the molecules can be best described as [Ag2]+X-. Silver dihalides are linear molecules; AgF2 has a 2Sg ground state, while all of the other silver dihalides have a ground state of 2Pg symmetry. The potential energy surface (PES) of all silver trihalides has been investigated. Neither of these molecules has a D3h symmetric trigonal planar geometry, due to their Jahn-Teller distortion. The minimum energy structure of AgF3 is a T-shaped structure with C2v symmetry. For AgCl3, AgBr3 and AgI3, the global minimum is an L-shaped structure, which lies outside the Jahn-Teller PES. This structure can be considered as a donor-acceptor system, with X2 acting as donor and AgX as acceptor. Thus, except for AgF3, in the other three silver trihalides, silver is not present in the formal oxidation state 3. Hybrid density functional theory methods have been used to examine the reactivity of hexafluoro- and hexachlorocyclotriphosphazene with respect to single, multiple and complete substitution with water, ammonia, phosphoric and sulfuric acid. Geometries of both educts and all substitution products habe been optimized and their thermodynamic properties are discussed. Based on these results the thermodynamically most favorable reaction pathways have been determined. Starting from a basic unit, which consists of two phosphazene rings that are geminally linked by two hydrazine bridges, several possibilities to form double-stranded chains or helices containing cyclotriphosphazenes were examined by PM3 calculations.

Highly functionalized organometallic compounds are key intermediates for the preparation of polyfunctionalized molecules. Halogen-metal exchange reaction is a general and chemoselective method for preparing functionalized organometallics, such as organolithium or organomagnesium. However, more sensitive functionalities, such as ketones and aldehydes are usually not compatible with these reagents. To overcome these problems, we developed a halogen-copper exchange reaction by using two sterically hindered lithium dialkylcuprates, which allowed a practical preparation of functionalized lithium cuprate reagents. Various functionalized cuprate reagents were prepared by using this method like functionalized aryl, heteroaryl and alkenyl cuprate reagents. A wide range of functional groups were tolerated by this method, such as ketones, esters, cyanide, halides, methoxyl group, or even aldehyde. The resulting functionalized copper reagents reacted readily with various electrophiles leading to highly functionalized molecules. The application of functionalized aryl cuprates obtained via I/Cu-exchange reaction in stereoselective allylic substitution reaction was also described. Finally, a related new method was developed for the preparation of polyfunctional magnesium arylcuprates obtained by an I/Cu-exchange reaction. Especial interest was that the reaction proceeded in a very clean way with the only side-product cyclopentane. Various functionalized organocopper reagents were obtained by this new method.