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

Die vorliegende Dissertation untersucht mit Hilfe der Microarray-Technologie die Auswirkungen gängiger Zytostatika auf die globale Genexpression humaner Tumor- und Normalzellen und evaluiert deren Gemeinsamkeiten und Unterschiede. Darüber hinaus werden niedermolekulare Inhibitoren, die spezifisch gegen onkologische Targets gerichtet sind, in ähnlicher Weise analysiert und mit einer RNAi-vermittelten Herabregulierung desselben Zielmoleküls verglichen. Der Vergleich der Expressionsmuster lässt darauf schließen, inwieweit die Effekte verschiedener Substanzen übereinstimmen, und ermöglicht aufgrund dessen eine Aussage über ihre Spezifität.

Dynamic remodeling of chromatin or other persistent protein:DNA complexes is essential for genome expression and maintenance. Proteins of the SWI2/SNF2 family catalyze rearrangements of diverse protein:DNA complexes. Although SWI2/SNF2 enzymes exhibit a diverse domain organisation, they share a conserved catalytic ATPase domain that is related to superfamily II helicases through the presence of seven conserved sequence motifs. In contrast to helicases, SWI2/SNF2 enzymes lack helicase activity, but use ATP hydrolysis to translocate on DNA and to generate superhelical torsion into DNA. How these features implicate remodeling function or how ATP hydrolysis is coupled to these rearrangements is poorly understood and suffers from the lack of structural information regarding the catalytic domain of SWI2/SNF2 ATPase In this PhD thesis I characterized the catalytic domain of Sulfolobus solfataricus Rad54 homolog (SsoRad54cd). Like the eukaryotic SWI2/SNF2 ATPases, SsoRad54cd exhibits dsDNA stimulated ATPase activity, lacks helicase activity and has dsDNA translocation and distortion activity. These activities are thereby features of the conserved catalytic ATPase domain itself. Furthermore, the crystal structures of SsoRad54cd in absence and in complex with its dsDNA substrate were determined. The Sulfolobus solfataricus Rad54 homolog catalytic domain consists of two RecA-like domains with two helical SWI2/SNF2 specific subdomains, one inserted in each domain. A deep cleft separates the two domains. Fully base paired duplex DNA binds along the domain 1: domain 2 interface in a position, where rearrangements of the two RecA-like domains can directly be translated in DNA manipulation. The binding mode of DNA to SsoRad54cd is consistent with an enzyme that translocate along the minor groove of DNA. The structure revealed a remarkable similarity to superfamily II helicases. The related composite ATPase active site as well as the mode of DNA recognition suggests that ATP-driven transport of dsDNA in the active site of SWI2/SNF2 enzymes is mechanistically related to ATP-driven ssDNA in the active site of helicases. Based on structure-function analysis a specific model for SWI2/SNF2 function is suggested that links ATP hydrolysis to dsDNA translocation and DNA distortion. The represented results have structural implications for the core mechanism of remodeling factors. If SWI2/SNF2 ATPases are anchored to the substrate protein:DNA complex by additional substrate interacting domains or subunits, ATP-driven cycles of translocation could transport DNA towards or away from the substrate or generate torsional stress at the substrate:DNA interface. Finally, I provide a molecular framework for understanding mutations in Cockayne and X-linked mental retardation syndromes. Mapping of the mutations on the structure of SsoRad54cd reveal that the mutations colocalize in two surface clusters: Cluster I is located adjacent to a hydrophobic surface patch that may provide a macromolecular interaction site. Cluster II is situated in the domain 1 : domain 2 interface near the proposed pivot region and may interfere with ATP driven conformational changes between domain 1 and domain 2.

Im Rahmen der vorliegenden Arbeit werden die Eigenschaften, insbesondere die Oligomerisierung, von Organometallphosphaniden der Gruppe 13 untersucht und neue Liganden entwickelt, die einen niedrigen Oligomerisierungsgrad ermöglichen können. Die Synthese von Trialkylsilylphosphanylgallanen, –alanen und -indanen führt in Abhängigkeit der Größe der organischen Reste an den Metallen zu monomeren und dimeren Verbindungen, deren Eigenschaften durch quantenmechanische Rechnungen (DFT) untersucht wurden. Anders als allein durch sterische Effekte zur kinetischen Stabilisierung monomerer Spezies wird das freie Elektronenpaar in den neuen Phosphanid-Liganden durch Koordination einer Wolframpentacarbonyl-Einheit an das Phosphoratom abgesättigt. Damit steht es nicht mehr für eine Oligomerisierungsreaktion zur Verfügung. Eine neue, einstufige Synthese der Monowolframpentacarbonyl-Verbindung Li[W(CO)5PH2] und der Biswolframpentacarbonyl-Verbindung Li[(W(CO)5)2PH2] wurde entwickelt, wobei das Syntheseprodukt über die Wahl der Stöchiometrie gesteuert wird. Ausgehend von diesen Lithiumphosphaniden und Kalium-trialkylsilylphosphaniden konnten die Mono- und Biswolframpentacarbonyl-Liganden des Typs [W(CO)5P(H)SiR3]- und [(W(CO)5)2P(H)SiR3]- hergestellt werden. Zusammenfassend liefert die vorliegende Arbeit neue Erkenntnisse über Oligomerisierungsprozesse sowie deren Beeinflussung und Steuerbarkeit und stellt neue Liganden für die Organometallchemie zur Verfügung.

Since the beginning of life on earth, cells and their DNA have been exposed to sunlight. Short-wavelength UV-irradiation causes severe genomic damage as a result of the formation of photoproducts in DNA. The major UV-induced DNA lesion is the cis,syn cyclobutane pyrimidine dimer (CPD) which is formed between two adjacent pyrimidine bases, normally thymidines. These photolesions, if left unrepaired, can be mutagenic and carcinogenic. For this reason it is currently of great interest to learn about the detection and repair of UV-induced DNA lesions. In prokaryotes, plants and some vertebrates DNA photolyases are responsible for the repair of these photolesions. Photolyases are coenzyme-dependent repair enzymes which cleave the dimer into the monomers in a sunlight-initiated process thereby regenerating the intact DNA. Although three crystal structures of photolyases have been solved, it has been difficult to obtain a co-crystal structure of this enzyme together with the substrate: DNA containing CPD lesions. The structure of the photolyase/substrate complex is of great importance, as this facilitates the complete elucidation of the binding and repair mechanisms of the enzyme. Photolyases of the deazaflavin-class contain as a light harvesting cofactor 8-hydroxy-5-deazaflavin, the F0 cofactor. Since the F0 cofactor cannot be synthesized during the overexpression process of A. nidulans photolyase in E. coli, the F0 was synthesized chemically as a part of this work and then incorporated into the enzyme. For the DNA substrate, a thymidine dimer lesion with a formacetal bridge was incorporated into oligonucleotides by solid phase synthesis and then crystallised with the protein. In this Ph. D. thesis a co-crystal structure of A. nidulans photolyase in complex with CPD-containing duplex DNA was successfully elucidated. The 1.8 Å crystal structure shows the dimer lesion being completely flipped out of the duplex DNA into the active site of the enzyme and split into the two corresponding thymines by synchrotron radiation at 100 K. This process is accompanied by additional bending of the DNA to about 50 °. The structure apparently mimics a structural sub-state during light-driven DNA repair in which back flipping of the thymines into duplex DNA has not yet taken place. The co-crystal structure described in this thesis represents the first structure which can confirm the hitherto only postulated dinucleotide flipping mechanism of photolyases. Photolyases of the deazaflavin class possess as cofactors one flavin and one deazaflavin moiety. Due to their interesting chemical and physical properties even these cofactors themselves can form the basis of variable and fundamental investigations. In nature flavin-dependent enzymes play a crucial role in the catalysis of redox reactions. Due to growing interest in biocatalysis based on oligonucleotide structures, the incorporation of coenzymes into DNA or RNA is of high interest. The construction of flavin-dependent ribozymes with properties that extend into the domain of redox catalysis requires detailed knowledge of the fluorescence properties of coenzymes in an oligonucleotide environment. A riboflavin cofactor was incorporated into DNA which could potentially replace the protein environment of coenzyme-dependent enzymes. This thesis presents the first study of the complex fluorescence behaviour of an artificial flavin base in DNA. The coenzyme is surprisingly useful for the precise monitoring of nucleobases in its vicinity. The described results will be the basis for further investigations into flavin-containing bioanalytical devices. The deazaflavin cofactor is another very common coenzyme with differing functions. It acts as redox cofactor F420 in methanogenic bacteria and as F0 it is a light harvesting photoantenna in many photolyases. In this work a deazaflavin building block was synthesized which was incorporated into DNA. For the first time a deazaflavin has been incorporated within an oligonucleotide via phosphodiester bonds. Together with an embedded flavin cofactor and pyrimidine lesions, a new complete model system of photolyase with DNA as a fixed pre-organising template can be generated. This will allow systematic investigations into the distance dependence of energy transfer processes between deazaflavin and flavin, and into repair processes. The results from this thesis yielded fascinating insights into one of the most important genome repair processes in nature.

Im Rahmen der Dissertation "Metall-katalysierte enantioselektive Synthese von Propargylaminen und ihre Anwendungen in der Synthese" wurde eine Kupfer-katalysierte enantioselektive Additionsreaktion von Alkinen an in situ generierte Iminium-Ionen untersucht. Zunächst wurde eine neuartige Eintopf-Synthese entwickelt, die eine besonders atomökonomische und synthetisch wertvolle Methode zur Darstellung der Propargylamne darstellt. Dabei wurden equimolare Mengen von terminalen Alkinen, Aldehyden und sekundären Aminen in Gegenwart von katalytischen Mengen Kupferbromid und Quinap als chiralem Ligand umgesetzt. Die Methode toleriert Variationen und die Einführung von funktionellen Gruppen in alle drei Komponenten. Insbesondere die Ausweitung der Synthese auf aromatische Aldehyde ist erwähnenswert. Die auf diesem Wege erhaltenen Propargylamine wurden weiter funktionalisiert und so in synthetisch nützliche Derivate überführt. Unter anderem wurde die synthetische Anwendbarkeit in der enantioselektiven Totalsynthese des Alkaloids Coniin demonstriert. Im zweiten Teil der Dissertation wurde der Einfluß der Stickstoff-Schutzgruppen auf die Enantioselektivität der Reaktion untersucht. Dabei wurden verschiedene neue Schutzgruppen entwickelt. Der Einsatz von Di(2-phenallyl)amin in der Propargylaminsynthese erlaubte nach einer Palladium-katalysierten allylischen Substitution an den allyl-geschützten Propargylaminen die Darstellung primärer Propargylamine, die bis dahin nicht in guten Enantioselektivitäten zugänglich waren. Im abschließenden Teil der Dissertation wurde ein neuartiger, chiraler P,N-Ligand ausgehend von chiralen Propargylalkoholen entwickelt und in der asymmetrischen Synthese (Palladium-katalysierte allylische Substitution) getestet.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor dysfunctions resulting from massive and selective loss of dopaminergic neurons. Mutations in the human parkin gene, which encodes an E3 ubiquitin ligase, are the most frequent causes of hereditary PD, leading to autosomal-recessive juvenile Parkinsonism. However, the cell biological role of Parkin and the molecular pathogenic mechanisms by which mutations cause PD are unclear. In this study, the Caenorhabditis elegans parkin ortholog, pdr-1, was identified and characterized in detail. PDR-1 is functionally conserved, since it physically associates and cooperates with enzymes of the ubiquitylation/degradation system to mediate ubiquitin conjugation. Strikingly, in contrast to pdr-1 loss-of-function mutants, the in-frame deletion mutant protein PDR-1(delaa24-247) still interacts with its co-enzymes, and moreover, the corresponding mutant pdr-1(lg103) is hypersensitive towards misfolded protein conditions. In this mutant, both cytosolic stress conferred by overexpression of mutant human alpha-synuclein, a gene linked to autosomal-dominant forms of PD, as well as endoplasmatic reticulum (ER)-derived folding stress result in severe developmental defects and lethality. Although expression of pdr-1 is regulated by all three activators of the unfolded protein response (UPR), IRE-1, PEK-1, and ATF-6, genetic analyses established a function of PDR-1 in parallel to IRE-1 signalling. Therefore, PDR-1/Parkin plays an essential role in the regulation of different proteotoxic stress pathways: it contributes to the ER-specific UPR, but also participates in the cytosolic detoxification of protein aggregates, including alpha-synuclein. The truncated protein PDR-1(delaa24-247) seems to mediate a toxic misfunction by sequestering critical components of the protein folding/degradation machinery, which is related to the stress hypersensitivity in the pdr-1(lg103) mutant. In this study, an experimental animal system was established which is well suited to identify modifiers of toxicity and relevant compounds. Such studies might allow to dissect the molecular and cellular pathways involved in the pathogenesis of PD and to identify potential therapeutic drug targets.

RNA polymerase II (Pol II) is the central enzyme, that synthetizes all mRNA in eukaryotic cells. In this work, I solved the structure of the complete, initiation-competent 12-subunit yeast RNA polymerase II at 3.8 Å. I also solved the structure of the Pol II subcomplex of Rpb4/7 alone at 2.3 Å resolution. These structures reveal the details of Pol II assembly from 12 subunits and give important insights into the initiation of transcription. The refined, atomic model of the complete 12-subunit Pol II enabled homology modeling of the two other nuclear RNA polymerases. In Pol I and Pol III, 65 % and 77 % of the Pol II fold are conserved, respectively. Together with a recent structure of a Pol II elongation complex, these results show that the basic mechanism of transcription applies also to the two other nuclear RNA polymerases

Ischemia-reperfusion injury (IRI), which is unavoidable in liver transplantation, is a multifactorial process that can cause non-function or dysfunction of the graft. These complications are the major cause for retransplantation and mortality. An understanding of the mechanisms involved in IRI is essential for the design of therapeutic strategies to prevent IRI and thus improve the outcome of liver transplantation. In recent years, the Atrial Natriuretic Peptide (ANP) has been demonstrated to posses potential in protection against IRI. Nevertheless, many questions remain to be answered about signaling pathways involved in ANP-mediated effects and the processes responsible for its protective properties. The isolated perfused rat liver is a well-investigated model for examination of treatment-mediated effects during ischemia and reperfusion (IR) in the liver. Using this setting, Kiemer et al. demonstrated that ANP-preconditioning increases the activity of p38 MAPK in isolated perfused livers during IR. Functional consequences of an activation of p38 MAPK are widely unknown. This protein kinase has been shown to participate in the regulation of cytoskeletal structures in various cells. Thus, our field of interest was to find a causal connection between ANP-mediated effects and possible cytoskeletal changes during IR in this model. In respect of IRI in the liver, ANP was evidenced to exhibit protective effects also in vivo, but so far nothing is known about signaling pathways responsible for this action. Apototic cell death in the liver during IR is discussed controversial and ANP was demonstrated to mediate anti-apoptotic effects in the isolated perfuse rat liver. We therefore aimed to determine whether ANP mediates effects in the liver in vivo regarding apoptotic death during IR. ANP has not only been shown to have protective properties in IRI but also anti-inflammatory effects in vitro. In previous studies we were able to show that ANP prevents TNF-α production in murine macrophages and whole human blood after LPS-stimulation. Moreover, we ddemonstrated that ANP also inhibits TNF-α-induced aactivation of NF-κB in human endothelial cells and reduces LPS-induced TNF-α secretion in Kupffer cells (KC). With regard to inflammatory processes during LPS-induced endotoxemia TNF-α has been demonstrated to be one of the prototypic pro-inflammatory cytokines mediating many of the immunopathological features of this disease. During sepsis endogenous ANP is suggested to be a regulatory mediator, as the level of its precursor Pro-ANP in blood from septic patients has been demonstrated to be an important rognostic marker for the outcome of sepsis. Another topic discussed in this work is therefore the disease pattern of sepsis. This life-threatening disorder results from a harmful host response to infection and is the leading cause of death for patients in intensive care units. Despite intensive research on the mechanisms involved in the fatal outcome of this disease, few is known about potential therapies preventing death of patients suffering from sepsis. Based on the knowledge of ANP-mediated protective effects during deleterious processes in IRI and its anti-inflammatory properties we therefore aimed to answer the following questions: 1. Does pretreatment with ANP cause cytoskeletal changes in the liver during IR in the isolated perfused rat liver? Which cell type is affected? What are the underlying signaling mechanisms for these changes? 2. Is apoptosis of liver cells during liver transplantation influenced by ANP? Which anti-apoptotic pathway is involved? 3. Are anti-inflammatory properties of ANP able to maintain survival after LPS-induced septic shock? How does ANP influence inflammatory processes during endotoxaemia?

In der vorliegenden Arbeit wurden erstmals Vertreter der Chaperoninklassen I und II (MmGroEL/MmGroES und MmThs/MmPfd) eines Archaeon untersucht. Das Chaperonin MmGroEL und sein Kofaktor MmGroES aus Methanosarcina mazei sind oligomere Komplexe, die jeweils aus identischen Untereinheiten aufgebaut sind. MmGroEL ist ein Homotetradecamer mit einer heptameren Doppelringstruktur. MmGroEL zeigt die für Chaperone typische Eigenschaft indem es ungefaltetes Substratprotein bindet und eine Aggregatbildung verhindert. Eine effiziente ATP-abhängige Rückfaltung denaturierter Proteine im Zusammenspiel mit MmGroES durch MmGroEL wird nur unter bestimmten Bedingungen vermittelt. Die Ergebnisse belegen, dass Ammoniumsulfat im Falle für das Modellsubstrat Malat-Dehydrogenase unerlässlich für den funktionellen Ablauf der Reaktivierung ist. Zwar kommt es auch in Abwesenheit von diesem Salz zu einer Faltung, wie dies im Falle des monomeren Substrates Rhodanese nachgewiesen werden konnte. Jedoch bewirkt im Fall der Malat-Dehydrogenase nur eine Zugabe von Ammoniumsulfat die mechanistisch notwendige Einschließung des Substrates in die cis-Kavität des GroEL. Das Gruppe II Chaperonin MmThs ist ein aus drei Untereinheiten bestehender hochmolekularer Komplex. Durch Immunpräzipitationen konnte nachgewiesen werden, dass im endogenen MmThs die Ths-Untereinheiten in einem Verhältnis von 2:1:1 (α:β:γ) vorliegen. Der MmThs Kofaktor MmPfd ist ein hochmolekularer Chaperon-Komplex mit einer heteromeren Untereinheiten-Zusammensetzung von α2β4, ohne jedoch eine ATP Hydrolyseaktivität aufzuweisen. MmPfd hat die für Chaperone typische Eigenschaft der Aggregationsprävention denaturierter Proteine und stabilisiert sie in einen faltungskompetenten Zustand. MmPfd ist zudem in der Lage denaturierte Proteine an MmThs und MmGroEL weiterzugeben.