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

Inhalational anthrax is an acute infectious disease caused by exposure of the lungs to B. anthracis spores. Alveolar macrophages engulf spores causing them to germinate to the vegetative form of B. anthracis, which secretes edema toxin (ET)and lethal toxin (LT). The pathogenesis of inhalational anthrax is characterized by flu-like symptoms, respiratory distress, meningitis and shock, which is fatal in almost all cases. The mechanism behind the respiratory distress is not well understood. Therefore, our goal was to determine the effects of lethal toxin in the human lung epithelium. To study alterations in a more physiological setting, we developed a differentiated, polarized lung epithelial system. Lethal toxin exposure disrupted the lung barrier function and wound healing. Assembly defects of junction proteins and additional multicellular junction sites resulted in a higher permeability. Pretreatment with keratinocyte growth factor (KGF) and dexamethasone increased the viability, resulting in the rescue of the permeability changes. Upon LT treatment, a more rigid cytoskeleton was observed, evidenced by enhanced actin stress fiber formations and tubulin stabilization. Cytoskeleton and adhesion alterations prevented the epithelial cells from polarization, directed migration, and wound healing. The MAPK pathway and Cdc42 activity might be partially responsible for these motility defects. Lethal toxin is known to induce rapid cell death in murine macrophages. In contrast, human epithelial cells are more resistant to the cytotoxic effect of LT. By following the growth of epithelial cells after LT treatment, we observed inhibited cell proliferation due to a cell cycle arrest in the G1 phase. Surprisingly, biotinylated lethal factor did not induce cytotoxicity in murine macrophages. This is not due to an internalization or proteolytic activity defect; instead changes in the mitochondrial potential and proteasome activity were observed. Biotinylated LT did not reduce proteasome activity as seen in LT treated cells and caused hypopolarization of the mitochondria. However, it is possible that biotinylation of lethal toxin could prevent interaction of LT with proteins that induce cell death. The major challenge for anthrax treatment is to find a treatment, which can act faster, is easy to use and can bring patient out of the dangerous physiological state in late pathogenesis. Our study has implications in saving the viability and barrier function of lung epithelial cells. One can devise better dosage and delivery of KGF and dexamethasone as treatment modality for post anthrax exposure to reduce respiratory distress. Furthermore, overcoming the cell cycle arrest by the development of a drug would reduce the damage of lung epithelial cells and induce proliferation. The discovery that biotinylated LT is non-toxic to murine macrophages could revolutionize treatment of anthrax infection. Exploring the types of posttranslational modifications of LT that decrease toxicity and finding the mechanism behind it might, lead to therapies that directly counteract the effects of the lethal toxin in vivo.

Kohlenhydrate gehören zu den Biomolekülen mit dem mengenmäßig größten Anteil in der Natur, jedoch wurden sie lange Zeit fast ausschließlich als Energiespeicher, Stütz- und Gerüstsubstanzen betrachtet. Die in den letzten Jahren stetig wachsenden Erkenntnisse ihrer essentiellen Bedeutung in wichtigen biologischen Prozessen und die damit verbundenen vielversprechenden Anwendungsmöglichkeiten als Impfstoffe und Medikamente gegen eine Reihe von Krankheiten wie zum Beispiel Krebs, Aids, Diabetes oder Alzheimer führen jedoch zu einem immer größer werdenden Interesse dieser Stoffklasse. Die vorliegende Dissertation gibt einen umfassenden und systematischen Einblick in die Koordinationsmöglichkeiten von Kohlenhydraten an Palladium(II). Dabei erweist sich Palladium(II) in besonderem Maße als ein Zentralatom, das in der Lage ist, die Isomeren- und Konformerenverteilung von Monosacchariden drastisch zu verschieben und so Formen zugänglich zu machen, die ohne Komplexierung nur schwer oder gar nicht nachweisbar sind. Palladium(II) ermöglicht nicht nur stabile Komplexe sowohl mit Pyranosen als auch mit Furanosen in den verschiedensten Bindungsmodi aufzubauen, sondern dient darüber hinaus, durch die in Abhängigkeit des jeweiligen Bindungsmodus auftretenden charakteristischen CIS-Werte der 13C-NMR-Signale, als „Sonde“ für die Koordination an ein bestimmtes Kohlenhydratisomer. Die erhaltenen Ergebnisse erweitern grundlegend die Kenntnisse der Koordinationschemie auf diesem Gebiet und können auch aus pharmazeutischer und katalytischer Sicht von Bedeutung sein.

Synthesis of ribosomal RNA by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. In this thesis a reproducible large-scale purification protocol for Pol I from S. cerevisiae could be developed. Crystals were obtained, diffraction to < 4 Å could be recorded, however, the enormously complex non-crystallographic symmetry impeded structure solution. Switching to cryo-electron microscopy, the structure of the complete 14-subunit enzyme could be solved to 12 Å resolution, a homology model for the core enzyme could be generated, and the crystal structure of the subcomplex A14/43 could be solved. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor, and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3’-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2, and apparently enables rRNA proofreading and 3’-end trimming.

This study characterized a panel of NSCLC cell lines as well as a stably transfected cell model expressing wild-type and mutated EGFR variants in terms of response to the EGFR-targeting drugs, gefitinib and cetuximab. The examinations support the notion that response to gefitinib is neither exclusive nor strictly determined by the presence of EGFR kinase mutations. Yet, cells expressing EGFR kinase domain mutations tended to generally respond better to treatments with gefitinib compared to those with wild-type EGFR. On the other hand, preliminary studies suggesting that cellular sensitivity to cetuximab is determined by factors other than EGFR kinase domain mutations could be substantiated through a robust set of data. Moreover, several promising candidate genes differentially expressed in gefitinib sensitive and resistant NSCLC cell lines were revealed by a global mRNA expression analyses. In addition, though statistically questionable, several biologically interesting genes that are possibly involved in determining in vitro response of NSCLC cells to cetuximab have been postulated. In this work, four cancer cell models, which are long-term exposed to gefitinib or cetuximab were established and characterized in terms of gain-of-resistance towards EGFR-targeting compounds, as well as in regard to biological and molecular alterations caused by long-term treatments. It was found that gefitinib long-term treatment of primary sensitive A431 cells confered growth-resistance to this TKI, but not to cetuximab. This observation may have clinical implications for patients that relapsed on gefitinib as it suggests that they might still profit from cetuximab therapy. On the other hand long-term exposure of A431 cells to cetuximab did not render cells resistant to neither the antibody nor gefitinib in regard to in vitro growth-inhibition. Furthermore, it appeared that long-term gefitinib-treated A431 cells downregulate overall EGFR levels, while long-term cetuximab exposed cells displayed decreased EGFR surface levels but constant overall expression. In addition, a candidate-based approach identified genes that are differentially expressed in cancer cells with primary or secondary resistance to gefitinib or cetuximab. Finally, this study for the first time provided evidence that cetuximab may block metastasis-facilitating epithelial-mesenchymal-transition (EMT) in an epithelial cell line. Moreover, it was suggested that activation of the HGFR may compensate for cetuximab-mediated blockage of EGFR. This was also reflected by an increased response of this population towards treatment with HGFR inhibitors.

DNA damage poses a considerable threat to genomic integrity and cell survival. One of the most harmful forms of DNA damage are double-strand breaks that arise spontaneously during regular DNA processing like replication or meiosis. In addition, they can also be induced by a variety of DNA damaging agents like UV light, cell toxins or anti-cancer drugs. Failure of the rapid repair of these breaks can lead to chromosomal rearrangements and ultimately tumorigenesis in humans. In response to these genomic threats, a highly developed DNA repair network of protein factors has evolved, where the Mre11/Rad50/Nbs1 (MRN) complex is sought to play a key role in sensing, processing and repair of DNA double-strand breaks. Orthologs of Mre11 and Rad50, but not Nbs1, are found in all taxonomic kingdoms of life, suggesting that Mre11 and Rad50 form the core of this complex. In this work structural studies were performed to decipher the overall architecture and the interaction of SbcC and SbcD, the bacterial orthologs of Rad50 and Mre11. Using X-ray crystallographic and small angle X-ray scattering techniques the crystal as well as the in solution structures of the Thermotoga maritima SbcC ATPase domain in complex with full-length SbcD were solved. The crystal and in solution structure match well fortifying the calculated models that reveal an open, elongated complex with dimensions of approximately 210 Å * 75 Å * 65 Å. The heterotetrameric protein assembly consists of two SbcD molecules that homodimerize at domains I to form the central portion of the complex. Located at the outer areas of this homodimer domains II are arranged close to lobe II of SbcC building a small protein-protein interface. The C-terminal domains III of SbcD are connected to domains II via a flexible linker and associate through hydrophobic interactions with the coiled-coils of SbcC. These arrangements in combination with earlier findings lead to a model where upon ATP-binding the complex performs a conformational switch resulting in a ring-shaped structure. This conformation would bear a central cavity to harbor DNA strands that can be processed by the inwards oriented nuclease active sites of SbcD.

RseB from Escherichia coli has been crystallized and crystal structures were determined at 2.4 Å and at 2.8 Å resolution. The structure of cytoplasmic expressed RseB revealed that it consists of two domains; an N-terminal large and a C-terminal small domain. The large domain resembles an unclosed β-barrel that is structurally remarkably similar to a protein family (LolA, LolB) capable of binding the lipid anchor of lipoproteins. Detailed structural comparison of RseB and LolA led to the hypothesis that RseB might be a sensor for mislocalized lipoproteins. The small C-terminal domain, connected to the large domain by a partially unstructured loop, was identified to mediate interaction with RseA. A peptide comprised of a putative helix of RseA was shown to constitute the binding site for RseB. Structure based results presented in this thesis indicate a new role of RseB in acting as a sensor for periplasmic stress: it detects mislocalized lipoproteins in the periplasm and propagates the signal to induce σE-response.

Replication of the genome is strongly inhibited when high fidelity DNA polymerases encounter unrepaired DNA lesions, which can not be processed. The highly stringent active sites of these polymerases are unable to accommodate damaged bases and therefore DNA lesions block the replication fork progression. In order to overcome this problem, cells have evolved mechanisms for either repairing the damage, or synthesising past it with specially adapted polymerasases. Eukaryotic DNA polymerase eta (Pol eta), belonging to the Y-family of DNA polymerases, is outstanding in its ability to replicate through a variety of highly distorting DNA lesions such as cyclobutane pyrimidine dimers (CPDs), which are the main UV-induced lesions. Also cisplatin induced 1,2-d(GpG) adducts (Pt-GGs), which are formed in a typical cancer therapy with cisplatin can be processed by Pol eta. The bypass of such intrastrand crosslinks by high fidelity DNA polymerases is particularly difficult because two adjacent coding bases are simultaneously damaged. Thus, replication by Pol eta allows organisms to survive exposure to sunlight or, in the case of cisplatin, gives rise to resistances against cisplatin treatment. Mutations in the human POLH gene, encoding Pol eta, causes the variant form of xeroderma pigmentosum (XP V), characterized by the failure to copy through CPDs. This leads to strongly increased UV sensitivity and skin cancer predisposition. This thesis describes mechanistic investigations of the translesion synthesis (TLS) process by S. cerevisiae DNA Pol eta at atomic resolution, which were undertaken in collaboration with the Hopfner group. To study this process, cisplatin lesioned DNA had to be prepared first. Once this technique was established, the catalytic fragment of Pol eta was crystallized as ternary complex with incoming 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) and an primer - template DNA containing a site specific Pt-GG adduct. The first obtained structure shows the ddCTP positioned in a loosely bound conformation in the active site, hydrogen bonded to the templating base. Realizing the importance of the 3’ hydroxy group for positioning the NTP and the DNA correctly inside the polymerase, the complex was crystallized again with a 2’-deoxynucleoside 5’-triphosphate (dNTP). To prevent nucleotidyl transfer, primer strands which terminate at the 3’-end with a 2’,3’ dideoxy ribose were prepared by reverse DNA synthesis and used for cocrystallization. The resulting crystals diffracted typically to 3.1-3.3Å resolution at a synchrotron light source. A Pol eta specific arginine (Arg73 in yeast Pol eta) was identified for its importance to position the dNTP correctly in the active site and was shown to be necessary for lesion bypass. In contrast to the fixed preorientation of the dNTP in the active site, the damaged DNA is bound flexibly in a rather open DNA binding cleft. Nucleotidyl transfer requires a revolving of the DNA, energetically driven by hydrogen bonding of the templating base to the dNTP. For the 3’dG of the Pt-GG, this step is accomplished by bona fide Watson-Crick base pairs to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5’dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP. Structurally, this can be attributed to misalignment of the templating 5’dG due to the rigid Pt crosslink. In cooperation with the Cramer group the structural reasons for the blockage of RNA Polymerase II (RNAP II) by the cisplatin lesion were elucidated. Using structural as well as biochemical methods it could be shown that stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according to an 'A-rule' known for DNA polymerases. RNAP II can bypass a cisplatin lesion that is artificially placed beyond the translocation barrier, even in the presence of a G A mismatch. Thus, the barrier prevents transcriptional mutagenesis.