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SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.
Diversity-oriented synthesis (DOS) is a rich source for novel lead structures in Medicinal Chemistry. In this study, we present a DOS-compatible method for synthesis of compounds bearing a free thiol moiety. The procedure relies on Rh(II)-catalyzed coupling of dithiols to diazo building blocks. The synthetized library was probed against metallo-β-lactamases (MBLs) NDM-1 and VIM-1. Biochemical and biological evaluation led to identification of novel potent MBL inhibitors with antibiotic adjuvant activity.
B-cell acute lymphoblastic leukaemia (B-ALL) is characterized by the overproduction of lymphoblasts in the bone marrow (BM), and it is the most common cancer in children while being comparatively uncommon in adults. On the other hand, in chronic myeloid leukaemia (CML), 70% of cases are found in patients older than 50 years, making it uncommon in children. All CML cases and up to 3% of paediatric B- ALL (and 25% of adult B-ALL) cases are due to fusion gene BCR-ABL1, which gives rise to the cytoplasmatic, constitutively active oncoprotein, tyrosine kinase BCR-ABL1 through a reciprocal translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase leads to deregulation of different signal transduction pathways such as cell growth, proliferation and cell survival. The role of the bone marrow microenvironment (BMM) can mediate disease initiation (only in mice), progression, therapy resistance, and relapse, as has been increasingly recognized over the last two decades. In general, the BMM is a very complex arrangement of various cell types such as osteoblasts, osteoclasts, endothelial cells, adipocytes, mesenchymal stromal cells, macrophages and several others. In addition, the BMM is composed of multiple chemical and mechanical factors and extra cellular matrix (ECM) proteins which contribute to the BMM’s features influencing leukaemia behaviour. Considering the incidence of B-ALL and CML in children and in adults respectively, we hypothesized that the young and/or an aged BMM might also play a previously unrecognized role in the aggressiveness of B-ALL and CML. We proposed that BM, transduced with BCR-ABL1-expressing retrovirus in the murine transduction/transplantation model of B-ALL, transplanted into young versus old recipient mice would lead to a more aggressive disease in young mice, and similarly CML would be more aggressive in old recipient mice. In close recapitulation with the human incidence, induction of CML led to a significantly shorted survival in old recipient mice. On the other hand, induction of B-ALL showed a shortened survival in young compared to old syngeneic mice, as well as in a xenotransplantation model. Among the highly heterogenous composition of the BMM, we implicate young BM macrophages as a supportive niche for B-ALL cells. The results were found to be mostly due to potential soluble factors differentially secreted from young and old macrophages. Therefore, we hypothesized that the chemokine CXCL13, which has been demonstrated to play a role in B cell migration and act as a diagnostic marker in the cerebrospinal fluid of patients with neuroborreliosis, might be responsible for the observed phenotype. CXCL13 was found to be more highly expressed in healthy and leukaemic young mice as well as in conditioned medium of young macrophages. Using a variety of in vitro experiments, CXCL13 showed to significantly increase the proliferation and the migration of leukaemia cells when exposed to young macrophages, and the phenotype was rescued while using a CXCL13 neutralizing antibody. The CXCL13 role was also confirmed in vivo, since macrophage ablation led to a prolongation of survival in young mice and a reduction of CXCL13 levels. The use of an additional mouse model, leukaemia cells with CXCR5 deficiency, led to a significant prolongation of survival of young mice, confirming the importance of the CXCL13-CXCR5 axis in B-ALL. In line with our murine results, we found that human macrophages and CXCL13 levels were higher in pediatric B-ALL patients than in adults. Consistent with our murine data, the expression level of CXCR5 may act as a prognostic marker in B-ALL, as well as a predictive marker for central nervous system relapse in human B-ALL. The overall findings show that a young BMM, and in particular macrophages, influences B-ALL progression. We specifically identified CXCL13, secreted by young macrophages, as a promoter of proliferation of B-ALL cells, influencing survival in B-ALL via CXCR5. The CXCR5-CXCL13 axis may be relevant in human B-ALL, and higher CXCR5 expression in human B-ALL may act as a predictive marker.
Precise control of blood clotting and rapid reversal of anticoagulation are essential in many clinical situations. We were successful in modifying a thrombin-binding aptamer with a red-light photocleavable linker derived from Cy7 by Cu-catalyzed Click chemistry. We were able to show that we can successfully deactivate the modified aptamer with red light (660 nm) even in human blood—restoring the blood's natural coagulation capability.
In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.
The intriguing (μ-hydrido)diboranes(4) with their prominent pristine representative [B2H5]− have mainly been studied theoretically. We now describe the behavior of the planarized tetraaryl (μ-hydrido)diborane(4) anion [1H]− in cycloaddition reactions with the homologous series of heterocumulenes CO2, iPrNCO, and iPrNCNiPr. We show that a C=O bond of CO2 selectively activates the B−B bond of [1H]−, while the μ-H ligand is left untouched ([2H]−). The carbodiimide iPrNCNiPr, in contrast, neglects the B−B bond and rather adds the B-bonded H− ion to its central C atom to generate a formamidinate bridge across the B2 pair ([3]−). As a hybrid, the isocyanate iPrNCO combines the reactivity patterns of both its congeners and gives two products: one of them ([4H]−) is related to [2H]−, the other ([5]−) is an analog of [3]−. We finally propose a mechanistic scenario that rationalizes the individual reaction outcomes and combines them to a coherent picture of B–B vs. B–H bond activation.
The crystal structures of sodium ethoxide (sodium ethanolate, NaOEt), sodium n-propoxide (sodium n-propanolate, NaOnPr), sodium n-butoxide (sodium n-butanolate, NaOnBu) and sodium n-pentoxide (sodium n-amylate, NaOnAm) were determined from powder X-ray diffraction data. NaOEt crystallizes in space group P421m, with Z = 2, and the other alkoxides crystallize in P4/nmm, with Z = 2. To resolve space-group ambiguities, a Bärnighausen tree was set up, and Rietveld refinements were performed with different models. In all structures, the Na and O atoms form a quadratic net, with the alkyl groups pointing outwards on both sides (anti-PbO type). The alkyl groups are disordered. The disorder becomes even more pronounced with increasing chain length. Recrystallization from the corresponding alcohols yielded four sodium alkoxide solvates: sodium ethoxide ethanol disolvate (NaOEt·2EtOH), sodium n-propoxide n-propanol disolvate (NaOnPr·2nPrOH), sodium isopropoxide isopropanol pentasolvate (NaOiPr·5iPrOH) and sodium tert-amylate tert-amyl alcohol monosolvate (NaOtAm·tAmOH, tAm = 2-methyl-2-butyl). Their crystal structures were determined by single-crystal X-ray diffraction. All these solvates form chain structures consisting of Na+, –O− and –OH groups, encased by alkyl groups. The hydrogen-bond networks diverge widely among the solvate structures. The hydrogen-bond topology of the iPrOH network in NaOiPr·5iPrOH shows branched hydrogen bonds and differs considerably from the networks in pure crystalline iPrOH.
Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion.
Lack of efficacy of a partial adenosine A1 receptor agonist in neuropathic pain models in mice
(2021)
Previous studies suggest that adenosine A1 receptors (A1R) modulate the processing of pain. The aim of this study was to characterize the distribution of A1R in nociceptive tissues and to evaluate whether targeting A1R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A1R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A1R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03–1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A1R agonist. Despite expression of A1R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A1R agonists might be a valuable approach for the treatment of neuropathic pain.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found “not guilty”. Now, new surprising evidence repeats its request to be the “smoking gun” which reopens the criminal trial revealing the crucial involvement of this tripeptide.
In this thesis, we characterized megasynthases such as fatty acid synthases (FASs) and polyketide synthases. The obtained insights into structure and function were used to engineer such systems to produce new-to-nature compounds.
The in vitro characterization of megasynthases requires reproducible access to these enzymes in high quality. Therefore, we established purification strategies for the yeast FAS and the methylsalicylic acid synthase (MSAS) from Saccharopolyspora erythraea (SerMSAS) and applied the latter one on MSAS from Penicillium patulum (PenPaMSAS) and on 6-deoxyerythronolide B synthase (DEBS) module 6. With the purified samples, we were able to obtain initial structural data for SerMSAS and solve the complete structure of the yeast FAS (PDB: 6TA1). On the example of the yeast FAS, we could show that the sample can suffer from adsorption to the water-air interface during the grid preparation for electron microscopy and presented how the use of graphene-based grids can overcome this problem. The combined structural and functional analysis of the yeast FAS showed that the structural domains trimerization module and dimerization module 2 are not essential for the assembly of the whole system. Therefore, they can potentially be used for domain exchange approaches. The in-depth functional analysis of SerMSAS revealed that not SerMSAS itself releases the product, but a 3-oxoacyl-(acyl-carrier protein) synthase like enzyme within the gene cluster transfers 6-methyl salicylic acid from SerMSAS to another carrier protein for subsequent modifications. In contrast, we showed that PenPaMSAS can release its product by hydrolysis and that non-native substrates can be incorporated although at significantly slower turnover rates compared to the native starter substrate. Our further investigation demonstrated that the substrate specificity of the acyltransferase (AT) is a critical factor for the incorporation of non-native substrates.
With the insight from the functional and structural characterization, we engineered megasynthases for the biosynthesis of natural product derivatives. We targeted the AT of PenPaMSAS for active site mutagenesis and discovered a mutant which can transfer non-native substrates significantly faster (~200-300%). Additionally, the malonyl/acetyl transferase (MAT) of the mammalian FAS was used as a promising target for protein engineering because of its previously reported properties including polyspecificity, fast transfer kinetics, robustness, and plasticity. We showed that the MAT can transfer fluorinated substrates and accept the acyl carrier protein of DEBS module 6. By exchanging the substrate specific AT of DEBS with the polyspecific MAT of the mammalian FAS, we demonstrated an efficient DEBS/FAS hybrid and an optimal truncation site for the applied ATs. In contrast to the wild type system, the DEBS/FAS enzyme was able to synthesize demethylated and fluorinated derivatives. The production and purification of a fluoro-methyl-disubstituted polyketide was of particular interest, as it has a high potential for the generation of new drugs and shows the potential of protein engineering. Furthermore, the incorporation of the disubstituted substrate had important implication in the mechanistic details of the ketosynthase-mediated C-C bond formation.
Specialized transporter proteins facilitate controlled uptake and extrusion of molecules across biological membranes that would otherwise be impermeable to them. The superfamily of solute carriers (SLC) comprises the second largest group of membrane proteins in humans, acting on a variety of small polar and non-polar molecules and ions. Because of their central role in metabolism, malfunctioning of these proteins often is pathogenic. The interest in SLC transporters as drug targets – as well as for drug delivery – has therefore increased in the past years. For many SLC subfamilies, however, structural and functional information remains scarce to date.
The here presented data provides important insights into different aspects of the transport mechanism of the SLC23 and SLC26 protein families. Importantly, we show that SLC23 nucleobase transporters, in contrast to what was been previously reported, work as uniporters rather than as proton-coupled symporters. In order to do so, we developed the first and only in vitro transport assay for the SLC23 family, which enables investigation of protein function in a defined environment. Moreover, we provide a hypothesis on the role of the extremely conserved negative charged substrate binding site residue found not only in the SLC23, but also SLC4 and SLC26 families. Based on a detailed analysis of binding and transport we conclude that this conserved negative charged has a relevance for protein stability rather than for substrate binding, which explains its conservation for all three protein families that otherwise differ in their substrate specificities and modes of transport. Lastly, we investigated the relevance of oligomerization for the SLC23 and SLC26 families, highlighting the importance of the STAS domain for forming active dimers in the SLC26 anion transporter family.
In this thesis, molecular dynamics (MD) simulations are used to study the interaction of different proteins with lipid bilayers. MD simulations can be used as a “computational microscope” to gain atomistic insights into the interactions between proteins and lipids that can barely be accessed in such detail by experimental methods. The different chapters of this thesis address the lipid sensing functionality of amphipathic helices (AHs) when bound to membranes, the folding of AHs at lipid-water interfaces as well as the conformational dynamics of the HIV-1 Env glycoproteins in viral-like and experimental bilayers. In the last chapter the possibilities to enhance the performance of MD simulations are explored, leading to a more efficient usage of computational resources.
Leukemia patients bearing the t(4;11)(q21;q23) translocations can be divided into two subgroups: those expressing both reciprocal fusion genes, and those that have only the MLL-AF4 fusion gene. Moreover, a recent study has demonstrated that patients expressing both fusion genes have a better outcome than patients that are expressing the MLL-AF4 fusion protein alone. All this may point to a clonal process where the reciprocal fusion gene AF4-MLL could be lost during disease progression, as this loss may select for a more aggressive type of leukemia. Therefore, we were interested in unraveling the decisive role of the AF4-MLL fusion protein at an early timepoint of disease development. We designed an experimental model system where the MLL-AF4 fusion protein was constitutively expressed, while an inducible AF4-MLL fusion gene was induced for only 48 h. Subsequently, we investigated genome-wide changes by RNA- and ATAC-Seq experiments at distinct timepoints. These analyses revealed that the expression of AF4-MLL for only 48 h was sufficient to significantly change the genomic landscape (transcription and chromatin) even on a longer time scale. Thus, we have to conclude that the AF4-MLL fusion protein works through a hit-and-run mechanism, probably necessary to set up pre-leukemic conditions, but being dispensable for later disease progression.
Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15–34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.
Für jeden Betroffenen ist die Diagnose Krebs ein schwerwiegender Einschnitt in der Lebensqualität und -führung, da die Behandlung oftmals mit langen Chemotherapien einhergeht. Moderne Durchbrüche in der Krebsbehandlung stammen aus dem Forschungsbereich der zielgerichteten Molekulartherapie oder aus dem Gebiet der Immuntherapien, die zu beachtlichen Erfolgen bei der Behandlung von Krebspatienten führten. Trotzdem bleiben auf dem Gebiet der Onkologie weiterhin Fragen zu den grundlegenden biologischen Prozessen unbeantwortet.
Zu den Onkoproteinen, die das Tumorwachstum in Leukemiezellen stark beeinflussen, gehören die Proteine der Klasse der mixed lineage leukemia (MLL) Histonmethyltransferasen. Genetische Fusionen des mll Gens, sogenannte Rearragments, führen zu MLL-fusion Produkten, die erheblich zum Verlauf der aggressiven akuten myeloischen Leukämie (AML) beitragen. Ein weiteres Onkoprotein, das für den Krankheitsverlauf vieler Krebsarten relevant ist, ist die Transkriptionsfaktorfamilie MYC. Überexprimierung von MYC wurde in einem Drittel aller humanen Tumore beobachtet. Zahlreiche Studien belegen, dass hohe MYC Level die Expression von Genen regulieren, die essentiell für den Transformationsprozess und somit das Tumorwachstum sind. Da der Transkriptionsfaktor weder eine sabile tertiäre Proteinstruktur noch eine für Inhibitoren adressierbare Bindetasche aufweist, gilt MYC bis heute als undruggable.
Sowohl die Histonmethyltransferase MLL1, als auch der Transkriptionsfaktor MYC interagieren mit einem ca. 37 kDa Protein namens WD40-repeat containing Protein 5 (WDR5), das durch seine propellerförmige Struktur eine Oberfläche mit insgesamt zwei Bindestellen aufweist. Mehrere Studien zeigten, dass WDR5 die Stabilität und somit die Funktion epigenetischer Proteinkomplexe wie SET/ MLL und NSL gewährleistet. In diesem Kontext wurde WDR5 als relevantes Target für die MLL-rearragend akute lymphatische Leukämie (ALL) postuliert. Weitere Studien zeigten zusätzliche Rollen von WDR5, wie die Interaktion zwischen WDR5 und dem Onkoprotein MYC sowie dessen Rekrutierung zum Chromatin. Seit 2015 wurden erfolgreich mehrere niedermolekulare Wirkstoffe für die Inhibierung von WDR5 entwickelt. Dabei zielten die meisten der literaturbekannten Inhibitoren auf die Argininmotiv-erkennende WDR5-interacting (Win) Bindestelle, eine große, hydrophobe Bindetasche im Zentrum des WDR5-Propellers. Die Resultate der besser erforschten Win Inhibitoren zeigten, dass WDR5 ein erfolgsversprechendes Target zur Inhibierung von leukämischen (MLL-r-abhängigen) und neuroblastomatischen (MYC-abhängigen) Zellwachstum ist.
Da beide Bindestellen des WDR5 Proteins Interaktionen mit onkologisch bedeutsamen Faktoren eingehen, würde eine einseitige Inhibierung nur die Effekte der jeweiligen Bindestelle aufzeigen. Diese Limitierung könnte jedoch durch die Entwicklung von WDR5 PROTACs (Proteolysis targeting chimeras) aufgehoben werden, da alle Gerüstfunktionen des Proteins und Protein-Protein-Interaktionen durch die Degradierung von WDR5 entfernt werden würden. Dabei induzieren die heterobifunktionellen Moleküle den Abbau des Zielproteins über das zelleigene Ubiquitin-Proteasom-System, statt die Enzymfunktion zu inhibieren. Nach dem zelleigenen Abbau des Zielproteins wird der PROTAC freigesetzt und kann einen neuen Zyklus der Proteindegradation einleiten, was die erforderliche Menge an Wirkstoff verringert.
Diese Dissertation beschäftigte sich mit dem Design, der Synthese sowie der biophysikalischen und biologischen Evaluierung von WDR5 PROTACs. Ausgehend von literaturbekannten WDR5 Liganden wurden zwei verschiedene PROTAC Typen entworfen. Diese beiden Molekültypen besitzen einen unterschiedlichen geometrischen Austrittswinkel, wodurch die Chance auf eine erfolgreiche Komplexbildung zwischen WDR5, PROTAC und E3 Ligase erhöht wird. Als Leitstruktur fungierten die Verbindungen OICR-9429 sowie DDO-2117 und ausgehend von Ligand (6d) wurden heterobifunktionelle Moleküle mit verschiedenen Linkersystemen ([PEG]- und alkyl-basiert, sowie aromatisch verbrückt) und verschiedenen E3 Ligase Liganden (Cereblon, VHL und MDM2) synthetisiert. Die anschließenden biochemischen und biophysikalischen Evaluierungen der verschiedenen PROTACs durch Thermofluor (DSF) und ITC zeigten eine hohe in vitro Affinität einiger Moleküle. Die zelluläre Permeabilität der großen Moleküle wurde in einem hier etablierten BRET Assay untersucht. Zur Assay-Etablierung wurden drei Tracer (21a-c), basierend auf BODIPY Konjugaten, synthetisiert und getestet, bevor die PROTACs in intakten und lysierten Zellen vermessen wurden. Während die zellulären Affinitäten von Cereblon- und VHL-adressierenden PROTACs sich im niedrigen μM Bereich bewegten, wurden die nicht zellgängigen MDM2 PROTACs von weiteren Experimenten ausgeschlossen.
Die Degradierungeffizienz der WDR5 PROTACs (7a-e) und (8a-j) wurden in der Leukämie Zellinie MV4-11 untersucht, da diese die am meisten auftretende MLL fusion Mutation AF4 birgt. Dabei wurde der Proteinabbau von WDR5 über den HiBiT Assay sowie Western Blots nachgewiesen. ...
Inducing cell death in tumor cells is a major goal of anti-cancer therapy. However, the preferable mode of cell death to induce is under debate. Apoptosis is known to be an anti-inflammatory and pro-resolving type of programmed cell death, whereas necroptosis results in the release of danger-associated molecular patterns (DAMPs) and is pro-inflammatory. Efferocytosis of apoptotic cells by macrophages results in a pro-resolving switch of macrophages polarization and is required to induce resolution of inflammation. This impact of apoptotic cells on macrophages is a non-desired consequence of cell death in tumors, which are often characterized by an overshooting wound healing response. Moreover, apoptosis resistance is frequently observed in cancer cells. To overcome apoptosis resistance in cancer cells, necroptosis can be induced as an alternative mechanism for cancer treatment. Interferons (IFNs) play an important role in tumor immune responses and act by inducing the expression of IFN-stiumlated genes (ISGs). Furthermore, IFNs were shown to be able to induce necroptosis together with Smac-mimetics when caspases are inhibited in different cancer cell lines. Necroptosis is induced by phosphorylation and activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like (MLKL).
In my thesis, we first identified MLKL as an ISG in various cancer cell lines. MLKL upregulation was found to be a general feature of IFN signaling since both type I and type II IFNs increase the expression of MLKL. IFNy was able to upregulate MLKL at messenger ribonucleic acid (mRNA) and protein level indicating that MLKL is elevated transcriptionally. Indeed, Actinomycin D chase experiments showed that inhibition of transcription abolished MLKL upregulation upon IFN treatment. Both, knockdown of the IFNy-activated transcription factors interferon regulatory factor 1 (IRF1) and signal transducer and activator of transcription 1 (STAT1) as well as knockout of IRF1 significantly dampened MLKL mRNA upregulation, demonstrating that STAT1 and especially IRF1 are necessary to induce MLKL expression. This first part of the study highlights the upregulation of MLKL by IFNy as valuable tool to sensitize cells towards necroptosis and by that overcome apoptosis resistance in cancers.
When compared to apoptosis, the immune response to necroptotic cells and the polarization of macrophages phagocytosing necroptotic cells is not well studied. In most studies, cell death was induced by biological or chemical compounds, which may lead to artifacts by affecting the macrophages and triggering of unrelated signaling pathways. Therefore, in the second part of my thesis we used a pure cell death system of NIH 3T3 cells expressing either dimerizable caspase 8 or oligomerizable RIPK3 to induce cell death. Addition of B/B-Homodimerizer (dimerizer) to the cells resulted in apoptosis or necroptosis, which was confirmed by caspase 3/7 activation, phosphorylation of MLKL and inhibitor experiments, respectively. We analyzed the effect of dying cells on peritoneal macrophages by establishing a co-culture in a transwell system. The genetic profile of macrophages co-cultured with dying cells was evaluated by whole transcriptome RNA sequencing. In macrophages co-cultured with necroptotic cells genes corresponding to chemotaxis and hypoxia pathways were upregulated. A significant proportion of hypoxia-related pathways are mediated by hypoxia-inducible factor 1-alpha (HIF-1α), which also induces metabolic changes in polarized macrophages. We could show that macrophages co-cultured with necroptotic cells showed a decreased mitochondrial respiration, indicating an inflammatory (M1) polarization. Protein levels of chemokine C-X-C motif ligand 1 (CXCL1), which was increased in the RNA sequencing data, were also upregulated in supernatant of co-cultured macrophages and of necroptotic cells, demonstrating that necroptotic cells both secrete CXCL1 and induce gene expression of CXCL1 in peritoneal macrophages. This may influence the recruitment of neutrophils as inhibition of necroptosis during Zymosan-A-induced peritonits in mice decreased the levels of neutrophils at day 1 of this model of self-resolving inflammation.
Furthermore, RNA sequencing revealed an unexpected impact of apoptotic cells on macrophage biology as cell cycle and cell division pathways were increased. Enhanced proliferation of macrophages was confirmed by two functional assay with peritoneal macrophages isolated from mice and IC-21 macrophages. Inhibition of apoptosis during Zymosan-A-induced peritonits in mice demonstrated decreased mRNA levels of cell cycle mediators in peritoneal macrophages. Simultaneously with cell cycle activation, gene sets of prostaglandin E2 (PGE2) signaling were upregulated during RNA sequencing. In the second part of my thesis we could demonstrate, that apoptotic cells induce transcription of cell cycle genes and proliferation of macrophages and necroptotic cells are able to influence the chemokine profile of macrophages and thereby the recruitment of neutrophils.
Chronic rhinosinusitis (CRS) is often treated by functional endoscopic paranasal sinus surgery, which improves endoscopic parameters and quality of life, while olfactory function was suggested as a further criterion of treatment success. In a prospective cohort study, 37 parameters from four categories were recorded from 60 men and 98 women before and four months after endoscopic sinus surgery, including endoscopic measures of nasal anatomy/pathology, assessments of olfactory function, quality of life, and socio-demographic or concomitant conditions. Parameters containing relevant information about changes associated with surgery were examined using unsupervised and supervised methods, including machine-learning techniques for feature selection. The analyzed cohort included 52 men and 38 women. Changes in the endoscopic Lildholdt score allowed separation of baseline from postoperative data with a cross-validated accuracy of 85%. Further relevant information included primary nasal symptoms from SNOT-20 assessments, and self-assessments of olfactory function. Overall improvement in these relevant parameters was observed in 95% of patients. A ranked list of criteria was developed as a proposal to assess the outcome of functional endoscopic sinus surgery in CRS patients with nasal polyposis. Three different facets were captured, including the Lildholdt score as an endoscopic measure and, in addition, disease-specific quality of life and subjectively perceived olfactory function.