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Copper perchlorophthalocyanine (CuPcCl16, CuC32N8Cl16, Pigment Green 7) is one of the commercially most important green pigments. The compound is a nanocrystalline fully insoluble powder. Its crystal structure was first addressed by electron diffraction in 1972 [Uyeda et al. (1972). J. Appl. Phys. 43, 5181–5189]. Despite the commercial importance of the compound, the crystal structure remained undetermined until now. Using a special vacuum sublimation technique, micron-sized crystals could be obtained. Three-dimensional electron diffraction (3D ED) data were collected in two ways: (i) in static geometry using a combined stage-tilt/beam-tilt collection scheme and (ii) in continuous rotation mode. Both types of data allowed the crystal structure to be solved by direct methods. The structure was refined kinematically with anisotropic displacement parameters for all atoms. Due to the pronounced crystal mosaicity, a dynamic refinement was not feasible. The unit-cell parameters were verified by Rietveld refinement from powder X-ray diffraction data. The crystal structure was validated by many-body dispersion density functional theory (DFT) calculations. CuPcCl16 crystallizes in the space group C2/m (Z = 2), with the molecules arranged in layers. The structure agrees with that proposed in 1972.
Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase β (IKKβ). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.
The DNA damage response (DDR) is a vast network of molecules that preserves genome integrity and allow the faithful transmission of genetic information in human cells. While the usual response to the detection of DNA lesions in cells involves the control of cell-cycle checkpoints, repair proteins or apoptosis, alterations of the repair processes can lead to cellular dysfunction, diseases, or cancer. Besides, cancer patients with DDR alterations often show poor survival and chemoresistance. Despite the progress made in recent years in identifying genes and proteins involved in DDR and their roles in cellular physiology and pathology, the question of the involvement of DDR in metabolism remains unclear. It remains to study the metabolites associated with specific repair pathways or alterations and to investigate whether differences exist depending on cellular origin. The identification of DDR-related metabolic pathways and of the pathways that cause metabolic reprogramming in DDR-deficient cells may produce new targets for the development of new therapies.
In this thesis, nuclear magnetic resonance spectroscopy (NMR) was used to assess the metabolic consequence of the loss of two central DNA repair proteins with importance in diseases context, ATM and RNase H2, in haematological cells. An increase in intracellular taurine was found in RNase H2- and ATM-deficient cells compared to wild-type cells for these genes and in cells after exposition to a source of DNA damage. The rise in taurine does not appear to result from an increase in its biosynthesis from cysteine, but more likely from other cellular processes such as degradation pathways.
Overall, evidence for metabolic reprogramming in haematological cells with faults in DNA repair resulting from ATM or RNase H2 deficiencies or upon exposition to a source of DNA damage is presented in this study.
Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection
(2021)
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.
Chronic inflammation is characterized by persisting leukocyte infiltration of the affected tissue, which is enabled by activated endothelial cells (ECs). Chronic inflammatory diseases remain a major pharmacotherapeutic challenge, and thus the search for novel drugs and drug targets is an ongoing demand. We have identified the natural product vioprolide A (vioA) to exert anti-inflammatory actions in vivo and in ECs in vitro through inhibition of its cellular target nucleolar protein 14 (NOP14). VioA attenuated the infiltration of microglia and macrophages during laser-induced murine choroidal neovascularization and the leukocyte trafficking through the vascular endothelium in the murine cremaster muscle. Mechanistic studies revealed that vioA downregulates EC adhesion molecules and the tumor necrosis factor receptor (TNFR) 1 by decreasing the de novo protein synthesis in ECs. Most importantly, we found that inhibition of importin-dependent NF-ĸB p65 nuclear translocation is a crucial part of the action of vioA leading to reduced NF-ĸB promotor activity and inflammatory gene expression. Knockdown experiments revealed a causal link between the cellular target NOP14 and the anti-inflammatory action of vioA, classifying the natural product as unique drug lead for anti-inflammatory therapeutics.
Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection
(2021)
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.
The vascular endothelium is a monolayer of endothelial cells that builds the inner lining of the blood vessels and constitutes a regulatory organ within the physiological system to sustain homeostasis. Endothelial cells participate in physiological processes including inflammation and angiogenesis. Dysregulation of these processes, however, can evoke or maintain pathological disorders, including cardiovascular and chronic inflammatory diseases or cancer. Although pathological inflammation and angiogenesis represent treatable conditions, current pharmacotherapeutic approaches are frequently not satisfying since their long-term application can evoke therapy resistance and thus reduced clinical efficacy. Consequently, there is an ongoing demand for the discovery of new therapeutic targets and drug leads. Considering that endothelial cells play a critical role in both angiogenesis and inflammation, the vascular endothelium represents a promising target for the treatment of diseases.
Vioprolide A is a secondary metabolite isolated from the myxobacterium Cystobacter violaceus Cb. vi35. Recently, vioprolide A was identified to interact with NOP14, a nucleolar protein involved in ribosome biogenesis. Ribosome biogenesis is an indispensable cellular event that ensures adequate homeostasis. Abnormal alterations in the ribosome biogenesis, referred to as ribosomopathies, however, can lead to an overall increase in the risk of developing cancer. Accordingly, several studies have outlined the involvement of NOP14 in cancer progression and metastasis, and vioprolide A has been demonstrated to exert anti-cancer effects in vitro. However, the impact of vioprolide A and NOP14 on the endothelium has been neglected so far, although endothelial cells are crucially involved in inflammation and angiogenesis under both physiological and pathological conditions.
In the present study, the effect of vioprolide A on inflammatory and angiogenic actions was analysed. In vivo, the laser-induced choroidal neovascularization (CNV) assay outlined a strong inhibitory effect of vioprolide A on both inflammation and angiogenesis. Furthermore, intravital microscopy of the cremaster muscle in mice revealed that vioprolide A strongly impaired the TNF-induced leukocyte-endothelial cell interaction in vivo.
In further experiments, the specific effect of vioprolide A on activation processes of primary human umbilical vein endothelial cells (HUVECs) was examined. According to the in vivo results, vioprolide A decreased the leukocyte-endothelial cell interaction in vitro through downregulating the cell surface expression and total protein expression of ICAM-1, VCAM-1 and E-selectin. Vioprolide A evoked its anti-inflammatory actions via a dual mechanism: On the one hand, the expression of pro-inflammatory proteins, including TNFR1 and cell adhesion molecules, was lowered through a general downregulation of de novo protein synthesis. The inhibition of de novo protein synthesis is most likely linked to the interaction with and inhibition of NOP14 by vioprolide A in HUVECs. On the other hand, the natural product prevented the nuclear translocation and promotor activity of the pro-inflammatory transcription factor NF-ĸB. Interestingly, most anti-inflammatory compounds that interfere with the NF-ĸB signaling pathway prevent NF-ĸB nuclear translocation through recovering or stabilizing the inhibitory IĸB proteins. Vioprolide A, however, decreased rather than stabilized the IĸB proteins and prevented NF-ĸB nuclear translocation through interfering with its importin-dependent nuclear import. By performing siRNA-mediated knockdown experiments, we evaluated the role of NOP14 in inflammatory processes in HUVECs and could establish a causal link between the anti-inflammatory actions of vioprolide A and the deletion of NOP14.
Besides exerting anti-inflammatory actions, we found that vioprolide A potently decreased the angiogenic key features proliferation, migration and sprouting of endothelial cells. Mechanistically, the natural product interfered with pro-angiogenic signaling pathways. Vioprolide A reduced the protein level of growth factor receptors, including VEGFR2, which is the most prominent receptor responsible for angiogenic signaling in endothelial cells. This effect was based on the general inhibition of de novo protein synthesis by the natural product. Downregulation of growth factor receptors impaired the activation of downstream signaling intermediates, including the MAPKs ERK, JNK and p38. To our surprise, however, activation of Akt, another downstream effector of VEGFR2, was increased rather than decreased. Furthermore, vioprolide A lowered the nuclear translocation of the transcriptional coactivator TAZ, which is regulated by the evolutionary conserved Hippo signaling pathway. Interestingly, however, and in contrast to NF-ĸB, TAZ nuclear translocation in mammalian cells seems to be independent of importins. In this context, we found that vioprolide A reduced both the protein level and nuclear localization of MAML1, which is needed to retain TAZ in the nucleus after its successful translocation.
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Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
Sphingosin 1 Phosphat (S1P) ist ein wichtiger Lipidmediator, der über G Protein gekoppelte Rezeptoren und intrazelluläre Wirkungen vielfältige Wirkungen auslöst und eine Rolle bei der Lymphozytenzirkulation, der Erhaltung der endothelialen Barriere, bei Entzündungsprozessen und Tumorwachstum spielt. Die S1P Lyase (Sgpl1) katalysiert den irreversiblen Abbau von S1P und damit den letzten Schritt des Sphingolipidkatabolismus‘. Ein Fehlen der Sgpl1 bewirkt eine Akkumulation von S1P und anderen Sphingolipiden im Blut und Gewebe, was multiple Organschäden zur Folge hat. Menschen mit S1P Lyase Insuffizienz Syndrom (SPLIS) leiden insbesondere unter steroidresistentem nephrotischem Syndrom, Nebennierenrinden-insuffizienz und neurologischen Störungen. Weitere mögliche Symptome sind Lymphopenie, Hautveränderungen und Dyslipidämien. S1P Lyase defiziente Mäuse weisen sehr ähnliche Organschädigungen auf.
An Sgpl1 Knockoutmäusen war zuerst die massive Akkumulation nicht nur von Sphingolipiden, sondern auch von Cholesterin und Triglyceriden in Blut und Leber aufgefallen. Auch bei SPLIS Patienten wurde eine Hypercholesterinämie beobachtet. Um die Kreuzregulation des Sphingolipid- und Cholesterinmetabolismus besser zu verstehen, sollte die Rolle der Sgpl1 in der Leber, dem Hauptort des Lipidmetabolismus, untersucht werden. Hierzu sollte ein Mausmodell mit einem hepatozytenspezifischen Sgpl1 Knockout (Sgpl1HepKO) etabliert und charakterisiert werden. Dies wurde durch Kreuzen von Sgpl1fl/fl-Mäusen mit Mäusen, welche die Cre-Rekombinase unter dem Albuminpromoter exprimierten, erreicht. Die basale Charakterisierung zeigte, dass diese Mäuse im Gegensatz zu globalen Sgpl1 Knockoutmäusen sowohl im Alter von acht Wochen, als auch im Alter von acht Monaten einen unauffälligen Phänotyp aufwiesen. Das äußere Erscheinungsbild inklusive Leber und Körpergewicht, das Blutbild, die Leberenzyme sowie die Histologie der Leber waren unverändert. Die Analyse der Leberlipide mit Hilfe von Hochleistungsflüssigkeits-chromatographie gekoppelt mit einer Tandem Massenspektrometrie zeigte eine signifikante Akkumulation (≈1,5 2 fach) von S1P, Sphingosin und Ceramiden, aber nicht von Glucosylceramiden und Sphingomyelin in der Leber. Messungen im Plasma zeigten eine Erhöhung mehrerer Ceramide, während der S1P Spiegel normal war. Ferner zeigten Untersuchungen der Galle signifikant erhöhte Konzentrationen an S1P, Dihydro S1P und Glucosylceramiden, jedoch unveränderte Ceramide. Die Ergebnisse legen folgende Schlussfolgerungen nahe: 1. In der Leber kann mit Hilfe von Ceramidsynthasen akkumulierendes Sphingosin in Ceramide umgewandelt werden, welche anschließend ins Blut sezerniert und letztendlich vermutlich von anderen Zellen verstoffwechselt werden. Außerdem ist nicht ausgeschlossen, dass S1P ebenfalls ins Blut sezerniert und dort effektiv abgebaut wird, so dass die S1P Konzentration im Plasma unverändert bleibt. 2. S1P sowie Glucosylceramide werden an die Galle abgegeben und ausgeschieden. 3. Die Sgpl1 in der Leber ist nicht essentiell für die Regulation des Plasma S1Ps, was zuvor vermutet worden war
Eine Analyse der Sterole zeigte in Sgpl1HepKO Mäusen erhöhte Spiegel an Cholesterin und Desmosterol in der Leber. In Übereinstimmung mit der erhöhten Proteinexpression des low density lipoprotein (LDL ) Rezeptors und erniedrigten Konzentrationen des LDL Cholesterins im Plasma, deuten diese Daten auf eine erhöhte Aufnahme von LDL Cholesterin durch die Leber hin. Untersuchungen in der Leber sowie mit primären Hepatozyten zeigten im Gegensatz zu globalen Sgpl1 Knockoutmäusen keine Veränderungen der Peroxisomen-Proliferator-aktiviertem Rezeptor γ Expression. Weitere Gene mit zentraler Rolle wie der Liver X receptor oder die Fettsäuresynthase, waren ebenfalls nicht reguliert. Dieser im Vergleich zu globalen Sgpl1-Knockoutmäusen milde Phänotyp lässt sich durch die deutlich geringere Akkumulation von Sphingolipiden aufgrund der oben beschriebenen Kompensations-mechanismen in Sgpl1HepKO Mäusen erklären.
In weiteren Untersuchungen sollten die Auswirkungen einer Sgpl1-Defizienz an Fibroblasten untersucht werden. Hierzu standen embryonale Fibroblasten aus Sgpl1 Knockoutmäusen zur Verfügung (Sgpl1-/- MEFs). In einer Kooperation mit Dr. Janecke von der Universität Innsbruck standen außerdem humane Fibroblasten eines SPLIS Patienten zur Verfügung.
An Sgpl1-/- MEFs war zuvor eine gestörte Calciumhomöostase festgestellt worden, welche sich durch eine erhöhte zytosolische Calciumkonzentration und vermehrte Calciumspeicherung im Endoplasmatischen Retikulum und in Lysosomen auszeichnete. Die Plasmamembran-Calcium ATPase (PMCA) trägt an Fibroblasten entscheidend zur Regulation der zytosolischen Calciumkonzentration bei. Ihre Expression auf Proteinebene war jedoch in Sgpl1-/- MEFs nicht verändert. Im Rahmen dieser Arbeit wurde durch eine Immunfärbung erstmals festgestellt, dass die PMCA in Sgpl1-/- MEFs nicht vollständig an der Plasmamembran lokalisiert war. Dies könnte der Grund für die erhöhte zytosolische Calciumkonzentration in den Zellen sein. ...