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Die Aufdeckung krankheitsbedingter Unterschiede und die Identifizierung neuer Biomarker sind essenziell für Diagnose und Behandlung verschiedener Erkrankungen. Unterschiede zwischen Erkrankungen können u.a. durch Analyse des Lipidprofils aufgedeckt werden, da dieses eng mit dem Phänotyp verknüpft ist. Ein unvoreingenommenes Screening gewährt einen umfassenderen Einblick in den metabolischen Zustand als eine gezielte Untersuchung weniger Analyten und kann neue Hypothesen generieren. Deshalb wurde im Rahmen dieser Arbeit eine Screening-Methode zur untargeted Untersuchung des Lipidoms in biologischen Proben entwickelt. Durch die Kombination aus Umkehrphasenchromatographie und hochauflösender Massenspektrometrie mit datenabhängiger Aufnahme von MS/MS-Spektren konnten in Humanplasma 440 Lipide aus mehr als 15 Lipidklassen identifiziert werden. Die mehrstufige Identifizierung der Analyten, basierend auf der exakten Masse ±5 ppm, der Isotopenverteilung, der MS/MS-Fragmentierungsmuster in beiden Ionisationsmodi sowie der chromatographischen Auftrennung von Isomeren und Isobaren, erfolgte mit hoher Selektivität. Mit der vorgestellten Methode können sowohl Lipidklassen als auch einzelne Lipide relativ zu den internen Standards quantifiziert werden.
Der Probendurchsatz wurde erhöht, um den Einsatz der Methode im Rahmen größerer klinischer Studien zu ermöglichen und vorhandene Ressourcen effizient einzusetzen. Dabei wurden die Inkubationszeiten während der Flüssig-Flüssig-Extraktion mit MTBE:Methanol deutlich reduziert und die Handhabung vereinfacht bei gleichbleibend hoher Wiederfindung. Der hohe Probendurchsatz wird weiter unterstützt durch die kurze chromatographische Laufzeit von 17 min pro Ionisationsmodus. Die Auswertung der Ergebnisse ist der heikelste und zeitintensivste Schritt bei der Entwicklung und Anwendung von Screening-Methoden, deshalb wurde der Arbeitsablauf zur univariaten Analyse durch Entwicklung von R Skripten vereinfacht und beschleunigt.
Die Qualität und Reproduzierbarkeit der Ergebnisse sind essenziell. Aus diesem Grund wurde die Qualität der entwickelten Methode, angelehnt an den strikten Vorgaben der FDA und EMA zur Validierung von quantitativen Methoden, sichergestellt, obwohl eine Methodenüberprüfung im Bereich von untargeted Methoden nicht verbreitet ist. Die Reproduzierbarkeit der relativen Lipidkonzentrationen konnte z.B. durch die Messung von Kontrollplasmaproben über einen Zeitraum von 10 Monaten gezeigt werden. Außerdem wurde die Linearität der Verdünnung von Plasmaproben bestätigt und eine Verschleppung in darauffolgende Proben ausgeschlossen. Die Stabilität der Proben muss in jeder Messphase inklusive der Präanalytik durch geeignete Untersuchungen und Maßnahmen sichergestellt werden. Anhand einer Studie zur präanalytischen Stabilität humaner Blutproben konnte ein Protokoll zur Probennahme und -vorbereitung für weitere klinische Studien erarbeitet werden. Die Stabilität des Lipidoms in Vollblut und Plasma konnte durch den Einsatz von Natriumfluorid/Citrat als Antikoagulans verbessert werden. Auch die Stabilität der Proben während der Lipidextraktion und Messung konnte gezeigt werden. Es wurden 16 verschiedene Probenarten analysiert, darunter Plasmaproben, verschiedene Mausgewebe und Zellpellets.
Mit der entwickelten Methode wurden die Unterschiede im Lipidprofil im Plasma und Gewebe von Mäusen mit einer akuten Entzündung durch LPS bzw. Zymosan-Injektion aufgedeckt. Dabei wurden die Ether-Phosphatidylcholine als potenzielle Entzündungsmarker identifiziert. Die entwickelte Methode wurde außerdem erfolgreich im Rahmen anderer Arbeiten für die Untersuchung verschiedener Erkrankungen angewendet.
In der vorliegenden Arbeit wird demnach eine schnelle, reproduzierbare und vor allem selektive LC-MS-Screening-Methode vorgestellt, die Veränderungen des Lipidstoffwechsels aufdecken und potenzielle Biomarker identifizieren kann.
Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.
Ubiquitination is regarded as one of the key post-translational modifications in nearly all biological processes, endowed with numerous layers of complexity. Deubiquitinating enzymes (DUBs) dynamically counterbalance ubiquitination events by deconjugating ubiquitin signals from substrates. Dysregulation of the ubiquitin code and its negative regulators drive various pathologies, such as neurological disorders and cancer.
The DUB ubiquitin-specific peptidase 22 (USP22) is well-known for its essential role in the human Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, mediating the removal of monoubiquitination events from Histone 2A and 2B (H2A and -B), thereby regulating gene transcription. In cancer, USP22 was initially described as a part of an 11-gene expression signature profile, predicting tumor metastasis, reoccurrence and death after therapy in a wide range of tumor cells. However, novel roles for USP22 have emerged recently, accrediting USP22 essential roles in regulating tumor development as well as apoptotic cell death signaling.
One of the hallmarks of cancer is the evasion of cell death, especially apoptosis, a form of programmed cell death (PCD). Necroptosis, a regulated form of necrosis, is regarded as an attractive therapeutic strategy to overcome apoptosis-resistance in tumor cells, although a profound understanding of the exact signaling cascade still remains elusive. Nevertheless, several ubiquitination and deubiquitination events are described in fine-tuning necroptotic signaling.
In this study, we describe a novel role for USP22 in regulating necroptotic cell death signaling in human tumor cell lines. USP22 depletion significantly delayed TNFa/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFa-induced nuclear factor-kappa B (NF-KB) signaling or TNFa-mediated extrinsic apoptosis. Intriguingly, re-expression of USP22 wildtype in the USP22 knockout background could re-sensitize HT-29 cells to TBZ-induced necroptosis, whereas re-constitution with the catalytic inactive mutant USP22 Cys185Ser did not rescue susceptibility to TBZ-induced necroptosis, confirming the USP22 DUB-function a pivotal role in regulating necroptotic cell death. USP22 depletion facilitated ubiquitination and unexpectedly also phosphorylation of Receptor-interacting protein kinase 3 (RIPK3) during necroptosis induction, as shown by Tandem Ubiquitin Binding Entities (TUBE) pulldowns and in vivo (de)ubiquitination immunoprecipitations. To substantiate our findings, we performed mass-spectrometric ubiquitin remnant profiling and identified the three novel USP22-regulated RIPK3 ubiquitination sites Lysine (K) 42, K351 and K518 upon TBZ-induced necroptosis. Further assessment of these ubiquitination sites unraveled, that mutation of K518 in RIPK3 reduced necroptosis-associated RIPK3 ubiquitination and additionally affected RIPK3 phosphorylation upon necroptosis induction. At the same time, genetic knock-in of RIPK3 K518R sensitizes tumor cells to TNFa-induced necroptotic cell death and amplified necrosome formation.
In summary we identified USP22 as a new regulator of TBZ-induced necroptosis in various human tumor cell lines and further unraveled the distinctive role of DUBs and (de)ubiquitination events in controlling programmed cell death signaling.
Two salts of the 6,6-difluoro-6H-dibenzo[c,e][1,2]oxaborinin-6-ide anion with different cations
(2020)
The crystal structures are reported of the 6,6-difluoro-6H-dibenzo[c,e][1,2]oxaborinin-6-ide (or 9,9-difluoro-10-oxa-9-boraphenanthren-9-ide) anion with two different cations, namely, potassium 6,6-difluoro-6H-dibenzo[c,e][1,2]oxaborinin-6-ide, K+·C12H8BF2O−, (II), featuring a polymeric structure, and bis(tetraphenylphosphonium) bis(6,6-difluoro-6H-dibenzo[c,e][1,2]oxaborinin-6-ide) acetonitrile trisolvate, 2C24H20P+·2C12H8BF2O−·3CH3CN, (III), which is composed of discrete cations, anions and acetonitrile solvent molecules linked by C—H...O, C—H...N and C—H...F hydrogen bonds. There are only minor differences in the geometrical parameters of the anions in these structures.
Transfer RNA fragments replace microRNA regulators of the cholinergic poststroke immune blockade
(2020)
Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a “changing of the guards” between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level.
Transfer RNA fragments replace microRNA regulators of the cholinergic post-stroke immune blockade
(2020)
Stroke is a leading cause of death and disability. Recovery depends on balance between inflammatory response and immune suppression, which can be CNS-protective but may worsen prognosis by increasing patients’ susceptibility to infections. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Therefore, we sought small RNA balancers of the cholinergic anti-inflammatory pathway in peripheral blood from ischemic stroke patients. Using RNA-sequencing and RT-qPCR, we discovered in patients’ blood on day 2 after stroke a “change of guards” reflected in massive decreases in microRNAs (miRs) and increases in transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by RT-qPCR validated the top 6 upregulated tRFs in a separate cohort of stroke patients, and independent small RNA-sequencing datasets presented post-stroke enriched tRFs as originating from lymphocytes and monocytes. In these immune compartments, we found CD14+ monocytes to express the highest amounts of cholinergic transcripts. In-depth analysis of CD14+ regulatory circuits revealed minimally overlapping subsets of transcription factors carrying complementary motifs to miRs or tRFs, indicating different roles for the stroke-perturbed members of these small RNA species. Furthermore, LPS-stimulated murine RAW264.7 cells presented dexamethasone-suppressible upregulation of the top 6 tRFs identified in human patients, indicating an evolutionarily conserved and pharmaceutically treatable tRF response to inflammatory cues. Our findings identify tRF/miR subgroups which may co-modulate the homeostatic response to stroke in patients’ blood and open novel venues for establishing RNA-targeted concepts for post-stroke diagnosis and therapeutics.
Transfer RNA fragments replace microRNA regulators of the cholinergic post-stroke immune blockade
(2020)
Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced two days after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by RT-qPCR validated the top 6 upregulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes upregulated the top 6 stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using an ssRNA mimic induced downregulation of immune regulator Z-DNA binding protein 1 (Zbp1). In summary, we identified a “changing of the guards” between RNA types that may systemically affect homeostasis in post-stroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein- and RNA-level.
Significance Statement Ischemic stroke triggers peripheral immunosuppression, increasing the susceptibility to post-stroke pneumonia that is linked with poor survival. The post-stroke brain initiates intensive communication with the immune system, and acetylcholine contributes to these messages; but the responsible molecules are yet unknown. We discovered a “changing of the guards,” where microRNA levels decreased but small transfer RNA fragments (tRFs) increased in post-stroke blood. This molecular switch may re-balance acetylcholine signaling in CD14+ monocytes by regulating their gene expression and modulating post-stroke immunity. Our observations point out to tRFs as molecular regulators of post-stroke immune responses that may be potential therapeutic targets.
Certain electron-rich 1,4-diborabenzene derivatives efficiently activate single, double, and triple bonds and thereby increasingly compete with transition metals in homogeneous catalysis. This review compares the activation of three model substrates (H2, H2C=CH2, CO2) by (i) 9,10-dihydro-9,10-diboraanthracene dianions, (ii) their neutral carbene-stabilized congeners, (iii) 1,3,2,5-diazadiborinines, and (iv) 1,4,2,5-diazadiborinines. Distinct structure-properties relationships become apparent, the most influential factors being (i) the steric demands of the B-bonded substituents, (ii) the charges on the B-doped (hetero)arenes, (iii) charge polarization as a result of additional N-doping, and (iv) the energies and nodal structures of the frontier orbitals. The observed reactions are explained by a transition metal-like activation mechanism. If the two boron atoms are chemically inequivalent, contributions of a B(+I)/B(+III) mixed-valence state determine the observed regioselectivities when polar substrates are added. The lessons learned from the conversions of the model substrates are subsequently used to rationalize the behavior of the B2 heterocycles also toward more sophisticated substrate molecules. Finally, catalytic cycles based on H2- and H−-transfers, hydroboration reactions, and CO2 reductions will be covered.
Background: Zolpidem is a non-benzodiazepine hypnotic agent which has been shown to be effective in inducing and maintaining sleep in adults and is one of the most frequently prescribed hypnotics in the world. For drugs that are used to treat sleeping disorders, the time to reach the maximum concentration (Tmax) of the drug in plasma is important to achieving a fast onset of action and this must be maintained when switching from one product to another.
Objectives: The main objective of the present work was to create a PBPK/PD model for zolpidem and establish a clinically relevant “safe space” for dissolution of zolpidem from the commercial immediate release (IR) formulation. A second objective was to analyze literature pharmacokinetic data to verify the negative food effect ascribed to zolpidem and consider its ramifications in terms of the “safe space” for dissolution.
Methods: Using dissolution, pharmacokinetic and pharmacodynamic data, an integrated PBPK/PD model for immediate release zolpidem tablets was constructed in Simcyp®. This model was used to identify the clinically relevant dissolution specifications necessary to ensure efficacy.
Results: According to the simulations, as long as 85% of the drug is released in 45 minutes or less, the impact on the PK and PD profiles of zolpidem would be minimal. According to the FDA, the drug has to dissolve from the test and reference products at a similar rate and to an extent of 85% in not more than 30 minutes to pass bioequivalence via the BCS-biowaiver test. Thus, the BCS-biowaiver specifications are somewhat more stringent than the “safe space” based on the PBPK/PD model. Published data from fasted and fed state pharmacokinetic studies suggest but do not prove a negative food effect of zolpidem.
Conclusions: A PBPK/PD model indicates that current BCS biowaiver criteria are more restrictive for immediate release zolpidem tablets than they need to be. In view of the close relationship between PK and PD, it remains advisable to avoid taking zolpidem tablets with or immediately after a meal, as indicated by the Stilnox® labeling.
Currently, due to the misuse of antibiotics, we are facing a major public health problem. The resistance to antibiotics of certain bacterial strains makes the treatment of infections very complex.
In this context, the present thesis project concerns the study of a bacterial efflux complex capable of transporting antibiotics from the cytoplasm to the outside of the cell. This complex is composed of an inner-membrane Major Facilitator Superfamily (MFS) transporter (EmrB, E. coli multidrug resistance), a channel of the outer membrane TolC (Tolerance to Colicin E1) and a periplasmic adapter (EmrA, E. coli multidrug resistance). Unlike RND-type efflux systems (such as AcrAB-TolC), little is known about the MFS-type EmrAB-TolC system. It is therefore important to study the entire complex on a structural and functional level, to analyse the marked differences between these two types of transport systems. The goal of my thesis project was to study at least one EmrAB-TolC complex from a structural point of view. For my studies the aim was to isolate the complex directly from bacteria overexpressing the three protein partners. In a first step, 15 homologous EmrAB-TolC systems were identified and their corresponding genes amplified from genomic DNA of different Gram-negative bacteria. Among the genes of the 15 systems, the genes coding for the E. coli and V. cholerae systems were further studied. The expression vectors encoded fluorescent markers for the monitoring of the expression levels of different proteins and for studying the formation of complexes. In a first step, the different protein expression levels (EmrB-mRFP1 and EmrA-sfGFP) were studied for several expression strains of E. coli by measuring the red and green fluorescence levels and by Western blot (anti-His, Myc, and Strep for EmrB, EmrA, and TolC). The E. coli strain C41(DE3) was best suited for co-expression of EmrAB-TolC. In a second step, the FSEC (Fluorescence detection Size Exclusion Chromatography) methodology was used to identify a complex suitable for structural study. Thus this method enabled the observation that the EmrAB-TolC complex of E. coli was produced in higher amount than that of V. cholerae. The final co-purification protocol consists in perfoming a gentle lysis of the bacteria using lysozyme, then after solubilization with DDM, the purification is started by a Ni2+-NTA affinity chromatography step followed by a size exclusion chromatography step. Finally, the fractions containing the three protein partners are used for the detergent-exchange by amphipol A8-35 before the structural study by electron microscopy. Negative stain EM-micrographs displayed elongated objects with a length of 33 nm in side view. An average image of EmrAB-TolC shows similarities to that of the AcrAB-TolC complex observed under similar conditions. Similarities included the characteristic densities of TolC. Whereas differences were found in the lower part of EmrAB which is thinner than the lower part of AcrAB. The densities visible above the amphipol-ring correspond to EmrA, which displays a channel-like structure as in AcrA. The channel however seems to extend further towards the amphipol belt. Since EmrB does not have an extended periplasmic domain as the RND proteins have, these densities are therefore solely assigned to EmrA. EmrA, on the other side, contacts TolC akin to the interaction of AcrA/MexA to their cognate outer membrane channels (TolC/OprM) in a ‘tip-to-tip’ fashion.