1,721,054 research outputs found
How to Tell an N from an O: Controlling the Chemoselectivity of Methyltransferases
No full textS-Adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) are important enzymes in numerous biological pathways. They share a common SN2 mechanism but act on different nucleophilic substrates in vivo. Therefore, MTs have a specific chemoselectivity to transfer CH3 onto the correct atom type and substrate. Caffeate O-MT from Prunus persica (PpCaOMT) and anthranilate N-MT from Ruta graveolens (RgANMT) share a high similarity regarding their amino acid sequence (>74%). Nevertheless, the physiological substrates (caffeate vs anthranilate) and attacking nucleophiles (hydroxyl vs amino group) are strikingly different. We demonstrate that the differing chemoselectivity is governed by different conformational states of the two enzymes. O-Methylation catalyzed by CaOMTs requires a “closed” conformation, whereas ANMTs perform N-methylation in an “open” state. We rationally designed seven variants for both PpCaOMT and RgANMT, which changed their original nucleophile preference to different extents, up to a full inversion. Interestingly, the generated O-selective ANMT variant catalyzes O-methylation considerably faster than wildtype CaOMT. Molecular dynamics (MD) simulations and hydrogen/deuterium exchange mass spectrometry (HDX-MS) experiments showed that the mutations induced changes in the conformational dynamics of the enzyme variants and by modulating the open/closed transitions impact the corresponding chemoselectivity. Our data show that the selectivity of the methyl transfer reaction is not solely governed by the key residues directly involved in the methyl transfer but is rather synergistically modulated by the conformational dynamics of the enzyme and reaction conditions
Modifying the makers: release factor hydroxylation, non-canonical nucleosides, and their influence on translation
No full textTranslation is a central process in cell metabolism and growth; tightly regulated, it involves a multitude of factors acting in a concerted manner to ensure accurate protein synthesis. With the majority of cellular participants and their respective roles in the process identified, research has increasingly shifted towards modifications of the translational machinery, both innate and exogenously introduced.Part one of this thesis further characterises the influence of Jumonji-domain containing protein 4 (JMJD4)-mediated eukaryotic release factor 1 (eRF1) hydroxylation on translation termination: Prior reports have posited an accuracy-enhancing effect due to lysyl hydroxylation of the stop codon recognising domain of the release factor. Using bicistronic reporter assays in vivo, the proposed unidirectional effect irrespective of the termination sequence was refuted in favour of a sequence-dependent role. Additionally, JMJD4 activity was confirmed in vitro through direct detection of the hydroxylated product by means of mass spectrometry-coupled liquid chromatography (LC-MS). Potential inhibitors were identified computationally and using surrogate assays, the methods establishedfor confirmation of enzymatic activity were then refined to test the identified substances.In the second part, non-canonical nucleotides were used for in vitro transcriptions and their incorporation by a viral RNA polymerase was tested. To this end, an LC-MS method amenable to adaptation for a wide variety of modified nucleosides was developed; its application revealed successful incorporation of three non-canonical nucleotides in substantial amounts: 7-deazaadenosine (7-Deaza-A), N6-methyladenosine (m6A), and 2-fluoroadenosine (2-F-A). Their effects on translational accuracy were tested using newly designed bicistronic reporter vectors for ribosomal frameshifting and sense codon misrecognition in addition to a previously generated readthrough reporter. The observed reduction in frameshifting rates for 7-Deaza-A were further elucidated via circular dichroism spectroscopy, revealing a potentially causative, destabilising effect on the RNA pseudoknot necessary for efficient frameshifting.In the context of the recently developed mRNA-based COVID-19 vaccines containing a non-canonical nucleotide, N1-methylpseudouridine, the translatability of mRNA transcribed with 7-Deaza-A and 2-F-A was tested in human cells. To this end, new reporter vectors encoding enhanced green fluorescent protein (EGFP) were developed and tested in vitro, followed by the transfection of the modified mRNA. Production of EGFP in cellulo was confirmed and analysis of extracted cellular nucleic acids gave insight into the metabolic fate of the modified mRNA.The results presented in this work highlight several possibilities to influence the process of translation, while the methods developed serve as important tools to identify additional avenues.Translation ist ein zentraler Prozess des Zellmetabolismus und -wachstums. Sie wird streng reguliert und umfasst eine Vielzahl von Faktoren, die im Zusammenspiel miteinander eine präzise Proteinproduktion gewährleisten. Nach Identifikation der meisten Beteiligten und deren jeweiliger Rolle rücken deren in- und extrinsische Modifikationen zunehmend in den Fokus der Forschung.Für den ersten Teil dieser Arbeit wurde der Einfluss der JMJD4-vermittelten Hydroxylierung des eukaryotischen Releasefaktors 1 (eRF1) auf die Terminationsgenauigkeit näher untersucht: In vorangegangenen Arbeiten wurde eine stets unidirektionale Steigerung der Akkurazität durch Lysylhydroxylierung der Pseudoanticodon-Domäne von eRF1 postuliert; im Gegensatz dazu konntehier mit bicistronischen Reporterassays eine sequenzabhängige Änderung festgestellt werden. Sich daran anschließende in vitro Arbeiten zielten auf den Nachweis der enzymatischen Aktivität von JMJD4 mittels direkter Detektion des hydroxylierten Produkts ab. Die hierfür entwickelte Methode für Flüssigkeitschromatografie mit Massenspektrometrie-Kopplung (LC-MS) wurde dann dazu adaptiert, zuvor ausgewählte Substanzen auf deren inhibitorische Wirkung auf die enzymatische Aktivität von JMJD4 zu testen.Im zweiten Teil wurden nicht-kanonische Nukleotide (nc-NTP) als Substrat für in vitro Transkriptionen mittels einer viralen RNA-Polymerase genutzt. Mit einer eigens entwickelten, versatilen LC-MS-Methode konnte der Einbau dreier nc-NTP nachgewiesen werden: 7-Deazaadenosin (7-Deaza-A), N6-Methyladenosin (m6A) und 2-Fluoradenosin (2-F-A). Potenzielle Auswirkungen der Modifikationen auf die Akkurazität der Translation wurden mit eigens entworfenen bicistronischen Reportervektoren für ribosomales Frameshifting und Fehlererkennung von Stopp- und Sensecodons untersucht. Die dabei beobachtete Reduktion der Frameshifting-Häufigkeit im Falle von 7-Deaza-A wurde mit Hilfe der Circulardichroismus-Spektroskopie weiter untersucht, wobei eine potenziell ursächliche, destabilisierende Wirkung auf die für Frameshifting erforderliche RNA-Struktur festgestellt wurde. Vor dem Hintergrund der mRNA-basierten COVID-19-Impfstoffe, die das nicht-kanonisches Nukleotid N1-Methylpseudouridin enthalten, wurde die Translatierbarkeit der mit 7-Deaza-A und 2-F-A erzeugten mRNA in menschlichen Zellen getestet. Zu diesem Zweck wurden neue Reportervektoren entwickelt, die für grün fluoreszierendes Protein (EGFP) kodieren, und zunächst in vitro getestet.Anschließend konnte die Bildung von EGFP nach Transfektion von Zellen mit modifizierter mRNA bestätigt und die Verstoffwechselung der modifizierten mRNA nachvollzogen werden.Die in dieser Arbeit vorgestellten Ergebnisse verdeutlichen die vielfältigen Möglichkeiten zur Beeinflussung des Translationsprozesses, die hier entwickelten Methoden können dabei als Werkzeuge für weitere Forschung dienen
Targeting the deacylases SmSirt2 and hSirt2: inhibitors discovery and optimization
No full textThe NAD+ dependent lysine deacylase Sirtuin family catalyzes the removal of various modifications from histone and non histone proteins, playing a wide spectrum of biological roles which have been correlated with several diseases including cancer. Besides a robust deacetylase activity, Sirtuin2 (hSirt2) shows both in vitro and in cells defatty acylase activity, which it exerts with a higher catalytic efficiency than deacetylation. A number of inhibitors, able to address both hSirt2 mediated deacetylation and defatty acylation, have shown anticancer effects compared to selective deacetylase inhibition. Such beneficial effects presumably arise from the additional inhibition of hSirt2 defatty acylation. However, its biological implications are still under debate for therapeutic application. Together with other epigenetic regulators, Sirtuins play a vital role in the life-cycle of parasites like Schistosoma mansoni and targeting these enzymes has been considered for potential treatment of schistosomiasis. Among the five isotypes identified, SmSirt2 appeared as a promising target, and the first SmSirt2 inhibitors showing antiparasitic activity have been reported.This work presents Structure Activity Relationship (SAR) studies conducted with collaboration partners of an indole based scaffold TCMDC 143159 previously identified as a SmSirt2 inhibitor. This led to optimized derivatives with enhanced potency against the parasitic target (IC50 values in the low micromolar range). A selectivity towards the human ortholog and low cytotoxicity were also observed for optimized SmSirt2 inhibitors investigated, as generally required for antiparasitic agents. A number of analogs were also active against hSirt2 deacetylation or demyristoylation, potentially serving for the future development of hSirt2 inhibitors with anticancer activity.A 1,2,4 oxadiazole based compound TCMDC 143362 had been also identified as SmSirt2 inhibitor, displaying even a higher potency against hSirt2. Therefore, further studies have been carried out to discover more potent and potential anticancer molecules against hSirt2, as well as to eventually develop selective inhibitors of SmSirt2. Computer aided designed analogs have been synthesized and their inhibitory activity evaluated in vitro. All analogs appeared to be optimized inhibitors of hSirt2 deacetylation with IC50 values in the submicromolar range and displayed promising activity (IC50 between 10-20 µM) against hSirt2 demyristoylation. According to docking experiments, supported by kinetic analyses, 1,2,4 oxadiazoles seemed to exert their inhibition via a substrate competitive and NAD+ non competitive binding mode, which does not appear to be affected by the stereochemistry of the scaffold. In correlation with the inhibition of hSirt2 observed in vitro and in cells, a selection of tested compounds reduced cell viability and inhibited cancer cell migration.From the screening of the “Kinetobox library”, the diphenyl urea scaffold TCMDC 143175 exhibited inhibition of hSirt2 mediated demyristoylation with a selectivity towards hSirt2 deacetylation, as well as the other robust deacetylase isotypes hSirt1 and hSirt3 in vitro. Further SAR studies led to a scaffold optimization (IC50 values in the low micromolar range). To explain this unprecedented case of selective inhibition of long chain deacylation, docking experiments suggested a ligand binding mode dependent on substrate hSirt2 bound conformations. These observations could then guide more efficiently future structural development
New inositol pyrophosphate prometabolites: specific organelle targeting, tissue penetration, and stable isotope labelling
No full textInositol pyrophosphates (PP-IP) are a group of highly phosphorylated signalling molecules in eukaryotes. Together with the less densely phosphorylated inositol phosphates (IP), they constitute a complex metabolic network. Research of the last three decades showed, that PP-IPs play a role in a vast variety of metabolic processes like apoptosis, vesicular traffic, mRNA degradation and have an important impact on energy homeostasis. The structural peculiarities of different densely phosphorylated isomers renders chemical synthesis approaches challenging. However, modified derivatives of PP-IPs can be highly valuable tools to gain new insights into their function and help to identify their role in complex signalling cascades.The prometabolite approach (originated form prodrugs) is a way to increase the messenger’s concentration in a controlled way in cellulo and trigger a response. The metabolic degradation and cellular function of the PP-IP is blocked by a photo labile protecting group, a so called photocage, until it is removed by light, which enables a controlled release after uptake. Cellular uptake is enhanced by masking the negative charges with lipophilic and biolabile protecting groups. The currently used coumarin based photocage DEACM requires UV light to be cleaved, which was replaced in this thesis by redshifted alternatives. Longer wavelengths are less phototoxic and have better tissue penetrating properties. Apart from blocking the messenger, the photocage can also serve as a reversible anchor point for further modifications. The alkyne modified and redshifted photocage DEAC450 was used to synthesize a new generation of prometabolites. Photocleavage was achieved with a cheap commercial purchased flashlight. Polycationic PAMAM dendrons were coupled via click chemistry to the prometabolite to promote cellular uptake and potentially improve the distribution in tissues. Furthermore, specific designed targeting octapeptoides (guanidiniumproline) were coupled to the photocage to achieve a specific localization in the nucleolus. The click chemistry approach to introduce modifications in the final step makes prometabolites easily adjustable to the need of the application and enable to answer new questions.To label biomolecules with stable isotopes is an efficient tool for observing their metabolism in vivo or generate standards for mass spectrometric analysis. A new group of 18O-phosphoramidites were used to synthesize isotope labelled PP-IPs and prometabolites. These references are important to assign and quantify inositol phosphates and pyrophosphates
Funktionelle Charakterisierung der cobalaminabhängigen Radical SAM Methyltransferase QCMT aus Methanoculleus thermophilus
No full textCharacterisation of family 2 polyphosphate kinases
No full textPolyphosphate kinases (PPK) are enzymes catalysing the reversible phosphorylation of nucleotides. PPK1 have initially been described to use nucleoside triphopshates (NTPs) such as adenosine triphosphate (ATP) to synthesise the long-chain-polymer polyphosphate (polyP) while PPK2 catalyse the reverse reaction. PPK2 are further classified based on phylogenetic analysis and their preferred substrate; PPK2-I phosphorylate nucleoside diphosphates, PPK2-II nucleoside monophosphates, and PPK2-III catalyse both phosphorylations.In the first part of the presented thesis, the structure-function interaction of PPK2 enzymes was investigated. Four enzymes of the PPK2-II subclass were biochemically characterised. Two of the enzymes were crystallised and the structures solved; for one of the enzymes, additional substrate bound structures could be obtained. The structures were assigned to stages of catalysis and allowed to hypothesise a catalytic cycle.In the second part, the thermodynamic characteristics of the ADP phosphorylation were investigated. To optimise conditions for the reaction, a proper knowledge of the thermodynamics of the reaction have to be at hand. Therefore, representative enzymes of PPK1 and PPK2-I were studied regarding their ability to synthesise ATP from ADP and vice versa. The observed thermodynamic equilibrium matched for all enzymes and Christoph Held from the TU Dortmund could qualitatively describe the reaction outcome using perturbation theory. To quantitatively apply such ePC-SAFT predictions, precisely described reactions with triphosphate and tetraphosphate were performed to generate data to adapt and improve future predictions of polyP containing reactions.Lastly, different PPK2 enzymes were combined with a nucleoside kinase (NK) to convert various natural and unnatural nucleosides to the corresponding NTPs. The cascade efficiently converted adenosine analogues and the rate of conversions were comparable to the single enzyme reaction. When converting cytidine and its modifications however, mediocre conversion was observed. This sets future perspectives for advancing research to further connect structure and functionality of the enzymes.Polyphosphatkinasen (PPK) sind Enzyme, die die reversible Phosphorylierung von Nukleotiden katalysieren. PPK1 wurden ursprünglich beschrieben als Enzyme, welche Nukleosidtriphosphate (NTPs), wie zum Beispiel Adenosintriphosphat (ATP), für die Synthese des langkettigen Polymers Polyphosphat (polyP) verwenden, wohingegen PPK2 die umgekehrte Reaktion katalysieren. PPK2 werden basierend auf einer phylogenetischen Analyse und des präferierten Substrates, weiter unterteilt; PPK2-I phosphorylieren Nukleosiddiphosphate, PPK2-II Nukleosidmonophosphate und PPK2-III katalysieren beide Phosphorylierungen.Im ersten Teil der vorliegenden Arbeit wurden die Struktur-Funktions Beziehungen von PPK2 Enzymen untersucht. Vier Enzyme der PPK2-II Unterklasse wurden biochemisch charakterisiert. Zwei der Enzyme konnten kristallisiert und deren Struktur gelöst werden, für eines konnten zusätzliche substratgebundene Strukturen erhalten werden. Diese Strukturen wurden Katalyseschritten zugeordnet und erlaubten einen Katalysezyklus vorherzusagen.Im zweiten Teil wurden die thermodynamischen Charakteristika der Reaktion untersucht. Um die Bedingungen der Phosphorylierungsreaktion zu optimieren, bedarf es entsprechendem Wissen über die Thermodynamik der Reaktion. Hierfür wurden repräsentative Enzyme der PPK1 und PPK2-I verwendet und hinsichtlich ihrer Fähigkeit untersucht, ATP aus ADP zu synthetisieren und vice versa. Das beobachtete thermodynamische Gleichgewicht war identisch für alle Enzyme und konnte von Christoph Held mittels Störungstheorie qualitativ beschrieben werden. Um solche ePC-SAFT Vorhersagen auch quantitativ anzuwenden, wurden genau definierte Experimente mit Triphosphat und Tetraphosphat durchgeführt, um notwendige Daten zu erhalten und zukünftige Vorhersagen für polyP beinhaltende Reaktionen zu verbessern.Schlussendlich wurden verschiedene PPK2 mit einer Nukleosidkinase (NK) kombiniert um diverse natürliche und unnatürliche Nukleoside in die entsprechenden NTPs zu konvertieren. Die Kaskade konnte effizient Adenosin und Adenosinderivate mit einer, zu der Einzel-Enzym-Reaktion vergleichbaren Effizienz, umsetzen. Bei der Umsetzung von Cytidin und dessen Analoga wurde jedoch eine eingeschränkte Umsetzung beobachtet. Das bildet die Grundlage für weitere Untersuchungen um Zusammenhänge von Struktur und Funktionalität der Enzyme zu verstehen
Using the CovPDB database as a basis for the HyperCys machine learning algorithm to simplify covalent drug design
No full textIn recent years, the drug discovery paradigm has shifted toward compounds that covalently modify disease-associated nucleophilic proteins, because they tend to possess high potency, selectivity, and duration of action. The rational design of novel targeted covalent inhibitors (TCIs) typically starts from resolved macromolecular structures of nucleophilic proteins in their apo or holo forms. However, the existing TCI databases contain only a paucity of covalent protein–ligand (cP–L) complexes. In this respect, CovPDB was developed, the first database dedicated to high-resolution cocrystal structures of biologically relevant cP–L complexes, curated from the Protein Data Bank. For these curated complexes, the chemical structures and warheads of pre-reactive electrophilic ligands, as well as the covalent bonding mechanisms to their target nucleophilic proteins, were expertly manually annotated. Totally, CovPDB contains 733 proteins and 1,501 ligands, relating to 2,294 cP–L complexes, 93 reactive warheads, 14 targetable residues, and 21 covalent mechanisms. In addition, the application of the “quantum chemical electrophilicity index” (ω) for covalent warhead reactivity was investigated. In this work, a method was established that applies the concept of electrophilicity index to estimate the reactivity potential of covalent compounds targeting cysteine. For leadlike molecules (molecular weight > 250 Da), a truncation algorithm was applied before reactivity calculations. Whereas for compounds with molecular weight (MW) below 250 Da, the electrophilicity index was directly used to estimate compound reactivity. A total of 148 covalent compounds with 8 different warheads were analyzed. This analysis demonstrated that the electrophilicity index calculated for compounds containing vinyl carbonyl, aldehyde, nitrile, and ketone warheads showed no correlation with binding affinity in our dataset. This suggested that while electrophilicity is an important factor in determining the reactivity of covalent warheads, it may not be the direct factor affecting binding affinity. Among the 14 targetable residues in CovPDB database, the cysteine side chain has a free thiol group, making it the amino acid residue most often covalently modified by TCIs, thereby prolonging on-target residence time and reducing the risk of idiosyncratic drug toxicity. Hence, to identify targetable cysteines, a novel ensemble stacked machine learning (ML) model to predict hyper-reactive druggable cysteines, called HyperCys was developed. It is anticipated that HyperCys will be an effective tool for discovering new potential reactive cysteines in a wide range of nucleophilic proteins and will provide an essential contribution to designing TCIs with high potency and selectivity
Nuclear actin assembly is required for chromatin reorganization after mitosis as well as androgen receptor signaling
No full textActin is one of the most abundant and ubiquitously expressed proteins in eukaryotic cells. With its rapid dynamics alongside versatile protein interactions actin mediates a variety of different cytoskeletal functions. Next to canonical cytoplasmic functions reaching from cell migration or cell adhesion to vesicle trafficking, actin dynamics and assembly inside the nuclear compartment has emerged as a novel cell biology process. Our work has described nuclear actin assembly for cell cycle-dependent processes such as for chromatin reorganization during nuclear volume expansion in postmitotic daughter cell nuclei. Furthermore, we identified alpha actinin 4 (ACTN4) as a nuclear actin filament bundling factor required for nuclear volume expansion. Nevertheless, a lot of actin binding proteins present in the nucleus are associated with human diseases and the roles of the nuclear actin cytoskeleton for nuclear architecture and organization are still mostly unclear. To this end, exome sequencing revealed two individual patients with partial androgen-insensitivity syndrome (PAIS) that displayed mutations in the actin nucleator Disheveled associated activator of morphogenesis 2 (DAAM2), which led us to investigate a potential causative role for actin polymerization in androgen signaling. Using biochemical approaches, we found DAAM2 being essential for dihydrotestosterone (DHT)-dependent androgen receptor (AR)-activity. Furthermore in vitro co-sedimentation assays suggest a direct interaction of F-actin and the C-terminus of the AR. To elucidate this in more detail, we applied 3D structured illumination microscopy (SIM) on fixed cells isolated from patients in comparison to the respective control genital skin fibroblasts. We could visualize endogenous nuclear DAAM2 droplets that colocalized with the AR after androgen signaling, while this was significantly impaired in the patient samples. DHT-stimulated control cells were sensitive to low concentrations of the aliphatic alcohol 1,6-hexanediol as well as pharmacological interference with the actin depolymerizing drugs swinholide and latrunculin, arguing for a role of hydrophobic interactions as well as dynamic actin assembly, respectively. DAAM2 and AR droplets colocalized with active RNA Pol II in a DHT-dependent manner to form transcriptionally active biomolecular condensates that were again sensitive to hexanediol and swinholide. Live cell imaging with high spatiotemporal resolution revealed highly dynamic formin mediated nuclear actin assembly directly at the AR droplets after androgen signaling. Quantification of the transcriptional activity by PSA reporter gene assays displayed nuclear actin dependence and si-RNA resistant expression of wt DAAM2 partially rescued AR activity. Our data uncover cell cycle dependent F-actin assembly for chromatin reorganization as well as signal regulated transcriptional activity of a steroid hormone receptor
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