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ß-Hydroxybutyrate (BHB) is a ketone body formed in high amounts during lipolysis and fasting. Ketone bodies and the ketogenic diet were suggested as neuroprotective agents in neurodegenerative disease. In the present work, we induced transient ischemia in mouse brain by unilaterally occluding the middle cerebral artery for 90 min. BHB (30 mg/kg), given immediately after reperfusion, significantly improved the neurological score determined after 24 h. In isolated mitochondria from mouse brain, oxygen consumption by the complexes I, II and IV was reduced immediately after ischemia but recovered slowly over 1 week. The single acute BHB administration after reperfusion improved complex I and II activity after 24 h while no significant effects were seen at later time points. After 24 h, plasma and brain BHB concentrations were strongly increased while mitochondrial intermediates (citrate, succinate) were unchanged in brain tissue. Our data suggest that a single administration of BHB may improve mitochondrial respiration for 1–2 days but not for later time points. Endogenous BHB formation seems to complement the effects of exogenous BHB administration.
1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10
(2020)
The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.
The FUBP1-FUSE complex is an essential component of a transcription molecular machinery that is necessary for tight regulation of expression of many key genes including c-Myc and p21. FUBP1 utilizes its four articulated KH modules, which function cooperatively, for FUSE nucleotide binding. To understand molecular mechanisms fundamental to the intermolecular interaction, we present a set of crystal structures, as well ssDNA-binding characterization of FUBP1 KH domains. All KH1-4 motifs were highly topologically conserved, and were able to interact with FUSE individually and independently. Nevertheless, differences in nucleotide binding properties among the four KH domains were evident, including higher nucleotide-binding potency for KH3 as well as diverse nucleotide sequence preferences. Variations in amino acid compositions at one side of the binding cleft responsible for nucleobase resulted in diverse shapes and electrostatic charge interaction, which might feasibly be a contributing factor for different nucleotide-binding propensities among KH1-4. Nonetheless, conservation of structure and nucleotide-binding property in all four KH motifs is essential for the cooperativity of multi KH modules present in FUBP1 towards nanomolar affinity for FUSE interaction. Comprehensive structural comparison and ssDNA binding characteristics of all four KH domains presented here provide molecular insights at a fundamental level that might be beneficial for elucidating the mechanisms of the FUBP1-FUSE interaction.
Bislang sind die strukturellen Voraussetzungen für die Selektivität von Agonisten an den Retinoid Rezeptor Subtypen RXRα, RXRβ und RXRγ kaum erforscht, obwohl RXR-Modulatoren, die eine Subtypen-Präferenz aufweisen, aufgrund der unterschiedlichen Expressionsmuster der Subtypen Gewebe-spezifische Effekte vermitteln und somit Nebenwirkungen verringern könnten. Der Grund dieser Forschungslücke liegt teilweise darin, dass die Entwicklung Subtypen-selektiver RXR-Agonisten aufgrund der enormen strukturellen Ähnlichkeit der Ligandbindestellen in den RXR-Subtypen - alle Aminosäuren, die die Bindungsstellen bilden sind identisch - als unerreichbar angesehen wurde. Die Entdeckung des Naturstoffs Valerensäure als RXR-Agonist mit ausgeprägter Präferenz für den RXRβ-Subtyp hat jedoch gezeigt, dass Subtypen-selektive RXR-Modulation möglich ist249 und SAR-Studien an unterschiedlichen RXR-Ligand-Chemotypen haben in der Folge bestätigt, dass die Entwicklung von RXR-Liganden mit Subtypen-Präferenz erreicht werden kann.
Auf der Basis von Valerensäure und der in früheren Arbeiten entwickelten RXR-Agonisten wurden in dieser Arbeit Strukturmodifikationen identifiziert, die zu einer RXR-Subtypen-Präferenz beitragen. Durch die Verschmelzung dieser Strukturelemente ist es gelungen, einen neuen RXR-Agonist-Chemotyp (A) zu entwerfen, der durch strategische Methylierung und weitere Strukturmodifikationen zur Präferenz für jeden Subtyp optimiert werden konnte.
In einem Adipozyten-Differenzierungsexperiment konnte gezeigt werden, dass RXRα der wichtigste Heterodimer-Partner von PPARγ während der Adipogenese ist. Ferner unterstrich diese biologische Untersuchung das Potenzial von 99, 103 und 105 als Subtyp-präferentielle RXR-Agonisten in vitro Experimenten zu dienen.
Auf der Grundlage dieser Ergebnisse wurde eine mögliche Rolle von Acrylsäurepartialstrukturen natürlicher RXR-Liganden basierend auf dem zuvor entwickelten Chemotyp untersucht. Hierzu wurden das α-Methylacrylsäuremotiv des Naturstoffs Valerensäure (18) und das β-Methylacrylsäuremotiv des endogenen RXR-Agonisten 9-cis-Retinsäure in den Chemotyp A integriert (Chemotyp B), um die Rolle dieser Acrylsäuregruppen bei der Vermittlung der RXR-Subtypen-Selektivität zu untersuchen. Die Strukturmodifikationen an B zeigten, dass nur die α-Methyl-substituierte Acrylsäurekette toleriert bzw. von RXRβ präferiert wurde, was die RXR-Präferenz der Valerensäure (18) unterstützte.
In dieser Arbeit konnte gezeigt werden, dass RXR-Liganden mit Subtypen-Präferenz realisierbar sind und durch gezielte Strukturmodifikationen in ihrer Präferenz gesteuert werden können. Die Erkenntnisse zu den Struktur-Wirkungs-Beziehungen der neuen RXR-Agonist-Chemotypen A und B erweitern den Wissenstand über die strukturellen Voraussetzungen von RXR-Liganden für die Subtypen-Präferenz deutlich.
This cumulative dissertation examines learning in chemistry laboratories, focusing on the challenges and benefits of problem-based learning (PBL) for novices in the lab. It addresses the lack of consistent understanding about what should be learned in labs and why it's important. The research aims to understand what students learn, how they learn, and how lab learning can be improved.
A central concept in PBL labs is Information Literacy, defined as a sociocultural practice enabling learners to identify and use information sources within a specific context as legitimized by the practice community.
The first publication, Wellhöfer and Lühken (2022a), investigates the relationship between PBL and learner motivation. It identifies factors that can foster students' intrinsic motivation in a PBL lab. Autonomy is found to be a key factor, increasing student motivation and presenting a model of the autonomous scientific process. This model involves four steps: information acquisition, designing and applying experimental procedures, experimental feedback, and autonomous process optimization. The results suggest that intrinsic motivation in PBL labs can be enhanced by enabling students to independently execute these steps.
The second publication, Wellhöfer and Lühken (2022b), examines the information process students undergo during their first PBL lab. Using a sociocultural framework, it explores Information Literacy to understand students' handling of information and their perceptions of the information process. The findings reveal that in PBL labs, developing a practical, applicable experimental procedure is crucial for problem-solving and significantly shapes the information-acquisition process. This process is iterative, influenced by new information, leading to more precise information needs. Students assess information quality based on its usefulness for their problem, implementability (considering cognitive understanding, available equipment, and psychomotor skills), and safety.
Furthermore, the role of privileged knowledge forms in evaluating the quality of text sources is explored. Students viewed non-scientific sources as "poor" and scientific sources as "good," yet used both for information gathering. There were discrepancies between their assessment of source quality and actual use, indicating that perception of source quality doesn't always affect their practical decisions.
The third publication, Wellhöfer, Machleid, and Lühken (2023), investigates students' information practices in the lab, focusing on discourse between novice learners and experienced assistants. It shows that theoretical knowledge isn't sufficient for independent practical action, and students need actionable social information from experienced community members. The results highlight that information literacy in the lab for newcomers to a community of practice has distinctive features, and physical experience and tacit knowledge are crucial for learning the methods and group-specific knowledge of the practice community. The article demonstrates how learning information literacy in a practice community requires a social and physical experience and provides insights on how educators can support this process.
Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.
Publicly available compound and bioactivity databases provide an essential basis for data-driven applications in life-science research and drug design. By analyzing several bioactivity repositories, we discovered differences in compound and target coverage advocating the combined use of data from multiple sources. Using data from ChEMBL, PubChem, IUPHAR/BPS, BindingDB, and Probes & Drugs, we assembled a consensus dataset focusing on small molecules with bioactivity on human macromolecular targets. This allowed an improved coverage of compound space and targets, and an automated comparison and curation of structural and bioactivity data to reveal potentially erroneous entries and increase confidence. The consensus dataset comprised of more than 1.1 million compounds with over 10.9 million bioactivity data points with annotations on assay type and bioactivity confidence, providing a useful ensemble for computational applications in drug design and chemogenomics.
Dimerization of Taspase1 activates an intrinsic serine protease function that leads to the catalytic Thr234 residue, which allows to catalyze the consensus sequence Q−3X−2D−1⋅G1X2D3D4, present in Trithorax family members and TFIIA. Noteworthy, Taspase1 performs only a single hydrolytic step on substrate proteins, which makes it impossible to screen for inhibitors in a classical screening approach. Here, we report the development of an HTRF reporter assay that allowed the identification of an inhibitor, Closantel sodium, that inhibits Taspase1 in a noncovalent fashion (IC50 = 1.6 μM). The novel inhibitor interferes with the dimerization step and/or the intrinsic serine protease function of the proenzyme. Of interest, Taspase1 is required to activate the oncogenic functions of the leukemogenic AF4-MLL fusion protein and was shown in several studies to be overexpressed in many solid tumors. Therefore, the inhibitor may be useful for further validation of Taspase1 as a target for cancer therapy.
Unraveling the activation mechanism of taspase1 which controls the oncogenic AF4–MLL fusion protein
(2015)
We have recently demonstrated that Taspase1-mediated cleavage of the AF4–MLL oncoprotein results in the formation of a stable multiprotein complex which forms the key event for the onset of acute proB leukemia in mice. Therefore, Taspase1 represents a conditional oncoprotein in the context of t(4;11) leukemia. In this report, we used site-directed mutagenesis to unravel the molecular events by which Taspase1 becomes sequentially activated. Monomeric pro-enzymes form dimers which are autocatalytically processed into the enzymatically active form of Taspase1 (αββα). The active enzyme cleaves only very few target proteins, e.g., MLL, MLL4 and TFIIA at their corresponding consensus cleavage sites (CSTasp1) as well as AF4–MLL in the case of leukemogenic translocation. This knowledge was translated into the design of a dominant-negative mutant of Taspase1 (dnTASP1). As expected, simultaneous expression of the leukemogenic AF4–MLL and dnTASP1 causes the disappearance of the leukemogenic oncoprotein, because the uncleaved AF4–MLL protein (328 kDa) is subject to proteasomal degradation, while the cleaved AF4–MLL forms a stable oncogenic multi-protein complex with a very long half-life. Moreover, coexpression of dnTASP1 with a BFP-CSTasp1-GFP FRET biosensor effectively inhibits cleavage. The impact of our findings on future drug development and potential treatment options for t(4;11) leukemia will be discussed.
Over the last 15 years the Diagnostic Center of Acute Leukemia (DCAL) at the Frankfurt University has diagnosed and elucidated the Mixed Lineage Leukemia (MLL) recombinome with >100 MLL fusion partners. When analyzing all these different events, balanced chromosomal translocations were found to comprise the majority of these cases (~70%), while other types of genetic rearrangements (3-way-translocations, spliced fusions, 11q inversions, interstitial deletions or insertion of chromosomal fragments into other chromosomes) account for about 30%. In nearly all those complex cases, functional fusion proteins can be produced by transcription, splicing and translation. With a few exceptions (10 out of 102 fusion genes which were per se out-of-frame), all these genetic rearrangements produced a direct MLL fusion gene, and in 94% of cases an additional reciprocal fusion gene. So far, 114 patients (out of 2454 = ~5%) have been diagnosed only with the reciprocal fusion allele, displaying no MLL-X allele. The fact that so many MLL rearrangements bear at least two fusion alleles, but also our findings that several direct MLL fusions were either out-of-frame fusions or missing, raises the question about the function and importance of reciprocal MLL fusions. Recent findings also demonstrate the presence of reciprocal MLL fusions in sarcoma patients. Here, we want to discuss the role of reciprocal MLL fusion proteins for leukemogenesis and beyond.
Mechanistic characterization of photoisomerization reactions in organic molecules and photoreceptors
(2023)
In dieser Arbeit wurden verschiedene Einflüsse auf die Dynamik von Photoisomerisierungen in Phytochromen und indigoiden Photoschaltern untersucht. Beide Forschungsgebiete teilen wesentliche Aspekte wie die Kontrolle durch sterische Wechselwirkungen und den starken Einfluss der Polarität oder der ionischen Umgebung.
Auf dem Gebiet der Phytochrome wurde die relative Positionierung der knotenlosen Phytochrome innerhalb der Superfamilie der Phytochrome in Bezug auf ihre Photodynamik und den Effekt von Grundzustandsheterogenität herausgearbeitet. Es wurde anhand von ultraschnellen, zeitaufgelösten Anrege-Abtast-Experimenten der einzelnen GAF-Domäne All2699g1 im Vergleich mit dem vollständigen knotenlosen Phytochrom All2699g1g2 und dem strukturell ähnlichen knotenlosen Phytochrom SynCph2 gezeigt, dass knotenlose Phytochrome in ihrer Vorwärtsdynamik eine komplexe mehrphasige Kinetik mit einem langlebigen angeregten Zustand (~100 ps) aufweisen. Die beobachtete mehrphasige Kinetik konnte einer initialen Chromophordynamik sowie einer nicht exponentiellen Reorganisation der chromophor-umgebenden Proteinmatrix zugeordnet werden. Dies steht im starken Kontrast zur im Gebiet der Phytochrome etablierten Beschreibung derartiger mehrphasiger Kinetiken mittels heterogener Grundzustände. Stattdessen wurde ein konserviertes kinetisches Muster identifiziert, welches die mehrphasige Dynamik beschreibt und in allen in dieser Arbeit untersuchten Phytochrome beobachtet wurde. Zudem konnte dieses Muster in einem Phytochrom der Gruppe I und einem Phytochrom der Gruppe III, die einen ähnlichen Pr Dunkelzustand aufweisen, gezeigt werden, was eine breite Anwendbarkeit des damit verbundenen Mechanismus vermuten lässt. Weiterhin konnte die zentrale Rolle eines konservierten Tyrosins in der Photoisomerisierung anhand von Mutationsstudien in All2699g1 herausgearbeitet werden. Diese konservierte Aminosäure muss im Rahmen der Reorganisation der Proteinmatrix vom Chromophor weggezogen werden, damit die sterische Blockade abgebaut werden kann, die die Isomerisierung des Chromophors zunächst verhindert. Da diese Bewegung von diversen Faktoren in der den Chromophor umgebenden Proteinmatrix abhängt, weist sie eine nicht exponentielle Kinetik auf, die je nach Phytochrom, der spezifischen Flexibilität und dem vorhandenen Raum in der Bindetasche unterschiedliche Lebenszeiten aufweist.
Die Rückreaktion knotenloser Phytochrome konnte ebenfalls im Rahmen dieser Arbeit charakterisiert werden, welche im Pikosekundenbereich abläuft, und damit signifikant schneller ist als die Vorwärtsreaktion. Im Gegensatz zur Vorwärtsreaktion nimmt Grundzustandsheterogenität in der Rückreaktion eine weitaus bedeutendere Rolle ein. Hier weisen die in All2699g1 vorhandenen heterogenen Grundzustandspopulationen jeweils eine eigene Kinetik ihres angeregten Zustands auf, während die homogenen Grundzustände von All2699g1g2 und SynCph2 jeweils nur einen Zerfall des angeregten Zustands zeigen. Der Ursprung dieser Heterogenität konnte im Wasserstoffbrückennetzwerk des Chromophors lokalisiert und mit dem konservierten Tyrosin und einem konservierten Serin in der PHY-Domäne verknüpft werden. Die Anwesenheit der PHY-Domäne sorgt demnach für eine Verringerung der Grundzustandsheterogenität und des vorhandenen Raums in der Bindetasche, wodurch die Effizienz der Photoreaktion optimiert wird.
Zuletzt konnte die Millisekundendynamik knotenloser Phytochrome und der Einfluss der PHY-Domäne auf diese aufgeklärt werden. Die PHY-Domäne sorgt hierbei durch den verringerten Raum in der Bindetasche dafür, dass die zunächst stattfindende thermische Relaxation des Chromophors signifikant verlangsamt wird, während spätere Änderungen im Photozyklus nur wenig beeinflusst werden.
Auf dem Gebiet der indigoiden Photoschalter konnte, anhand eines sterisch überladenen Hemithioindigo Photoschalters, der Photoisomerisierungsmechanismus des Hula-Twists beobachtet und eine starke Lösungsmittelabhängigkeit der entsprechenden Kinetik aufgezeigt werden. Aus den durchgeführten zeitaufgelösten Anrege-Abtast-Experimenten in verschiedenen Lösungsmitteln konnte ein Modell für die Photodynamik des verwendeten Hemithioindigo Photoschalters entwickelt werden. In unpolaren Lösungsmitteln muss eine hohe Barriere zur produktiven konischen Durchschneidung überwunden werden, was zu Lebenszeiten des angeregten Zustands im Nanosekundenbereich führt. Der Weg zur produktiven konischen Durchschneidung folgt dabei dem Hula-Twist Mechanismus. Dieser Pfad ist in polaren Lösungsmitteln unerreichbar, weshalb eine schnelle Relaxation über eine unproduktive konische Durchschneidung stattfindet.
Im zweiten Projekt auf dem Gebiet der indigoiden Photoschalter wurde anhand der neuartigen Klasse der Iminothioindoxyl Photoschalter ein Schwingungsenergiedonor für Schwingungsenergietransferstudien entwickelt. Das daraus entwickelte Modellsystem, bestehend aus einer künstlichen Aminosäure auf Basis des Iminothioindoxyl Photoschalters und einem daran gekoppelten Schwingungsenergiesensor, wurde charakterisiert und die primäre Photoreaktion untersucht. Es konnte gezeigt werden, dass der angeregte Zustand des Modellsystems kurzlebig ist und unter Abgabe von großen Mengen an Schwingungsenergie zerfällt, unabhängig von der Anregungswellenlänge und dem verwendeten Lösungsmittel. Somit zeigt das entwickelte System vorteilhafte Eigenschaften für Schwingungsenergietransferstudien.
Insgesamt konnten somit die Mechanismen der Photoisomerisierungsreaktionen in knotenlosen Phytochromen und indigoiden Photoschaltern aufgeklärt und daraus die Relevanz der Umgebung für derartige Reaktionen herausgearbeitet werden.
Glucose hypometabolism, mitochondrial dysfunction, and cholinergic deficits have been reported in early stages of Alzheimer’s disease (AD). Here, we examine these parameters in TgF344-AD rats, an Alzheimer model that carries amyloid precursor protein and presenilin-1 mutations, and of wild type F344 rats. In mitochondria isolated from rat hippocampi, we found reductions of complex I and oxidative phosphorylation in transgenic rats. Further impairments, also of complex II, were observed in aged (wild-type and transgenic) rats. Treatment with a “cocktail” containing magnesium orotate, benfotiamine, folic acid, cyanocobalamin, and cholecalciferol did not affect mitochondrial activities in wild-type rats but restored diminished activities in transgenic rats to wild-type levels. Glucose, lactate, and pyruvate levels were unchanged by age, genetic background, or treatment. Using microdialysis, we also investigated extracellular concentrations of acetylcholine that were strongly reduced in transgenic animals. Again, ACh levels in wild-type rats did not change upon treatment with nutrients, whereas the cocktail increased hippocampal acetylcholine levels under physiological stimulation. We conclude that TgF344-AD rats display a distinct mitochondrial and cholinergic dysfunction not unlike the findings in patients suffering from AD. This dysfunction can be partially corrected by the application of the “cocktail” which is particularly active in aged rats. We suggest that the TgF344-AD rat is a promising model to further investigate mitochondrial and cholinergic dysfunction and potential treatment approaches for AD.
Cytochrome c oxidase catalyzes the reduction of oxygen to water. This process is accompanied by the vectorial transport of protons across the mitochondrial or bacterial membrane (“proton pumping”). The mechanism of proton pumping is still a matter of debate. Many proposed mechanisms require structural changes during the reaction cycle of cytochrome c oxidase. Therefore, the structure of the cytochrome c oxidase was determined in the completely oxidized and in the completely reduced states at a temperature of 100 K. No ligand exchanges or other major structural changes upon reduction of the cytochrome coxidase from Paracoccus denitrificans were observed. The three histidine CuB ligands are well defined in the oxidized and in the reduced states. These results are hardly compatible with the “histidine cycle” mechanisms formulated previously.
The potential of a protein-engineered His tag to immobilize macromolecules in a predictable orientation at metal-chelating lipid interfaces was investigated using recombinant 20 S proteasomes His-tagged in various positions. Electron micrographs demonstrated that the orientation of proteasomes bound to chelating lipid films could be controlled via the location of their His tags: proteasomes His-tagged at their sides displayed exclusively side-on views, while proteasomes His-tagged at their ends displayed exclusively end-on views. The activity of proteasomes immobilized at chelating lipid interfaces was well preserved. In solution, His-tagged proteasomes hydrolyzed casein at rates comparable with wild-type proteasomes, unless the His tags were located in the vicinity of the N termini of α-subunits. The N termini of α-subunits might partly occlude the entrance channel in α-rings through which substrates enter the proteasome for subsequent degradation. A combination of electron micrographs and atomic force microscope topographs revealed a propensity of vertically oriented proteasomes to crystallize in two dimensions on fluid lipid films. The oriented immobilization of His-tagged proteins at biocompatible lipid interfaces will assist structural studies as well as the investigation of biomolecular interaction via a wide variety of surface-sensitive techniques including single-molecule analysis.
ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic Gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a Km of 0.9 mm and kcat of 9 s−1 at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.
The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 μm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.
The ABC transporter Mdl1p, a structural and functional homologue of the transporter associated with antigen processing (TAP) plays an important role in intracellular peptide transport from the mitochondrial matrix of Saccharomyces cerevisiae. To characterize the ATP hydrolysis cycle of Mdl1p, the nucleotide-binding domain (NBD) was overexpressed in Escherichia coli and purified to homogeneity. The isolated NBD was active in ATP binding and hydrolysis with a turnover of 25 ATP per minute and a Km of 0.6 mm and did not show cooperativity in ATPase activity. However, the ATPase activity was non-linearly dependent on protein concentration (Hill coefficient of 1.7), indicating that the functional state is a dimer. Dimeric catalytic transition states could be trapped either by incubation with orthovanadate or beryllium fluoride, or by mutagenesis of the NBD. The nucleotide composition of trapped intermediate states was determined using [alpha-32P]ATP and [gamma-32P]ATP. Three different dimeric intermediate states were isolated, containing either two ATPs, one ATP and one ADP, or two ADPs. Based on these experiments, it was shown that: (i) ATP binding to two NBDs induces dimerization, (ii) in all isolated dimeric states, two nucleotides are present, (iii) phosphate can dissociate from the dimer, (iv) both nucleotides are hydrolyzed, and (v) hydrolysis occurs in a sequential mode. Based on these data, we propose a processive-clamp model for the catalytic cycle in which association and dissociation of the NBDs depends on the status of bound nucleotides.
The purification and functional reconstitution of a five-component oligopeptide ATP-binding cassette transporter with a remarkably wide substrate specificity are described. High-affinity peptide uptake was dependent on liganded substrate-binding protein OppA, which interacts with the translocator OppBCDF with higher affinity than unliganded OppA. Transport screening with combinatorial peptide libraries revealed that (i) the Opp transporter is not selective with respect to amino acid side chains of the transported peptides; (ii) any peptide that can bind to OppA is transported via Opp, including very long peptides up to 35 residues long; and (iii) the binding specificity of OppA largely determines the overall transport selectivity.
The transporter associated with antigen processing (TAP) plays a key role in the adaptive immune response by pumping antigenic peptides into the endoplasmic reticulum for subsequent loading of major histocompatibility complex class I molecules. TAP is a heterodimer consisting of TAP1 and TAP2. Each subunit is composed of a transmembrane domain and a nucleotide-binding domain, which energizes the peptide transport. To analyze ATP hydrolysis of each subunit we developed a method of trapping 8-azido-nucleotides to TAP in the presence of phosphate transition state analogs followed by photocross-linking, immunoprecipitation, and high resolution SDS-PAGE. Strikingly, trapping of both TAP subunits by beryllium fluoride is peptide-specific. The peptide concentration required for half-maximal trapping is identical for TAP1 and TAP2 and directly correlates with the peptide binding affinity. Only a background level of trapping was observed for low affinity peptides or in the presence of the herpes simplex viral protein ICP47, which specifically blocks peptide binding to TAP. Importantly, the peptide-induced trapped state is reached after ATP hydrolysis and not in a backward reaction of ADP binding and trapping. In the trapped state, TAP can neither bind nor exchange nucleotides, whereas peptide binding is not affected. In summary, these data support the model that peptide binding induces a conformation that triggers ATP hydrolysis in both subunits of the TAP complex within the catalytic cycle.
The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for functional reconstitution of human TAPL was developed. By intensive screening of detergents, ideal solubilization conditions were evolved with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with high purity and, subsequently, reconstituted into proteoliposomes. The peptide transport activity of reconstituted TAPL strongly depends on the lipid composition. With the help of combinatorial peptide libraries, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones.
The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticular lumen for subsequent loading onto major histocompatibility complex (MHC) class I molecules. These peptide-MHC complexes are inspected at the cell surface by cytotoxic T-lymphocytes. Assembly of the functional peptide transport and loading complex depends on intra- and intermolecular packing of transmembrane helices (TMs). Here, we have examined the membrane topology of human TAP1 within an assembled and functional transport complex by cysteine-scanning mutagenesis. The accessibility of single cysteine residues facing the cytosol or endoplasmic reticular lumen was probed by a minimally invasive approach using membrane-impermeable, thiol-specific fluorophores in semipermeabilized “living” cells. TAP1 contains ten transmembrane segments, which place the N and C termini in the cytosol. The transmembrane domain consists of a translocation core of six TMs, a building block conserved among most ATP-binding cassette transporters, and a unique additional N-terminal domain of four TMs, essential for tapasin binding and assembly of the peptide-loading complex. This study provides a first map of the structural organization of the TAP machinery within the macromolecular MHCI peptide-loading complex.
P2X1 receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X1receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn284 remains unused because of a proline in the +4 position. The four other sites (Asn153, Asn184, Asn210, and Asn300) carryN-glycans, but solely Asn300 located only eight residues upstream of the predicted reentry loop of P2X1acquires complex-type carbohydrates. Like parent P2X1, glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn153 or Asn210 diminishes or increases functional expression levels, respectively. In addition, elimination of Asn210 causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X1 receptors is severely impaired or abolished, respectively. We conclude that at least oneN-glycan per subunit of either position is absolutely required for the formation of P2X1 receptors and that individual N-glycans possess marked positional effects on expression levels (Asn154, Asn210) and ATP potency (Asn210).
One current goal in native mass spectrometry is the assignment of binding affinities to noncovalent complexes. Here we introduce a novel implementation of the existing laser-induced liquid bead ion desorption (LILBID) mass spectrometry method: this new method, LILBID laser dissociation curves, assesses binding strengths quantitatively. In all LILBID applications, aqueous sample droplets are irradiated by 3 µm laser pulses. Variation of the laser energy transferred to the droplet during desorption affects the degree of complex dissociation. In LILBID laser dissociation curves, laser energy transfer is purposely varied, and a binding affinity is calculated from the resulting complex dissociation. A series of dsDNAs with different binding affinities was assessed using LILBID laser dissociation curves. The binding affinity results from the LILBID laser dissociation curves strongly correlated with the melting temperatures from UV melting curves and with dissociation constants from isothermal titration calorimetry, standard solution phase methods. LILBID laser dissociation curve data also showed good reproducibility and successfully predicted the melting temperatures and dissociation constants of three DNA sequences. LILBID laser dissociation curves are a promising native mass spectrometry binding affinity method, with reduced time and sample consumption compared to melting curves or titrations.
Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRβ subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5–40 amino acids, are subject to alternative splicing (C1–C7, C4′–C6′). Since one of the cassettes, C5′, has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRβ loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6′, already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5′ was coisolated mainly from liver. Notably C5′-containing gephyrin bound to the GlyRβ loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.
Background: In the past, protease inhibitors (PIs) and the reverse transcriptase inhibitor abacavir were identified increasing the risk for thromboembolic complications and cardiovascular events (CVE) of HIV infected patients taking a combination antiretroviral therapy (cART). Results of the previous HIV-PLA I-study lead to the assumption that platelet activation could play a substantial role in increasing CVE risks.
Methods: The open label, monocentric HIV-PLA II-study investigated HIV-1-infected, therapy-naïve adults (n=45) starting with cART, consisting either of boosted PI (atazanavir, n= 6, darunavir, n=11), NNRTI (efavirenz, n=14) or integrase inhibitor (raltegravir, n=14), each plus tenofovir/emtricitabine co-medication. Main exclusion criteria were tobacco smoking, the intake of NSAIDs or abacavir or past CVE. Platelet adhesive molecule p-selectin (CD62P) and FITC anti-human Integrin α-IIb/Integrin β-3 (CD41/CD61) antibody (PAC-1) binding, monocyte CD11b/monocyte-associated CD41 expression and the endogenous thrombin potential (ETP) were assessed ex vivo-in vitro at baseline, weeks 4, 12 and 24. Therapy regimens were blinded to the investigators for laboratory and statistical analyses.
Results: CD11b and ETP showed no significant changes or differences between all study groups. In contrast, the mean + SD mean fluorescence units (MFI) of CD62P and PAC-1 increased significantly in patients taking PI, indicating an enhanced potential for thrombocyte activation and aggregation.
Conclusion: CD62P expression, detecting the ɑ-platelet degranulation of pro-inflammatory and pro-thrombotic factors and adhesive proteins, and PAC-1 expression, representing a marker for conformation changes of the GIIb/IIIa receptor, increased significantly in patients taking HIV protease inhibitors. The findings of this study revealed a yet unknown pathway of platelet activation, possibly contributing to the increased risk for CVE under HIV protease inhibitor containing cART.
Clinical Trial Registration No.: DRKS00000288.
Oligonucleotide-based therapeutics have made rapid progress in clinical treatment of a variety of disease indications. Since most therapeutic oligonucleotides serve more than just one function and tend to have a prolonged lifetime, spatio-temporal control of these functions would be desirable. Photoswitches like azobenzene have proven themselves as useful tools in this matter. Upon irradiation, the photoisomerization of the azobenzene moiety causes destabilization in adjacent base pairs, leading to a decreased hybridization affinity. Since the way the azobenzene is incorporated in the oligonucleotide is of utmost importance, we synthesized locked azobenzene C-nucleosides and compared their photocontrol capabilities to established azobenzene C-nucleosides in oligonucleotide test-sequences by means of fluorescence-, UV/Vis-, and CD-spectroscopy.
Electronic circular dichroism unravels atropisomers of a broadly absorbing fulgide derivative
(2022)
We prepared and studied six atropisomers with different chiroptical properties emerging from a single, robust, broadly-absorbing fulgide photoswitch. After separation of the different atropisomers via HPLC on a chiral column, their isomerization processes at room temperature and the energy barriers of the different species were investigated in detail using spectroscopic and theoretical methods.
In the development of photolabile protecting groups, it is of high interest to selectively modify photochemical properties with structural changes as simple as possible. In this work, knowledge of fluorophore optimization was adopted and used to design new coumarin- based photocages. Photolysis efficiency was selectively modulated by inactivating competitive decay channels, such as twisted intramolecular charge transfer (TICT) or hydrogen-bonding, and the photolytic release of the neurotransmitter serotonin was demonstrated. Structural modifications inspired by the fluorophore ATTO 390 led to a significant increase in the uncaging cross section that can be further improved by the simple addition of a double bond. Ultrafast transient absorption spectroscopy gave insights into the underlying solvent-dependent photophysical dynamics. The chromophores presented here are excellently suited as new photocages in the visible wavelength range due to their simple synthesis and their superior photochemical properties.
We synthesized two green-light activatable 5’-caps for oligonucleotides based on the BODIPY and coumarin scaffold. Both bear an alkyne functionality allowing their use in numerous biological applications. They were successfully incorporated in oligonucleotides via solid-phase synthesis. Copper-catalyzed alkyne-azide cycloaddition (CuAAC) using a bisazide photo-tether gave cyclic oligonucleotides that could be relinearized by activation with green light and were shown to exhibit high stability against exonucleases. Chemical ligation as another example for bioconjugation yielded oligonucleotides with an internal strand break site. Irradiation at 530 nm or 565 nm resulted in complete photolysis of both caging groups.
Purpose: The quality testing and approval procedure for most pharmaceutical products is a streamlined process with standardized procedures for the determination of critical quality attributes. However, the evaluation of semisolid dosage forms for topical drug delivery remains a challenging task. The work presented here highlights confocal Raman microscopy (CRM) as a valuable tool for the characterization of such products.
Methods: CRM, a laser-based method, combining chemically-selective analysis and high resolution imaging, is used for the evaluation of different commercially available topical acyclovir creams.
Results: We show that CRM enables the spatially resolved analysis of microstructural features of semisolid products and provides insights into drug distribution and polymorphic state as well as the composition and arrangement of excipients. Further, we explore how CRM can be used to monitor phase separation and to study skin penetration and the interaction with fresh and cryopreserved excised human skin tissue.
Conclusion: This study presents a comprehensive overview and illustration of how CRM can facilitate several types of key analyses of semisolid topical formulations and of their interaction with their biological target site, illustrating that CRM is a useful tool for research, development as well as for quality testing in the pharmaceutical industry.
Non-alcoholic steatohepatitis (NASH) - a hepatic manifestation of the metabolic syndrome - is a multifactorial disease with alarming global prevalence. It involves steatosis, inflammation and fibrosis in the liver, thus demanding multiple modes of action for robust therapeutic efficacy. Aiming to fuse complementary validated anti-NASH strategies in a single molecule, we have designed and systematically optimized a scaffold for triple activation of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor (PPAR) α and PPARδ. Pilot profiling of the resulting triple modulator demonstrated target engagement in native cellular settings and in mice, rendering it a suitable tool to probe the triple modulator concept in vivo. In DIO NASH in mice, the triple agonist counteracted hepatic inflammation and reversed hepatic fibrosis highlighting the potential of designed polypharmacology in NASH.
Antigen presentation via major histocompatibility complex class I (MHC I) molecules is essential to mount an adaptive immune response against pathogens and cancerous cells. To this end, the transporter associated with antigen processing (TAP) delivers snippets of the cellular proteome, resulting from proteasomal degradation, into the ER lumen. After peptide loading and editing by the peptide-loading complex (PLC), stable peptide-MHC I complexes are released for cell surface presentation. Since the process of MHC I trafficking is poorly defined, we established an approach to control antigen presentation by introduction of a photo-caged amino acid in the catalytic ATP-binding site of TAP. By optical control, we initiate TAP-dependent antigen translocation, thus providing new insights into TAP function within the PLC and MHC I trafficking in living cells. Moreover, this versatile approach has the potential to be applied in the study of other cellular pathways controlled by P-loop ATP/GTPases.
Despite all advances in drug delivery, the limitations of the analytical technologies involved in the characterization of next-generation nanomedicines are still impeding further progress of an emerging market. Discriminating between different formulations and batches, drug release is one of the most important quality criteria in development and quality control of pharmaceutics. Unfortunately, there are only few methods available to sensitively measure this important parameter for nanosized carriers. With the development of the dispersion releaser (DR) technology our group has set up a dialysis-based technique that was tested with a number of nanocarrier and nanocrystal formulations such as liposomes and polymeric nanoparticles. By supporting formulation development with a more reliable methodology to assess the drug release from nanosized carriers, a first step has been made to improve future products.
Quinone methide precursors protected with alkyldithiomethyl groups have been synthesized and converted into PNA conjugates. Stable in the absence of reducing agents, the electrophilic quinone methide is released by glutathione in concentrations typical for the cytosol. Self-alkylation then occurs or crosslinking of RNA when hybridized with complementary strands. Fastest reactions are seen for the sterically least hindered compound.
The Mycobacterium tuberculosis tyrosine-specific phosphatase MptpA and its cognate kinase PtkA are prospective targets for anti-tuberculosis drugs as they interact with the host defense response within the macrophages. Although both are structurally well-characterized, the functional mechanism regulating their activity remains poorly understood. Here, we investigate the effect of post-translational oxidation in regulating the function of MptpA. Treatment of MptpA with H2O2/NaHCO3, mimicking cellular oxidative stress conditions, leads to oxidation of the catalytic cysteine (C11) and to a conformational rearrangement of the phosphorylation loop (D-loop) by repositioning the conserved tyrosine 128 (Y128) and generating a temporarily inactive preclosed state of the phosphatase. Thus, the catalytic cysteine in the P-loop acts as a redox switch and regulates the phosphatase activity of MptpA.
The p53 protein family is the most studied protein family of all. Sequence analysis and structure determination have revealed a high
similarity of crucial domains between p53, p63 and p73. Functional studies, however, have shown a wide variety of different tasks in
tumor suppression, quality control and development. Here we review the structure and organization of the individual domains of
p63 and p73, the interaction of these domains in the context of full-length proteins and discuss the evolutionary origin of this
protein family.
FACTS:
● Distinct physiological roles/functions are performed by specific isoforms.
● The non-divided transactivation domain of p63 has a constitutively high activity while the transactivation domains of p53/p73
are divided into two subdomains that are regulated by phosphorylation.
● Mdm2 binds to all three family members but ubiquitinates only p53.
● TAp63α forms an autoinhibited dimeric state while all other vertebrate p53 family isoforms are constitutively tetrameric.
● The oligomerization domain of p63 and p73 contain an additional helix that is necessary for stabilizing the tetrameric states.
During evolution this helix got lost independently in different phylogenetic branches, while the DNA binding domain became
destabilized and the transactivation domain split into two subdomains.
OPEN QUESTIONS:
● Is the autoinhibitory mechanism of mammalian TAp63α conserved in p53 proteins of invertebrates that have the same function
of genomic quality control in germ cells?
● What is the physiological function of the p63/p73 SAM domains?
● Do the short isoforms of p63 and p73 have physiological functions?
● What are the roles of the N-terminal elongated TAp63 isoforms, TA* and GTA?
tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.
Cytochrome P450 enzymes are a large superfamily of membrane-bound heme-containing monooxygenases. They are essential for the oxidative metabolism of endogenous substrates such as steroids and fatty acids, and biotransformation of xenobiotic substrates such as pollutants and drugs. Although the highest expression of CYPs is found in the liver, their cardiovascular expression is not negligible with CYP450 subfamilies being responsible for the production of vasoactive lipids. Of importance, the enzymatic activity of all microsomal CYP450 isoenzymes is dependent on the cytochrome P450 reductase (POR), an electron donor.
In the first part of this work, the role of cytochrome P450 monooxygenases on the biotransformation of organic nitrates was investigated. Recombinant SupersomesTM were selected and incubated with NTG and PETN, where nitrite release was measured as a nitric oxide (NO) footprint. The capacity of the recombinant POR/CYP450 system to release nitrite from NO prodrugs was shown to be CYP-specific and dose-dependent. To study the involvement of CYP450 enzymes in the vascular biotransformation of organic nitrates in vivo, a smooth muscle-cell specific, inducible knockout model of POR (smcPOR-/-) was generated. Organ chamber experiments revealed that the vascular POR/CYP450 system had no impact on the dilator response of NTG and PETN. In line with previous publications, inhibition of ALDH2, known as the main enzyme responsible for the activation of NTG and PETN, and/or abolishment of the endogenous NO production did not reveal a contribution of the POR/CYP450 system to the dilator response of NTG and PETN. To better understand these results, we looked at the expression of the hepatic and vascular expression of the POR/CYP450 system where the hepatic was increased by 10- to 40-fold as shown by Western blot analysis. We concluded that due to insufficient vascular expression of CYP450 enzymes their contribution to the bioactivation of NTG and PETN is only minor.
The second part of this work focused on the cardiac relevance of endothelial isoenzymes. For that purpose, an endothelial cell-specific, tamoxifen-inducible knockout model of POR was generated and characterized in the present study. RNA-sequencing of the heart of healthy mice revealed that the CYP450 expression is cell-specific with cardiac endothelial cells (ECs) exhibiting an enrichment in the expression of the Cyp4 family (ω-oxidation of fatty acids) and of the Cyp2 family (production of EETs). Under non-stredded conditions (i.e. 30 days after inducing the knockout by tamoxifen feeding), endothelial deletion of POR was associated with cardiac remodelling as observed by an increase in the ratio of heart weight to body weight and an increase in the cardiomyocyte area. RNA-sequencing of cardiac ECs suggested that loss of POR might alter ribosomal biogenesis and protein synthesis, which could potentially affect the cardiac contractility in ecPOR-/- mice. Metabolomics from cardiac tissue of CTL and ecPOR-/- mice were not indicative for an important metabolic function of the endothelial POR/CYP450 system in the heart. The combination of transverse aortic constriction (TAC) with endothelial deletion of POR accelerates the development of heart failure in mice as detected by a reduction in cardiac output and stroke volume. These effects were mediated most likely by a reduction in vascular EETs production, which increases vascular stiffness, resulting in cardiac remodeling.
Several lines of evidence suggest the ligand-sensing transcription factor Nurr1 as a promising target to treat neurodegenerative diseases. Nurr1 modulators to validate and exploit this therapeutic potential are rare, however. To identify novel Nurr1 agonist chemotypes, we have employed the Nurr1 activator amodiaquine as template for microscale analogue library synthesis. The first set of analogues was based on the 7-chloroquiolin-4-amine core fragment of amodiaquine and revealed superior N-substituents compared to diethylaminomethylphenol contained in the template. A second library of analogues was subsequently prepared to replace the chloroquinolineamine scaffold. The two sets of analogues enabled a full scaffold hop from amodiaquine to a novel Nurr1 agonist sharing no structural features with the lead but comprising superior potency on Nurr1. Additionally, pharmacophore modeling based on the entire set of active and inactive analogues suggested key features for Nurr1 agonists.
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential applications in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of the wild-type CbFDH at cryogenic and ambient temperatures as well as Val120Thr mutant at cryogenic temperature determined at the Turkish Light Source "Turkish DeLight". The structures reveal new hydrogen bonds between Thr120 and water molecules in the mutant CbFDH's active site, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, our findings provide invaluable insights into future protein engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
Candida boidinii NAD+-dependent formate dehydrogenase (CbFDH) has gained significant attention for its potential applications in the production of biofuels and various industrial chemicals from inorganic carbon dioxide. The present study reports the atomic X-ray crystal structures of the wild-type CbFDH at cryogenic and ambient temperatures as well as Val120Thr mutant at cryogenic temperature determined at the Turkish Light Source "Turkish DeLight". The structures reveal new hydrogen bonds between Thr120 and water molecules in the mutant CbFDH's active site, suggesting increased stability of the active site and more efficient electron transfer during the reaction. Further experimental data is needed to test these hypotheses. Collectively, our findings provide invaluable insights into future protein engineering efforts that could potentially enhance the efficiency and effectiveness of CbFDH.
BH3 mimetics are promising novel anticancer therapeutics. By selectively inhibiting BCL-2, BCL-xL, or MCL-1 (i.e. ABT-199, A-1331852, S63845) they shift the balance of pro- and anti-apoptotic proteins in favor of apoptosis. As Bromodomain and Extra Terminal (BET) protein inhibitors promote pro-apoptotic rebalancing, we evaluated the potential of the BET inhibitor JQ1 in combination with ABT-199, A-1331852 or S63845 in rhabdomyosarcoma (RMS) cells. The strongest synergistic interaction was identified for JQ1/A-1331852 and JQ1/S63845 co-treatment, which reduced cell viability and long-term clonogenic survival. Mechanistic studies revealed that JQ1 upregulated BIM and NOXA accompanied by downregulation of BCL-xL, promoting pro-apoptotic rebalancing of BCL-2 proteins. JQ1/A-1331852 and JQ1/S63845 co-treatment enhanced this pro-apoptotic rebalancing and triggered BAK- and BAX-dependent apoptosis since a) genetic silencing of BIM, BAK or BAX, b) inhibition of caspase activity with zVAD.fmk and c) overexpression of BCL-2 all rescued JQ1/A-1331852- and JQ1/S63845-induced cell death. Interestingly, NOXA played a different role in both treatments, as genetic silencing of NOXA significantly rescued from JQ1/A-1331852-mediated apoptosis but not from JQ1/S63845-mediated apoptosis. In summary, JQ1/A-1331852 and JQ1/S63845 co-treatment represent new promising therapeutic strategies to synergistically trigger mitochondrial apoptosis in RMS.
Currently, a wide variety of complex non-oral dosage forms are entering the global healthcare market. Although many assays have been described in recent research, harmonized procedures and standards for testing their in vitro performance remain widely unexplored. Among others, dialysis-based techniques such as the Pharma Test Dispersion Releaser are developed for testing the release of drugs from nanoparticles, liposomes, or extracellular vesicle preparations. Here, we provide advanced strategies and practical advice for the development and validation of dialysis-based techniques, including documentation, analysis, and interpretation of the raw data. For this purpose, key parameters of the release assay, including the hydrodynamics in the device at different stirring rates, the selectivity for particles and molecules, as well as the effect of excipients on drug permeation were investigated. At the highest stirring rate, a more than twofold increase in the membrane permeation rate (from 0.99 × 10−3 to 2.17 × 10−3 cm2/h) was observed. Additionally, we designed a novel computer model to identify important quality parameters of the dialysis experiment and to calculate error-corrected release profiles. Two hydrophilic creams of diclofenac, Voltaren® Emulgel, and Olfen® gel, were tested and provide first-hand evidence of the robustness of the assay in the presence of semisolid dosage forms.
During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self-replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel (Science 2000, 289, 448–452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self-cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in-line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure-function relation of ribozyme function in this neutral network.
Polo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors.
Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ~70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated trans-membrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.