Identification and Phenotypic Characterization of Hsp90 Phosphorylation Sites That Modulate Virulence Traits in the Major Human Fungal Pathogen Candida albicans

The highly conserved, ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. In human pathogenic fungi, which kill more than 1.6 million patients each year worldwide, Hsp90 governs cellular morphogenesis, drug resistance, and virulence. Yet, our understanding of the regulatory mechanisms governing fungal Hsp90 function remains sparse. Post-translational modifications are powerful components of nature’s toolbox to regulate protein abundance and function. Phosphorylation in particular is critical in many cellular signaling pathways and errant phosphorylation can have dire consequences for the cell. In the case of Hsp90, phosphorylation affects its stability and governs its interactions with co-chaperones and clients. Thereby modulating the cell’s ability to cope with environmental stress. Candida albicans, one of the leading human fungal pathogens, causes ~750,000 life-threatening invasive infections world-wide with unacceptably high mortality rates. Yet, it remains unknown if and how Hsp90 phosphorylation affects C. albicans virulence traits. Here, we show that phosphorylation of Hsp90 is critical for expression of virulence traits. We combined proteomics, molecular evolution analyses and structural modelling with molecular biology to characterize the role of Hsp90 phosphorylation in this non-model pathogen. We demonstrated that phosphorylation negatively affects key virulence traits, such as the thermal stress response, morphogenesis, and drug susceptibility. Our results provide the first record of a specific Hsp90 phosphorylation site acting as modulator of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitations as antifungal drug targets

Differential sensing with arrays of de novo designed peptide assemblies

Differential sensing attempts to mimic the mammalian senses of smell and taste to identify analytes and complex mixtures. In place of hundreds of complex, membrane-bound G-protein coupled receptors, differential sensors employ arrays of small molecules. Here we show that arrays of computationally designed de novo peptides provide alternative synthetic receptors for differential sensing. We use self-assembling α-helical barrels (αHBs) with central channels that can be altered predictably to vary their sizes, shapes and chemistries. The channels accommodate environment-sensitive dyes that fluoresce upon binding. Challenging arrays of dye-loaded barrels with analytes causes differential fluorophore displacement. The resulting fluorimetric fingerprints are used to train machine-learning models that relate the patterns to the analytes. We show that this system discriminates between a range of biomolecules, drink, and diagnostically relevant biological samples. As αHBs are robust and chemically diverse, the system has potential to sense many analytes in various settings

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries

LOTSE - Library Online Tour & Self-paced Education:Ein multimediales Navigationssystem als Einführung in die hybride Bibliothek : Schlussbericht

Teil 1 umfasst die Aufgabenstellung des Projektes LOTSE, die Voraussetzungen, Planung und Ablauf des Projektes, state of the art und Hinweise zur Zusammenarbeit mit anderen Stellen. Teil 2 schildert die Ergebnisse des Projektes LOTSE, den Nutzen für Anwender (Wissenschaftler, Studierende, Bibliothekare) und Verwertbarkeit in einer NAchnutzung (durch Bibliotheken). Zukunftsperspektiven (Transferprojekt NRW) bis zum Dauerbetrieb werden dargelegt. Der technische Fortschritt anderer Stellen wird kurz berichtet. Öffentlichkeitsarbeit wird geschilert. Teil 3 legt den Beitrag dar, den LOTSE zu den Förderzielen von Global info leistet. Das wissenschaftlich-technische Ergebnis und die gesammelten Erfahrungen werden dargestellt. Fortschreibung und Zukunfstperspektiven (Nachfolgeprojekt) werden skizziert, ebenso Öffentlichkeitsarbeit/Präsentation für Nutzer. Das Projekt wurde Gefördert durch das Bundesministerium für Bildung und Forschung (BMBF

Molecular Assembly and Encapsulation Directed by Hydrogen-Bonding Preferences and the Filling of Space

Multiple copies of a molecule, held together in finite aggregates, give rise to properties and functions that are unique to their assembled states. Because these aggregates are held together by weak forces operating over short distances, a premium is placed on complementarity: The molecular surfaces must facilitate specific interactions that direct the assembly to one aggregate rather than another. Hydrogen-bonding preferences can be combined with molecular curvature to favor the assembly of four self-complementary subunits into a pseudo-spherical capsule. Filling the capsule with smaller, complementary molecules provides the final instruction for the assembly process.Peer reviewe

Molecular Recognition with Biological Receptors: Structure-Based Design of Thrombin Inhibitors

Molecular recognition is at the center of biological function. Consequently, a profound understanding of the underlying nonbonding interactions is required to intervene in a rational way in biological processes. Such detailed knowledge can be gained in studies with designed artificial receptors or, more directly, with biological receptors such as the enzyme thrombin. X-ray structural information on this key enzyme in the blood coagulation cascade has guided the structure-based design of a class of active and selective non-peptidic, reversibly binding low molecular weight inhibitors. These compounds feature a conformationally rigid bi- or tricyclic core structure from which side chains diverge into the four major binding pockets (distal D, proximal P, recognition or selectivity S1, and oxyanion hole) at the thrombin active site. With their rigid central core, all inhibitors prefer similar modes of association to thrombin, and detailed information on the strength of individual intermolecular bonding interactions and their incremental contribution to the overall free enthalpy of complexation is generated in correlative binding studies. Phenylamidinium is the side chain of choice for the S1-pocket. Attempts to replace this group with less basic functional groups, which cannot undergo bidentate ionic H-bonding to the carboxylate of Asp189 at the bottom of this pocket, were unsuccessful. The P-pocket is occupied by an isopropyl group, in analogy to the natural substrate fibrinogen, which uses the side chain of a valine residue to fill this site. The large hydrophobic D-pocket was found to accommodate one and even two aromatic residues. Attempts to direct side chains bearing H-bond acceptor groups into the oxyanion hole are described. The most active inhibitor prepared in this investigation showed a Ki value for thrombin inhibition of 9 nM and a 800-fold selectivity for binding thrombin over trypsin

Characterization of Self-Assembling Encapsulation Complexes in the Gas Phase and Solution

The interaction of quaternary ammonium ion guests with self-assembling hosts was examined by 1H NMR and ESI-MS experiments. The hosts consist of four identical, self-complementary subunits, reversibly joined in a capsular assembly through hydrogen bonding. Both approaches show the ammonium ions to be encapsulated, held within the hollow shell of the capsule. Competition experiments with a series of different guest ions reveal a characteristic size selectivity. The NMR and MS methods are complementary:  MS easily reveals the formation of heterotetramers from different subunits that could not be determined by NMR, while NMR allowed competitive encapsulations of neutral and ionic guests that were not possible to detect with MS. These competition experiments gave a lower limit of ca. 3.6 kcal/mol for the contribution of cation−π interactions involved in the encapsulation of the ionic guests.Peer reviewe