Chemoselective Labeling and Immobilization of Phosphopeptides with Phosphorimidazolide Reagents

Protein phosphorylation is one of the most ubiquitous post-translational modifications, regulating numerous essential processes in cells. Accordingly, the large-scale annotation of phosphorylation sites continues to provide central insight into the regulation of signaling networks. The global analysis of the phosphoproteome typically relies on mass spectrometry analysis of phosphopeptides, with an enrichment step necessary due to the sub-stoichiometric nature of phosphorylation. Several affinity-based methods and chemical modification strategies have been developed to date, but the choice of enrichment method can have a considerable impact on the results. Here, we show that a biotinylated, photo-cleavable phosphorimidazolide reagent permits the immobilization and subsequent cleavage of phosphopeptides. The method is capable of the capture and release of phosphopeptides of varying characteristics, and this mild and selective strategy expands the current repertoire for phosphopeptide chemical modification with the potential to enrich and identify new phosphorylation sites in the future.MS facilityNMR facilitypeptide facility of the FMPFiedler labPeer Reviewe

Previous Mental Load and Incentives Influence Anticipatory Arousal as Indexed by the Baseline Pupil Diameter in a Speech-in-Noise Task

Listening effort and fatigue are common experiences when conversing in noisy environments. Much research has investigated listening effort in relation to listening demand using the speech-in-noise paradigm. Recent conceptualizations of listening effort postulate that mental fatigue should result in decreased arousal and a reluctance to invest further effort, particularly when the effort is not worthwhile. The aim of the study was to investigate the influence of fatigue on listening effort, in interaction with listening demands and motivation. To induce fatigue 30 adults with normal hearing completed a 40-minute long speech-in-noise task (“load sequence”). Pre- and post-load sequence listening effort was probed in easy and hard listening demands (individually adjusted signal-to-noise ratios); with high and low motivation (manipulated with monetary incentives). Subjective effort, estimated performance, and tendency to quit listening were collected using rating scales. Baseline pupil diameter and mean pupil dilation were recorded as indices of anticipatory arousal and objective effort. Self-reported effort and mean pupil dilation were overall larger during hard SNR as compared to easy SNR. Baseline pupil diameter declined from pre- to post-load sequence, suggesting an overall decrease in arousal. Monetary incentives had no influence on the baseline pupil diameter for the easy SNR condition, but for the hard SNR condition larger incentives led to larger baseline pupil diameter. These results suggest that anticipatory arousal may be influenced by fatigue and motivation effects. Models of listening effort should account for the independent influence of motivation and previous load on anticipatory arousal and effort in distinct parameters

Inositol Pyrophosphate-Controlled Kinetochore Architecture and Mitotic Entry in S. pombe

Inositol pyrophosphates (IPPs) comprise a specific class of signaling molecules that regulate central biological processes in eukaryotes. The conserved Vip1/PPIP5K family controls intracellular IP8 levels, the highest phosphorylated form of IPPs present in yeasts, as it has both inositol kinase and pyrophosphatase activities. Previous studies have shown that the fission yeast S. pombe Vip1/PPIP5K family member Asp1 impacts chromosome transmission fidelity via the modulation of spindle function. We now demonstrate that an IP8 analogue is targeted by endogenous Asp1 and that cellular IP8 is subject to cell cycle control. Mitotic entry requires Asp1 kinase function and IP8 levels are increased at the G2/M transition. In addition, the kinetochore, the conductor of chromosome segregation that is assembled on chromosomes is modulated by IP8. Members of the yeast CCAN kinetochore-subcomplex such as Mal2/CENP-O localize to the kinetochore depending on the intracellular IP8-level: higher than wild-type IP8 levels reduce Mal2 kinetochore targeting, while a reduction in IP8 has the opposite effect. As our perturbations of the inositol polyphosphate and IPP pathways demonstrate that kinetochore architecture depends solely on IP8 and not on other IPPs, we conclude that chromosome transmission fidelity is controlled by IP8 via an interplay between entry into mitosis, kinetochore architecture, and spindle dynamics.Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)Medical Research Council (MRC)Peer Reviewe

Fluorination Influences the Bioisostery of Myo‐Inositol Pyrophosphate Analogs

Inositol pyrophosphates (PP−IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP−IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP−IP5 is the most abundant PP−IP in human cells. To investigate the function and regulation by PP−IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP−IP5. Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.Swiss National Science Foundation http://dx.doi.org/10.13039/501100001711German Academic Exchange Service http://dx.doi.org/10.13039/100021828Wellcome Trust http://dx.doi.org/10.13039/100010269Peer Reviewe

Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network

The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers involved in a myriad of cellular processes. Despite their centrality, our understanding of human InsP metabolism is incomplete because the available analytical toolset to characterize and quantify InsPs in complex samples is limited. Here, we have synthesized and applied symmetrically and unsymmetrically 13C-labeled myo-inositol and inositol phosphates. These probes were utilized in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to investigate InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR─down to individual enantiomers─which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme contributing to cellular homeostasis. Detailed characterization of MINPP1 activity in vitro and in cells showcased the unique reactivity of this phosphatase. Our results demonstrate that metabolic labeling with stable isotopomers in conjunction with NMR spectroscopy and CE-MS constitutes a powerful tool to annotate InsP networks in a variety of biological contexts

Ontologies for Models and Algorithms in Applied Mathematics and Related Disciplines

In applied mathematics and related disciplines, the modeling-simulation-optimization workflow is a prominent scheme, with mathematical models and numerical algorithms playing a crucial role. For these types of mathematical research data, the Mathematical Research Data Initiative has developed, merged and implemented ontologies and knowledge graphs. This contributes to making mathematical research data FAIR by introducing semantic technology and documenting the mathematical foundations accordingly. Using the concrete example of microfracture analysis of porous media, it is shown how the knowledge of the underlying mathematical model and the corresponding numerical algorithms for its solution can be represented by the ontologies.Comment: Preprint of a Conference Paper to appear in the Proceeding of the 17th International Conference on Metadata and Semantics Researc

Conversion of dietary inositol into propionate and acetate by commensal Anaerostipes associates with host health

Here, the authors report an anaerobic metabolic pathway from the dominant gut butyrogen Anaerostipes, showing several strains of this genus to be capable of producing propionate from dietary myo-inositol that associates with reduced fasting-glucose levels in mice. We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [C-13]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [C-13]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.Peer reviewe

Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8–SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors

Gelöste organische Stickstoffverbindungen in Seen - Vorkommen und Wirkung

Die Konzentration von Gesamt-DON betrug in den vier Modellseen im Jahresdurchschnitt 0.54 mg L-1 und übertraf damit die mittleren Konzentrationen von DIN (0.11-0.35 mg L-1) in allen Modellseen außer der Unterhavel. Huminstoffe und hochmolekulare Substanzen machen mit 80 - 93% den größten Anteil von DON aus, gefolgt von Harnstoff mit 6 - 16%. Die Konzentrationen von Aminosäuren sind so gering, dass ihnen im Folgenden keine weitere Bedeutung zugemessen wird. Die DON-Verbindungen sind innerhalb der einzelnen Gewässer über alle vier Jahreszeiten nahezu konstant. Dagegen zeigen die DIN-Verbindungen, besonders Nitrat, ein saisonales Muster. Sie erreichen im Winter ihre höchsten Konzentrationen und liegen im Sommer im Bereich der Nachweisgrenze. Daher ist der relative Anteil von DON-Verbindungen an der TN-Konzentration im Sommer deutlich höher. Von den DIN-Verbindungen Nitrat und Ammonium weiß man, dass sie von Algen für ihr Wachstum gut genutzt werden können. Sie werden beim Aufbau der Algenbiomasse bis zum Sommer nahezu aufgebraucht, weshalb sie im Sommer häufig nicht mehr nachweisbar sind. Daher tritt nach unseren bisherigen Untersuchungen besonders im Sommer in vielen Gewässern eine Begrenzung der Algen-biomasse durch Stickstoff auf (NITROLIMIT Diskussionspapier, Band 1). Allerdings könnten die Algen von den weiterhin reichlich vorhandenen DON-Verbindungen profitieren, wenn sie diese nutzen können. Unsere Studie zeigte, dass die verschiedenen DON-Verbindungen in unterschiedlichen Konzentrationen vorliegen und unterschiedlich durch Algen nutzbar sind. Mit zunehmender Komplexität der DON-Verbindungen nimmt die Nutzbarkeit durch Algen ab. Als am besten nutzbar erwies sich Harnstoff. Dessen relativ konstante Konzentration im Jahresverlauf trotz seiner guten Nutzbarkeit ist überraschend und am ehesten durch rasche Nachlieferung, etwa durch bakteriellen Abbau höhermolekularer N-Verbindungen, erklärbar. Die Nutzbarkeit von Huminstoffen hängt laut unseren ersten Untersuchungen auch von deren Ursprung und Alter ab. Während die frisch aus der Kläranlage Waßmannsdorf kommenden Huminstoffe von Algen für ihr Wachstum genutzt wurden, waren die schon länger in den untersuchten drei Seen befindlichen Huminstoffe kaum noch nutzbar. Der hohe Anteil von Huminstoffen und hochmolekularen Substanzen am TN könnte eine Ursache für die große Variabilität der TN-Zielwerte zum Erreichen des guten ökologischen Zustandes sein (NITROLIMIT Diskussionspapier, Band 1). Diese Substanzen können überwiegend nicht gut von Algen zum Aufbau von Biomasse genutzt werden und führen daher bei der Analyse des Zusammenhangs zwischen Biomasse und TN zu einer erhöhten Streuung (der TN-Zielwert wird hierbei über eine Regressionsanalyse zwischen Biomasse und der TN-Konzentration abgeleitet). Theoretisch sollten die Konzentrationen aller DON-Verbindungen, die nicht durch Algen nutzbar sind, von der TN-Konzentration subtrahiert werden. Die so kalkulierten Konzentrationen von durch Algen nutzbarem Stickstoff sollten einen besseren statistischen Zusammenhang mit der Algenbiomasse aufweisen als TN. Damit könnte die Streuung der Stickstoff-Zielwerte vermindert werden. Allerdings werden derartige Auswertungen erst möglich, wenn die Datenbasis zu algenverfügbaren DON-Verbindungen auf ähnlichem Niveau ist wie die zu DIN und TN. Zu letzteren liegen Daten für hunderte von Seen über mehrere Jahre vor, während Daten zu DON hier erstmals für vier Gewässer erhoben wurden