Structural response of G protein binding to the cyclodepsipeptide inhibitor FR900359 probed by NMR spectroscopy.

The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of Gq proteins by blocking GDP/GTP exchange. The inhibitor binding site of Gq has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects.

The Bacterial Gq Signal Transduction Inhibitor FR900359 Impairs Soil-Associated Nematodes.

The cyclic depsipeptide FR900359 (FR) is derived from the soil bacterium Chromobacterium vaccinii and known to bind Gq proteins of mammals and insects, thereby abolishing the signal transduction of their Gq protein-coupled receptors, a process that leads to severe physiological consequences. Due to their highly conserved structure, Gq family of proteins are a superior ecological target for FR producing organisms, resulting in a defense towards a broad range of harmful organisms. Here, we focus on the question whether bacteria like C. vaccinii are important factors in soil in that their secondary metabolites impair, e.g., plant harming organisms like nematodes. We prove that the Gq inhibitor FR is produced under soil-like conditions. Furthermore, FR inhibits heterologously expressed Gαq proteins of the nematodes Caenorhabditis elegans and Heterodera schachtii in the micromolar range. Additionally, in vivo experiments with C. elegans and the plant parasitic cyst nematode H. schachtii demonstrated that FR reduces locomotion of C. elegans and H. schachtii. Finally, egg-laying of C. elegans and hatching of juvenile stage 2 of H. schachtii from its cysts is inhibited by FR, suggesting that FR might reduce nematode dispersion and proliferation. This study supports the idea that C. vaccinii and its excreted metabolome in the soil might contribute to an ecological equilibrium, maintaining and establishing the successful growth of plants.

Feature-Based Molecular Networking for the Targeted Identification of Gq-Inhibiting FR900359 Derivatives.

Both the soil bacterium Chromobacterium vaccinii and the bacterial endosymbiont Candidatus Burkholderia crenata of the plant Ardisia crenata are producers of FR900359 (FR). This cyclic depsipeptide is a potent and selective Gq protein inhibitor used extensively to investigate the intracellular signaling of G protein coupled receptors (GPCRs). In this study, the metabolomes of both FR producers were investigated and compared using feature-based molecular networking (FBMN). As a result, 30 previously unknown FR derivatives were identified, one-third being unique to C. vaccinii. Guided by MS, a novel FR derivative, FR-6 (compound 1), was isolated, and its structure unambiguously established. In a whole-cell biosensing assay based on detection of dynamic mass redistribution (DMR) as readout for Gq inhibition, FR-6 suppressed Gq signaling with micromolar potency (pIC50 = 5.56). This functional activity was confirmed in radioligand binding assays (pKi = 7.50). This work demonstrates the power of molecular networking, guiding the way to a novel Gq-inhibiting FR derivative and underlining the potency of FR as a Gq inhibitor.

Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

Outlining where humans live, the World Settlement Footprint 2015.

Human settlements are the cause and consequence of most environmental and societal changes on Earth; however, their location and extent is still under debate. We provide here a new 10 m resolution (0.32 arc sec) global map of human settlements on Earth for the year 2015, namely the World Settlement Footprint 2015 (WSF2015). The raster dataset has been generated by means of an advanced classification system which, for the first time, jointly exploits open-and-free optical and radar satellite imagery. The WSF2015 has been validated against 900,000 samples labelled by crowdsourcing photointerpretation of very high resolution Google Earth imagery and outperforms all other similar existing layers; in particular, it considerably improves the detection of very small settlements in rural regions and better outlines scattered suburban areas. The dataset can be used at any scale of observation in support to all applications requiring detailed and accurate information on human presence (e.g., socioeconomic development, population distribution, risks assessment, etc.).

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity.

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.

Antimicrobial Dialkylresorcins from Marine-Derived Microorganisms: Insights into Their Mode of Action and Putative Ecological Relevance.

Zobellia galactanivorans has been reported as a seaweed-associated or marine-derived species with largely unknown secondary metabolites. The combination of bioinformatic analysis and MS- and bioactivity guided separation led to the isolation of a new antibiotically active dialkylresorcin from the marine bacterium Z. galactanivorans. The antibiotic profile of the new dialkylresorcin zobelliphol (1: ) was investigated and compared with related and naturally occurring dialkyresorcins (i.e., stemphol (2: ) and 4-butyl-3,5-dihydroxybenzoic acid (3: )) from the marine-derived fungus Stemphylium globuliferum. Bacterial reporter strain assays provided insights into the mode of action of this antibiotic compound class. We identified an interference with bacterial DNA biosynthesis for the dialkylresorcin derivative 1: . In addition, the putative biosynthetic gene cluster corresponding to production of 1: was identified and a biosynthetic hypothesis was deduced.

The impact of livestock grazing on plant diversity: an analysis across dryland ecosystems and scales in southern Africa.

A general understanding of grazing effects on plant diversity in drylands is still missing, despite an extensive theoretical background. Cross-biome syntheses are hindered by the fact that the outcomes of disturbance studies are strongly affected by the choice of diversity measures, and the spatial and temporal scales of measurements. The aim of this study is to overcome these weaknesses by applying a wide range of diversity measures to a data set derived from identical sampling in three distinct ecosystems. We analyzed three fence-line contrasts (heavier vs. lighter grazing intensity), representing different degrees of aridity (from arid to semiarid) and precipitation regimes (summer rain vs. winter rain) in southern Africa. We tested the impact of grazing intensity on multiple aspects of plant diversity (species and functional group level, richness and evenness components, alpha and beta diversity, and composition) at two spatial scales, and for both 5-yr means and interannual variability. Heavier grazing reduced total plant cover and substantially altered the species and functional composition at all sites. However, a significant decrease in species alpha diversity was detected at only one of the three sites. By contrast, alpha diversity of plant functional groups responded consistently across ecosystems and scales, with a significant decrease at heavier grazing intensity. The cover-based measures of functional group diversity responded more sensitively and more consistently than functional group richness. Beta diversity of species and functional types increased under heavier grazing, showing that at larger scales, the heterogeneity of the community composition and the functional structure were increased. Heavier grazing mostly increased interannual variability of alpha diversity, while effects on beta diversity and cover were inconsistent. Our results suggest that species diversity alone may not adequately reflect the shifts in vegetation structure that occur in response to increased grazing intensity in the dryland biomes of southern Africa. Compositional and structural changes of the vegetation are better reflected by trait-based diversity measures. In particular, measures of plant functional diversity that include evenness represent a promising tool to detect and quantify disturbance effects on ecosystems.

[Self-selection processes in the choice of the therapeutic training approach: differences in therapeutic attitudes, personality traits and attributional complexity]. / Selbstselektionsprozesse bei der Wahl des therapeutischen Ausbildungsverfahrens: Unterschiede in therapeutischen Haltungen, Persönlichkeitseigenschaften und dem Mentalisierungsinteresse.

AIM: Treatment approaches differ to a great extent in terms of basic psychological assumptions and practical procedures. This creates questions about the fitting of therapist and therapeutic approach. This paper examines the influence of therapeutic attitudes, mentalization interest and personality traits on the decision for an approach. METHODS: 184 participants of training programs in one of the 3 licensed treatment approaches in Germany were examined with questionnaires at the beginning of their training. RESULTS: Participants significantly differed in terms of therapeutic attitudes and the metallization interest but not in personality traits except openness. Satisfaction with training was not related to the individual fit of participants to the therapeutic attitudes typical for their approach. CONCLUSION: Therapeutic attitudes, the extent of mentalization interest, and openness may play a role in self-selection processes in the choice of the approach.