35 research outputs found

    Ecological and Chemical Studies on the Gq-protein Inhibitor FR900359

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    The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, displays a strong and selective inhibition of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as corroborated by in vivo experiments on mice, affinity to insect Gq proteins and insect toxicity studies. The uncultured endosymbiont of A. crenata, 'Candidatus Burkholderia crenata' was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. In this study we provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time, paving the way for a biotechnological production of FR independent from time- and work-intensive plant cultivation, harvesting and extraction. Direct targeting of intracellular Gα subunits is a challenging task in pursuit of chemical tools for pharmacological studies and for developing novel therapeutic approaches. We isolated four new FR analogs (1-4) from A. crenata and elucidated their structures by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS2 analysis. Next, we analyzed all currently known inhibitors of Gq protein including YM-254890, FR900359, above mentioned novel FR-derivatives from A. crenata, and synthetic cyclic peptides to devise a strategy for the elucidation of characteristics that determine interaction with Gq. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocycles that govern specific binding to and inhibition of Gq. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed biological activity of FR or YM. This raises the question of whether nature has optimized these depsipeptides for specific inhibition of Gq. Thus, rather than attempting to enhance Gq activity of newly synthesized inhibitors, future synthetic efforts on FR/YM-analogs should target Gα subunits other than Gq. Additionally, FR was detected from leaves of five other Ardisia species, among them the non-nodulated A. lucida as well as from a soil bacterium, implicating a much broader distribution of FR as originally anticipated. Furthermore the first reported fluorescent FR analogs were synthesized, biologically evaluated and applied to study the mechanism of cellular uptake of FR

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

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    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 G subunits, we here set out to rationally design G16 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 Gq/G11-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 G proteins harboring engineered FR-binding sites

    A community resource for paired genomic and metabolomic data mining

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    Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.Peer reviewe

    MassIVE MSV000090957 - Marine fungi - metabolomics and native metabolomics

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    MassIVE MSV000092685 - MassQL-guided_MN_new_cyclic_tetrapeptides

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    MassIVE MSV000090955 - Native MS of Gproteins 12_2022

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    The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis

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    A majority of Ardisia species harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted hereditarily and have not yet been cultured outside of their host. Because the plants cannot develop beyond the seedling stage without their symbionts, the symbiosis is considered obligatory. We sequenced for the first time the genome of Candidatus Burkholderia crenata (Ca. B. crenata), the leaf nodule symbiont of Ardisia crenata. The genome of Ca. B. crenata is the smallest Burkholderia genome to date. It contains a large amount of insertion sequences and pseudogenes and displays features consistent with reductive genome evolution. The genome does not encode functions commonly associated with plant symbioses such as nitrogen fixation and plant hormone metabolism. However, we identified unique genes with a predicted role in secondary metabolism in the genome of Ca. B. crenata. Specifically, we provide evidence that the bacterial symbionts are responsible for the synthesis of compound FR900359, a cyclic depsipeptide with biomedical properties previously isolated from leaves of A. crenata
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