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Diazirine photo-immobilisation for target profiling of natural products and co-factors: pulldown data

ABSTRACT: Finding the targets of natural products is of key importance in both chemical biology and drug discovery, and deconvolution of cofactor interactomes contribute to the functional annotation of the proteome. Identifying the proteins that underlie natural compound activity in phenotypic screens help to validate the respective targets and, potentially, expand the druggable proteome. Here, we present a generally applicable protocol for the photoactivated immobilisation of unmodified and microgram quantities of natural products on diazirine-decorated beads and their use for systematic affinity-based proteome profiling. We show that amongst 31 molecules of very diverse reported activity and biosynthetic origin, 25 could indeed be immobilised. Dose-response competition binding experiments using lysates of human or bacterial cells followed by quantitative mass spectrometry recapitulated <100 µM targets of 9 molecules. Among them, immobilisation of coenzyme A produced a tool to interrogate proteins containing a HotDog domain. Surprisingly, immobilisation of the cofactor flavin adenine dinucleotide (FAD) led to the identification of nanomolar interactions with dozens of RNA-binding proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Cell Suspension Culture, Epithelial Cell, Cell Culture, Escherichia Coli Metabolite

DISEASE(S): Colorectal Adenocarcinoma,Acute Leukemia

SUBMITTER: Polina Prokofeva

LAB HEAD: Prof. Dr. Bernhard Kuster

PROVIDER: PXD033292 | Pride | 2022-10-06

REPOSITORIES: Pride

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Project description:Background: Earthworms are essential for a healthy soil ecosystem, performing key functions such as bioturbation. The soil ecosystem can be challenged by natural toxins such as isothiocyanates (ITC), secondary metabolites produced by many commercial crops. This study investigated the effects of the natural toxin, 2-phenylethyl isothiocyanate on the earthworm Eisenia andrei using an ecotoxicogenomics approach that combined standardised ecotoxicological testing with gene expression analyses. Results and discussion: Exposure to 2-Phenylethyl ITC reduced both survival and reproduction of E. andrei in a dose-dependent manner, with an EC50 (reproduction) value of 556 nmol/g soil. Cross-species comparative genomic hybridisation validated the applicability of an existing 4x44k E. fetida microarray to E. andrei. Gene expression profiles revealed the importance of metallothionein (mt) as an early warning signal when E. andrei is exposed to low concentrations of 2-phenylethyl ITC. Allignment of these mt genes with the mt-2 gene of Lumbricus rubellus showed that there must at least have 2 mt genes in the Eisenia sp. genome. At high exposure concentrations of 2-phenylethyl ITC gene expression was mainly affected by inhibiting chitinase activities, inducing an oxidative stress response, and stimulating energy metabolism. KEGG pathway analysis, furthermore, implied that the high concentration may cause impaired light sensitivity, angiogenesis (e.g. wound healing), olfactory perception (olfaction), learning and memory. Conclusions: Increased levels of isothiocyanates may be found in the field in the near future. Risks of such compounds should be carefully investigated before allowing them to enter the soil on a large scale. This is the first study to investigate the effects of an isothiocyanate on an earthworm both on the ecotoxicological and genomic level. Three-exposure experiment; Acetone control vs.EC10 vs.EC50 exposed animals. Biological replicates: 4 replicates for each exposure. Technical replicates: 2 replicates for each exposure

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Diazirine photo-immobilisation for target profiling of natural products and co-factors: pulldown data

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