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Smrckova, H.

Publications and source records attributed to Smrckova, H..

2 recordsLinked to original sources
bioRxiv2024

Discovery and isolation of novel capsaicinoids and their TRPV1-related activity

Chilis contain capsaicin and other structurally related molecules known as capsaicinoids. Capsaicins target protein, the transient receptor potential cation channel subfamily V member 1 (TRPV1), has been linked to many post-activation effects, including changes in metabolism and pain sensation. Capsaicinoids also bind to TRPV1, but current studies often disregard non-capsaicin interactions. To fill in these gaps, we screened 40 different chili varieties derived from four Capsicum species by means of untargeted metabolomics and a rat TRPV1 (rTRPV1) calcium influx activation assay. The resulting capsaicinoid profiles were specific to each variety but only partially corresponded with species delimitations. Based on rTRPV1 activation elicited by crude chili extracts, capsaicinoids act in an additive manner and a capsaicinoid profile can serve as a gauge of this activation. In addition, we isolated eighteen capsaicinoids, including five previously unreported ones, and confirmed their structure by NMR and MS/MS. We then tested rTRPV1 activation by 23 capsaicinoids and three related compounds. This testing revealed that even slight deviations from the structure of capsaicin reduce the ability to activate the target, with a mere single hydroxylation on the acyl tail reducing potency towards rTRPV1 by more than 100-fold. In addition, we tested how rTRPV1 activity changes in the presence of capsaicin together with non-activating capsaicin analogs and weakly activating capsaicinoids and found both classes of molecules to positively modulate the effects of capsaicin. This demonstrates that even such compounds have measurable pharmacological effects, making a case for the use and study of natural chili extracts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/620944v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1d56d02org.highwire.dtl.DTLVardef@e7a66dorg.highwire.dtl.DTLVardef@5f409forg.highwire.dtl.DTLVardef@18b88f7_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Smith, J. (2025) https://BioRender.com/a61h668 HighlightsO_LI5 novel capsaicinoids (vanilloids) and 13 other capsaicinoids were isolated from chilis. C_LIO_LIThe slightest deviation in the capsaicin structure results in decreased TRPV1 activity. C_LIO_LIA single hydroxylation can reduce potency to TRPV1 100-fold. C_LIO_LINon-pungent vanilloids modulate capsaicin-TRPV1 activity. C_LI

pharmacology and toxicology↗
bioRxiv2023

POMBOX: a fission yeast toolkit for molecular and synthetic biology

Schizosaccharomyces pombe is a popular model organism in molecular biology and cell physiology. With its ease of genetic manipulation and growth, supported by in-depth functional annotation in the PomBase database and genome-wide metabolic models, S. pombe is an attractive option for synthetic biology applications. However, S. pombe currently lacks modular tools for generating genetic circuits with more than one transcriptional unit. We have developed a toolkit to address this issue. Adapted from the MoClo- YTK plasmid kit for Saccharomyces cerevisiae and using the same Golden Gate grammar, our POMBOX toolkit is designed to facilitate the fast, efficient and modular construction of genetic circuits in S. pombe. It allows for interoperability when working with DNA sequences that are functional in both S. cerevisiae and S. pombe (e.g. protein tag, antibiotic resistance cassette, coding sequences). Moreover, POMBOX enables the modular assembly of multi-gene pathways and increases possible pathway length from 6 to 12 transcriptional units. We also adapted the stable integration vector homology arms to Golden Gate assembly and tested the genomic integration success rate depending on different sequence sizes, from four to twenty-four kilobases. We included fourteen S. pombe promoters that we characterized for two fluorescent proteins, in both minimal defined media (EMM2) and complex media (YES). Then we tested six S. cerevisiae and six synthetic terminators in S. pombe. Finally, we used the POMBOX kit for a synthetic biology application in metabolic engineering and expressed plant enzymes in S. pombe to produce specialized metabolite precursors, namely methylxanthine, amorpha-4,11-diene and cinnamic acid from the purine, mevalonate and amino acid pathways.

synthetic biology↗