9 research outputs found
Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening
AbstractSmall molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29μM), retinoic acid, eosin (IC50 27μM) and erythrosin (IC50 36μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads
Label-Free Confocal Raman Mapping of Transportan in Melanoma Cells
Cell-penetrating peptides (CPPs)
are promising vectors for the
intracellular delivery of a variety of membrane-impermeable bioactive
compounds. The mechanisms by which CPPs cross the cell membrane, and
the effects that CPPs may have on cell function, still remain to be
fully clarified. In this work, we employed confocal Raman microscopy
(CRM) and atomic force microscopy (AFM) to study the infiltration
and physiological effects of the amphipathic CPP transportan (Tp)
on the metastatic melanoma cell line SK-Mel-2. CRM enabled the detection
of label-free Tp within the cells. Raman maps of live cells revealed
rapid entry (within 5 min) and widespread distribution of the peptide
throughout the cytoplasm and the presence of the peptide within the
nucleus after ∼20 min. Principal component analysis of the
CRM data collected from Tp-treated and untreated cells showed that
Tp Raman bands were not positively correlated with lipid Raman bands,
indicating that Tp entered the cells via a nonendocytic mechanism.
Analysis of intracellularly recovered Tp by mass spectrometry showed
that Tp remained intact in SK-Mel-2 cells for up to 24 h. The Raman
spectroscopic data also showed that, although Tp was predominantly
unstructured (random coil) in aqueous solution, it accumulated to
high densities within the cells with mostly β-sheet and α-helical
structures. AFM was employed to measure the effect of Tp treatment
on cell stiffness. These data showed that Tp induced a significant
increase in cell stiffness within the first hour of treatment, which
was partially abated after 2 h. It is hypothesized that the increase
in cell stiffness was the result of cytoskeletal changes triggered
by Tp