A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom <i>Phaeodactylum tricornutum</i>

<div><p>Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom <i>Phaeodactylum tricornutum</i> in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.</p></div

Revealing the macromolecular targets of complex natural products

Natural products have long been a source of useful biological activity for the development of new drugs. Their macromolecular targets are, however, largely unknown, which hampers rational drug design and optimization. Here we present the development and experimental validation of a computational method for the discovery of such targets. The technique does not require three-dimensional target models and may be applied to structurally complex natural products. The algorithm dissects the natural products into fragments and infers potential pharmacological targets by comparing the fragments to synthetic reference drugs with known targets. We demonstrate that this approach results in confident predictions. In a prospective validation, we show that fragments of the potent antitumour agent archazolid A, a macrolide from the myxobacterium Archangium gephyra, contain relevant information regarding its polypharmacology. Biochemical and biophysical evaluation confirmed the predictions. The results obtained corroborate the practical applicability of the computational approach to natural product 'de-orphaning'

Confident target proteins found by 2D GE. ¹

<p>¹Proteins in this table were found in at least two of the three gels, a full list of labeled proteins can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140927#pone.0140927.s007" target="_blank">S1 Information</a>. OOO—only one protein per excised gel spot was found, OO—identification of probe labeled protein besides other unlabeled proteins in a gel spot, O—more than one labeled protein per excised gel spot, X—no hit.</p><p>^aNames are temporarily ascribed to an open reading frame (ORF) by a sequencing project [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140927#pone.0140927.ref041" target="_blank">41</a>].</p><p>Confident target proteins found by 2D GE. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140927#t001fn001" target="_blank">¹</a></p

Fluorescence intensity of <i>P</i>. <i>tricornutum</i> cells treated under different conditions.

<p>Cells were either incubated with TAMRA-PUA, TAMRA-N₃, TAMRA-SA for one hour or kept under identical conditions without probe. For each treatment three microscope slides with four cells each were measured. Unmodified raw data are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140927#pone.0140927.s004" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140927#pone.0140927.s005" target="_blank">S2</a> Folders. Fluorescence intensities were recorded as mean gray value per pixel after data treatment as described in the main text. Averaged mean gray values per pixel of cells of each treatment are presented as bars ±SD. One way Anova comparing results of different microscope slides within one treatment revealed no statistical difference (p>0.05). Kruskal-Wallis one way analysis of variance on ranks revealed differences in the median values among the treatment groups (H = 42.436, p<0.001) and Tukey’s HSD test (p<0.05) allowed classification into three groups: (a) TAMRA-PUA with the highest mean gray value per pixel of 3661±809, (b) TAMRA-SA with 800±140 and (c) TAMRA-N₃ and control with almost no emission signals (20±10 and 35±9); these controls were not significantly different to each other (Tukey’s HSD test p>0.05).</p

Schematic <i>in vivo</i> application of the probe.

<p>Living cells of <i>P</i>. <i>tricornutum</i> were incubated with the PUA-derivative DDY. After removal of excess DDY cell lysis followed by CuAAC enables attachment of the fluorescent reporter to covalently labeled proteins. 1D GE quickly allows detection of labeled proteins (not shown). Identification of protein targets is enabled by 2D GE. Therefore, a second <i>P</i>. <i>tricornutum</i> sample was treated with Cy5 NHS ester to label the whole proteome. The combined samples were separated using DIGE, labeled proteins were digested using trypsin and the resulting peptides were separated and analyzed by LC-MS/MS.</p

Fluorescence microscopy of <i>P</i>. <i>tricornutum</i> cells.

<p>3D (left) and 2D (right) images of a TAMRA-PUA (A) and a TAMRA-SA (B) treated cell. Images were taken in 3D SIM mode. A 561nm laser was used for excitation, and fluorescence was filtered by a band pass filter (BP 570-620nm) which opens up above 750nm. Fluorescence is visible in the entire cells, which confirms that both probes were taken up.</p

Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol