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

Abstract

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 Phaeodactylum tricornutum 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.

Fig 1. Synthesis of the probe TAMRA-PUA and the control TAMRA-SA.

For details on the synthesis see [35].

Fig 2. Schematic in vivo application of the probe.

Living cells of P. tricornutum 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 P. tricornutum 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.

Fig 3. Fluorescence intensity of P. tricornutum cells treated under different conditions.

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 S1 and S2 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).

Fig 4. Fluorescence microscopy of P. tricornutum cells.

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.