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. 2022 May 10;11:e73913. doi: 10.7554/eLife.73913

Figure 3. (S)-DACs lipoxidize multiple cellular proteins, triggering their association with cellular membranes.

(A) U2OS cells were incubated for 2 h with 2 µM (S)- or (R)–9, proteins were extracted and DAC-modified proteins were detected by CuAAC-mediated ligation of azido-AlexaFluor-647 to clickable molecules, separation by SDS-PAGE, transfer to a membrane which was scanned for fluorescence. (B) Landscape of proteins modified in U2OS cells by clickable DAC (S)–9 computed from three independent experiments. Fold enrichment (FC) as compared to the clickable (R)–9 is computed and color-coded as depicted. Box size corresponds to -log(p) computed as described in the materials and methods section. (C) PSMD2 or control immunoprecipitations (Ctrl) were performed from extracts of U2OS cells treated 2 h with 2 µM clickable DAC (S)- or (R)–9. DAC-modified proteins were detected by CuAAC-mediated ligation of azido-AlexaFluor-647 to clickable molecules, separation by SDS-PAGE, transfer to a membrane, which was scanned for fluorescence. PSMD2 was subsequently visualized by immunoblotting. (D) PSMD2 immunoprecipitations were performed from extracts of wild-type or DAC-resistant HAP-1 cells treated or not for 2 h with 2 µM clickable DAC (S)–9. DAC-modified proteins were detected by CuAAC-mediated ligation as in (C). PSMD2 was subsequently visualized by immunoblotting. (E) U2OS cells were treated 2 h with 0.5 µM DAC, fixed, permeabilized, and clickable molecules were detected by click with AlexaFluor488 azide. (F) U2OS expressing GFP-BRAT1 were treated 2 h with 1 µM (S)–3, pre-extracted, fixed and processed for analysis by fluorescence microscopy.

Figure 3—source data 1. Source data related to Figure 3A.
The tiff files correspond to an uncropped picture of the AlexaFluor647 fluorescence signal, acquired on an Odyssey LI-COR, and of a scan of the membrane stained with Ponceau S. The regions used to generate the figure are highlighted by back squares in the jpg file.
Figure 3—source data 2. Source data related to Figure 3C.
The tiff files correspond to uncropped pictures of the AlexaFluor647 and PSMD2 fluorescence signal, both detected on a LI-COR Odyssey. The jpg file combines both pictures and can be used to locate the protein ladders.
Figure 3—source data 3. Source data related to Figure 3D.
The tiff files correspond to uncropped pictures of the AlexaFluor647 and PSMD2 fluorescence signal, both detected on a LI-COR Odyssey. The jpg file combines both pictures and can be used to locate the protein ladders.

Figure 3.

Figure 3—figure supplement 1. Parallel between protein palmitoylation and protein lipoxidation by the DAC 3.

Figure 3—figure supplement 1.

The addition of a C16 lipidic anchor resulting from protein palmitoylation is sufficient to promote the association of some intracellular proteins to membranes. Lipoxidation by a C17 DAC is likely to have the same effect.
Figure 3—figure supplement 2. DAC (S)–9 is bioactivated into protein-reactive species.

Figure 3—figure supplement 2.

(A) Cell viability analysis of U2OS, wild-type HAP-1 or DAC resistant HAP-1 clone A4 treated for 72 h with clickable DAC (S)–9 or (R)–9. (B) Analysis by immunoblotting of GFP levels in U2OS stably expressing GFP (Ctrl), and GFP-tagged PLIN3, BRAT1, PSMD2 and TK1. (C) U2OS depicted in B were incubated for 2 h with 2 µM (S)–9 or (R)–9, proteins were extracted and DAC-modified proteins were detected by CuAAC-mediated ligation of azido-AlexaFluor-647 to clickable molecules, separation by SDS-PAGE, transfer to a membrane which was scanned for fluorescence. (D) PSMD2 or control immunoprecipitations were performed from extracts of U2OS cells and beads were treated with (S)–9 or DACone 10. After washes, DAC/DACone-modified proteins were detected by CuAAC-mediated ligation of azido-AlexaFluor-647 to clickable molecules, separation by SDS-PAGE, transfer and scanning membrane fluorescence. PSMD2 was subsequently visualized by immunoblotting. (E) Wild-type or (S)-DAC-resistant HAP-1, clone A1 or A4, cells were untreated or treated 2 h with 2 µM clickable DAC (S)–9. DAC-modified proteins were detected by CuAAC-mediated ligation of azido-AlexaFluor-647 to clickable molecules, separation by SDS-PAGE, transfer to a membrane which was scanned for fluorescence. PSMD2 was subsequently visualized by immunoblotting.
Figure 3—figure supplement 2—source data 1. Source data related to Figure 3—figure supplement 1B.
The tiff files correspond to uncropped pictures of the IRDye800CW fluorescence signal acquired on a LI-COR Odyssey. The regions used to generate the figure are highlighted by back squares in the jpg file.
Figure 3—figure supplement 2—source data 2. Source data related to Figure 3—figure supplement 2C.
The tiff files correspond to uncropped pictures of the AlexaFluor647 fluorescence signal and of the IRDye800CW signal (GFP), both acquired on an Odyssey LI-COR, and a scan of the membrane stained with Ponceau S. The jpg file combines all pictures and can be used to locate the protein ladders.
Figure 3—figure supplement 2—source data 3. Source data related to Figure 3—figure supplement 2D.
The tiff files correspond to uncropped pictures of the AlexaFluor647 and PSMD2 fluorescence signal, both detected on a LI-COR Odyssey. The jpg file combines both pictures and can be used to locate the protein ladders.
Figure 3—figure supplement 2—source data 4. Source data related to Figure 3—figure supplement 2E.
The tiff files correspond to an uncropped picture of the AlexaFluor647 fluorescence signal, acquired on an Odyssey LI-COR, and of a scan of the membrane stained with Ponceau S. The jpg file combines both pictures and can be used to locate the protein ladders.
Figure 3—figure supplement 3. Glutathione can be modified by DACones in vitro but does not impact on (S)-DAC 3 cytotoxic activity. (A).

Figure 3—figure supplement 3.

(A) Structure of reduced glutathione. (B) Absorption spectra of Nα-acetyl-lysine (NAK), N-acetyl-cysteine (NAC), reduced glutathione (GSH) or DACone 8 incubated alone or together at pH 7. (C) Structure of the 8-GSH adduct (top) with the related 1H-13C HSQC spectra (bottom). (D) Cell viability analysis of U2OS cells incubated for 24 h with 5 mM cell permeable glutathione (reduced glutathione monoethyl ester, GSHe) and subsequently co-treated with (S)–3 for 72 h. (E) Cell viability analysis of A549 cells pre-treated for 24 h with the indicated concentration of glutathione S-transferase inhibitor ethacrynic acid (GSTi) and subsequently co-treated with (S)–3 for 72 h.
Figure 3—figure supplement 4. The clickable DAC (S)–9 staining colocalizes with the nucleus, ER and mitochondria.

Figure 3—figure supplement 4.

(A-C) U2OS cells were treated 2 h with 0.5 µM of DAC (S/R)–9. (A) After fixation, CuAAC was used to label the DACs with AlexaFluor594-azido, while the ER membranes were labelled using Concanavalin A-AlexaFluor488 (Conc. A). (B) 30 min before the end of DAC treatment, the cells were incubated with MitoTracker Red CMXRos to label mitochondria. After fixation, click chemistry was used to label clickable DACs with AlexaFluor488-azido. (C) After fixation, CuAAC was used to label clickable DACs with AlexaFluor488-azido while immunofluorescence with an anti-COXIV antibody coupled with AlexaFluor594 was used to label mitochondria. On all pictures, DAPI was used to stain DNA. White scale bar = 10 µm.
Figure 3—figure supplement 5. The DAC (S)–3 triggers ER-swelling and mitochondrial fission.

Figure 3—figure supplement 5.

U2OS stably co-expressing a GFP variant addressed and retained in the endoplasmic reticulum and mCherry addressed to mitochondria were treated with 1 µM (S)–3 and monitored by live imaging.