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

Figure 2. DACones are protein reactive species.

(A) Reaction catalyzed by HSD17B11. (B) Clickable DACs and DACones used in the study. (C) Viability analysis of U2OS cells treated in PBS for 1 h with (S)–3 or DACones and incubated for an additional 72 h after drug washout. (D) FBS or purified BSA were incubated 40 min at 30 °C with clickable DAC (S)–9 or clickable DACone 10. After reaction, CuAAC was used to ligate an azido-AlexaFluor647 to clickable molecules. Modified proteins were detected by scanning membrane fluorescence after SDS-PAGE and transfer. Ponceau S stains total proteins. (E) BSA or BLG were incubated with the indicated DACs or DACones, as in (D). After reaction, modified proteins were detected as in (D). Coomassie stains total proteins. (F) WT or S172L HSD17B11-GFP were immunoprecipitated from complemented U2OS KO HSD17B11 cells and incubated with clickable DAC 9 and BLG. After reaction, modified proteins were detected in the supernatant (BLG) or on the beads (HSD17B11-GFP) as in (D). GFP immunoblotting confirmed that equal amounts of WT and S172L proteins were used. (G) Analysis by direct-infusion mass spectrometry of purified BLG (mixture of isoform A and B) modified or not by DACone 10. Cyan and green arrows indicate the formation of a first and second adduct, respectively. (H) % of each amino acid detected as modified by DACones 10 or 11 in U2OS extracts as determined using an isoDTB-ABPP-based framework. (I) Proposed reactions of DACones with cysteine and lysine side chains in proteins.

Figure 2—source data 1. Source data related to Figure 2D.
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 2—source data 2. Source data related to Figure 2E.
The tiff files correspond to uncropped pictures of the AlexaFluor647 fluorescence, acquired in gel on an Odyssey LI-COR, and of the gels after staining with Coomassie blue scanned with the BioRad Chemidoc. Two different gels were used (respectively labeled upper and lower). Each jpg file combines the two pictures used to generate upper and lower parts of the figure.
Figure 2—source data 3. Source data related to Figure 2F.
The tiff files correspond to uncropped pictures of the AlexaFluor647 fluorescence signal, acquired on an Odyssey LI-COR, of a scan of the membrane stained with Ponceau S, for the upper part (supernatant), and of the chemiluminescent signal acquired using autoradiographic films for the lower part (beads). The jpg file combines the pictures used to generate the figure and can be used to locate the protein ladders.

Figure 2.

Figure 2—figure supplement 1. DACones are protein-reactive species.

Figure 2—figure supplement 1.

(A) Purified BSA, BLG, carbonic anhydrase (CANH) and concanavalin A (ConcA) were incubated with clickable DAC (S)–9 or DACone 10. Adducts on proteins were detected by scanning membrane fluorescence after CuAAC-mediated ligation of a AlexaFluor647 fluorophore, SDS-PAGE and transfer on a nitrocellulose membrane. Total proteins were visualized by Ponceau S staining. (B) Absorption spectra of BLG, DAC (S)–3 or DACones incubated alone or together at pH 7.4. (C) Absorbance spectra of purified BSA, DAC (S)–3 or DACones incubated alone or together as indicated. (D,E,F) Number of peptide spectrum matches (PSMs) in the analysis of the U2OS extracts treated with MeCN (solvent control, D), 100 µM of the DACone 10 (E) or 11 (F). An Open Search in MSFragger was used to identify the masses of modification. The peaks highlighted in red are the expected modifications (Δmexp) resulting from lipoxidation and modification with the light and heavy isoDTB tags, respectively. Further modification of the lipoxidated peptides by reaction with water ( + 18) or DTT ( + 154) are also indicated. (G) Absorption spectra of Nα-acetyl-lysine (NAK), N-acetyl-cysteine (NAC), N-acetyl-glycine (NAG), or DACone 8 incubated alone or together at pH 7 or at pH 10. (H,I) NAC (H) or NAK (I) were incubated at pH10 (KOH) with the DACone 8 to allow the formation of the covalent linkage between the DACone and the thiol/amine group of NAC/NAK, as monitored by formation of the ~323 nm absorbance band. The pH was neutralized with diluted HCl and the reaction products were incubated at 30 °C at neutral pH (H2O) or with TFA at 0.1% (pH~2) for the indicated time. Acquisitions of UV-visible spectra were performed to monitor the presence of NAC-DACone (H) or NAK-DACone (I) covalent linkages.
Figure 2—figure supplement 1—source data 1. Source data related to Figure 2—figure supplement 1A.
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 the pictures used to generate and can be used to locate the protein ladders.
Figure 2—figure supplement 2. Molecular docking of (S)- and (R)–3 on 17-βHSD.

Figure 2—figure supplement 2.

(A) AlphaFold2-multimer model of the HSD17B11 homodimer. NAD+ was added to one HSD17B11 subunit only. (B) Close-up on (S)–3 docked into HSD17B11-NAD+ model. The hydrogen bonds are shown in green and the NAD+ in pink. (C) Representation of the contacts between (S)–3 and HSD17B11. (D) Close up on (R)–3 docked into the HSD17B11 model. (E) Representation of the contacts between (R)–3 and HSD17B11. (F) Table summarizing for each of the 17β-HSD17 SDRs its expression in U2OS and HAP1, its reactional polarity, its preferred cofactor(s), its percentage of sequence identity with HSD17B11 and the number of high stringency conformers identified by molecular docking between them and (S)- and (R)–3. (G) Analysis by immunoblotting of the expression levels of GFP tagged proteins in U2OS KO for HSD17B11 stably complemented by GFP, HSD17B11-GFP or HSD17B13-GFP. (H) Cell viability analysis of U2OS inactivated for HSD17B11, complemented as shown in (G) and treated for 72 h with DAC (S)–3.
Figure 2—figure supplement 2—source data 1. Source data related to Figure 1—figure supplement 2G.
The tiff files correspond to uncropped pictures the chemiluminescent signal acquired on a BioRad Chemidoc. The regions used to generate the figure are highlighted for each immunoblot by back squares in the jpg file, which also contains at the bottom an overlay with a picture of the membrane to locate the protein ladder positions.
Figure 2—figure supplement 3. Sequence context of modified cysteines and lysines.

Figure 2—figure supplement 3.

(A,B) A 11 amino acids window centered on the cysteines (A) or lysines (B) modified by the DACone 10 was used to generate a sequence logo. This highlighted that the DACone-modified lysines are preferentially surrounded by hydrophobic amino acids, while no clear enrichment could be identified for the modified cysteines.
Figure 2—figure supplement 4. Characterization of DACone reaction products with NAC and NAK.

Figure 2—figure supplement 4.

(A) 1H NMR spectra of DACone 8-NAC adduct (lower spectrum) or NAC alone (upper spectrum). (B)1H-13C HSQC spectrum of DACone 8-NAC adduct. (C)1H NMR spectra of DACone 8-NAK adduct (lower spectrum) or NAK alone (upper spectrum). (D)1H-13C HSQC spectrum of DACone 8-NAK adduct. (E) Proposed Michael addition reaction of NAC or NAK with the short DACone 8.