Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation

Abstract

S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.

Keywords: Activity-based protein profiling; Palmitoylation.

Figure 1.. Structures and initial screening of palmostatin analogs.

a, Structures of Palmostatin M (Palm M) and analogs. b, Effect of palmostatin analogs on dynamic protein palmitoylation as detected by pulse-chase metabolic labeling of ON and ONK cells with the 17-ODYA probe followed by CuAAC conjugation to a Rh-N₃ reporter tag, SDS-PAGE, and in-gel fluorescence scanning. Compounds were screened at 20 μM (Palm M control screened at 10 μM). c, ABHD17 inhibitory activity of palmostatin analogs measured by gel ABPP of HEK293T cells stably expressing ABHD17B. Compounds, including HDFP, were screened at 20 μM and incubated with cells in situ for 4 h prior to lysis, treatment with FP-Rh (1 μM, 1 h), SDS-PAGE, and in-gel fluorescence scanning. The screens in b and c were performed once. Note that the order of compounds 9–13 is differently arranged in b and c.

Figure 2.. Palmostatin analog effects on the dynamic palmitoylation of N-Ras.

a, b, Effect of active (12) and inactive (1) palmostatin analogs on N-Ras (a, b) and global protein (a) palmitoylation as detected by pulse-chase metabolic labeling of ON cells (or ONK control cells) with the 17-ODYA probe. Data shown are representative of three independent experiments. For b, N-Ras was immunoprecipitated using anti-GFP antibodies prior to visualizing palmitoylation by CuAAC conjugation to a Rh-N₃ reporter tag (top panel). Total N-Ras content was measured by Western blotting of GFP enrichments (bottom panel).c, Quantification of palmostatin analog effects on N-Ras palmitoylation; data represent average values ± s.d. from three independent biological replicates, where densitometry was performed on N-Ras signals from anti-GPF immunoprecipitations as shown in b (top panel).

Figure 3.. Concentration dependent effects of palmostatin analog 12 on N-Ras and global protein palmitoylation.

a, Effect of 12 on N-Ras (a, b) and global protein (a) palmitoylation as detected by pulse-chase metabolic labeling of ON cells (or ONK control cells) with the 17-ODYA probe. Data shown are representative of three independent experiments. For b, N-Ras was immunoprecipitated using anti-GFP antibodies prior to visualizing palmitoylation by CuAAC conjugation to a Rh-N₃ reporter tag (top panel). Total N-Ras content was measured by Western blotting of GFP enrichments (bottom panel). c, Quantification of concentration-dependent effects of 12 on N-Ras palmitoylation; data represent average values ± s.d. from two independent biological replicates, where densitometry was performed on N-Ras signals from anti-GFP immunoprecipitations as shown in b (top panel).

Figure 4.. Serine hydrolase engagement profiles for palmostatin analogs determined by MS-ABPP using the FP-biotin probe.

Venn diagram depicting serine hydrolases showing substantial engagement (> 50%) by Palm M (10 μM), 12 (20 μM), and/or 1 (20 μM) in OCI-AML3 cells as determined by MS-ABPP (also see Supp. Table 1). Palm M targets listed from previously published dataset. MS-ABPP data represent average values from independent experiments (n = 3 for 12, n = 2 for 1).

Figure 5.. Protein engagement profiles for Palmostatin M as determined by MS-ABPP using the Palm M-yne probe.

a, Structure of the Palm M-yne probe. b, Effect of Palm M-yne probe on N-Ras palmitoylation as detected by pulse-chase metabolic labeling of ON cells (or ONK control cells) with the 17-ODYA probe. N-Ras was immunoprecipitated using anti-GFP antibodies prior to visualizing palmitoylation by CuAAC conjugation to a Rh-N₃ reporter tag (top panel). Total N-Ras content was measured by Western blotting of GFP enrichments (bottom panel). The concentration-dependent analysis of Palm M and Palm M-yne blockade of N-Ras depalmitoylation was performed once. c, Heatmap summarizing results from competitive MS-ABPP experiments with Palm M (10 μM; n = 3), HDFP (20 μM; n = 2), and ABD957 (500 nM; n = 1) in ON cells treated in situ for 2 h prior to lysis, fractionation, and treatment of the particulate proteome with Palm M-yne (10 μM, 1 h). Values plotted represent percent of blockade of Palm M-yne enrichment by competitor ligands for each protein compared to DMSO control. Heatmap shows only targets that were enriched > 5-fold by the Palm M-yne probe (compared to DMSO control for enrichment; n = 2) and showing > 50% blockade of enrichment by Palm M (compared to DMSO control for competition). For full dataset, see Supp. Table 1. Grey color marks proteins that were not detected or failed to pass quality filters in the indicated experiments. Insufficient numbers of unique peptides were obtained to quantify ABHD17C in this experiment. MS-ABPP data represent average values from independent experiments, as indicated for each condition.