Profiling targets of the irreversible palmitoylation inhibitor 2-bromopalmitate

Abstract

2-Bromohexadecanoic acid, or 2-bromopalmitate, was introduced nearly 50 years ago as a nonselective inhibitor of lipid metabolism. More recently, 2-bromopalmitate re-emerged as a general inhibitor of protein S-palmitoylation. Here, we investigate the cellular targets of 2-bromopalmitate through the synthesis and application of click-enabled analogues. In cells, 2-bromopalmitate is converted to 2-bromopalmitoyl-CoA, although less efficiently than free palmitate. Once conjugated to CoA, probe reactivity is dramatically enhanced. Importantly, both 2-bromopalmitate and 2-bromopalmitoyl-CoA label DHHC palmitoyl acyl transferases (PATs), the enzymes that catalyze protein S-palmitoylation. Mass spectrometry analysis of enriched 2-bromopalmitate targets identified PAT enzymes, transporters, and many palmitoylated proteins, with no observed preference for CoA-dependent enzymes. These data question whether 2-bromopalmitate (or 2-bromopalmitoyl-CoA) blocks S-palmitoylation by inhibiting protein acyl transferases, or by blocking palmitate incorporation by direct covalent competition. Overall, these findings highlight the promiscuous reactivity of 2BP and validate clickable 2BP analogues as activity-based probes of diverse membrane associated enzymes.

Figure 1. Metabolic labeling with 2BPN3 allows click-enabled detection of cellular targets

(A) Schematic of 2BPN₃ alkylation of cysteine thiolates on proteins by attack of the α-halo-carbonyl group, followed by click chemistry detection of labeled proteins. (B) Concentration-dependent metabolic labeling with 2BPN₃ in 293T cells. (C) Time-dependent metabolic labeling with 2BPN₃ in 293T cells. (D) Pre-incubation with 2BP attenuated 2BPN₃ labeling at higher concentrations. Each inhibitor was added for 1 hour at the described concentrations. For each gel, lysates were reacted with rhodamine-alkyne and separated by SDS-PAGE gel followed by fluorescence analysis.

Figure 2. 2BPN₃ is conjugated to CoA in cells, resulting in an increase in probe reactivity

(A) Comparison of 2BPN₃ metabolic labeling and in vitro labeling. Cells or lysates were labeled for 1 hour with 2BPN₃. Lower probe concentrations were required for equivalent labeling in vitro compared to metabolic labeling. (B) 2BP is marginally converted to 2BP-CoA in cells. Control samples were collected immediately after transfer from RPMI to ringer’s solution. The remaining cells were left for 30 minutes in the presence of fatty acid free BSA, BSA bound to 50 µM palmitic acid, or BSA bound to 50 µM 2BP. Cells were quenched and metabolites were extracted and analyzed in quadruplicate by high-resolution LC-MS. Samples were run as biological quadruplicates and standard errors are shown. Synthetic palmitoyl-CoA and 2BP-CoA were used to generate a standard curve to calculate endogenous metabolite concentrations. (C) Labeling of cell lysates with increasing concentrations of 2BPN₃-CoA in vitro. The control lane was lysate incubated with 50 µM 2BP-CoA, and is not labeled following the click chemistry reaction. (D) Comparison of 2BPN₃, 2BPN₃-CoA, and 2-iodoacetamide-rhodamine (2IA-Rh) shows probe-specific labeling in vitro. The control lanes were incubated with 50 µM 2BP (left) and 2.5 µM 2BP-CoA (center). 2BPN₃-CoA labeling is more reactive, but labels a similar pattern of proteins. 2IA-Rh labels a distinct pattern of proteins.

Figure 3. 2BPN₃ and 2BPN₃-CoA are activity-based probes for DHHC2

(A) 2BPN₃ labels FLAG epitope-tagged DHHC2, but not DHHS2 (C157S) by metabolic labeling in live cells. Labeling is resistant to hydroxylamine, demonstrating the absence of a stable acyl-intermediate. (B) 2BPN₃ and 2BPN₃-CoA both label DHHC2, but not DHHS2 (C157S) in vitro. Anti-FLAG western blots show recombinant protein expression levels.