Profiling Targets of the Irreversible Palmitoylation Inhibitor 2-Bromopalmitate

Abstract

2-bromohexadecanoic acid, or 2-bromopalmitate, was introduced nearly 50 years ago as a non-selective 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.

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

“…Although 2-BP clearly inhibits the palmitoyltransferases, there is evidence that it inhibits the thioesterases as well (44,52,53). 2-BP also inhibits metabolic enzymes, including fatty acid CoA ligase, which reduces the cellular content of the palmitoylation substrate palmitoyl-CoA (44,52,53). We also observed a decrease in TER after treatment of mCCD cl1 cells with 2-BP, which may reflect reduced palmitoylation of claudins normally found within the tight junctions (54 -56).…”

“…6). To determine whether palmitoylation has a role in activating endogenous ENaCs in mCCD cl1 cells, we treated polarized cultures of mCCD cl1 cells mounted in an Ussing chamber with either 2-bromopalmitate (2-BP), a general irreversible inhibitor of protein palmitoylation, or DMSO (vehicle control) (42)(43)(44). We observed a significant decrease in short circuit current (I sc ) within 5 min after apical addition of 2-BP that reached a new steady state I sc after 30 min (Fig.…”

“…However, the reduced currents could also reflect changes in palmitoylation of additional proteins that either alter ENaC trafficking or gating. The palmitate analog 2-BP has been regularly used in cultured cells to inhibit palmitoylation of proteins, thereby providing a complementary in vivo approach to study the function of palmitate addition to specified proteins (44,52). Although 2-BP clearly inhibits the palmitoyltransferases, there is evidence that it inhibits the thioesterases as well (44,52,53).…”

“…The palmitate analog 2-BP has been regularly used in cultured cells to inhibit palmitoylation of proteins, thereby providing a complementary in vivo approach to study the function of palmitate addition to specified proteins (44,52). Although 2-BP clearly inhibits the palmitoyltransferases, there is evidence that it inhibits the thioesterases as well (44,52,53). 2-BP also inhibits metabolic enzymes, including fatty acid CoA ligase, which reduces the cellular content of the palmitoylation substrate palmitoyl-CoA (44,52,53).…”

Emerging roles for protein S-palmitoylation in Toxoplasma biology

“…Many of these proteins are broadly conserved across the phylum Apicomplexa and for some of them, palmitoylation is essential for their function. In support of this, treatment of T. gondii and Plasmodium falciparum with 2-bromopalmitate (2-BP), an inhibitor of palmitoylation, is deleterious to parasite survival (Alonso et al, 2012;Jones et al, 2012), although these findings should be interpreted with caution, given that 2-BP is considered to be poorly specific and highly toxic (Davda et al, 2013).…”