Profiling targets of the irreversible palmitoylation inhibitor 2-bromopalmitate.
Profiling targets of the irreversible palmitoylation inhibitor 2-bromopalmitate.
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DOI：
10.1021/cb400380s
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发表时间：
2013-09-20
影响因子：
4
通讯作者：
Martin BR
中科院分区：
文献类型：
--
作者：
Davda D;El Azzouny MA;Tom CT;Hernandez JL;Majmudar JD;Kennedy RT;Martin BR
2-溴代十六烷酸或2-溴代棕榈酸酯在近50年前作为脂质代谢的非选择性抑制剂引入。最近，2-溴棕榈酸酯作为蛋白S-棕榈酰化的一般抑制剂重新出现。在这里，我们调查的细胞目标2-溴棕榈酸酯通过点击启用类似物的合成和应用。在细胞中，2-溴棕榈酸被转化为2-溴棕榈酰-CoA，尽管效率低于游离棕榈酸。一旦与CoA缀合，探针反应性显著增强。重要的是，2-溴棕榈酸和2-溴棕榈酰辅酶A都标记DHHC棕榈酰酰基转移酶（PAT），这种酶催化蛋白质S-棕榈酰化。质谱分析富集2-溴棕榈酸目标确定PAT酶，转运蛋白，和许多棕榈酰化的蛋白质，没有观察到的CoA依赖性酶的偏好。这些数据质疑2-溴棕榈酸酯（或2-溴棕榈酰-CoA）是否通过抑制蛋白酰基转移酶或通过直接共价竞争阻断棕榈酸酯掺入来阻断S-棕榈酰化。总之，这些发现突出了2BP的混杂反应性，并验证了可点击的2BP类似物作为不同膜相关酶的基于活性的探针。
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.
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