Meng L, Sin N, Crews C M
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
Biochemistry. 1998 Jul 21;37(29):10488-92. doi: 10.1021/bi9804479.
Dynamic protein palmitoylation has been proposed to regulate GTP-binding proteins by controlling their membrane association and thus their access to key signaling proteins. While the palmitoyl protein thioesterase(s) responsible for depalmitoylation of plasma membrane-associated signaling proteins has (have) not been identified, the lysosomal palmitoyl protein thioesterase 1 (PPT1) has proven useful in in vitro studies of membrane localization requirements of GTP-binding proteins. We have previously reported the binding of the antiproliferative cyclic depsipeptide didemnin B to PPT1. To investigate the nature of this binding and its possible effects on PPT1 enzymatic activity, human PPT1 was expressed in an insect cell baculoviral system, and inhibition assays were performed using both [3H]palmitoyl Ha-Ras and myristoyl-CoA as PPT1 substrates. Didemnin B was shown to inhibit recombinant human PPT1 with a Ki of 92 nM. Kinetic analysis of this inhibition revealed that didemnin B inhibits PPT1 uncompetitively. Providing biochemical support for an uncompetitive mode of inhibition, in vitro binding studies of PPT1 and didemnin indicate that the natural product binds preferentially to the enzyme-substrate complex PPT1-palmitoyl-CoA. As the first described inhibitor of PPT1, didemnin B may prove to be a useful tool in the investigation of protein palmitoylation regulation.
动态蛋白质棕榈酰化被认为可通过控制GTP结合蛋白与膜的结合，进而控制其与关键信号蛋白的接触来调节GTP结合蛋白。虽然负责使质膜相关信号蛋白去棕榈酰化的棕榈酰蛋白硫酯酶尚未被鉴定，但溶酶体棕榈酰蛋白硫酯酶1（PPT1）已被证明在GTP结合蛋白膜定位需求的体外研究中很有用。我们之前报道了抗增殖环缩肽didemnin B与PPT1的结合。为了研究这种结合的性质及其对PPT1酶活性的可能影响，在昆虫细胞杆状病毒系统中表达了人PPT1，并使用[3H]棕榈酰化的Ha-Ras和肉豆蔻酰辅酶A作为PPT1底物进行了抑制试验。结果表明didemnin B以92 nM的Ki抑制重组人PPT1。对这种抑制作用的动力学分析表明，didemnin B以非竞争性方式抑制PPT1。PPT1与didemnin的体外结合研究为非竞争性抑制模式提供了生化支持，表明该天然产物优先结合酶 - 底物复合物PPT1 - 棕榈酰辅酶A。作为首次描述的PPT1抑制剂，didemnin B可能被证明是研究蛋白质棕榈酰化调节的有用工具。
