Creaser Steffen P, Peterson Blake R
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
J Am Chem Soc. 2002 Mar 20;124(11):2444-5. doi: 10.1021/ja017671x.
The localization of oncogenic Src and Ras proteins to cellular plasma membranes is critical for the proliferation of specific cancers. In addition to other lipid modifications, these proteins require posttranslational palmitoylation of specific cysteine residues by the enzyme palmitoyl acyltransferase (PAT) in order to be stably anchored at plasma membranes. Hence, the identification of inhibitors of protein palmitoylation has significant potential to define a new class of antitumor agents. However, studies of protein palmitoylation have been hindered by the dynamic and reversible nature of cysteine acylation and the lack of sensitive and convenient assays of PAT activity. To facilitate the rapid identification of compounds that affect protein palmitoylation, we report the solid-phase synthesis of a fluorescent cell-permeable palmitoyl acyltransferase substrate that mimics the N-terminus of Src family proteins. Metabolic radiolabeling and epifluorescence microscopy of Jurkat lymphocytes treated with this Src-mimetic lipopeptide revealed that this compound is palmitoylated intracellularly, which confers localization at cellular plasma membranes. Addition of the palmitoylation inhibitor 2-bromopalmitic acid to substrate-treated cells blocked palmitoylation and diminished substrate-mediated plasma membrane fluorescence. Analysis of inhibition of palmitoylation by flow cytometry revealed that this fluorescent lipopeptide substrate represents a highly sensitive molecular probe of palmitoyl acyltransferase activity that enables unprecedented high-throughput assays of protein palmitoylation.
致癌性Src和Ras蛋白定位于细胞质膜对于特定癌症的增殖至关重要。除了其他脂质修饰外，这些蛋白还需要通过棕榈酰酰基转移酶（PAT）对特定半胱氨酸残基进行翻译后棕榈酰化，以便稳定锚定在质膜上。因此，鉴定蛋白质棕榈酰化抑制剂具有定义一类新型抗肿瘤药物的巨大潜力。然而，蛋白质棕榈酰化的研究受到半胱氨酸酰化的动态和可逆性质以及缺乏灵敏且便捷的PAT活性检测方法的阻碍。为了便于快速鉴定影响蛋白质棕榈酰化的化合物，我们报道了一种模仿Src家族蛋白N端的荧光细胞可渗透棕榈酰酰基转移酶底物的固相合成。用这种模拟Src的脂肽处理的Jurkat淋巴细胞的代谢放射性标记和落射荧光显微镜检查表明，该化合物在细胞内被棕榈酰化，这使其定位于细胞质膜。向底物处理的细胞中添加棕榈酰化抑制剂2-溴棕榈酸可阻断棕榈酰化并减少底物介导的质膜荧光。通过流式细胞术分析棕榈酰化抑制作用表明，这种荧光脂肽底物代表了一种高度灵敏的棕榈酰酰基转移酶活性分子探针，能够实现前所未有的蛋白质棕榈酰化高通量检测。
