Adachi Naoko, Hess Douglas T, Ueyama Takehiko
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan.
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
J Lipid Res. 2025 Feb;66(2):100743. doi: 10.1016/j.jlr.2025.100743. Epub 2025 Jan 10.
At least 10% of proteins constituting the human proteome are subject to S-acylation by a long-chain fatty acid, thioesterified to a Cys thiol side chain. Fatty S-acylation (prototypically, S-palmitoylation) operates across eukaryotic phylogeny and cell type. S-palmitoylation is carried out in mammalian cells by a family of 23-24 dedicated zDHHC palmitoyl transferase enzymes, and mutation of zDHHCs is associated with a number of human pathophysiologies. Activation of the zDHHCs by auto-S-palmitoylation, the transthioesterification of the active site Cys by fatty acyl coenzyme A, is the necessary first step in zDHHC-mediated protein S-palmitoylation. Most prior in vitro assessments of zDHHC activation have utilized purified zDHHCs, a time- and effort-intensive approach, which removes zDHHCs from their native membrane environment. We describe here a facile assay for zDHHC activation in native membranes. We overexpressed hemagglutinin-tagged wild-type or mutant zDHHCs in cultured HEK293 cells and prepared a whole membrane fraction, which was incubated with fluorescent palmitoyl CoA (NBD-palmitoyl-CoA) followed by SDS-PAGE, fluorescence imaging, and Western blotting for hemagglutinin. We show by mutational analysis that, as assayed, zDHHC auto-S-palmitoylation by NBD-palmitoyl-CoA is limited to the active site Cys. Application of the assay revealed differential effects on zDHHC activation of posttranslational zDHHC modification and of zDHHC mutations associated with human disease, in particular cancer. Our assay provides a facile means of assessing zDHHC activation, and thus of differentiating the effects of zDHHC mutation and posttranslational modification on zDHHC activation versus secondary effects on zDHHC functionality including altered zDHHC interaction with substrate palmitoyl-proteins.
构成人类蛋白质组的蛋白质中，至少10%会被长链脂肪酸进行S-酰化，该脂肪酸硫酯化为半胱氨酸（Cys）的硫醇侧链。脂肪酸S-酰化（典型的是S-棕榈酰化）在整个真核生物进化谱系和细胞类型中都存在。在哺乳动物细胞中，S-棕榈酰化由一个包含23 - 24种专门的zDHHC棕榈酰转移酶的家族来完成，zDHHCs的突变与多种人类病理生理学状况相关。zDHHCs通过自身S-棕榈酰化激活，即活性位点半胱氨酸被脂肪酰辅酶A进行硫酯交换，这是zDHHC介导的蛋白质S-棕榈酰化的必要第一步。之前大多数对zDHHC激活的体外评估都使用纯化的zDHHCs，这是一种耗时费力的方法，会使zDHHCs脱离其天然膜环境。我们在此描述一种用于在天然膜中检测zDHHC激活的简便方法。我们在培养的HEK293细胞中过表达带有血凝素标签的野生型或突变型zDHHCs，并制备全膜组分，将其与荧光棕榈酰辅酶A（NBD-棕榈酰辅酶A）孵育，然后进行SDS-PAGE、荧光成像以及针对血凝素的蛋白质免疫印迹分析。我们通过突变分析表明，如所检测的那样，NBD-棕榈酰辅酶A对zDHHC的自身S-棕榈酰化作用仅限于活性位点半胱氨酸。该检测方法的应用揭示了翻译后zDHHC修饰以及与人类疾病（特别是癌症）相关的zDHHC突变对zDHHC激活的不同影响。我们的检测方法提供了一种评估zDHHC激活的简便手段，从而能够区分zDHHC突变和翻译后修饰对zDHHC激活的影响与对zDHHC功能的次级影响，包括zDHHC与底物棕榈酰化蛋白质相互作用的改变。
