Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
J Biol Chem. 2013 Jul 5;288(27):19816-29. doi: 10.1074/jbc.M112.431676. Epub 2013 May 16.
Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) γ-2, TARP γ-8, cornichon-2, Ca(2+)/calmodulin-dependent protein kinase IIα, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.
蛋白质棕榈酰化是一种常见的翻译后脂质修饰，在蛋白质运输和功能中发挥重要作用。最近开发的棕榈酰蛋白质组学方法鉴定了许多新的底物。然而，棕榈酰化底物的全貌尚未阐明。在这里，我们使用 CSS-Palm 2.0 程序（一种用于预测棕榈酰化位点的免费软件）进行了全局计算机筛选，并选择了 17 个候选物作为新的棕榈酰化底物。在 17 个候选物中，有 10 种蛋白质，包括 6 种突触蛋白（Syd-1、跨膜 AMPA 受体调节蛋白 (TARP) γ-2、TARP γ-8、cornichon-2、钙/钙调蛋白依赖性蛋白激酶 IIα和神经软骨素 (Ncdn)/诺宾)、一种粘着斑蛋白 (zyxin)、两种离子通道 (TRPM8 和 TRPC1) 和一种 G 蛋白偶联受体 (食欲素 2 受体)，被棕榈酰化。使用 DHHC 棕榈酰化酶文库，我们发现所有测试的底物均由高尔基体定位的 DHHC3/7 亚家族棕榈酰化。Ncdn 是神经突生长和突触可塑性的调节剂，被 DHHC1/10（zDHHC1/11；z1/11）亚家族强烈棕榈酰化，其底物尚未报道。正如 CSS-Palm 2.0 预测的那样，Cys-3 和 Cys-4 是 Ncdn 的棕榈酰化位点。Ncdn 特异性定位于大鼠培养神经元的体树突区，而不在轴突中。受激发射损耗显微镜显示，Ncdn 以棕榈酰化依赖的方式定位于 Rab5 阳性早期内体，DHHC1/10（z1/11）也分布于此。DHHC1、-3 或 -10（z11）的敲低导致 Ncdn 从 Rab5 阳性内体中丢失。因此，通过计算机筛选，我们证明 Ncdn 和 DHHC1/10（z1/11）和 DHHC3/7 亚家族是新的棕榈酰化底物-酶对，Ncdn 的棕榈酰化在其特定的内体靶向中起关键作用。
