Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
Mol Cell Proteomics. 2011 Oct;10(10):M110.006007. doi: 10.1074/mcp.M110.006007. Epub 2011 Jul 23.
S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.
S-棕榈酰化作用，即脂肪酸棕榈酸可逆地酰化特定半胱氨酸残基，促进了几种生物途径中蛋白质的膜锚定和亚细胞定位。尽管抑制棕榈酰化作用有望成为一种操纵蛋白质靶向的手段，但该领域的进展受到对棕榈酰化酶学生物学和共识基序的有限理解的阻碍。为了确定巨噬细胞中 S-酰化蛋白的成分，我们用羟胺处理 RAW 264.7 巨噬细胞膜，以切割酰基硫酯，然后用新暴露的巯基进行生物素化，并进行链霉亲和素琼脂糖亲和层析。在通过 LC-MS/MS 鉴定的 1183 种蛋白质中，通过谱计数来评估在羟胺与模拟处理条件下的富集情况，从而确定 S-酰化状态。在四个独立实验中，有 80 种蛋白质在 S-酰化方面具有统计学意义（假发现率 = 0.05），有 101 种蛋白质在 S-酰化方面具有统计学意义（假发现率 = 0.10）。从几个功能类别中鉴定出候选 S-酰基蛋白，包括膜运输、信号转导、转运蛋白和受体。其中包括 29 种先前通过生化方法证实为棕榈酰化的蛋白质、45 种先前在蛋白质组筛选中报道为假定的 S-酰基蛋白、24 种先前未与棕榈酰化相关的蛋白质和 3 种假定的假阳性蛋白。候选蛋白中有近一半是我们以前在巨噬细胞去污剂抗性膜中鉴定的，这表明棕榈酰化促进了蛋白质在巨噬细胞中的脂筏定位。候选新型 S-酰基蛋白之一是磷脂翻转酶 3（Plscr3），它通过重塑线粒体膜来调节细胞凋亡。通过 3H-棕榈酸标记证实了 Plscr3 的棕榈酰化。此外，五个半胱氨酸簇（Cys159-161-163-164-166）的定点突变消除了棕榈酰化，导致 Plscr3 从线粒体易位到细胞核，并减少了依托泊苷诱导的巨噬细胞凋亡，这表明 Plscr3 的棕榈酰化在该位点对于线粒体靶向和促凋亡功能具有重要作用。综上所述，我们提出通过重新编程蛋白质定位来操纵蛋白质棕榈酰化，为干预巨噬细胞生物学提供了巨大潜力。
