Peng Tao, Hang Howard C
Laboratory of Chemical Biology and Bacterial Pathogenesis, The Rockefeller University , New York, New York 10065, United States.
J Am Chem Soc. 2015 Jan 21;137(2):556-9. doi: 10.1021/ja502109n. Epub 2015 Jan 12.
Studying the functions of S-palmitoylated proteins in cells can be challenging due to the membrane targeting property and dynamic nature of protein S-palmitoylation. New strategies are therefore needed to specifically capture S-palmitoylated protein complexes in cellular membranes for dissecting their functions in vivo. Here we present a bifunctional fatty acid chemical reporter, x-alk-16, which contains an alkyne and a diazirine, for metabolic labeling of S-palmitoylated proteins and photo-cross-linking of their involved protein complexes in mammalian cells. We demonstrate that x-alk-16 can be metabolically incorporated into known S-palmitoylated proteins such as H-Ras and IFITM3, a potent antiviral protein, and induce covalent cross-linking of IFITM3 oligomerization as well as its specific interactions with other membrane proteins upon in-cell photoactivation. Moreover, integration of x-alk-16-induced photo-cross-linking with label-free quantitative proteomics allows identification of new IFITM3 interacting proteins.
由于蛋白质S-棕榈酰化的膜靶向特性和动态性质，研究细胞中S-棕榈酰化蛋白的功能具有挑战性。因此，需要新的策略来特异性捕获细胞膜中的S-棕榈酰化蛋白复合物，以剖析其在体内的功能。在此，我们展示了一种双功能脂肪酸化学报告分子x-alk-16，它含有一个炔烃和一个重氮甲烷，用于S-棕榈酰化蛋白的代谢标记及其在哺乳动物细胞中相关蛋白复合物的光交联。我们证明，x-alk-16可以代谢整合到已知的S-棕榈酰化蛋白中，如H-Ras和一种有效的抗病毒蛋白IFITM3，并在细胞内光激活后诱导IFITM3寡聚化的共价交联及其与其他膜蛋白的特异性相互作用。此外，将x-alk-16诱导的光交联与无标记定量蛋白质组学相结合，可以鉴定新的IFITM3相互作用蛋白。
