Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine (SJTU-SM) , Shanghai 200025, China.
J Proteome Res. 2012 Mar 2;11(3):1773-81. doi: 10.1021/pr2010204. Epub 2012 Feb 10.
FBOX6 ubiquitin ligase complex is involved in the endoplasmic reticulum-associated degradation pathway by mediating the ubiquitination of glycoproteins. FBXO6 interacts with the chitobiose in unfolded N-glycoprotein, pointing glycoproteins toward E2 for ubiquitination. Although the glycoprotein-recognizing mechanism of FBXO6 is well documented, its bona fide interacting glycoproteins are largely unknown. Here we utilized a protein purification approach combined with LC-MS to systematically identify the FBXO6-interacting glycoproteins. Following identification of 39 proteins that specifically interact with FBXO6 in all three different cell lines, 293T, HeLa and Jurkat cells, we compared the protein complex organization between wild-type FBXO6 and its mutant, which fails to recognize glycoproteins. Combining these databases, 29 highly confident glycoproteins that interact with FBXO6 in an N-glycan dependent manner are identified. Our data provide valuable information for the discovery of the interacting glycoproteins of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other FBAs (F-box associated regions) containing F-box proteins.
FBOX6 泛素连接酶复合物通过介导糖蛋白的泛素化参与内质网相关降解途径。FBXO6 与未折叠 N-糖蛋白中的二糖结合，将糖蛋白指向 E2 进行泛素化。尽管 FBXO6 的糖蛋白识别机制已有详细记录，但其真正的相互作用糖蛋白在很大程度上仍是未知的。在这里，我们利用蛋白质纯化方法结合 LC-MS 系统地鉴定了 FBXO6 的相互作用糖蛋白。在三种不同的细胞系（293T、HeLa 和 Jurkat 细胞）中，鉴定出 39 种特异性与 FBXO6 相互作用的蛋白质后，我们比较了野生型 FBXO6 及其突变体（无法识别糖蛋白）的蛋白质复合物组织。将这些数据库结合起来，确定了 29 种高度可信的糖蛋白，它们以 N-糖基化依赖的方式与 FBXO6 相互作用。我们的数据为发现 FBXO6 的相互作用糖蛋白提供了有价值的信息，也证明了这些方法作为包含 F-box 蛋白的其他 FBA（F-box 相关区域）相互作用糖蛋白的全局发现的通用平台的潜力。
