Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, USA.
Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA.
Nat Commun. 2017 Jan 30;8:14286. doi: 10.1038/ncomms14286.
Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6.
蛋白质泛素化是由泛素激活酶 E1、泛素结合酶 E2 和泛素连接酶 E3 依次介导的。Uba1 曾被认为是唯一的 E1，直到最近才发现 Uba6。为了区分 Uba1 和 Uba6 的生物学功能，我们应用正交泛素转移（OUT）技术在哺乳动物细胞中对它们的泛素化靶标进行了分析。通过表达为专门相互作用而设计的一对工程化泛素和工程化 Uba1 或 Uba6，我们鉴定了 697 个潜在的 Uba6 靶标和 527 个潜在的 Uba1 靶标，其中有 258 个重叠。生物信息学分析揭示了涉及 Uba1 和 Uba6 特异性靶标的途径存在显著差异。我们证明 ezrin 和 CUGBP1 的多泛素化和蛋白酶体降解需要 Uba6，但不需要 Uba1，并且 Uba6 参与 ezrin 定位和上皮形态发生的控制。这些数据表明，Uba1 和 Uba6 存在不同的底物池，反映了 Uba6 的非冗余生物学作用。
