Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160, Derio, Spain.
IFOM ETS, The AIRC Institute of Molecular Oncology, Milan, Italy.
Nat Commun. 2023 Nov 23;14(1):7656. doi: 10.1038/s41467-023-43326-8.
Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.
数百种 E3 连接酶在识别特定的泛素（Ub）修饰底物方面发挥着关键作用。将 E3 的真正靶标与 E3 相互作用体区分开来仍然是一个挑战。我们提出了 BioE3，这是一种将底物与感兴趣的 Ub E3 连接酶匹配的强大方法。使用 BirA-E3 连接酶融合物和生物 Ub，特定 E3 的 Ub 修饰底物的定点生物素化促进了蛋白质组学鉴定。我们表明，BioE3 可识别两种 RING 型 E3 连接酶（RNF4（DNA 损伤反应，PML 体）和 MIB1（内吞作用，自噬，中心体动力学）的已知和新靶标。多功能的 BioE3 可识别一种细胞器特异性 E3（MARCH5）和一种相对未表征的 E3（RNF214）的靶标。此外，BioE3 与 NEDD4（一种 HECT 型 E3）一起使用，可鉴定与囊泡运输相关的新靶标。BioE3 可检测到对化学物质的特异性改变，为靶向蛋白降解开辟了途径，并且可能适用于其他 Ub 样（UbL，例如 SUMO）和 E3 类型。BioE3 的应用揭示了复杂的 UbL 网络对细胞调控的影响。
