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首页> 外文期刊>Molecular and Cellular Biology >Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase
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Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase

机译：底物捕获蛋白质组学揭示了βTrCP2/ FBXW11泛素连接酶的目标。

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Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1–CUL1–F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCFFBXW11 E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCFFBXW11 bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.
机译：定义每种泛素连接酶的底物的完整补体仍然是一个重要的挑战。质谱仪器和计算以及蛋白质生物化学方法的改进导致了几种新的泛素连接酶底物鉴定方法。在这里，我们使用并行衔接子捕获（PAC）蛋白质组学方法来研究βTrCP2/ FBXW11，这是SKP1-CUL1-F-box（SCF）E3泛素连接酶复合物的底物适配器。泛素化反应的持续性需要在FBXW11及其底物之间进行短暂的物理相互作用，从而使与FBXW11结合的底物的生化纯化变得困难。使用基于PAC的方法，我们抑制了蛋白酶体“捕获” SCF FBXW11 E3复合体上的泛素化底物。蛋白酶体抑制前后免疫纯化的FBXW11蛋白复合物的比较质谱分析显示21种已知底物和23种推定的新型底物。在重点研究中，我们发现SCF FBXW11 结合，多泛素化和不稳定的RAPGEF2，一种鸟嘌呤核苷酸交换因子，可激活小GTPase RAP1。高RAPGEF2蛋白质水平促进细胞间融合，并因此促进多核化。令人惊讶地，这独立于鸟嘌呤核苷酸交换因子（GEF）催化活性和RAP1的存在而发生。我们的数据建立了RAPGEF2的新功能，可能有助于癌症的非整倍性。更广泛地说，该报告支持继续使用底物捕获蛋白质组学来全面确定E3泛素连接酶的靶标。所有蛋白质组学数据都可以通过ProteomeXchange获得，其标识符为PXD001062。

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