Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are >600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes.

中文翻译：

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泛素环 E3 连接酶的基于光交联活性的探针。

基于活性的蛋白质分析是研究酶生物学和促进治疗剂开发的宝贵技术。泛素 E3 连接酶 (E3) 是最大的酶家族之一，可调节许多（病理）生理过程。最大的子类型是 RING E3，其成员超过 600 个。RING E3 具有类似适配器的活性，可以受到多种调节机制的影响，并已成为有吸引力的药物靶点。不存在用于测量 RING E3 活动的基于活动的探针 (ABP)。在这里，我们重新设计了泛素带电的 E2 结合酶以产生光交联 ABP。我们展示了两种不同的癌症相关环 E3、RNF4 和 c-Cbl 的活动依赖性分析，以响应它们的天然激活信号。我们还展示了响应表皮生长因子 (EGF) 刺激的内源性 RING E3 连接酶激活分析。这些光交联 ABP 应该会促进 E3 连接酶研究和针对这类重要酶的选择性调节剂的开发。