Comprehensive Identification of Substrates for F-box Proteins by Differential Proteomics Analysis

Although elucidation of enzyme–substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases.尽管阐明酶与底物的相互关系是推动生物学发展的核心基础，但目前尚未建立起针对特定酶的底物进行系统性鉴定的通用方法。鉴于大多数泛素连接酶的底物在与酶结合后会迅速被泛素化并降解，因此鉴定这类酶的底物尤为困难。本文报道了一种用于发现泛素连接酶底物的新型方法DiPIUS（differential proteomics-based identification of ubiquitylation substrates，即基于差异蛋白质组学的泛素化底物鉴定法）的开发过程。我们将DiPIUS应用于Fbxw7α、Skp2和Fbxl5这三个研究最为透彻的F-box蛋白，并成功鉴定出包括已有已知靶点在内的候选底物。因此，DiPIUS是一种可用于对泛素连接酶底物进行无偏倚、系统性筛选的高效工具。