Persaud Avinash, Rotin Daniela
Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.
Methods Mol Biol. 2011;759:215-24. doi: 10.1007/978-1-61779-173-4_13.
Ubiquitin-protein ligases (E3s) are responsible for target recognition and subsequent modification of selected substrates within the ubiquitin proteasomal system (UPS). Substrates of this pathway are covalently modified by the attachment of ubiquitin usually onto Lys residues. As a result, these modified proteins can be targeted for degradation, endocytosis, protein sorting, subnuclear trafficking, or other fates. Despite the advancements in understanding the underlying mechanisms of the ubiquitin system, the substrates of most E3 enzymes remain largely unknown. Here, we describe the development of a high-throughput method to identify in vitro substrates for E3 ligases on a global proteomic scale. The enzymatic activity (ubiquitylation) and binding of ubiquitin ligases to their substrates are performed using protein (proteome) microarrays as the experimental platform, and using Nedd4/Rsp5 family members as examples of HECT E3 ligases. The in vitro ubiquitylation and binding substrates identified in these screens can provide invaluable insight into the cellular pathways in which E3 ligases participate.
泛素蛋白连接酶（E3s）负责在泛素蛋白酶体系统（UPS）中识别靶标并随后对选定的底物进行修饰。该途径的底物通常通过泛素连接到赖氨酸残基上而被共价修饰。结果，这些修饰的蛋白质可被靶向降解、内吞、蛋白质分选、核内运输或其他命运。尽管在理解泛素系统的潜在机制方面取得了进展，但大多数E3酶的底物在很大程度上仍然未知。在这里，我们描述了一种高通量方法的开发，用于在全球蛋白质组规模上鉴定E3连接酶的体外底物。使用蛋白质（蛋白质组）微阵列作为实验平台，以Nedd4/Rsp5家族成员作为HECT E3连接酶的示例，进行泛素连接酶的酶活性（泛素化）及其与底物的结合。在这些筛选中鉴定出的体外泛素化和结合底物可以为E3连接酶参与的细胞途径提供宝贵的见解。
