An ORFeome of rice E3 ubiquitin ligases for global analysis of the ubiquitination interactome.
An ORFeome of rice E3 ubiquitin ligases for global analysis of the ubiquitination interactome.
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水稻E3泛素连接酶的ORF组用于泛素化相互作用组的全局分析。
DOI：
10.1186/s13059-022-02717-8
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发表时间：
2022-07-11
期刊：
影响因子：
12.3
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中科院分区：
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泛素化在真核生物的许多细胞过程中是必不可少的，包括26 S蛋白酶体依赖的蛋白质降解、细胞周期进程、转录调控和信号转导。虽然许多泛素化蛋白已被经验性地确定，其同源泛素E3连接酶仍然在很大程度上未知。本研究构建了一个完整的水稻泛素E3连接酶开放阅读框架（UbE 3-ORFeome）文库，该文库包含了水稻1515个E3连接酶基因的98.94%。基因组在四种已知泛素化蛋白的测试筛选中，我们鉴定了已知和新的E3。在体外和体内证实了几种E3与其底物之间的相互作用和降解。此外，我们确定的F盒E3连接酶OsFBK 16作为枢纽相互作用的蛋白质的苯丙氨酸解氨酶家族OsPAL 1-OsPAL 7。我们证明OsFBK 16促进OsPAL 1、OsPAL 5和OsPAL 6的降解。值得注意的是，我们发现OsPAL 1或OsPAL 6的过表达以及OsFBK 16的功能丧失在水稻中显示出增强的稻瘟病抗性，表明OsFBK 16降解OsPAL以负调节水稻免疫。水稻UbE 3-ORFeome是植物中第一个完整的E3连接酶文库，为植物中泛素化蛋白同源E3连接酶的快速鉴定和功能性E3-底物相互作用组的建立提供了有力的蛋白质组学资源。在线版本包含补充材料，可通过10.1186/s13059-022-02717-8获得。
Ubiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown. Here, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice (Oryza sativa L.) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1–OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity. The rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3–substrate interactome in plants. The online version contains supplementary material available at 10.1186/s13059-022-02717-8.