靶向蛋白质降解（TPD）代表了一种有效的化学生物学范例，它利用细胞降解机制从药理学上消除感兴趣的特定蛋白质。尽管已发现多种 E3 连接酶可促进 TPD，但仍迫切需要使可用于此类应用的 E3 连接酶库多样化。在这里，我们描述了一种基于簇规则间隔短回文重复序列（CRISPR）的转录激活筛选，重点关注人类E3连接酶，目的是鉴定可以促进异双功能化合物介导的靶标降解的E3连接酶。通过这种方法，我们鉴定了一种候选蛋白水解靶向嵌合体（PROTAC）22-SLF，当FBXO22基因的转录被激活时，它会诱导FK506结合蛋白12的降解。随后的机制研究表明，22-SLF 与 F-box 蛋白 22 (FBXO22) 中的 C227 和/或 C228 相互作用以实现目标降解。最后，我们通过有效降解其他内源蛋白（包括含溴结构域蛋白 4 和棘皮动物微管相关蛋白样 4-间变性淋巴瘤激酶融合蛋白），证明了基于 FBXO22 的 PROTAC 的多功能性。© 2024。作者，获得 Springer Nature America, Inc. 的独家许可。

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein.© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.