Grohmann Christoph, Magtoto Charlene M, Walker Joel R, Chua Ngee Kiat, Gabrielyan Anna, Hall Mary, Cobbold Simon A, Mieruszynski Stephen, Brzozowski Martin, Simpson Daniel S, Dong Hao, Dorizzi Bridget, Jacobsen Annette V, Morrish Emma, Silke Natasha, Murphy James M, Heath Joan K, Testa Andrea, Maniaci Chiara, Ciulli Alessio, Lessene Guillaume, Silke John, Feltham Rebecca
The Walter and Eliza Hall Institute for Medical Research, 1G Royal Parade, Parkville, Melbourne, VIC, 3052, Australia.
Department of Medical Biology, University of Melbourne, Parkville, VIC, 3052, Australia.
Nat Commun. 2022 Apr 19;13(1):2073. doi: 10.1038/s41467-022-29670-1.
Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12 (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4 complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.
使用靶向FKBP12的标签靶向蛋白降解剂（dTAGs）或HaloTag7（HaloPROTACs）来调节蛋白质丰度是临床前靶点验证的有力方法。互换标签和标签靶向降解剂对于实现有效的底物降解很重要，但可用的降解剂/标签对有限，且尚未进行并排比较。为了扩展标签靶向蛋白降解（tTPD）方法，我们开发了催化性靶向纳米荧光素酶的PROTAC（NanoTACs），以劫持CRL4复合物并降解纳米荧光素酶标记的底物，从而实现基于发光的快速降解筛选。为了将NanoTACs与现有的tTPD系统进行比较，我们使用了一种可互换的报告系统来比较测试最佳降解剂/标签对。总体而言，我们发现dTAG系统表现出更好的降解效果。为了使标签诱导的降解与生理学相匹配，我们证明NanoTACs可限制由混合谱系激酶结构域样蛋白（MLKL）驱动的坏死性凋亡。在这项工作中我们将tTPD平台扩展到包括NanoTACs，为tTPD研究增加了灵活性，并对每个tTPD系统进行了比较，以突出针对每种底物比较每个系统时进行比较的重要性。
