PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network
PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network
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DOI：
10.1016/j.molcel.2019.12.013
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发表时间：
2020-03-05
期刊：
影响因子：
16
通讯作者：
Kirkpatrick, Donald S.
中科院分区：
文献类型：
--
作者：
Reichermeier, Kurt M.;Straube, Ronny;Kirkpatrick, Donald S.
利用 Cullin4 RING 泛素连接酶 (CRL4) 来诱导降解致病蛋白正在成为一种有前景的治疗策略。尽管人们付出了巨大的努力来合理设计与 CRL4 结合的降解分子，但有关这些连接酶的组织和调节的许多信息仍然难以捉摸。在这里，我们建立了蛋白质相互作用动力学和化学计量估计（PIKES）分析，这是一个系统的蛋白质组分析平台，集成了细胞工程、亲和纯化、化学稳定和定量质谱分析，以研究可互换多蛋白质复合物的动力学。使用 PIKES，我们发现 Cullin4 与各个底物受体的连接酶组装体的丰度差异高达 200 倍，并且 Cand1/2 充当底物受体交换因子。此外，降解剂分子可以诱导其同源 CRL4 的组装，并且相关底物受体的较高表达可增强降解剂的效力。除了 CRL4 网络之外，我们还展示了 PIKES 如何揭示对药物开发很重要的细胞蛋白质网络的系统级生物化学。
Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.