PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network

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. [Display omitted] •Cand1/2 act as substrate receptor exchange factors (SREFs) for CRL4s•CRL4s assemble ∼10% of all DDB1⋅DCAF modules and differ up to 200-fold in abundance•Substrate availability shapes the composition of the CRL4 ligase network•Potency of degrader molecules depends on expression levels of cognate DCAFs Co-opting CRL4 ligases for targeted protein degradation is considered a promising therapeutic strategy. Reichermeier et al. develop the systematic proteomic profiling tool PIKES to generate quantitative snapshots of cellular CRL4s in action. Degrader molecules can induce Cand1/Nedd8-driven ligase assembly, and higher expression levels of cognate substrate receptors increase their potency.