PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.
Mol Cell
; 77(5): 1092-1106.e9, 2020 03 05.
Article
in En
| MEDLINE
| ID: mdl-31973889
ABSTRACT
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.
Key words
Full text:
1
Database:
MEDLINE
Main subject:
Chromatography, High Pressure Liquid
/
Spectrometry, Mass, Electrospray Ionization
/
Proteomics
/
Ubiquitin-Protein Ligases
/
Tandem Mass Spectrometry
Limits:
Humans
Language:
En
Journal:
Mol Cell
Journal subject:
BIOLOGIA MOLECULAR
Year:
2020
Type:
Article