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

Mol Cell. 2020 Mar 5;77(5):1092-1106.e9. doi: 10.1016/j.molcel.2019.12.013. Epub 2020 Jan 20.

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.

Keywords: CRL4; DCAFs; Nedd8; PIKES; QconCAT; cullin-RING ligase; mass spectrometry; quantitative proteomics; targeted protein degradation; ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid*
  • Cullin Proteins / genetics
  • Cullin Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Kinetics
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism
  • NEDD8 Protein / genetics
  • NEDD8 Protein / metabolism
  • Protein Interaction Maps
  • Proteolysis
  • Proteomics / methods*
  • Signal Transduction
  • Spectrometry, Mass, Electrospray Ionization*
  • Tandem Mass Spectrometry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • CAND1 protein, human
  • CAND2 protein, human
  • CUL4A protein, human
  • CUL4B protein, human
  • Cullin Proteins
  • Muscle Proteins
  • NEDD8 Protein
  • NEDD8 protein, human
  • Transcription Factors
  • Ubiquitin-Protein Ligases
  • CRL4 protein, human