An interactome-based framework for DDB1- and CUL4-associated factor prioritization in targeted protein degradation

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The DDB1- and CUL4-associated factor (DCAF) family are crucial components of Cullin4-RING (CRL4) ubiquitin ligases, regulating targeted protein degradation (TPD).
• Many DCAFs' functions, substrates, and complex formations are not well understood, limiting their application in TPD strategies for challenging targets.
• CRL4-based TPD is a significant therapeutic strategy for modulating proteins previously considered undruggable.

Purpose of the Study:

• To systematically characterize the interactome and functional associations of individual DCAFs.
• To identify substrates for model DCAFs, COP1 and DCAF3, using advanced proteomic techniques.
• To establish reliable methods for evaluating DCAF degradation activity and identify high-activity DCAFs for TPD applications.

Main Methods:

• Proximity biotinylation-based interactome analysis in HEK293T cells to map DCAF interactions.
• Proximity biotinylation coupled with multi-omics approaches to identify DCAF substrates.
• Biochemical and cell-based assays to assess CRL4 complex formation and DCAF autodegradation as indicators of activity.

Main Results:

• Systematic annotation of interactors and functional associations for individual DCAFs.
• Identification of specific protein substrates for the DCAFs COP1 and DCAF3.
• Establishment of CRL4 complex formation and DCAF autodegradation as tractable metrics for DCAF activity.
• Proposal of a set of high-activity DCAFs based on experimental evaluation.

Conclusions:

• The study provides a comprehensive resource for understanding DCAF biology and function.
• Developed methods offer a framework for evaluating and prioritizing DCAFs for targeted protein degradation applications.
• This work facilitates the advancement of CRL4-based TPD strategies by identifying key DCAFs and their substrates.