Characterization of PROTAC specificity and endogenous protein interactomes using ProtacID.

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• PROTACs (proteolysis-targeting chimeras) are an emerging therapeutic modality.
• Identifying PROTAC-proximal proteins is crucial for target validation and understanding mechanism of action.
• Existing methods for identifying protein interactions can be limited in scope or require extensive sample manipulation.

Purpose of the Study:

• To introduce ProtacID, a novel proximity-dependent biotinylation-based method.
• To demonstrate the utility of ProtacID for validating PROTAC degradation targets.
• To showcase ProtacID's capability in identifying non-productive PROTAC-interacting proteins and characterizing native protein complexes.

Main Methods:

• Development and application of ProtacID, a flexible BioID-based technique.
• Analysis of VHL- and CRBN-recruiting PROTACs across diverse cellular compartments and cell lines.
• Characterization of endogenous multiprotein complexes without antibody or epitope tag modification.

Main Results:

• ProtacID successfully identified PROTAC-proximal proteins in living cells.
• The method validated PROTAC degradation targets and identified non-degraded interacting proteins.
• ProtacID enabled characterization of native protein complexes, demonstrating its broad applicability.

Conclusions:

• ProtacID is a versatile tool for advancing PROTAC technology.
• This method addresses a critical need in identifying functional and non-functional protein interactions in PROTAC development.
• ProtacID offers a powerful approach for studying endogenous protein complexes without genetic or antibody manipulation.