Molecular features defining the efficiency of bioPROTACs

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• BioPROTACs (biological Proteolysis-Targeting Chimeras) leverage target-binding moieties and ubiquitin-proteasome system (UPS) components for protein degradation.
• Understanding the biophysical design principles of the target-binding unit is crucial for enhancing bioPROTAC efficacy.
• Current knowledge on factors influencing bioPROTAC performance remains limited.

Purpose of the Study:

• To investigate the design principles governing the target-binding moiety of bioPROTACs.
• To identify key factors influencing the efficiency of protein degradation mediated by bioPROTACs.
• To establish strategies for characterizing and optimizing bioPROTAC systems.

Main Methods:

• Utilized a novel assay employing microinjection and live-cell microscopy to accurately measure protein degradation rates, independent of biosynthesis and transport.
• Employed DARPins (Designed Ankyrin Repeat Proteins) as target-binding units and a CHIP E3 ligase interaction domain.
• Tested the degradation of enhanced green fluorescent protein (eGFP) as a proof-of-principle target.

Main Results:

• Most DARPins effectively facilitated eGFP degradation, with two exceptions due to steric hindrance of E2 binding or overlap with the ubiquitination epitope.
• The affinity and thermodynamic stability of binders played a minor role in degradation efficiency.
• Constructed bioPROTACs demonstrated catalytic degradation of eGFP.
• DARPins alone accelerated GFP degradation non-catalytically via endogenous E3 ligases.

Conclusions:

• Efficient trans-acting bioPROTAC degradation critically depends on the correct complex orientation for target ubiquitination and proteasome presentation, requiring empirical determination.
• The developed strategies offer a robust pathway for characterizing and optimizing bioPROTAC design and function.
• BioPROTACs can achieve catalytic protein degradation, offering a powerful tool for molecular manipulation.