Multiplex design and discovery of proximity handles for programmable proteome editing

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Genome editing tools are advanced, but proteome manipulation lacks similar flexibility and specificity.
• Bifunctional molecules like PROTACs show promise for proteome editing but are limited by a scarcity of well-characterized effector handles.
• A gap exists in discovering novel handles for targeted protein degradation, stabilization, or relocalization.

Purpose of the Study:

• To address the limitations in proteome editing by developing a framework for discovering new effector handles.
• To accelerate the discovery of handles for intracellular protein degradation, stabilization, and relocalization using de novo protein design and multiplex screening.

Main Methods:

• Employed de novo protein design coupled with a multiplex screening framework (LABEL-seq) to discover effector handles.
• Screened 9,715 de novo designed candidate effector handles for their ability to recruit target proteins to the ubiquitin-proteasome system (UPS) or autophagy pathways.
• Validated findings using orthogonal assays to confirm changes in intracellular protein abundance.

Main Results:

• Discovered hundreds of de novo designed effector handles, including 277 for degradation and 204 for stabilization of a reporter protein.
• Identified effective handles for both UPS (194) and autophagy (287) pathways, demonstrating complementary routes for proteome editing.
• Demonstrated generalization to endogenous targets by reducing MCL1 levels and showed functional perturbation of mitochondria via targeted relocalization.

Conclusions:

• Established a scalable, low-cost platform linking deep learning-guided protein design to functional cellular readouts for proteome editing.
• Generated a diverse repertoire of effector handles for controlling protein abundance and localization.
• Paved the way for a general framework for programmable proteome editing.