Engineering synthetic adaptors and substrates for controlled ClpXP degradation

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• Targeted intracellular protein degradation is crucial for basic science and biotechnology.
• Redesigning adaptor proteins to link substrates to proteases like ClpXP is a promising strategy.
• Understanding the minimal functions for adaptor proteins is key to engineering degradation systems.

Purpose of the Study:

• To probe the minimal biochemical functions required for adaptor protein function in protein degradation.
• To engineer a synthetic system for facile control of targeted intracellular protein degradation.
• To establish a foundation for engineering highly specific degradation of target proteins regulated by small molecules.

Main Methods:

• Designed and characterized variant substrates, adaptors, and ClpX enzymes for the ClpXP protease system.
• Utilized heterologous interaction domains and a small bridging molecule (rapamycin) for substrate tethering.
• Assessed the sufficiency of simple tethering for synthetic adaptor function and proteolysis.

Main Results:

• Substrate tethering mediated by heterologous interaction domains and rapamycin effectively mimics wild-type substrate delivery.
• Simple tethering is sufficient for synthetic adaptor function in controlling protein degradation.
• The engineered system demonstrates rapamycin-dependent tethering and proteolysis, enabling small-molecule-regulated degradation.

Conclusions:

• Engineered simple tethering as a synthetic adaptor function for controlling protein degradation.
• Developed a rapamycin-regulated system for targeted protein degradation without new biosynthesis.
• This work provides a foundation for engineering specific protein degradation in cells using small molecules.