Targeting endogenous proteins for degradation through the affinity-directed protein missile system

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Targeted proteolysis offers advantages over CRISPR/Cas9 and RNA interference for protein manipulation.
• Existing methods like gene knockouts are irreversible, and RNA interference can have off-target effects and incomplete knockdown.
• A versatile system for selective protein degradation is needed for research and therapeutics.

Purpose of the Study:

• To develop and validate a novel affinity-directed protein missile (AdPROM) system for targeted degradation of endogenous proteins.
• To demonstrate the efficacy of AdPROM using synthetic monobodies and VHH nanobodies against specific target proteins.
• To investigate the biological consequences of AdPROM-mediated protein depletion.

Main Methods:

• Engineered AdPROM system by tethering the von Hippel-Lindau (VHL) protein to polypeptide binders.
• Utilized synthetic monobodies targeting protein tyrosine phosphatase SHP2.
• Employed a VHH nanobody targeting human ASC protein.
• Assessed degradation of SHP2 and ASC in human cell lines.
• Evaluated the impact of SHP2 depletion on cellular processes.

Main Results:

• Demonstrated highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC proteins in human cell lines.
• Showcased the ability of synthetic polypeptide binders to recruit target proteins to the CUL2-E3 ligase complex.
• Confirmed that AdPROM-mediated loss of SHP2 affects its known biological functions.
• Established the feasibility of using small polypeptide binders for targeted protein destruction.

Conclusions:

• The AdPROM system provides a powerful and adaptable tool for targeted protein degradation.
• Small polypeptide binders can be effectively utilized for selective recruitment and degradation of endogenous proteins.
• AdPROM offers a promising alternative to existing gene modulation techniques with potential therapeutic applications.