Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Post-translational modification by ubiquitin and ubiquitin-like proteins (Ubls) regulates eukaryotic cellular processes.
• Studying ubiquitylation is challenging due to difficulties in preparing homogeneous modified proteins and selectively triggering events in vivo.

Purpose of the Study:

• To develop a general tool for inducible protein ubiquitylation.
• To enable site-specific and ubiquitin-ligase-independent ubiquitylation in mammalian cells.

Main Methods:

• Combination of genetic-code expansion, bioorthogonal Staudinger reduction, and sortase-mediated transpeptidation.
• Generation of ubiquitin conjugates with native isopeptide bonds and resistance to deubiquitinases.
• Site-specific attachment of Ubls to nonrefoldable, multidomain proteins.

Main Results:

• Developed a tool for inducible protein ubiquitylation in mammalian cells.
• Generated ubiquitin conjugates resistant to deubiquitinases while retaining native function.
• Enabled temporal control over ubiquitylation events independent of endogenous ubiquitin ligases.

Conclusions:

• The developed method provides a powerful tool for dissecting the biological functions of ubiquitylation with temporal control.
• Facilitates the study of ubiquitylation on complex, nonrefoldable proteins.
• Overcomes limitations in current ubiquitylation research methodologies.