Targeted stress granule regulation by engineering a non-catalytic O-GlcNAc transferase

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Medicine

Background:

• Stress granules (SGs) are dynamic protein-RNA structures linked to various diseases.
• Understanding SG regulation is crucial for developing new therapeutic interventions.
• The role of O-GlcNAc transferase (OGT) in SG regulation beyond its enzymatic activity is not well understood.

Purpose of the Study:

• To investigate the non-catalytic functions of OGT in regulating stress granule assembly.
• To develop novel tools for modulating stress granule formation using OGT.
• To explore the potential of targeting protein material properties for therapeutic purposes.

Main Methods:

• Induced proximity experiments to control OGT localization and function.
• Biochemical assays to measure liquid-liquid phase separation (LLPS) of G3BP1.
• Genetic engineering to create modular OGT-based tools.
• Analysis of protein immobilization and material property changes under stress.

Main Results:

• OGT, when targeted to G3BP1 via induced proximity, suppresses G3BP1 LLPS and SG assembly independently of its catalytic activity.
• A modular tool derived from OGT's N-catalytic and intervening domains (NI) effectively inhibits SG formation.
• This strategy reduces the mobility and rigidifies G3BP1 under prolonged stress.
• The OGT-based tool is effective for other proteins with similar domain architectures, demonstrating broad applicability.

Conclusions:

• OGT possesses a cryptic, non-catalytic function in suppressing stress granule formation.
• Targeted protein immobilization using modular OGT domains is a viable strategy for regulating SG assembly.
• This approach provides a novel method for dissecting SG biology and offers potential therapeutic avenues for diseases involving aberrant SGs.