Targeted protein O-GlcNAc reveals transcriptional functions for O-GlcNAc

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• O-Linked β-N-acetylglucosamine (O-GlcNAc) is a crucial post-translational modification (PTM) found on numerous nucleocytoplasmic substrates.
• The functional significance of most O-GlcNAc modifications remains largely uncharacterized.
• Targeted PTM manipulation is vital for functional annotation and identifying therapeutic targets.

Purpose of the Study:

• To develop and validate an induced proximity method for targeted protein O-GlcNAc.
• To investigate the direct effects of O-GlcNAc on transcription factors.
• To elucidate the role of O-GlcNAc in cellular signaling pathways.

Main Methods:

• An induced proximity system utilizing a destabilized nanobody-O-GlcNAc transferase (OGT) fusion protein was engineered.
• The method was applied to achieve targeted O-GlcNAc on 21 different protein substrates.
• Cellular assays were performed to analyze the functional consequences of targeted O-GlcNAc, particularly on transcription factor activity.

Main Results:

• The developed method demonstrated general utility for site-specific protein O-GlcNAc modification.
• The direct impact of O-GlcNAc on the function of multiple transcription factors was successfully annotated.
• Specific findings include the inhibitory regulation of AP-1 transcriptional activation by O-GlcNAc on c-Fos and c-Jun.

Conclusions:

• A novel and generalizable induced proximity method enables rapid investigation of O-GlcNAc functions in cells.
• Targeted O-GlcNAc modification provides insights into nutrient-sensing mechanisms and transcriptional regulation.
• This approach accelerates the functional characterization of PTMs and opens avenues for therapeutic development.