Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and...

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• The RE-1 silencing transcription factor (REST/NRSF) is vital for neuronal gene repression.
• REST/NRSF defects are linked to neuropathies, neuron activity disorders, and autism.
• REST recruits Sin3B via its PAH1 domain, forming a complex essential for neuronal robustness.

Purpose of the Study:

• To establish protein-protein interaction assays for the Sin3B PAH1/REST binding surface.
• To reconstitute the PAH1/REST interaction using recombinant peptides.
• To develop high-throughput and high-dynamic-range assays for monitoring this interaction.

Main Methods:

• Utilized structural information of the PAH1/REST complex to select key amino acids.
• Employed surface plasmon resonance (SPR) to validate direct interaction and competitive binding of PAH1 peptides with REST.
• Developed and applied a novel time-resolved fluorescence energy transfer (TR-FRET) assay for monitoring the interaction.

Main Results:

• Confirmed direct interaction between biotinylated REST and GST-tagged PAH1.
• Demonstrated competitive binding of a PAH1-FLAG peptide with biotinylated REST using SPR.
• Successfully established a novel, high-quality TR-FRET assay for the REST-Sin3B interaction.
• Developed a novel SPR-based interaction assay.

Conclusions:

• Successfully established novel SPR and TR-FRET assays to study the REST-Sin3B interaction.
• These assays provide valuable tools for understanding the molecular mechanisms underlying REST-mediated gene repression.
• The developed methods can aid in the study of neurological disorders associated with REST/NRSF dysfunction.