Functional domain analysis of SOX18 transcription factor using a single-chain variable fragment-based approach.

Area of Science:

• Molecular Biology
• Protein-Protein Interactions
• Transcription Factor Regulation

Background:

• Antibodies are established tools for studying transcription factor (TF) activity through various in vitro and in vivo methods.
• Antibodies are also emerging as crystallization scaffolds for challenging proteins like TFs.
• A knowledge gap exists in designing antibodies to effectively modulate TF activity and transcription.

Purpose of the Study:

• To explore the potential of antibodies and antibody single-chain variable region fragments as molecular tools for studying TF mechanisms.
• To investigate the molecular mode of action of the transcription factor SOX18, a target in cancer metastasis.
• To demonstrate the efficacy of an engineered antibody in inhibiting SOX18 transcriptional activity.

Main Methods:

• Utilized antibody engineering to create a single-chain variable region fragment targeting SOX18.
• Investigated protein-DNA and protein-protein binding mechanisms involving SOX18.
• Focused on disrupting specific protein-protein interactions mediated by SOX18.

Main Results:

• Demonstrated that engineered antibodies/fragments are potent tools for analyzing TF binding mechanisms.
• Identified a specific 8-amino-acid motif in SOX18's C-terminal region crucial for its activity.
• Showed that the engineered antibody specifically inhibits SOX18 transcriptional activity by disrupting protein-protein interactions at this motif.

Conclusions:

• Antibodies can be effectively engineered to modulate transcription factor activity.
• This approach provides a novel framework for studying TF interactomes using antibodies.
• Targeting SOX18 with engineered antibodies offers a potential strategy for therapeutic intervention in conditions like cancer metastasis.