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Expanding the LexA Bacterial Two-Hybrid System for Human Protein-Protein Interaction Screening: Application to Tumor Suppressor PAR-4

Year: 2025
Type: dissertation
Abstract: Protein-protein interactions (PPI) are fundamental to cellular function, influencing processes such as signaling, structural organization, and disease progression. Disruptions in PPIs are implicated in cancer and neurodegenerative diseases, highlighting their importance as therapeutic targets. However, traditional methods for studying human PPIs, such as yeast two-hybrid and co-immunoprecipitation mass spectrometry, are costly and technically demanding. This dissertation explores the LexA-based bacterial two-hybrid (LexA-B2H) system as a cost effective and accessible alternative for investigating human PPIs. This study focuses on the interaction between glucose-regulated protein 78 (GRP78) and cleaved prostate apoptosis response-4 (cl-Par-4), a tumor suppressor protein involved in apoptosis and neurodegenerative disease pathways. Using the LexA-B2H system, βgalactosidase assays, and pull-down experiments, we confirm a direct interaction between GRP78 and cl-Par-4, demonstrating the system’s reliability for mammalian protein studies. Additionally, during cDNA library screening experiments, we discovered an unexpected self-association mode of cl-Par-4, distinct from the established coiled coil dimerization mechanism. These findings add to current knowledge about Par-4 self-association and suggest a previously unrecognized role of the SAC domain in homomeric interactions. Despite technical challenges, including difficulties with cDNA library handling and the co-purification of a potentially endogenous E. coli protein, this work underscores the adaptability of the LexA-B2H system for human PPI research. These findings expand our understanding of Par-4’s role in apoptotic regulation and highlight the potential of B2H systems for scalable, high-throughput PPI screening. Future optimizations should focus on improving B2H systems and library screening protocols, refining quantitative analysis, and validating interactions in mammalian systems. By broadening the applicability of B2H assays, this study contributes to advancing PPI research and therapeutic target discovery in biomedical sciences.
DOI: 10.25777/w1h6-j794
Language: English
Open Access status: green
OpenAlex ID: W6964628997
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