Exploring Protein-Protein Interactions of the TCS (Thrombospondin-I, Cysteine-rich, and Spacer domains) Region of ADAMTS7 through Yeast Two-Hybrid System

Background: Coronary artery disease (CAD) remains a leading cause of mortality and morbidity globally, imposing a significant burden on healthcare and individuals alike. The ADAMTS7 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 7) gene locus has been implicated in the progression and pathogenesis of atherosclerosis or CAD. A distinctive feature of ADAMTS7 is the presence of the Thrombospondin, Cysteine-rich, Spacer domain (TCS) within its structure. The TCS domain is believed to play a crucial role in substrate binding and specificity of ADAMTS7, yet the specific molecular interactions involving this region remain largely unexplored. This study aims to screen and identify potential biological interacting partners of the TCS-encoding region of ADAMTS7 using the Yeast Two-Hybrid (Y2H) system to understand the role of ADAMTS 7 in the progression of atherosclerosis.

Methods: The TCS region of ADAMTS7 was cloned into the plasmid pGBKT7 (bait) which encodes the GAL4 DNA-Binding Domain region. It was then transformed into the Yeast 2 Gold strain and mated with the Y187 library strain that contains the plasmid pGADT7(prey) which encodes for the GAL4 DNA-Activation Domain fused with heart cDNA isolated from CAD patients. After the mating procedure, the yeast cells were subjected to various screening processes using the dropout media containing X-alpha gal and aureobasidin to find the biological interacting partners. Subsequently, prey plasmid transformants were selected using the dropout media (DDO/X, QDO/X, SD/-Leu) and plasmids were extracted using a yeast plasmid extraction kit (Takara) and then sent for sequencing at Oklahoma State University DNA Protein Core Facility in Stillwater. The positive interactions were validated as genuine by using the recommended co-transformation procedure.

Results: Through Yeast two-hybrid screening, we identified six hundred possible interacting clones that encode for different substrates that need to be sequenced. Two potential substrates already have been identified to belong to the tetraspanin and heat shock protein families. Additional validation work is needed to ascertain the nature of interactions.

Conclusion: The Yeast Two-Hybrid System is a useful tool to screen protein interaction partners for a known protein. Our preliminary data are promising for possible biological interaction partners of the proatherogenic metalloprotease ADAMTS7.