Analysis of protein-protein interaction of ADAMTS7 with cardiomyocytes using the yeast two-hybrid system

Abstract

Background: Coronary artery disease (CAD) is one of the most prevalent diseases in the USA. About 20.1 million adults of age 20 and above have CAD which makes up 7.2% of the population. A disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS7) is found to be associated with many inflammatory diseases such as CAD and atherosclerosis. Previous studies have shown ADAMTS7 enzyme interacts with the COMP proteins present in the cardiac region and causes degradation of COMP protein which induces CAD in both knock-out and wild-type hyperlipidemic mouse models. This study focuses on finding the domain interaction of ADAMTS7 enzyme with cardiomyocytes using the Yeast 2 hybrid system (Y2H). The Y2H system is one of the powerful methods to find protein-protein interactions in in vitro studies. The study will be conducted to ascertain strong interaction of ADAMTS7 domains and variants with cardiomyocyte proteins causing CAD in humans.

Methods: Two yeast strains are grown, mated to generate a yeast 2 hybrid system used to determine the ADAMTS7 and CAD relationship. The ADAMTS7 encoding gene cloned into the frame of bait plasmid and the prey plasmid generated using cDNA of c-myc gene from a CAD patient. Following, the bait and prey plasmids transformed into Y2H Gold Yeast Strain and Y187 Yeast Strain vectors. After mating they are screened on DDO, DDO/X/A and QDO/X/A drop out media. Then transformed yeasts are plated on SD media containing QDO/X/A.

Results and Discussions: Yeast grown on DDO, DDO/X/A and QDO/X/A media and observed as blue or white colonies. Therefore, yeast containing interactive plasmids grew as blue colonies while the negative interaction colonies as white. The yeast strain employed contained a reporter gene whose regulatory regions contained DNA binding sites for protein X fusion (Bait) and the second hybrid protein Y fusion (Prey). Positive interaction between ADAMTS 7 and cardiomyocytes would result into bait and prey interactions activating upstream activating sequence (UAS). The interaction allows yeast to grow into blue colonies as it selectively expresses enzymes utilizing histidine amino acids. Therefore, if there is an ADAMTS7 enzyme interaction with cardiomyocytes then it confirms the relationship between ADAMTS7 and CAD.