Study On Factors Interacted With Angiotensin-(1-7) From Rat Cardiac Myocyte CDNA Library By Yeast Two-hybrid System

[Abstract] Objective: Accumulating evidence suggests that Ang-(1-7) be the antagonist of Ang II, showing several protecting effects in the pathophysiological changes of cardiovascular diseases correlated with RAS. It was also indicated that some factors other than Angiotensin subtype 1 receptor(ATl) or subtype 2 receptor(AT2) may mediate the effects of Ang-(l-7). But the proteins directly interacting with this seven amino acid peptide remain unclear. In current study, we used yeast two-hybrid system were to identify proteins interacting with angiotensin-(l-7). Methods: cDNA encoding the bait protein, wild-type Ang-(l-7) of rat, was synthesized. The recombinant bait plasmid renamed pBD-Ang-(l-7) was constructed by inserting the bait cDNA between the mutipule clone sites(MCS) EcoR I and Sal I of the pBD-GAL4 Cam phagemid vector. The bait vector was transformed into yeast strain YGR-2 to test pBD-Ang-(l-7) vector for no self-activation and non-specific activation. Transform yeast containing pBD-Ang-(l-7) with the rat myocardial cDNA library plasmids and plate the cells on SD without histidine, trptophan and leucine to assay the cotransformants for expression of HIS3 reporter gene. In order to distinguish between leaky expression of HIS3 reporter gene and specifically interacting proteins, detection of the expression of lacZ gene is determined by filter lift assay. Colonies with β-galactosidase (P-gal) activity were streaked again on new plates with selective media to select for His⁺ colonies, repeating filter lift assay to verify the presence of P-gal activity in lacZ⁺ colonies. Plasmid DNA were isolated from lacZ⁺ colonies then co-transformed into YGR-2 pairwisely with the control plasmids and bait vector to verify the specificity of the interaction between the bait and target protein. The DNAinserts of true positive target plasmids were analyzed by sequencing and basic local alignment sequence tool (BLAST). Results: The recombinant bait vector, pBD-Ang-(l-7) was successfully constructed and confirmed by sequencing. Bait vector was transformed into yeast strain YGR-2. The transformnants had no autonomously activated reporter genes and no toxicity in YGR-2. 6 proteins interacting with Ang-(l-7), obtained by screening of rat myocardial cDNA libraries, filter lift assay and verification of specificity of protein-protein interactions, were identified, including connective tissue growth factor, eukaryotic translation elongation factor 1 alpha, Fattyacid binding protein 4, TATA binding Protein associated factor, Phosphoglycerate Kinase 1. Conclusion: Our study indicated that Angiotensin-(l-7) may interact with factors, which may disturb balance between transcription factors, interfere with protein synthesis and cell metabolism. The antagonism of angiotensin-(l-7) on angiotensin II could be partially attribute to the interaction between angiotensin-(l-7) and some of these factors. This investigation provides molecular functional clue for further exploration of Ang-(l-7).