Structural characterization of the glyceraldehyde‐3‐phosphate dehydrogenase‐mediated regulation of endothelin‐1 expression

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) plays a key role in the regulatory pathways of endothelin‐1 (ET‐1), a vasoconstrictor critical for vascular homeostasis. Elevated ET‐1 levels are associated with certain vascular diseases, thus production and secretion of ET‐1 must be tightly regulated. This regulation occurs primarily via GAPDH‐mediated destabilization of ET‐1 mRNA. Recent studies suggest that GAPDH binds particular adenine‐uridine rich elements (AREs) in the 3′‐untranslated region (3′‐UTR) of the ET‐1 gene. However, the structural details underlying the GAPDH‐mediated control of ET‐1 expression are lacking. With the techniques of molecular biology, electrophoretic mobility shift assay (EMSA), and x‐ray crystallography, we seek to probe the structural interaction between GAPDH and the 3′‐UTR of ET‐1. To this end, we cloned, expressed, and purified human GAPDH. To identify the specific GAPDH binding sequences, we constructed small RNA transcripts of the implicated AREs using T7 RNA polymerase. We will next utilize EMSA to identify the smallest RNA fragments that bind to GAPDH. Ultimately, we will co‐crystallize these RNA fragments with GAPDH and solve the structure of the complexes. These studies will provide the first molecular basis for structure‐based drug design, leading to the development of novel molecules that specifically target ET‐1 mRNA destabilization.