Abstract
Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) plays a key role in vascular homeostasis by modulating levels of the vasoconstrictor endothelin‐1 (ET‐1). Elevated ET‐1 levels are associated with vascular diseases, and its expression is tightly regulated primarily via GAPDH‐mediated destabilization of its mRNA. Recent studies suggest GAPDH binding of adenine‐uridine rich elements in the 3′‐untranslated region of ET‐1 gene leads to decreased mRNA stability. However, structural and mechanistic details underlying the GAPDH‐mediated control of ET‐1 expression are lacking. We seek to probe the structural and mechanistic basis for the GAPDH‐induced destabilization of ET‐1 mRNA via electrophoretic mobility shift assay (EMSA) and x‐ray crystallography. To identify specific GAPDH binding sequences, we constructed short RNA transcripts of the putative mRNA binding region. Utilizing a modified EMSA with biotinylated RNA transcripts, we obtained association and dissociation constants. For transcripts yielding the greatest affinity for GAPDH, we obtained crystals. Ultimately, we aim to solve the crystal structures for these ET‐1 mRNA constructs with GAPDH. 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.Funding: UMBC DRIF SRAIS (EG).
Published Version
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