Duchenne Muscular Dystrophy (DMD) is a lethal genetic disease caused by malfunction of the protein dystrophin. The main function of dystrophin... Show moreDuchenne Muscular Dystrophy (DMD) is a lethal genetic disease caused by malfunction of the protein dystrophin. The main function of dystrophin is to connect the sarcolemma to the machinery of muscle function via specific binding domains near the C-terminus and N-terminus. These binding domains are connected by a large central rod region that had previously thought to have been biologically inert, but has recently been shown to contain binding sites for various biological partners, including a specific rod region that binds to nNOS, which is an important molecule in modulating vasodilation and increasing blood flow by relax vessel. Removal of this nNOS binding eliminates NO signaling during exercise, causing exercise-related ischemia leading to muscle damage. A major therapeutic strategy being studied to treat DMD is exon skipping, which results in modifications to the dystrophin protein. However, it remains unclear exactly where the binding sites on dystrophin protein and nNOS are, or how they interact with each other. This project is embarked upon more fully characterize the D1617 binding site on the nNOS PDZ domain. Previous studies show that, in neural tissue, PDZnNOS binds to two other proteins (PSD95 and N-methyl-D-aspartate receptor, NMDAR) forming a ternary complex in which the nNOS: PSD95 is a typical PDZ type interaction, but in which the nNOS :NMDAR binding occurred in an unusual fashion, through a unique “finger” region present in PDZnNOS but not in most other PDZ domains. Interestingly, in muscle cells, nNOS also interacts with two proteins, dystrophin and syntrophin (SNTA). Using this as an analogy, we hypothesize that in this case, the binding may also involve both the canonical and finger regions of PDZnNOS, one binding to each protein. We are testing this by constructing PDZnNOS variants with specific amino acid changes designed to xi disrupt each of these interactions (canonical and finger region) independently, and will then examine the impact on both the PDZSNTA and dystrophin D1617 interactions. It is hoped fully understanding the dystrophin nNOS interaction will allow therapies that require modification to dystrophin to conduct these modifications in a way that retains nNOS signaling required for proper muscle function. M.S. in Cell and Molecular Biology, July 2014 Show less
