Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy th rough unknown pathological mechanism(s). Dystrophin is a large extrasarcome ric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy, In addition, we have recently shown that dystrophin is pro teolytically cleaved by the Coxsackievirus protease 2A leading to functiona l impairment and morphological disruption. However, the mechanism of dystro phin cleavage and the exact cleavage site remained to be identified. Antibo dy epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network al gorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-direct ed mutagenesis, peptide sequencing, and fluorescence resonance energy trans fer assays with recombinant dystrophin, we demonstrate that this putative s ite in mouse and human dystrophin is a direct substrate for the Coxsackievi ral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that int eracts with the protease 2A and was found to have an IC50 of 550 nM in vitr o, Dystrophin is the first cellular substrate of the enteroviral protease 2 A that was identified using by a bioinformatic approach and for which the c leavage site was molecularly mapped within living cells.