Enteroviral Protease 2A Directly Cleaves Dystrophin and Is Inhibited by a Dystrophin-based Substrate Analogue

• Published in: The Journal of biological chemistry Vol. 275; no. 15; pp. 11191 - 11197
• Main Authors: Badorff, Cornel, Berkely, Neil, Mehrotra, Sanjiv, Talhouk, Jamil W., Rhoads, Robert E., Knowlton, Kirk U.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14.04.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Coxsackievirus B3; Coxsackievirus Infections - metabolism; Cysteine Endopeptidases - physiology; Dystrophin - chemistry; Dystrophin - metabolism; Enteroviral Proteinase 2A; Enterovirus; Epitope Mapping; HeLa Cells; Humans; Mice; Molecular Sequence Data; Protease Inhibitors - pharmacology; Rabbits; substrate analogue; Viral Proteins; z-LSTT-fmk
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.15.11191

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC50 of 550 nmin vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells.