title: Nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2A through S-nitrosylation: A protective mechanism against enteroviral cardiomyopathy.
abstract: BACKGROUND
  Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirus-induced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A.
  
  
  RESULTS
  In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC(50), 51 micromol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S:-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S:-nitrosylation of this site was confirmed with electron spin resonance spectroscopy.
  
  
  CONCLUSIONS
  These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S:-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.
authors.affiliation: Molecular Cardiology Unit, Department of Medicine IV, Goethe-University, Frankfurt, Germany. badorff@em.uni-frankfurt.de
authors.forename: C
authors.initials: C
authors.lastname: Badorff
authors.forename: B
authors.initials: B
authors.lastname: Fichtlscherer
authors.forename: R E
authors.initials: RE
authors.lastname: Rhoads
authors.forename: A M
authors.initials: AM
authors.lastname: Zeiher
authors.forename: A
authors.initials: A
authors.lastname: Muelsch
authors.forename: S
authors.initials: S
authors.lastname: Dimmeler
authors.forename: K U
authors.initials: KU
authors.lastname: Knowlton
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002311:Cardiomyopathy, Dilated / Q000378:metabolism* / Q000517:prevention & control; D020134:Catalytic Domain / Q000187:drug effects; D003545:Cysteine / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003720:Densitometry; D016189:Dystrophin / Q000378:metabolism*; D004769:Enterovirus Infections / Q000378:metabolism* / Q000517:prevention & control; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009589:Nitrogen Oxides; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D026423:S-Nitroso-N-Acetylpenicillamine; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics; D014764:Viral Proteins
chemical_list: D016189:Dystrophin; D004791:Enzyme Inhibitors; D006834:Hydrazines; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; C112653:PAPA NONOate; D013438:Sulfhydryl Compounds; D014760:Viral Fusion Proteins; D014764:Viral Proteins; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus; D010396:Penicillamine; D003545:Cysteine