Persistent susceptibility of cathepsin B to irreversible inhibition by nitroxyl (HNO) in the presence of endogenous nitric oxide.
Nitrosation of enzyme regulatory cysteines is one of the key posttranslational modification mechanisms of enzyme function. Frequently such modifications are readily reversible; however, cysteine proteases, such as cathepsin B, have been shown to be covalently and permanently inactivated by nitroxyl (HNO), the one-electron reduction product of NO. Owing to the high reactivity of HNO with NO, endogenous NO production could provide direct protection for the less reactive protein cysteines by scavenging HNO. Additionally, endogenous cellular production of NO could rescue enzyme function by protective nitrosation of cysteines prior to exposure to HNO. Thus, we studied the effect of endogenous NO production, induced by LPS or IFN-gamma, on inhibition of cysteine protease cathepsin B in RAW macrophages. Both LPS and IFN-gamma induce iNOS with generation of nitrate up to 9 muM in the media after a 24-h stimulation, while native RAW 264.7 macrophages neither express iNOS nor generate nitrate. After the 24-h stimulation, the HNO-releasing Angeli's salt (0-316 microM) caused dose-dependent and DTT-irreversible loss of cathepsin B activity, and induction of iNOS activity did not protect the enzyme. The lack of protection was also verified in an in vitro setup, where papain, a close structural analogue of cathepsin B, was inhibited by Angeli's salt (2.7 microM) in the presence of the NO donor DEA/NO (0-316 microM). This clearly showed that a high molar excess of DEA/NO (EC(50) 406 microM) is needed to protect papain from the DTT-irreversible covalent modification by HNO. Our results provide first evidence on a cellular level for the remarkably high sensitivity of active-site cysteines in cysteine proteases for modification by HNO.
Department of Pharmacology, Institute of Biomedicine, Biomedicum Helsinki, P.O. Box 63, FIN-00014 University of Helsinki, Finland.
Antti J
AJ
Väänänen
Pertteli
P
Salmenperä
Mika
M
Hukkanen
Katrina M
KM
Miranda
Ari
A
Harjula
Pekka
P
Rauhala
Esko
E
Kankuri
D000818:Animals; D015153:Blotting, Western; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D005978:Glutathione / Q000494:pharmacology; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology*; D009589:Nitrogen Oxides / Q000378:metabolism*
D008070:Lipopolysaccharides; D009589:Nitrogen Oxides; D009569:Nitric Oxide; D007371:Interferon-gamma; D002401:Cathepsin B; D005978:Glutathione; C039900:nitroxyl