title: Cathepsin B is a differentiation-resistant target for nitroxyl (HNO) in THP-1 monocyte/macrophages.
abstract: We previously showed that the one-electron reduction product of nitric oxide (NO), nitroxyl (HNO), irreversibly inhibits the proteolytic activity of the model cysteine protease papain. This result led us to investigate the differential effects of the nitrogen oxides, such as nitroxyl (HNO), NO, and in situ-generated peroxynitrite on cysteine modification-sensitive cellular proteolytic enzymes. We used Angeli's salt, diethylaminenonoate (DEA/NO), and 3-morpholinosydnoniminehydrochloride (SIN-1), as donors of HNO, NO, and peroxynitrite, respectively. In this study we evaluated their inhibitory activities on the lysosomal mammalian papain homologue cathepsin B and on the cytosolic 26S proteasome in THP-1 monocyte/macrophages after LPS activation or TPA differentiation. HNO-generating Angeli's salt caused a concentration-dependent (62 +/- 4% at 316 muM) inhibition of the 26S proteasome activity, resulting in accumulation of protein-bound polyubiquitinylated proteins in LPS-activated cells, whereas neither DEA/NO nor SIN-1 showed any effect. Angeli's salt, but not DEA/NO or SIN-1, also caused (94 +/- 2% at 316 muM) inhibition of lysosomal cathepsin B activity in LPS-activated cells. Induction of macrophage differentiation did not significantly alter the inhibitory effect of HNO on lysosomal cathepsin B activity, but protected the proteasome from HNO-induced inhibition. The protection awarded by macrophage differentiation was associated with induction of the GSH synthesis rate-limiting enzyme gamma-glutamylcysteine synthetase, as well as with increased intracellular GSH. In conclusion, HNO abrogates both lysosomal and cytosolic proteolysis in THP-1 cells. Macrophage differentiation, associated with upregulation of antioxidant defenses such as increased cellular GSH, does not protect the lysosomal cysteine protease cathepsin B from inhibition.
authors.affiliation: Department of Pharmacology, Institute of Biomedicine, Biomedicum Helsinki, P.O. Box 63, University of Helsinki 00014, Finland.
authors.forename: Antti J
authors.initials: AJ
authors.lastname: Väänänen
authors.forename: Pertteli
authors.initials: P
authors.lastname: Salmenperä
authors.forename: Mika
authors.initials: M
authors.lastname: Hukkanen
authors.forename: Pekka
authors.initials: P
authors.lastname: Rauhala
authors.forename: Esko
authors.initials: E
authors.lastname: Kankuri
mesh_terms: D002401:Cathepsin B / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D003602:Cytotoxicity, Immunologic; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009573:Nitrites / Q000494:pharmacology; D009589:Nitrogen Oxides / Q000378:metabolism / Q000494:pharmacology*; D010101:Oxygen Consumption / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000378:metabolism; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D008070:Lipopolysaccharides; D020030:Nitric Oxide Donors; D009573:Nitrites; D009589:Nitrogen Oxides; D061988:Proteasome Inhibitors; D025821:Polyubiquitin; D030421:Peroxynitrous Acid; C021229:oxyhyponitrite; D009569:Nitric Oxide; D009249:NADP; D002401:Cathepsin B; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C039900:nitroxyl; D013755:Tetradecanoylphorbol Acetate