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Titolo:
Inhibition of cathepsin K by nitric oxide donors: Evidence for the formation of mixed disulfides and a sulfenic acid
Autore:
Percival, MD; Ouellet, M; Campagnolo, C; Claveau, D; Li, C;
Indirizzi:
Merck Frosst Ctr Therapeut Res, Dept Biochem & Mol Biol, Pointe Claire, PQH9R 4P8, Canada Merck Frosst Ctr Therapeut Res Pointe Claire PQ Canada H9R4P8 P8, Canada Merck Frosst Ctr Therapeut Res, Dept Med Chem, Pointe Claire, PQ H9R 4P8, Canada Merck Frosst Ctr Therapeut Res Pointe Claire PQ Canada H9R 4P8 P8, Canada
Titolo Testata:
BIOCHEMISTRY
fascicolo: 41, volume: 38, anno: 1999,
pagine: 13574 - 13583
SICI:
0006-2960(19991012)38:41<13574:IOCKBN>2.0.ZU;2-G
Fonte:
ISI
Lingua:
ENG
Soggetto:
S-NITROSYLATION; REVERSIBLE INACTIVATION; ACTIVE-SITE; GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE; FUNCTIONAL EXPRESSION; NITROSOTHIOLS; ENZYME; PEROXYNITRITE; BONE; STABILIZATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
58
Recensione:
Indirizzi per estratti:
Indirizzo: Percival, MD Merck Frosst Ctr Therapeut Res, Dept Biochem & Mol Biol, POB 1005, Pointe Claire, PQ H9R 4P8, Canada Merck Frosst Ctr Therapeut Res POB 1005 Pointe Claire PQ Canada H9R 4P8
Citazione:
M.D. Percival et al., "Inhibition of cathepsin K by nitric oxide donors: Evidence for the formation of mixed disulfides and a sulfenic acid", BIOCHEM, 38(41), 1999, pp. 13574-13583

Abstract

The cysteine protease cathepsin K is believed to play a key role in bone resorption as it has collagenolytic activity and is expressed predominantly and in high levels in bone resorbing osteoclast cells. The addition of nitric oxide (NO) and NO donors to osteoclasts in vitro results in a reduction of bone resorption, although the mechanism of this effect is not fully understood. The S-nitroso derivatives of glutathione (GSNO) and N-acetylpenicillamine (SNAP) and the non-thiol NO donors NOR-1 and NOR-3 all inhibited theactivity of purified cathepsin K in a time- and concentration-dependent manner (IC50 values after 15 min of preincubation at pH 7.5 of 28, 105, 0.4, and 10 mu M, respectively). Cathepsin K activity in Chinese hamster ovary cells stably transfected with cathepsin K was also inhibited by the above NOdonors with similar potencies. GSNO at 100 mu M also completely inhibited the autocatalytic maturation at pH 4.0 of procathepsin K to cathepsin K. The inhibition of cathepsin K by GSNO was rapidly reversed by DTT, but inhibition by NOR-1 was not reversed by DTT, and analysis of the inhibited cathepsin K for S-nitrosylation using the Greiss reaction gave negative results in both cases. Analysis of the protein by electrospray liquid chromatography/mass spectrometry showed that the inhibition of cathepsin K by GSNO resulted in a mass increase of 306 +/- 2 Da, consistent with the formation of a glutathione adduct. Prior inhibition of cathepsin K by the active site thiol-modifying inhibitor E-64 blocked the modification by GSNO, indicating thatthe glutathione adduct is likely formed at the active site cysteine. Treatment of cathepsin K with NOR-1 resulted in a mass increase of between 30 and 50 Da, corresponding to the oxidation of a cysteine to sulfinic and sulfonic acids. Cotreatment of cathepsin K with NOR-1 plus the sulfenic acid reagent dimedone resulted in a mass increase of approximately 141 Da, which isconsistent with the formation of a dimedone adduct. This result demonstrates that the NOR-1-dependent formation of cathepsin K sulfinic and sulfonic acids occurs via a sulfenic acid. These results show that inhibition of cathepsin K activity and its autocatalytic maturation represent two potential mechanisms by which NO can exert its inhibitory effect on bone resorption. This work also shows that oxidative thiol modifications besides S-nitrosylation should be considered when the effects of NO and NO donors on critical thiol-containing proteins are investigated.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 26/09/20 alle ore 04:09:06