Inhibition of Cathepsin K by Nitric Oxide Donors:  Evidence for the Formation of Mixed Disulfides and a Sulfenic Acid

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 the activity of purified cathepsin K in a time- and concentration-dependent manner (IC(50) values after 15 min of preincubation at pH 7.5 of 28, 105, 0.4, and 10 microM, respectively). Cathepsin K activity in Chinese hamster ovary cells stably transfected with cathepsin K was also inhibited by the above NO donors with similar potencies. GSNO at 100 microM 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 that the 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 is consistent 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.

Cysteine-Based Redox Switches in Enzymes

“…Some of the earliest data directly linking the oxidation of cysteine residues to the regulation of activity were with papain, where sulfenic acid formation following H 2 O 2 treatment caused a reversible inhibition in activity (1). Similarly, caspases and cathepsins have been shown to be sensitive to reversible oxidative inhibition in vitro by H 2 O 2 , nitric oxide donors, and=or protein hydroperoxides through similar mechanisms (15,29,30,32,60).…”

Oxidation as a Post-Translational Modification that Regulates Autophagy

“…19 The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is inhibited by oxidation to sulfenic acid. 21 Oxidation to sulfenic acid also inhibits the activity of members of the papain super family of proteins, such as the lysosomal cathepsin K. 22 Notably, Atg4 shares a high level of structural homology with this family of proteins 23,24 and this protease is possibly also oxidized to sulfenic acid by H 2 O 2 . Alternatively, a disulfide bridge between the catalytic cysteine (Cys 77 or Cys 74 of HsAtg4A or HsAtg4B, respectively) and the regulatory cysteine residue (Cys 81 or Cys 78 of HsAtg4A or HsAtg4B, respectively) might be forming, though we could not detect such a bond.…”

A Simple Assay for Detection of Small-Molecule Redox Activity

“…9,10 Although this allows careful regulation in the cellular environment, it creates a challenge for drug discovery efforts with these targets, as compound-mediated oxidation is not considered an exploitable mechanism of action (MOA) for drug development. Small-molecule-mediated oxidation of targets in vitro has been documented only occasionally, 11,12 and importantly, an understanding of this mechanism as well as a method for their detection is lacking.…”