Biol. Chem., Vol. 389, pp. 1123–1126, August 2008 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.109 2008/391 Article in press - uncorrected proof Short Communication Regulation of cathepsin K activity by hydrogen peroxide Emmanuel Godat 1,a , Virginie Herve´ -Gre´ pinet 1,b , Florian Veillard 1 , Fabien Lecaille 1 , Maya Belghazi 2,c , Dieter Bro¨ mme 3 and Gilles Lalmanach 1, * 1 INSERM, U 618, Prote´ ases et Vectorisation Pulmonaires, and IFR 135 ‘Imagerie Fonctionnelle’, Universite´ Francois Rabelais, Faculte´ de Me´ decine, 10 ¸ Bd Tonnelle´, F-37032 Tours cedex, France 2 INRA-Centre de Tours, Service de Spectrome´ trie de Masse, Unite´ de Physiologie de la Reproduction et des Comportements, UMR INRA 85-CNRS 6175, F-37380 Nouzilly, France 3 Faculty of Dentistry and the Center for Blood Research, University of British Columbia, Vancouver V6T 1Z3, Canada * Corresponding author e-mail: gilles.lalmanach@univ-tours.fr Abstract Although cysteine cathepsins, including cathepsin K, are sensitive to oxidation, proteolytically active forms are found at inflammatory sites. Regulation of cathepsin K activity was analyzed in the presence of H 2 O 2 to gain an insight into these puzzling observations. H 2 O 2 impaired processing of procathepsin K and inactivated its mature form in a time- and dose-dependent mode. However, as a result of the formation of a sulfenic acid, as confirmed by trapping in the presence of 7-chloro-4-nitrobenzo-2- oxa-1,3-diazol, approximately one-third of its initial activ- ity was restored by dithiothreitol. This incomplete inactivation may partially explain why active cysteine cathepsins are still found during acute lung inflammation. Keywords: cysteine cathepsin; inflammation; oxidation; protease; proteolysis; sulfenic acid. During chronic lung inflammation associated with mucus hypersecretion, emphysema and/or fibrosis, both prote- ase/antiprotease systems and antioxidant/oxidant bal- ance are impaired (Lopez and Murray, 1998; Owen, 2005). Reactive oxygen species (ROS) including hydro- gen peroxide, a potent and archetypal marker of oxida- tion, initiate protein and lipid oxidation, participate in Present addresses: a Laboratoire de PhysioGe´ nomique, SBGM/DBJC, CEA/Saclay, F-91191 Gif-sur-Yvette, France b Centre INRA de Tours, UR 83 Recherches Avicoles, F-37380 Nouzillly, France c Centre d’Analyses Prote´ omiques de Marseille, IFR Jean Roche, F-13916 Marseille, France alteration of the extracellular matrix (ECM) and may induce apoptosis (Kharitonov and Barnes, 2001). For a long time, studies on pulmonary ECM-degrading enzymes have focused on serine proteases and matrix metalloproteases, with fewer studies exploring the role of lung lysosomal cysteine cathepsins that are expressed by macrophages, fibroblasts, pneumocytes and epithelial cells (Punturieri et al., 2000; Brasch et al., 2002; Gutten- tag et al., 2003; Veillard et al., 2008). Cysteine cathepsins are released extracellularly and participate in ECM deg- radation and/or remodeling (Wolters and Chapman, 2000; Turk et al., 2001; Lecaille et al., 2002b, 2007; Novi- nec et al., 2007). Buhling et al. (2004) proposed that cathepsin K may play a pivotal role in lung homeostasis owing to its potent collagenolytic and elastinolytic activity. Although cathepsin activity is normally tightly controlled by endogenous cathepsin inhibitors, cystatins (Abrahamson et al., 2003), an imbalance between cathepsins and cystatins may occur under pathophysio- logical conditions (Altiok et al., 2006); we have identified active cysteine cathepsins in bronchoalveolar lavage fluid (BALF) from patients with acute inflammation or silicosis (Lalmanach et al., 2006; Perdereau et al., 2006; Serveau- Avesque et al., 2006). Furthermore, the preservation of cysteine cathepsin activity in BALF remains perplexing because of the presence of an unfavorable oxidizing environment. As a result of the chemical characteristics of its thiol group, cysteine was identified as a key amino acid of enzyme function that exists in different oxidative states (for a review see Giles et al., 2003). In thiol-dependent cathepsins, the nucleophilic cysteinyl residue of the active site (Cys25, papain numbering; Storer and Menard, 1994) is sensitive to peptide and protein hydro- peroxides and NO donors, in contrast to non-thiol- dependent cathepsins D and G (Percival et al., 1999; Lockwood, 2002; Headlam et al., 2006). In the present study we conducted an in vitro investigation of cathepsin K inactivation by H 2 O 2 . Our aim was to gain an insight into the partial and unexpected resistance of cysteine cathepsins to oxidation that may occur during pulmonary inflammation (Veillard et al., 2008; E. Godat and G. Lal- manach, unpublished data). Cathepsin K was inactivated by H 2 O 2 in a time- and dose-dependent manner (Figure 1), indicating that the enzyme may remain temporarily active under oxidative conditions. Similar observations were reported for papain and caspases (Borutaite and Brown, 2001; Lockwood, 2004). Fitting of the reaction curves revealed that the kinetics of cathepsin K inactivation followed a second- order exponential decay function (data not shown). These observations suggest that inactivation of cathepsin K does not obey a simple chemical mechanism and that the oxidized active-site cysteine may be found in differ- Brought to you by | Dalhousie University Authenticated Download Date | 5/18/15 10:20 PM