Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues

Abstract

Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 M HNE lost ∼76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.Keywords: Oxidative damage; hydroxynonenal; cathepsin B; Michael adducts; mass spectrometry Oxidation of plasma low-density lipoprotein (oxLDL) generates lipid oxidation products that have been proposed to play a role in the pathogenesis of atherosclerosis by modulating the expression of inflammatory factors and by creating ligands for macrophage scavenger receptors (Witztum and Steinberg 1991;Podrez et al. 2000). When oxLDL is incubated with macrophages in culture, intracellular degradation appears to be deficient after receptor-mediated endocytosis (Lougheed et al. 1991;Jessup et al. 1992;Hoppe et al. 1994). Long-term incubation of macrophages with oxLDL leads to the accumulation of ceroid, an insoluble protein-lipid product that is a hallmark of foam cell lesions and advanced atherosclerotic plaques (Ball et al. 1986;Yin 1996). In vivo and in vitro inhibition of thiol proteases also can result in the formation of ceroid and lipofuscin-like substances (Ivy et al. 1990). The molecular mechanisms responsible for the poor degradation of oxidized macromolecules in macrophages are not fully understood. One mechanism proposes that cross-linking of substrate proteins by Abbreviations: BHT, butylated hydroxytoluene; BSA, bovine serum albumin; CLN, N␣-CBZ-L-lysine p-nitrophenyl ester; DTT, dithiothreitol; ESMS, electrospray mass spectrometry; HNE, 4-hydroxy-2-nonenal; LC ESMS, liquid chromatography electrospray mass spectrometry; LDL, lowd...

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