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

• Published in: Protein science Vol. 11; no. 4; pp. 831 - 840
• Main Authors: Crabb, John W., O'Neil, June, Miyagi, Masaru, West, Karen, Hoff, Henry F.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.04.2002
• Subjects: Aldehydes - pharmacology; Amino Acids - metabolism; Animals; BHT, butylated hydroxytoluene; Binding Sites; Blotting, Western; BSA, bovine serum albumin; cathepsin B; Cathepsin B - antagonists & inhibitors; Cathepsin B - metabolism; Cattle; CLN, Nα‐CBZ‐l‐lysine p‐nitrophenyl ester; Cysteine Proteinase Inhibitors - pharmacology; DTT, dithiothreitol; ESMS, electrospray mass spectrometry; Gas Chromatography-Mass Spectrometry; HNE, 4‐hydroxy‐2‐nonenal; hydroxynonenal; LC ESMS, liquid chromatography electrospray mass spectrometry; LDL, low‐density lipoprotein; Lipid Peroxidation; Macrophages - drug effects; Macrophages - enzymology; Macrophages - metabolism; mass spectrometry; Mice; Michael adducts; Oxidation-Reduction; Oxidative damage; Oxidative Stress; ox‐LDL, oxidized LDL; PBS, phosphate‐buffered saline; Protein Conformation; Spleen - drug effects; Spleen - enzymology; TCA, trichloroacetic acid
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.4400102

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 μM 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.