HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Crabb, J. W. Articles by Hoff, H. F. Search for Related Content PubMed PubMed Citation Articles by Crabb, J. W. Articles by Hoff, H. F. Social Bookmarking                   What's this?

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

¹ Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
² Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA

Reprint requests to: John W. Crabb, Ph.D., Cole Eye Institute (i31), Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA; e-mail: crabbj{at}ccf.org; fax: (216) 445-3670 or Henry F. Hoff, Ph.D., Department of Cell Biology (NC-10), Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA; e-mail: hoffh{at}ccf.org; fax: (216) 444-9404.

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.

Keywords: Oxidative damage; hydroxynonenal; cathepsin B; Michael adducts; mass spectrometry

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, low-density lipoprotein • ox-LDL, oxidized LDL • PBS, phosphate-buffered saline • TCA, trichloroacetic acid

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?