The biosynthesis of elastin and collagen involves post-translational, enzyme-catalyzed processing of each of the newly synthesized proteins. Among these essential processes, the oxidation of peptidyl lysine, catalyzed by lysyl oxidase, initiates the formation of intra- and intermolecular lysine-derived crosslinkages which stabilize and insolubilize extracellular fibers of these connective tissue proteins. Since this appears to be the last step in fiber synthesis, it is potentially a key point of biological control in the development of connective tissues. Moreover, lysyl oxidase is a rational chemotherapeutic target of anti-fibrotic agents designed to prevent of limit the development of fibrotic sequellae as in lung fibrosis and atherosclerosis and to limit scar formation. The present proposal intends to define key aspects of the mechanism of action and substrate specificity of lysyl oxidase and, based on such information, to design, synthesize and test at both test-tube and tissue levels new, mechanism- based, peptidyl and non-peptidyl inhibitors of this crosslinking enzyme. These efforts will involve analyses of the roles of the pyrroloquinoline quinone (PQQ) and copper ion cofactors, employing techniques as electron paramagnetic resonance, absorption and fluorescence spectrophotometry, and stopped flow kinetic analyses of the reductive and oxidative half reactions catalyzed by this enzyme. Specificity of the enzyme will be probed by kinetic analyses of oligopeptide substrates assembled by solid phase peptide synthesis. The peptide environment of the active site will be assessed by amino acid sequence analysis and mass spectroscopy of peptides isolated from the enzyme labelled with covalent, radioactive probes of the active site. Chemical and kinetic mechanisms of inhibition by new inhibitors directed at the cofactor or active site nucleophiles will be analyzed. The effect of such new inhibitors on the crosslinking of elastin in chick embryo aortae will be assessed in organ culture as an index of their potential as anti-fibrotic agents in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR018880-17
Application #
3481472
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1976-06-20
Project End
1993-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
17
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Boston University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Lucero, H A; Kagan, H M (2006) Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol Life Sci 63:2304-16
Kagan, Herbert M; Li, Wande (2003) Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. J Cell Biochem 88:660-72
Nellaiappan, K; Kagan, H M (1999) Proteins secreted by vascular smooth muscle cells as substrates of lysyl oxidase. Connect Tissue Res 40:123-9
Nagan, N; Callery, P S; Kagan, H M (1998) Aminoalkylaziridines as substrates and inhibitors of lysyl oxidase: specific inactivation of the enzyme by N-(5-aminopentyl)aziridine. Front Biosci 3:A23-6
Smith-Mungo, L I; Kagan, H M (1998) Lysyl oxidase: properties, regulation and multiple functions in biology. Matrix Biol 16:387-98
Li, W; Nellaiappan, K; Strassmaier, T et al. (1997) Localization and activity of lysyl oxidase within nuclei of fibrogenic cells. Proc Natl Acad Sci U S A 94:12817-22
Gacheru, S N; Thomas, K M; Murray, S A et al. (1997) Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: effects of TGF-beta 1 and serum deprivation. J Cell Biochem 65:395-407
Liu, G; Nellaiappan, K; Kagan, H M (1997) Irreversible inhibition of lysyl oxidase by homocysteine thiolactone and its selenium and oxygen analogues. Implications for homocystinuria. J Biol Chem 272:32370-7
Lazarus, H M; Cruikshank, W W; Narasimhan, N et al. (1995) Induction of human monocyte motility by lysyl oxidase. Matrix Biol 14:727-31
Kagan, H M; Reddy, V B; Narasimhan, N et al. (1995) Catalytic properties and structural components of lysyl oxidase. Ciba Found Symp 192:100-15;discussion 115-21

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