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Guarino, C.* ; Legowska, M.* ; Epinette, C.* ; Kellenberger, C.* ; Dallet-Choisy, S.* ; Sienczyk, M.* ; Gabant, G.* ; Cadène, M.* ; Zoidakis, J.* ; Vlahou, A.* ; Wysocka, M.* ; Marchand-Adam, S.* ; Jenne, D. ; Lesner, A.* ; Gauthier, F.* ; Korkmaz, B.*

New selective peptidyl di(chlorophenyl)-phosphonate esters to visualize and block neutrophil proteinase 3 in human diseases.

J. Biol. Chem. 289, 31777-31791 (2014)
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The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure: Ac-peptidylP(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl-phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect and control PR3 activity in a variety of inflammatory diseases.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Activity-based Probes ; Inflammation ; Inflammatory Diseases ; Neutrophil ; Protease ; Protease Inhibitor ; Proteinase 3 (myeloblastin), ; Serine Protease
Language english
Publication Year 2014
HGF-reported in Year 2014
ISSN (print) / ISBN 0021-9258
e-ISSN 1083-351X
Journal Journal of Biological Chemistry, The
Quellenangaben Volume: 289, Issue: 46, Pages: 31777-31791 Article Number: , Supplement: ,
Publisher American Society for Biochemistry and Molecular Biology
Reviewing status Peer reviewed
Institute(s) Institute of Lung Health and Immunity (LHI)
POF-Topic(s) 30202 - Environmental Health
Research field(s) Lung Research
PSP Element(s) G-501600-005
PubMed ID 25288799
DOI 10.1074/jbc.M114.591339
WOS ID WOS:000345314700010
Erfassungsdatum 2014-10-09
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