期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 289, 期 46, 页码 31777-31791出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.591339
关键词
Inflammation; Neutrophil; Protease; Protease Inhibitor; Serine Protease; Activity-based Probes; Inflammatory Diseases; Proteinase 3 (Myeloblastin)
资金
- Ministere de l'Enseignement Superieur et de la Recherche
- Region Centre
- Fonds Europeen de Developpement Regional (project INFINHI)
- Polish Ministry of Science and Higher Education [IP2012 0596 72]
- European Union [261382]
- European Union Commission [601933]
- Alexandre von Humboldt Foundation
Background: Proteinase 3 activity is poorly controlled by physiological inhibitors, and its biological function is not well understood. Results: We have designed irreversible phosphonate inhibitors based on structural differences between proteinase 3 and elastase. Conclusion: They selectively inhibit proteinase 3 in biological fluids and can act as activity-based probes. Significance: These inhibitors will help clarify proteinase 3 function. 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-peptidyl(P)(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 the 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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