Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates

Attucci, Sylvie; Korkmaz, Brice; Juliano, Luiz; Hazouard, E.; Girardin, Catherine; Brillard-Bourdet, Michèle; Réhault, Sophie; Anthonioz, P; Gauthier, Francis

doi:10.1042/bj20020321

Cited by 32 publications

(26 citation statements)

References 29 publications

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“…This latter peptide is derived from hCG protein substrate, the protease-activated thrombin receptor PAR-1 (26). Of interest, hCG-mediated cleavage at the F-S bond was conserved in mCG.…”

Section: Resultsmentioning

confidence: 99%

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

Kalupov

Brillard-Bourdet

Dadé

et al. 2009

Journal of Biological Chemistry

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It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases.Neutrophil serine proteases (NSPs), 3 neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (Pr3), are mainly stored in neutrophil primary granules in readily active forms. NSPs are structurally related and share the conserved chargerelay triad, His 57 -Asp , where Ser is the active residue (chymotrypsinogen numbering) (1). NSPs contribute to neutrophil oxygen-independent system-mediated protection of the host against invading pathogens (2). Indeed, NSPs serve a physiological role for killing of microbes (3). Activated neutrophils are also known to release NSPs in the setting of inflammation. In vitro, NSPs are capable of cleaving a panoply of substrates. These include extracellular matrix proteins, pro-inflammatory mediators, coagulation factors, and immunoglobulins (4). NE degrades pro-inflammatory mediators such as tumor necrosis factor-␣ and interleukin-1␤, hence could alter the inflammatory response. The enzyme is capable of inducing the secretion of granulocyte macrophage-colony stimulating factor and interleukin-8, which could amplify the inflammation. Consequently, the release of these potent proteinases in diseased situations could create a proteolytic environment where degradation of different host molecules might occur and result in inappropriate inflammatory response. Because of their large substrate repertoire, NSPs have been implicated in the pathogenesis of various inflammatory and tissue-destructive diseases, including acute lung injury, cystic fibrosis, and COPD (5-7).COPD is recognized as a major health problem whose worldwide incidence is increasing dramatically...

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“…This latter peptide is derived from hCG protein substrate, the protease-activated thrombin receptor PAR-1 (26). Of interest, hCG-mediated cleavage at the F-S bond was conserved in mCG.…”

Section: Resultsmentioning

confidence: 99%

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

Kalupov

Brillard-Bourdet

Dadé

et al. 2009

Journal of Biological Chemistry

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“…Free Pr3 and cat G were titrated with ␣ 1 -PI and ␣ 1 -antichymotrypsin, respectively (Attucci et al, 2002;Korkmaz et al, 2004). Activities were measured in 50 mM HEPES buffer, pH 7.4, 0.75 M NaCl, 0.05% (v/v) Igepal CA-630 for Pr3 and in 50 mM HEPES buffer, pH 7.4, 0.1 M NaCl, 0.01% (v/v) Igepal CA-630 for cat G, using Abz-VADCADQ-EDDnp and Abz-TPFSGQ-EDDnp that are specifically cleaved by Pr3 and cat G, respectively (Attucci et al, 2002;Korkmaz et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

Attucci¹,

Gauthier²,

Korkmaz³

et al. 2006

J Pharmacol Exp Ther

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EPI-hNE4 (depelstat) is a potent inhibitor of human neutrophil elastase derived from human inter-␣-trypsin inhibitor and designed to control the excess proteolytic activity in the sputum of cystic fibrosis patients. We analyzed its resistance to the proteolysis it is likely to encounter at inflammatory sites in vivo. EPI-hNE4 resisted hydrolysis by neutrophil matrix metalloproteases (MMPs) and serine proteases that are released from activated neutrophils in inflammatory lung secretions, including MMP-8 and MMP-9, and the elastase-related protease 3 and cathepsin G. It also resisted degradation by epithelial lung cell MMP-7 but was broken down by the Pseudomonas aeruginosa metalloelastase pseudolysin, when used in a purified system, but not when this protease competed with equimolar amounts of neutrophil elastase. We also investigated the inhibitory properties of EPI-hNE4 at the surface of purified blood neutrophils and in the sputum of cystic fibrosis patients where neutrophil elastase is in both a soluble and a gel phase. The elastase at the neutrophil surface was fully inhibited by EPI-hNE4 and formed soluble complexes. The elastase in cystic fibrosis sputum supernatants was inhibited by stoichiometric amounts of EPIhNE4, allowing titration of the protease. But the percentage of inhibition in whole sputum homogenates varied from 50 to 100%, depending on the sample tested. EPI-hNE4 was rapidly cleaved by the digestive protease pepsin in vitro. Therefore, EPI-hNE4 seems to be an elastase inhibitor suitable for use in aerosols to treat patients with cystic fibrosis.

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“…All three NSPs cleave PAR-1, which impairs their activation by thrombin (Renesto et al, 1997). The sequence containing the CG cleavage site (-EPF 55 2WEDEE-) (Renesto et al, 1997) has been used to raise a sensitive FRET substrate for this protease (Attucci et al, 2002). PAR-2 is expressed on endothelial cells and can be activated by trypsin (SKGR 34 2SLIGKV) (Nystedt et al, 1994(Nystedt et al, , 1995a but also by the three NSPs (Uehara et al, 2002(Uehara et al, , 2003(Uehara et al, , 2004.…”

Section: Processing and Activation Of Cellular Receptorsmentioning

confidence: 99%

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

et al. 2010

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Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates

Cited by 32 publications

References 29 publications

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

EPI-hNE4, a Proteolysis-Resistant Inhibitor of Human Neutrophil Elastase and Potential Anti-Inflammatory Drug for Treating Cystic Fibrosis

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

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