Secretory Leukocyte Protease Inhibitor (SLPI) Is, like Its Homologue Trappin-2 (Pre-Elafin), a Transglutaminase Substrate

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases

La justice des mineurs et les affaires de terrorisme

National audienceTerrorism-related cases were marginal in the juvenile justice system for many years but now occupy a growing proportion of its activity, because of the conflict in Syria and Iraq. In criminal matters, it must deal with the cases of minors who have gone to the war zones (or wanted to do so), or who have even planned or conducted attacks on national territory. In educational assistance, it must handle the situations of children who have returned from the war zones and whose parents are under investigation (and often imprisoned). In a context marked by concern and a focus on terrorism, the juvenile justice system has difficulty upholding its specific character and modes of action, and yet these are essential to understanding and addressing these issues.Longtemps anecdotiques pour la justice des mineurs, les affaires de terrorisme occupent désormais une place plus importante dans son activité, notamment en raison du conflit en Syrie et en Irak. Au pénal, elle est saisie de dossiers de mineur(e)s s'étant rendu(e)s sur place (ou ayant souhaité le faire), voire qui ont planifié ou mené des attaques sur le territoire national. En assistance éducative, elle traite les situations d'enfants revenus de ces zones de guerre et dont les parents sont mis en examen (et souvent incarcérés). Dans un contexte marqué par l'inquiétude et la prégnance des logiques antiterroristes, elle a du mal à faire prévaloir ses spécificités et ses modes d'actions, qui pourtant constituent un atout essentiel pour appréhender et traiterces questions

Développement d'une stratégie thérapeutique anti-inflammatoire en pathologie pulmonaire basée sur l'administration d'anti-protéases

Les travaux de cette thèse ont porté sur l étude des propriétés fonctionnelles d inhibiteurs recombinants polyvalents ciblant les trois protéases à sérine du neutrophile (élastase, protéase 3, cathepsine G), libérées massivement au cours des inflammations pulmonaires. Ces inhibiteurs, dérivés de molécules naturelles (élafine, trappine-2, SLPI), interagissent fortement avec les trois protéases à sérine, sous forme soluble ou liée aux membranes des neutrophiles et possèdent des propriétés anti-bactériennes indépendantes de leurs capacités inhibitrices. De plus, nous avons montré que ces inhibiteurs peuvent être ancrés à différentes protéines de structure par transglutamination tout en conservant leurs propriétés inhibitrices. Par leurs différentes propriétés, ces inhibiteurs représentent des molécules particulièrement attractives dans le cadre du développement d une stratégie thérapeutique basée sur l administration d anti-protéases par aérosol pour le traitement de maladies pulmonaires inflammatoires.In this study, we have analysed the properties of recombinant polyvalent inhibitors of the three neutrophil serine proteinases (elastase, proteinase 3, cathepsin G), massively released during inflammatory events in various lung diseases. These inhibitors, derived from natural endogeneous inhibitors (elafin, trappin-2, SLPI), interact tightly with both free and membrane-bound neutrophil serine proteinases and possess antimicrobial properties that are independent from their inhibitory capacity. We have also shown that these polyvalent inhibitors can be covalently bound to the extracellular matrix proteins by a transglutaminase and that they retain their inhibitory properties in these conditions. With their various biological properties, these inhibitors are attractive molecules for the development of an aerosol-based therapeutic strategy for the treatment of inflammatory lung diseases.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases

The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin-2 are endogeneous low-molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol-based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin-2 variant, trappin-2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin-2 A62L are tight-binding inhibitors of all three NSPs with subnanomolar Kis, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane-bound forms of all three NSPs. The trappin-2 A62L and elafin-SLPI chimeras, like wild-type elafin and trappin-2, can be covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti-inflammatory agents

Inhibition of neutrophil elastase and cathepsin G by inhibitors bound to fibronectin or elastin.

<p>Inhibitors (10⁻⁶ M) (SLPI, SLPI domain 2 (SLPI2), trappin-2 and Cem-SLPI2, a fusion protein containing SLPI2 with the cementoin domain of trappin-2 fused to it N-terminus) were first cross-linked to fibronectin or elastin in 96-well microplates by incubation with TGase or FXIIIa for 2 h at 37°C. The microplate wells were then washed thoroughly to remove unreacted products and incubated with HNE (1 nM) or cathepsin G (2 nM) for 15 min at 37°C to form the protease-inhibitor complexes. A fluorogenic substrate specific for each protease was then added to the wells to measure the residual enzyme activity. (A) Inhibition of HNE by inhibitors bound to fibronectin by TGase (product release expressed as fluorescence units/time). (B, C) Residual HNE or cathepsin G activities after incubation with inhibitors conjugated to fibronectin or elastin by TGase (B) or FXIIIa (C), calculated as the ratio of substrate hydrolysis rates with and without inhibitor (Control). Data are represented as means ± SEM (n = 6) of residual enzymatic activity and were compared to the control (***p≤0.001, **p≤0.005 and *p≤0.05) using the post hoc student test.</p

Location of TGase-reactive Gln and Lys residues in SLPI, trappin-2 and elafin.

<p>Structural organization of SLPI (A) with its two elafin-like domains, trappin-2 (B) and elafin (C). The amino acid sequence of each inhibitor is shown below each structure. The disulfide bond topology (plain lines) and the inhibitory loop (half black disc) of each inhibitor are also shown. Arrows show the TGase-reactive Gln residues in SLPI (17, 27, 70) and trappin-2 (10, 22, 40) and the reactive Lys residues in SLPI (14, 35, 46) and trappin-2 (44, 50, 81). The reactive residues in elafin (trappin-2 numbering) were the same as those in the elafin moiety of trappin-2. Asterisks above the amino acid sequence denote the location of the reactive residues in each inhibitor.</p