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Brice Korkmaz - One of the best experts on this subject based on the ideXlab platform.
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monitoring human neutrophil activation by a Proteinase 3 near infrared fluorescence substrate based probe
Bioconjugate Chemistry, 2021Co-Authors: Brice Korkmaz, Seda Seren, Ahlame Saidi, Mylene Wartenberg, Jeanbaptiste Madinier, Guy IlangoAbstract:A near-infrared fluorescent (NIRF) substrate-based probe (SBP) was conceived to monitor secreted human Proteinase 3 (hPR3) activity. This probe, called pro3-SBP, is shaped by a fused peptide hairpin loop structure, which associates a hPR3 recognition domain (Val-Ala-Asp-Nva-Ala-Asp-Tyr-Gln, where Nva is norvaline) and an electrostatic zipper (consisting of complementary polyanionic (d-Glu)5 and polycationic (d-Arg)5 sequences) in close vicinity of the N- and C-terminal FRET couple (fluorescent donor, sulfoCy5.5; dark quencher, QSY21). Besides its subsequent stability, no intermolecular fluorescence quenching was detected following its complete hydrolysis by hPR3, advocating that pro3-SBP could further afford unbiased imaging. Pro3-SBP was specifically hydrolyzed by hPR3 (kcat/Km= 440 000 ± 5500 M-1·s-1) and displayed a sensitive detection threshold for hPR3 (subnanomolar concentration range), while neutrophil elastase showed a weaker potency. Conversely, pro3-SBP was not cleaved by cathepsin G. Pro3-SBP was successfully hydrolyzed by conditioned media of activated human neutrophils but not by quiescent neutrophils. Moreover, unlike unstimulated neutrophils, a strong NIRF signal was specifically detected by confocal microscopy following neutrophil ionomycin-induced degranulation. Fluorescence release was abolished in the presence of a selective hPR3 inhibitor, indicating that pro3-SBP is selectively cleaved by extracellular hPR3. Taken together, the present data support that pro3-SBP could be a convenient tool, allowing straightforward monitoring of human neutrophil activation.
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inhibitors and antibody fragments as potential anti inflammatory therapeutics targeting neutrophil Proteinase 3 in human disease
Pharmacological Reviews, 2016Co-Authors: Brice Korkmaz, Christine Kellenberger, Francis Gauthier, Ulrich Specks, Adam Lesner, Carla Guarino, Magdalena Wysocka, Herve Watier, Dieter E JenneAbstract:Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.
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a monoclonal antibody mcpr3 7 interfering with the activity of Proteinase 3 by an allosteric mechanism
Journal of Biological Chemistry, 2013Co-Authors: Lisa C Hinkofer, Brice Korkmaz, Dieter E Jenne, Amber M Hummel, Susanne A I Seidel, Francisco Silva, Dieter Braun, Ulrich SpecksAbstract:Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-Proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-Proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1′ pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis. Background: Proteinase 3 is an abundant serine protease with high similarity to neutrophil elastase and a major autoimmune target in systemic vasculitis. Results: We identified a monoclonal antibody that inhibits PR3 activity. Conclusion: PR3-inhibiting antibodies can change its conformation and impair interactions with α1-Proteinase inhibitor. Significance: PR3-inhibiting antibodies may play a role in autoimmune vasculitis and could be exploited as highly selective inhibitors.
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Neutrophil Proteinase 3 and dipeptidyl peptidase I (cathepsin C) as pharmacological targets in granulomatosis with polyangiitis (Wegener granulomatosis)
Seminars in Immunopathology, 2013Co-Authors: Brice Korkmaz, Stephanie Letast, Yassir K. Mahdi, Sandrine Dallet-choisy, Christine Kellenberger, Marie-claude Viaud-massuard, Marielise Jourdan, Sylvain Marchand-adam, Adam Lesner, Dieter E JenneAbstract:Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine Proteinases. Neutrophil elastase, cathepsin G, Proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular Proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA.
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A selective reversible azapeptide inhibitor of human neutrophil Proteinase 3 derived from a high affinity FRET substrate.
Biochemical Pharmacology, 2012Co-Authors: Christophe Epinette, Christine Kellenberger, Marie-claude Viaud-massuard, Francis Gauthier, Sylvain Marchand-adam, Cécile Croix, Lucie Jaquillard, Gilles Lalmanach, Martine Cadene, Brice KorkmazAbstract:The biological functions of human neutrophil Proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex; it is a reversible competitive inhibitor with a K(i) comparable to the K(m) of the parent substrate. Low concentrations (60 μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h.
Francis Gauthier - One of the best experts on this subject based on the ideXlab platform.
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Human Proteinase 3 resistance to inhibition extends to alpha-2 macroglobulin
FEBS Journal, 2020Co-Authors: Koffi N'guessan, Christine Kellenberger, Renata Grzywa, Seda Seren, Guillaume Gabant, Maria Juliano, Marc Moniatte, Alain Dorsselaer, Joseph Bieth, Francis GauthierAbstract:Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), Proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including a2-macroglobulin (a2-M), serpins [a1-Proteinase inhibitor (a1-PI)], monocyte neutrophil elastase inhibitor (MNEI), a1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine Proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by a1-PI and MNEI but not by SLPI. a2-M is a large spectrum inhibitor that traps a variety of Proteinases in response to cleavage(s) in its bait region. We report here that a2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between a2-M and hPR3 is governed by a kass in the ≤ 10^5 M-1s-1 range. Since a2M-trapped Proteinases retain peptidase activity, we first predicted the putative cleavage sites within the a2-M bait region (residues 690–728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of a2-M (39pep-a2-M). Since the 39pep-a2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that a2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.
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inhibitors and antibody fragments as potential anti inflammatory therapeutics targeting neutrophil Proteinase 3 in human disease
Pharmacological Reviews, 2016Co-Authors: Brice Korkmaz, Christine Kellenberger, Francis Gauthier, Ulrich Specks, Adam Lesner, Carla Guarino, Magdalena Wysocka, Herve Watier, Dieter E JenneAbstract:Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.
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A selective reversible azapeptide inhibitor of human neutrophil Proteinase 3 derived from a high affinity FRET substrate.
Biochemical Pharmacology, 2012Co-Authors: Christophe Epinette, Christine Kellenberger, Marie-claude Viaud-massuard, Francis Gauthier, Sylvain Marchand-adam, Cécile Croix, Lucie Jaquillard, Gilles Lalmanach, Martine Cadene, Brice KorkmazAbstract:The biological functions of human neutrophil Proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex; it is a reversible competitive inhibitor with a K(i) comparable to the K(m) of the parent substrate. Low concentrations (60 μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h.
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A substrate-based approach to convert SerpinB1 into a specific inhibitor of Proteinase 3, the Wegener's granulomatosis autoantigen.
FASEB Journal, 2011Co-Authors: Gwenhael Jégot, Christine Kellenberger, Marielise Jourdan, Francis Gauthier, Chrystelle Derache, Sandrine Castella, Hichem Lahouassa, Elodie Pitois, Eileen Remold-o'donnell, Brice KorkmazAbstract:The physiological and pathological functions of Proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 10⁷ M⁻¹ * s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.
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neutrophil elastase Proteinase 3 and cathepsin g as therapeutic targets in human diseases
Pharmacological Reviews, 2010Co-Authors: Brice Korkmaz, Dieter E Jenne, Marshall S. Horwitz, Francis GauthierAbstract:Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, Proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound Proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefevre syndrome. Because of their roles in host defense and disease, elastase, Proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.
Thomas Hellmark - One of the best experts on this subject based on the ideXlab platform.
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natural autoantibodies to myeloperoxidase Proteinase 3 and the glomerular basement membrane are present in normal individuals
Kidney International, 2010Co-Authors: Zhao Cui, Mårten Segelmark, Minghui Zhao, Thomas HellmarkAbstract:Anti-neutrophil cytoplasmic antibodies (ANCAs) have a pathogenic role in ANCA-associated vasculitis. The origin of ANCAs and anti-glomerular basement membrane (GBM) antibodies, however, is unknown. In this study, we determined whether natural autoantibodies against myeloperoxidase (MPO), Proteinase 3 (PR3), and GBM were present in each of 10 healthy Chinese and Swedish individuals, negative for all three antigens by routine ELISA. Antibodies were purified from isolated IgG by antigen-specific affinity columns. Natural anti-GBM autoantibodies gave a linear staining pattern along the GBM of human renal sections. On ethanol-fixed granulocytes, both natural anti-MPO and anti-PR3 autoantibodies gave cytoplasmic staining. The titers of natural anti-MPO/PR3 autoantibodies were significantly lower than those from patients with vasculitis. In competition ELISA, the binding of natural anti-MPO autoantibodies could be inhibited by MPO, but not by PR3 or noncollagenous domains from type IV collagen. The same specificity results were found for natural anti-PR3 and anti-GBM autoantibodies. Overall, individuals of the Chinese origin had more natural autoantibodies than did those of the Swedish origin, but no other differences were found. Hence, our study shows that healthy individuals have masked circulating, noncross-reactive, antigen-specific natural autoantibodies against MPO, PR3, and GBM in their serum and IgG fractions. Further studies are needed to determine their role if any in the etiology of ANCA-associated vasculitis and anti-GBM disease.
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elevated neutrophil membrane expression of Proteinase 3 is dependent upon cd177 expression
Clinical and Experimental Immunology, 2010Co-Authors: Mohamed Abdgawad, Mårten Segelmark, Lena Gunnarsson, Anders A Bengtsson, Pierre Geborek, Lennart Nilsson, Thomas HellmarkAbstract:Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte–macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177–mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.
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epitope shift of Proteinase 3 anti neutrophil cytoplasmic antibodies in patients with small vessel vasculitis
Clinical and Experimental Immunology, 2010Co-Authors: Daina Selga, Mårten Segelmark, Lena Gunnarsson, Thomas HellmarkAbstract:Anti-neutrophil cytoplasmic antibodies against Proteinase 3 (PR3-ANCA) are used as diagnostic tools for patients with small vessel vasculitis (AASV). We have produced chimeric mouse/human PR3 molecules and investigate changes in reactivity over time and the possible relationship between epitope specificity and clinical course. Thirty-eight PR3-ANCA-positive patients diagnosed between 1990 and 2003 were followed until December 2005. Plasma was collected at each out-patient visit and older samples were retrieved retrospectively. Patients reacted with multiple epitopes at the time of diagnosis. At subsequent relapses 12 patients shifted reactivity, in 11 cases from epitopes located in the C-terminal towards epitopes in the N-terminal. Patients with reactivity against N-terminal parts of PR3 at diagnosis had a significantly lower relapse rate, 30% compared to 78% in the group with predominantly C-terminal reactivity (P = 0·04). The reactivity pattern did not correlate to outcome measured as death, end-stage renal disease or vasculitis activity index score (VDI) at 5 years. Further research is necessary to conclude if this is a general phenomenon.
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neutrophil derived Proteinase 3 induces kallikrein independent release of a novel vasoactive kinin
Journal of Immunology, 2009Co-Authors: Robin Kahn, Nasrin Akbari, Mihail Todiras, Kerstin Westman, Anders Christensson, Thomas Hellmark, Tor Olofsson, L Fredrik M Leeblundberg, Jorgen Wieslander, Michael BaderAbstract:The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived Proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH 2 -MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and α 1 -antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B 1 receptors, but did not bind to B 2 receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B 2 receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B 1 and B 2 receptors as demonstrated using wild-type and B 1 overexpressing rats as well as wild-type and B 2 receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.
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neutrophil derived Proteinase 3 induces kallikrein independent release of a novel vasoactive kinin
Journal of Immunology, 2009Co-Authors: Robin Kahn, Nasrin Akbari, Mihail Todiras, Kerstin Westman, Anders Christensson, Thomas Hellmark, Tor Olofsson, L Fredrik M Leeblundberg, Jorgen Wieslander, Michael BaderAbstract:The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived Proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH 2 -MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and α 1 -antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B 1 receptors, but did not bind to B 2 receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B 2 receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B 1 and B 2 receptors as demonstrated using wild-type and B 1 overexpressing rats as well as wild-type and B 2 receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.
Dieter E Jenne - One of the best experts on this subject based on the ideXlab platform.
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inhibitors and antibody fragments as potential anti inflammatory therapeutics targeting neutrophil Proteinase 3 in human disease
Pharmacological Reviews, 2016Co-Authors: Brice Korkmaz, Christine Kellenberger, Francis Gauthier, Ulrich Specks, Adam Lesner, Carla Guarino, Magdalena Wysocka, Herve Watier, Dieter E JenneAbstract:Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.
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Proteinase 3 dependent caspase 3 cleavage modulates neutrophil death and inflammation
Journal of Clinical Investigation, 2014Co-Authors: Fabien Loison, Dieter E Jenne, Kutay Karatepe, Haiyan Zhu, Anongnard Kasorn, Peng Liu, Jiaxi Zhou, Shannan Cao, Haiyan Gong, Eileen RemoldodonnellAbstract:Caspase-3–mediated spontaneous death in neutrophils is a prototype of programmed cell death and is critical for modulating physiopathological inflammatory responses; however, the underlying regulatory pathways remain ill defined. Here we determined that in aging neutrophils, the cleavage and activation of caspase-3 is independent of the canonical caspase-8– or caspase-9–mediated pathway. Instead, caspase-3 activation was mediated by serine protease Proteinase 3 (PR3), which is present in the cytosol of aging neutrophils. Specifically, PR3 cleaved procaspase-3 at a site upstream of the canonical caspase-9 cleavage site. In mature neutrophils, PR3 was sequestered in granules and released during aging via lysosomal membrane permeabilization (LMP), leading to procaspase-3 cleavage and apoptosis. Pharmacological inhibition or knockdown of PR3 delayed neutrophil death in vitro and consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in a murine model of peritonitis. Adoptive transfer of both WT and PR3-deficient neutrophils revealed that the delayed death of neutrophils lacking PR3 is due to an altered intrinsic apoptosis/survival pathway, rather than the inflammatory microenvironment. The presence of the suicide protease inhibitor SERPINB1 counterbalanced the protease activity of PR3 in aging neutrophils, and deletion of Serpinb1 accelerated neutrophil death. Taken together, our results reveal that PR3-mediated caspase-3 activation controls neutrophil spontaneous death.
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unopposed cathepsin g neutrophil elastase and Proteinase 3 cause severe lung damage and emphysema
American Journal of Pathology, 2014Co-Authors: Nicolas Guyot, Dieter E Jenne, Julien Wartelle, Laurette Malleret, Alexandre A Todorov, Gilles Devouassoux, Yves Pacheco, Azzaq BelaaouajAbstract:Cigarette smoking is a major factor for the development of pulmonary emphysema because it induces abnormal inflammation and a protease-rich local milieu that causes connective tissue breakdown of the lungs. As a result of its capacity to degrade lung tissue and the high risk of patients lacking α 1 -antitrypsin to develop emphysema, much interest has focused on neutrophil elastase (NE). Two similar neutrophil serine proteases (NSPs), cathepsin G and Proteinase 3, coexist with NE in humans and mice, but their potential tissue-destructive role(s) remains unclear. Using a gene-targeting approach, we observed that in contrast to their wild-type littermates, mice deficient in all three NSPs were substantially protected against lung tissue destruction after long-term exposure to cigarette smoke. In exploring the underlying basis for disrupted wild-type lung air spaces, we found that active NSPs collectively caused more severe lung damage than did NE alone. Furthermore, NSP activities unleashed increased activity of the tissue-destructive proteases macrophage elastase (matrix metalloProteinase-12) and gelatinase B (matrix metalloProteinase-9). These in vivo data provide, for the first time, compelling evidence of the collateral involvement of cathepsin G, NE, and Proteinase 3 in cigarette smoke–induced tissue damage and emphysema. They also reveal a complex positive feed-forward loop whereby these NSPs induce the destructive potential of other proteases, thereby generating a chronic and pathogenic protease-rich milieu.
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a monoclonal antibody mcpr3 7 interfering with the activity of Proteinase 3 by an allosteric mechanism
Journal of Biological Chemistry, 2013Co-Authors: Lisa C Hinkofer, Brice Korkmaz, Dieter E Jenne, Amber M Hummel, Susanne A I Seidel, Francisco Silva, Dieter Braun, Ulrich SpecksAbstract:Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-Proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-Proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1′ pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis. Background: Proteinase 3 is an abundant serine protease with high similarity to neutrophil elastase and a major autoimmune target in systemic vasculitis. Results: We identified a monoclonal antibody that inhibits PR3 activity. Conclusion: PR3-inhibiting antibodies can change its conformation and impair interactions with α1-Proteinase inhibitor. Significance: PR3-inhibiting antibodies may play a role in autoimmune vasculitis and could be exploited as highly selective inhibitors.
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Neutrophil Proteinase 3 and dipeptidyl peptidase I (cathepsin C) as pharmacological targets in granulomatosis with polyangiitis (Wegener granulomatosis)
Seminars in Immunopathology, 2013Co-Authors: Brice Korkmaz, Stephanie Letast, Yassir K. Mahdi, Sandrine Dallet-choisy, Christine Kellenberger, Marie-claude Viaud-massuard, Marielise Jourdan, Sylvain Marchand-adam, Adam Lesner, Dieter E JenneAbstract:Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine Proteinases. Neutrophil elastase, cathepsin G, Proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular Proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA.
Michael Bader - One of the best experts on this subject based on the ideXlab platform.
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neutrophil derived Proteinase 3 induces kallikrein independent release of a novel vasoactive kinin
Journal of Immunology, 2009Co-Authors: Robin Kahn, Nasrin Akbari, Mihail Todiras, Kerstin Westman, Anders Christensson, Thomas Hellmark, Tor Olofsson, L Fredrik M Leeblundberg, Jorgen Wieslander, Michael BaderAbstract:The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived Proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH 2 -MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and α 1 -antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B 1 receptors, but did not bind to B 2 receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B 2 receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B 1 and B 2 receptors as demonstrated using wild-type and B 1 overexpressing rats as well as wild-type and B 2 receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.
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neutrophil derived Proteinase 3 induces kallikrein independent release of a novel vasoactive kinin
Journal of Immunology, 2009Co-Authors: Robin Kahn, Nasrin Akbari, Mihail Todiras, Kerstin Westman, Anders Christensson, Thomas Hellmark, Tor Olofsson, L Fredrik M Leeblundberg, Jorgen Wieslander, Michael BaderAbstract:The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived Proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH 2 -MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and α 1 -antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B 1 receptors, but did not bind to B 2 receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B 2 receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B 1 and B 2 receptors as demonstrated using wild-type and B 1 overexpressing rats as well as wild-type and B 2 receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.
