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1.
J Biol Chem ; 299(4): 103072, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36849007

RESUMO

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Anticorpos Anticitoplasma de Neutrófilos , Mieloblastina , Animais , Humanos , Camundongos , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Mieloblastina/imunologia , Mieloblastina/metabolismo , Neutrófilos/metabolismo , Inibidores de Proteases/metabolismo , Superóxidos/metabolismo
2.
Int J Mol Sci ; 22(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562184

RESUMO

The deepest evolutionary branches of the trypsin/chymotrypsin family of serine proteases are represented by the digestive enzymes of the gastrointestinal tract and the multi-domain proteases of the blood coagulation and complement system. Similar to the very old digestive system, highly diverse cleavage specificities emerged in various cell lineages of the immune defense system during vertebrate evolution. The four neutrophil serine proteases (NSPs) expressed in the myelomonocyte lineage, neutrophil elastase, proteinase 3, cathepsin G, and neutrophil serine protease 4, collectively display a broad repertoire of (S1) specificities. The origin of NSPs can be traced back to a circulating liver-derived trypsin-like protease, the complement factor D ancestor, whose activity is tightly controlled by substrate-induced activation and TNFα-induced locally upregulated protein secretion. However, the present-day descendants are produced and converted to mature enzymes in precursor cells of the bone marrow and are safely sequestered in granules of circulating neutrophils. The potential site and duration of action of these cell-associated serine proteases are tightly controlled by the recruitment and activation of neutrophils, by stimulus-dependent regulated secretion of the granules, and by various soluble inhibitors in plasma, interstitial fluids, and in the inflammatory exudate. An extraordinary dynamic range and acceleration of immediate defense responses have been achieved by exploiting the high structural plasticity of the trypsin fold.


Assuntos
Linhagem da Célula , Monócitos/enzimologia , Células Mieloides/enzimologia , Serina Proteases/metabolismo , Animais , Catepsina G/metabolismo , Humanos , Elastase de Leucócito/metabolismo , Monócitos/citologia , Mieloblastina/metabolismo , Células Mieloides/citologia
3.
Proc Natl Acad Sci U S A ; 114(14): 3613-3618, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28265093

RESUMO

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3'-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis.


Assuntos
Histona Desacetilases/genética , Placa Aterosclerótica/complicações , Polimorfismo de Nucleotídeo Único , Proteínas Repressoras/genética , Acidente Vascular Cerebral/genética , alfa 1-Antitripsina/genética , Regiões 3' não Traduzidas , Medição da Troca de Deutério , Estudos de Associação Genética , Humanos , Elastase de Leucócito/metabolismo , Espectrometria de Massas , Placa Aterosclerótica/genética , Acidente Vascular Cerebral/etiologia , alfa 1-Antitripsina/metabolismo
4.
J Biol Chem ; 293(32): 12415-12428, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29925593

RESUMO

Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.


Assuntos
Catepsina C/antagonistas & inibidores , Membrana Celular/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Granulomatose com Poliangiite/patologia , Mieloblastina/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Granulomatose com Poliangiite/tratamento farmacológico , Granulomatose com Poliangiite/genética , Granulomatose com Poliangiite/metabolismo , Humanos , Masculino , Mieloblastina/genética , Neutrófilos/enzimologia , Proteólise , Adulto Jovem
5.
Pharmacol Rev ; 68(3): 603-30, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27329045

RESUMO

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.


Assuntos
Anti-Inflamatórios/farmacologia , Fragmentos de Imunoglobulinas/farmacologia , Fragmentos de Imunoglobulinas/uso terapêutico , Mieloblastina/antagonistas & inibidores , Neutrófilos/enzimologia , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Apoptose/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Terapia de Alvo Molecular , Mieloblastina/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia
6.
J Biol Chem ; 291(16): 8486-99, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26884336

RESUMO

The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.


Assuntos
Catepsina C/metabolismo , Pulmão/enzimologia , Neutrófilos/enzimologia , Pneumonia/enzimologia , Animais , Catepsina C/genética , Modelos Animais de Doenças , Células HL-60 , Humanos , Pulmão/patologia , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/patologia , Pneumonia/induzido quimicamente , Pneumonia/patologia , Ratos Sprague-Dawley , Escarro/metabolismo
7.
Clin Exp Rheumatol ; 35 Suppl 103(1): 98-101, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28281453

RESUMO

OBJECTIVES: To determine the prevalence of anti-myeloperoxidase (MPO) antibodies of IgA (IgA anti-MPO) isotype in patients with eosinophilic granulomatosis with polyangiitis (EGPA), and the association of the IgA antibodies with IgG anti-MPO and with disease activity. METHODS: Serum samples from patients with EGPA followed in a multicenter longitudinal cohort were tested by ELISA for the presence of IgA anti-MPO and IgG anti-MPO antibodies. Sera from 87 healthy controls were used to define a positive test. Sera from 168 patients with EGPA (298 samples) were tested. Frequencies of positive testing for IgA anti-MPO were compared between patients with active EGPA, patients in remission, and controls. RESULTS: IgA anti-MPO was detected in 10 of 168 (6%) patients with EGPA (11 of 298 serum samples) compared to 1 of 87 (1%) healthy controls (p=0.10). All 11 samples testing positive for IgA anti-MPO also tested positive for IgG anti-MPO. Ninety samples tested positive for IgG anti-MPO but negative for IgA. Samples taken during active EGPA were positive for IgA anti-MPO in 6/72 cases (8%), compared to 5/226 (2%) during remission (p=0.03). Among samples taken during moderate or high disease activity, 5/41 were positive (12%, p=0.01 compared to remission). CONCLUSIONS: Although IgA anti-MPO antibodies are detectable in some patients with EGPA and may be detectable more frequently during active disease, their presence seems unlikely to provide information beyond what is obtained from conventional IgG anti-MPO.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/sangue , Síndrome de Churg-Strauss/sangue , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Peroxidase/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Canadá , Estudos de Casos e Controles , Síndrome de Churg-Strauss/diagnóstico , Síndrome de Churg-Strauss/tratamento farmacológico , Síndrome de Churg-Strauss/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Indução de Remissão , Resultado do Tratamento , Estados Unidos , Adulto Jovem
9.
J Biol Chem ; 289(46): 31777-31791, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25288799

RESUMO

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.


Assuntos
Ésteres/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Mieloblastina/antagonistas & inibidores , Mieloblastina/química , Oligopeptídeos/química , Organofosfonatos/química , Animais , Apoptose , Biotinilação , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Hidrólise , Inflamação , Insetos , Espectrometria de Massas , Modelos Químicos , Mutação , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Peptídeos/química , Prolina/química , Inibidores de Proteases/química , Solventes
10.
Am J Pathol ; 184(8): 2197-210, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24929239

RESUMO

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 α1-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.


Assuntos
Catepsina G/metabolismo , Elastase de Leucócito/metabolismo , Mieloblastina/metabolismo , Enfisema Pulmonar/metabolismo , Fumar/efeitos adversos , Animais , Western Blotting , Modelos Animais de Doenças , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/patologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
J Autoimmun ; 59: 43-52, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25744251

RESUMO

Anti-neutrophil cytoplasmic antibodies (ANCA) with proteinase 3 (PR3) specificity are a useful laboratory biomarker for the diagnosis of Granulomatosis with Polyangiitis (GPA) and are believed to be implicated in the pathogenesis. It has been repeatedly suggested that disease activity of GPA is more closely related to the appearance and rise of PR3-inhibiting ANCA than to an increase of total ANCA. Previous studies on a limited number of patient samples, however, have yielded inconclusive results. To overcome the previous methodological limitations, we established a new ultrasensitive method to quantify the inhibitory capacity of PR3-ANCA using small volumes of plasma from patients with GPA. A large collection of longitudinally-collected samples from the Wegener Granulomatosis Etanercept Trial (WGET) became available to us to determine the functional effects of ANCA on PR3 in comparison to clinical disease manifestations. In these patient samples we not only detected PR3-ANCA with inhibitory capacity, but also PR3-ANCA with enhancing effects on PR3 activity. However no correlation of these activity-modulating PR3-ANCA with disease activity at either the time of enrollment or over the course of disease was found. Only patients with pulmonary involvement, especially patients with nodule formation in the respiratory tract, showed a slight, but not significant, decrease of inhibitory capacity. Epitope mapping of the activity-modulating PR3-ANCA revealed a binding on the active site surface of PR3. Yet these ANCA were able to bind to PR3 with an occupied active site cleft, indicating an allosteric mechanism of inhibition. The recently described signal ratio between the MCPR3-3 and MCPR3-2 capture ELISA was consistent with the binding of activity-modulating ANCA to the active site surface. Evidence for a shared epitope between activity-modulating PR3-ANCA and MCPR3-7, however, was very limited, suggesting that a majority of PR3-ANCA species do not inhibit PR3 by the same mechanism as previously reported for MCPR3-7.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Biomarcadores/metabolismo , Mapeamento de Epitopos/métodos , Granulomatose com Poliangiite/imunologia , Mieloblastina/metabolismo , Regulação Alostérica/imunologia , Sítios de Ligação de Anticorpos/imunologia , Progressão da Doença , Seguimentos , Granulomatose com Poliangiite/enzimologia , Humanos , Mieloblastina/imunologia
12.
J Immunol ; 191(5): 2700-7, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23904161

RESUMO

Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer-based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1', P2', P3, P4, and P2 positions were tested. This enabled us to construct an α1-proteinase inhibitor variant (Ile-Lys-Pro-Arg-/-Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefèvre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils.


Assuntos
Ativação de Neutrófilo/fisiologia , Neutrófilos/enzimologia , Doença de Papillon-Lefevre/enzimologia , Serina Endopeptidases/metabolismo , Adolescente , Sequência de Aminoácidos , Western Blotting , Catepsina C/genética , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Ensaio de Imunoadsorção Enzimática , Transferência Ressonante de Energia de Fluorescência , Haptoglobinas/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Doença de Papillon-Lefevre/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/química
13.
Proc Natl Acad Sci U S A ; 109(16): 6229-34, 2012 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-22474388

RESUMO

Neutrophil serine proteases (NSPs) in cytoplasmic granules of neutrophils are regarded as important antimicrobial defense weapons after engulfment and exposure of pathogens to the content of primary granules. Despite intensive studies on neutrophils during the last three decades, only three active serine proteases, neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) have been identified in these short-lived cells. Here, we report on the identification of a fourth serine protease (NSP4) with 39% identity to NE and PR3, but arginine specificity, yet sharing features like propeptide processing by dipeptidyl peptidase I, storage, and release as an active enzyme with the three active proteases. We established monoclonal antibodies against NSP4, excluded cross-reactivity to human granzymes, NE, CG, PR3, and azurocidin, and screened for NSP4 protein expression in various human tissues and blood leukocyte populations. Only granulocyte precursors and neutrophil populations from peripheral blood were positive. The content of NSP4 in neutrophil lysates, however, was about 20-fold lower compared with CG. Upon neutrophil activation, NSP4 was released into the supernatant. Profiling its specificity with peptide libraries from Escherichia coli revealed a preference for arginine in P1; it cleaved Tyr-Arg-Phe-Arg-AMC and Ala-Pro-Nva-thiobenzyl esters. NSP4 was inhibited by α(1)-proteinase inhibitor (α(1)-antitrypsin), C1 inhibitor, and most efficiently by antithrombin-heparin, but not by elafin, secretory leukocyte protease inhibitor, α(1)-antichymotrypsin, and monocyte-neutrophil elastase inhibitor. Functional specialization and preferred natural substrates of NSP4 remain to be determined to understand the biological interplay of all four NSPs during neutrophil responses.


Assuntos
Arginina/metabolismo , Neutrófilos/enzimologia , Elastase Pancreática/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antitrombinas/farmacologia , Western Blotting , Catepsina C/metabolismo , Células Cultivadas , Células HEK293 , Heparina/farmacologia , Humanos , Dados de Sequência Molecular , Neutrófilos/citologia , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/genética , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteólise , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Inibidores de Serina Proteinase/farmacologia , alfa 1-Antitripsina/farmacologia
14.
J Biol Chem ; 288(37): 26635-48, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23902773

RESUMO

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.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Anticorpos Monoclonais/imunologia , Mieloblastina/imunologia , Sítio Alostérico , Animais , Autoimunidade , Catálise , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ativação de Neutrófilo , Neutrófilos/metabolismo , Conformação Proteica , Proteínas Recombinantes/imunologia , Vasculite/imunologia
15.
Immunol Rev ; 235(1): 159-71, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20536562

RESUMO

Granzyme B (GzmB) is used by cytotoxic lymphocytes as a molecular weapon for the defense against virus-infected and malignantly transformed host cells. It belongs to a family of small serine proteases that are stored in secretory vesicles of killer cells. After secretion of these cytolytic granules during killer cell attack, GzmB is translocated into the cytosol of target cells with the help of the pore-forming protein perforin. GzmB has adopted similar protease specificity as caspase-8, and once delivered, it activates major executioner apoptosis pathways. Since GzmB is very effective in killing human tumor cell lines that are otherwise resistant against many cytotoxic drugs and since GzmB of human origin can be recombinantly expressed, its use as part of a 'magic bullet' in tumor therapy is a very tempting idea. In this review, we emphasize the peculiar characteristics of GzmB that make it suited for use as an effector domain in potential immunoconjugates. We discuss what is known about its uptake into target cells and the trials performed with GzmB-armed immunoconjugates, and we assess the prospects of its potential therapeutic value.


Assuntos
Antineoplásicos/uso terapêutico , Granzimas/uso terapêutico , Imunotoxinas/uso terapêutico , Células Matadoras Naturais/enzimologia , Linfócitos T Citotóxicos/enzimologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Terapia Genética , Granzimas/antagonistas & inibidores , Granzimas/química , Granzimas/genética , Granzimas/metabolismo , Humanos , Imunotoxinas/química , Imunotoxinas/genética , Imunotoxinas/metabolismo , Modelos Moleculares , Perforina/metabolismo , Conformação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/uso terapêutico , Serpinas/metabolismo
16.
J Immunol ; 186(2): 1131-9, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21160041

RESUMO

The regulation of neutrophil recruitment, activation, and disposal is pivotal for circumscribed inflammation. SHP1(Y208N/Y208N) mutant mice develop severe cutaneous inflammatory disease that is IL-1R dependent. Genetic reduction in neutrophil numbers and neutrophilic responses to infection is sufficient to prevent the spontaneous initiation of this disease. Neutrophils from SHP1(Y208N/Y208N) mice display increased pro-IL-1ß production due to altered responses to MyD88-dependent and MyD88-independent signals. The IL-1R-dependent inflammatory disease in SHP1(Y208N/Y208N) mice develops independently of caspase 1 and proteinase 3 and neutrophil elastase. In response to Fas ligand, a caspase 1-independent inducer of IL-1ß production, neutrophils from SHP1(Y208N/Y208N) mice produce elevated levels of IL-1ß but display reduced caspase 3 and caspase 7 activation. In neutrophils deficient in SHP1, IL-1ß induces high levels of pro-IL-1ß suggesting the presence of a paracrine IL-1ß loop. These data indicate that the neutrophil- and IL-1-dependent disease in SHP1(Y208N/Y208N) mice is a consequence of loss of negative regulation of TLR and IL-1R signaling.


Assuntos
Mediadores da Inflamação/fisiologia , Interleucina-1beta/biossíntese , Neutrófilos/imunologia , Neutrófilos/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Dermatopatias/patologia , Dermatopatias/prevenção & controle , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/prevenção & controle , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Neutrófilos/metabolismo , Comunicação Parácrina/genética , Comunicação Parácrina/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Índice de Gravidade de Doença , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Dermatopatias/imunologia , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/fisiologia
17.
Pharmacol Rev ; 62(4): 726-59, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21079042

RESUMO

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-Lefèvre 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.


Assuntos
Catepsina G/química , Catepsina G/fisiologia , Elastase de Leucócito/fisiologia , Terapia de Alvo Molecular , Mieloblastina/fisiologia , Animais , Domínio Catalítico , Catepsina G/antagonistas & inibidores , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/química , Pneumopatias/tratamento farmacológico , Pneumopatias/enzimologia , Mieloblastina/antagonistas & inibidores , Mieloblastina/química , Neutropenia/tratamento farmacológico , Neutropenia/enzimologia , Doença de Papillon-Lefevre/tratamento farmacológico , Doença de Papillon-Lefevre/enzimologia
18.
Arthritis Rheumatol ; 75(5): 748-759, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36515151

RESUMO

OBJECTIVE: Proteinase 3 (PR3) is the major antigen for antineutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3-targeting ANCAs (PR3-ANCAs) recognize different epitopes on PR3. This study was undertaken to study the effect of mutations on PR3 antigenicity. METHODS: The recombinant PR3 variants, iPR3 (clinically used to detect PR3-ANCAs) and iHm5 (containing 3 point mutations in epitopes 1 and 5 generated for epitope mapping studies) immunoassays and serum samples from patients enrolled in ANCA-associated vasculitis (AAV) trials were used to screen for differential PR3-ANCA binding. A patient-derived monoclonal ANCA 518 (moANCA518) that selectively binds to iHm5 within the mutation-free epitope 3 and is distant from the point mutations of iHm5 was used as a gauge for remote epitope activation. Selective binding was determined using inhibition experiments. RESULTS: Rather than reduced binding of PR3-ANCAs to iHm5, we found substantially increased binding of the majority of PR3-ANCAs to iHm5 compared to iPR3. This differential binding of PR3-ANCA to iHm5 is similar to the selective moANCA518 binding to iHm5. Binding of iPR3 to monoclonal antibody MCPR3-2 also induced recognition by moANCA518. CONCLUSION: The preferential binding of PR3-ANCAs from patients, such as the selective binding of moANCA518 to iHm5, is conferred by increased antigenicity of epitope 3 on iHm5. This can also be induced on iPR3 when captured by monoclonal antibody MCPR2. This previously unrecognized characteristic of PR3-ANCA interactions with its target antigen has implications for studying antibody-mediated autoimmune diseases, understanding variable performance characteristics of immunoassays, and design of potential novel treatment approaches.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Granulomatose com Poliangiite , Humanos , Mieloblastina/genética , Epitopos , Granulomatose com Poliangiite/genética , Anticorpos Monoclonais
19.
Cancers (Basel) ; 15(6)2023 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-36980752

RESUMO

Kirsten rat sarcoma virus (KRAS)-mutant cancers are frequent, metastatic, lethal, and largely undruggable. While interleukin (IL)-1ß and nuclear factor (NF)-κB inhibition hold promise against cancer, untargeted treatments are not effective. Here, we show that human KRAS-mutant cancers are addicted to IL-1ß via inflammatory versican signaling to macrophage inhibitor of NF-κB kinase (IKK) ß. Human pan-cancer and experimental NF-κB reporter, transcriptome, and proteome screens reveal that KRAS-mutant tumors trigger macrophage IKKß activation and IL-1ß release via secretory versican. Tumor-specific versican silencing and macrophage-restricted IKKß deletion prevents myeloid NF-κB activation and metastasis. Versican and IKKß are mutually addicted and/or overexpressed in human cancers and possess diagnostic and prognostic power. Non-oncogene KRAS/IL-1ß addiction is abolished by IL-1ß and TLR1/2 inhibition, indicating cardinal and actionable roles for versican and IKKß in metastasis.

20.
J Immunol ; 185(1): 387-99, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20530264

RESUMO

Anti-neutrophil cytoplasmic Abs (cANCAs) against conformational epitopes of proteinase 3 (PR3) are regarded as an important pathogenic marker in Wegener's granulomatosis (WG). Although the three-dimensional structure of PR3 is known, binding sites of mAbs and cANCAs have not been mapped to date. Competitive binding and biosensor experiments suggested the existence of four nonoverlapping areas on the PR3 surface. In this paper, we present an approach to identify these discontinuous surface regions that cannot be mimicked by linear peptides. The very few surface substitutions found in closely related PR3 homologs from primates, which were further varied by the construction of functional human-gibbon hybrids, resulted in the differential loss of three Ab binding sites, two of which were mapped to the N-terminal beta-barrel and one to the linker segment connecting the N- and C-terminal barrels of PR3. The sera from WG patients differed in their binding to gibbon PR3 and the gibbon-human PR3 hybrid, and could be divided into two groups with similar or significantly reduced binding to gibbon PR3. Binding of almost all sera to PR3-alpha1-protease inhibitor (alpha1-PI) complexes was even more reduced and often absent, indicating that major antigenic determinants overlap with the active site surface on PR3 that associates with alpha1-PI. Similarly, the mouse mAbs CLB12.8 and 6A6 also did not react with gibbon PR3 and PR3-alpha1-PI complexes. Our data strongly suggest that cANCAs from WG patients at least in part recognize similar surface structures as do mouse mAbs and compete with the binding of alpha1-PI to PR3.


Assuntos
Autoantígenos/metabolismo , Epitopos/metabolismo , Granulomatose com Poliangiite/enzimologia , Granulomatose com Poliangiite/imunologia , Mieloblastina/metabolismo , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Autoantígenos/imunologia , Sítios de Ligação de Anticorpos , Ligação Competitiva/imunologia , Linhagem Celular , Reações Cruzadas , Epitopos/imunologia , Humanos , Hylobates , Macaca mulatta , Camundongos , Dados de Sequência Molecular , Mieloblastina/imunologia , Pan troglodytes , Conformação Proteica , Inibidores de Serina Proteinase/imunologia , Inibidores de Serina Proteinase/metabolismo
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