RESUMO
Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with kinact/KI value over 2,000,000 [M-1s-1]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3.
Assuntos
Elastase de Leucócito , Serina Proteases , Humanos , Elastase de Leucócito/metabolismo , Catepsina G , Mieloblastina/química , Inibidores de Serina Proteinase/farmacologiaRESUMO
Members of the EAP family of Staphylococcus aureus immune evasion proteins potently inhibit the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin-G, and proteinase-3. Previously, we determined a 1.8 Å resolution crystal structure of the EAP family member EapH1 bound to neutrophil elastase. This structure revealed that EapH1 blocks access to the enzyme's active site by forming a noncovalent complex with this host protease. To determine how EapH1 inhibits other NSPs, we studied here the effects of EapH1 on cathepsin-G. We found that EapH1 inhibits cathepsin-G with a Ki of 9.8 ± 4.7 nm Although this Ki value is â¼466-fold weaker than the Ki for EapH1 inhibition of neutrophil elastase, the time dependence of inhibition was maintained. To define the physical basis for EapH1's inhibition of cathepsin-G, we crystallized EapH1 bound to this protease, solved the structure at 1.6 Å resolution, and refined the model to Rwork and Rfree values of 17.4% and 20.9%, respectively. This structure revealed a protease-binding mode for EapH1 with cathepsin-G that was globally similar to that seen in the previously determined EapH1-neutrophil elastase structure. The nature of the intermolecular interactions formed by EapH1 with cathepsin-G differed considerably from that with neutrophil elastase, however, with far greater contributions from the inhibitor backbone in the cathepsin-G-bound form. Together, these results reveal that EapH1's ability to form high-affinity interactions with multiple NSP targets is due to its remarkable level of local structural plasticity.
Assuntos
Proteínas de Bactérias/química , Catepsina G/química , Elastase de Leucócito/química , Mieloblastina/química , Neutrófilos/enzimologia , Inibidores de Serina Proteinase/química , Staphylococcus aureus/química , Cristalografia por Raios X , Humanos , Estrutura Quaternária de ProteínaRESUMO
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= 440â¯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.
Assuntos
Mieloblastina/metabolismo , Ativação de Neutrófilo/fisiologia , Neutrófilos/fisiologia , Sobrevivência Celular , Corantes Fluorescentes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Ionomicina , Microscopia Confocal , Estrutura Molecular , Mieloblastina/química , Neutrófilos/efeitos dos fármacos , Espectrofotometria InfravermelhoRESUMO
Proteinase 3 (PR3) is a neutrophil serine protease present in cytoplasmic granules but also expressed at the neutrophil surface where it mediates proinflammatory effects. Studies of the underlying molecular mechanisms have been hampered by the lack of inhibitors of the PR3 membrane anchorage. Indeed while there exist inhibitors of the catalytic activity of PR3, its membrane interfacial binding site (IBS) is distinct from its catalytic site. The IBS has been characterized both by mutagenesis experiments and molecular modeling. Through docking and molecular dynamics simulations we have designed d-peptides targeting the PR3 IBS. We used surface plasmon resonance to evaluate their effect on the binding of PR3 to phospholipid bilayers. Next, we verified their ability of binding to PR3 via fluorescence spectroscopy and isothermal titration calorimetry. The designed peptides did not affect the catalytic activity of PR3. A few peptides bound to PR3 hydrophobic pockets and inhibited PR3 binding to lipids. While the (KFF)3K d-peptide inconveniently showed a significant affinity for the lipids, another d-peptide (SAKEAFFKLLAS) did not and it inhibited the PR3-membrane binding site with IC50 of about 40µM. Our work puts forward d-peptides as promising inhibitors of peripheral protein-membrane interactions, which remain high-hanging fruits in drug design.
Assuntos
Membrana Celular/metabolismo , Mieloblastina/metabolismo , Neutrófilos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Biocatálise , Calorimetria/métodos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mieloblastina/química , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Ressonância de Plasmônio de SuperfícieRESUMO
Chronic Obstructive Pulmonary Disease (COPD) is a common, multifactorial lung disease which results in significant impairment of patients' health and a large impact on society and health care burden. It is believed to be the result of prolonged, destructive neutrophilic inflammation which results in progressive damage to lung structures. During this process, large quantities of neutrophil serine proteinases (NSPs) are released which initiate the damage and contribute towards driving a persistent inflammatory state.Neutrophil elastase has long been considered the key NSP involved in the pathophysiology of COPD. However, in recent years, a significant role for Proteinase 3 (PR3) in disease development has emerged, both in COPD and other chronic inflammatory conditions. Therefore, there is a need to investigate the importance of PR3 in disease development and hence its potential as a therapeutic target. Research into PR3 has largely been confined to its role as an autoantigen, but PR3 is involved in triggering inflammatory pathways, disrupting cellular signalling, degrading key structural proteins, and pathogen response.This review summarises what is presently known about PR3, explores its involvement particularly in the development of COPD, and indicates areas requiring further investigation.
Assuntos
Sistemas de Liberação de Medicamentos , Mieloblastina/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/enzimologia , Anti-Inflamatórios/administração & dosagem , Doença Crônica , Sistemas de Liberação de Medicamentos/tendências , Inibidores Enzimáticos/administração & dosagem , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Pneumopatias/tratamento farmacológico , Pneumopatias/enzimologia , Mieloblastina/antagonistas & inibidores , Mieloblastina/química , Estrutura Secundária de ProteínaRESUMO
Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Mieloblastina/metabolismo , Fosfatidilserinas/metabolismo , Animais , Apoptose , Linhagem Celular , Granulomatose com Poliangiite/enzimologia , Granulomatose com Poliangiite/etiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Mieloblastina/química , Neutrófilos/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ratos , Explosão RespiratóriaRESUMO
BACKGROUND AIMS: PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood-derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci. METHODS: We first determined the frequency of PR1-CTLs in HLA-A2(+) UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells. RESULTS: PR1-CTLs are detected at an average frequency of 0.14% within the CD8(+) population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8(+) population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines. CONCLUSIONS: This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Sangue Fetal/citologia , Antígeno HLA-A2/imunologia , Imunoterapia Adotiva , Leucemia Mieloide/terapia , Mieloblastina/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Células Cultivadas , Sangue Fetal/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Células K562 , Leucemia Mieloide/imunologia , Contagem de Linfócitos , Mieloblastina/química , Mieloblastina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células U937RESUMO
BACKGROUND AIMS: The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T cells to mediate anti-leukemia activity. METHODS: Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. RESULTS: Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. CONCLUSIONS: Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.
Assuntos
Sangue Fetal , Antígeno HLA-A2/imunologia , Leucemia Mieloide Aguda/terapia , Leucócitos Mononucleares/metabolismo , Mieloblastina/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Epitopos/imunologia , Sangue Fetal/citologia , Sangue Fetal/imunologia , Terapia Genética , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/imunologia , Mieloblastina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/imunologiaRESUMO
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 , SolventesRESUMO
PR1 is a HLA-A2-restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived APCs, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific CTLs (PR1-CTL) and the anti-PR1/HLA-A2 Ab 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an Ag become susceptible to therapies that target cross-presented Ags and suggest PR1 as a broadly expressed tumor Ag.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/terapia , Imunoterapia , Elastase de Leucócito/imunologia , Melanoma/terapia , Mieloblastina/imunologia , Neoplasias Cutâneas/terapia , Anticorpos/farmacologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Apresentação Cruzada , Feminino , Antígeno HLA-A2/imunologia , Humanos , Elastase de Leucócito/química , Melanoma/imunologia , Melanoma/patologia , Terapia de Alvo Molecular , Mieloblastina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T Citotóxicos/imunologia , Células Tumorais CultivadasRESUMO
Molecular surfaces provide a useful mean for analyzing interactions between biomolecules; such as identification and characterization of ligand binding sites to a host macromolecule. We present a novel technique, which extracts potential binding sites, represented by cavities, and characterize them by 3D graphs and by amino acids. The binding sites are extracted using an implicit function sampling and graph algorithms. We propose an advanced cavity exploration technique based on the graph parameters and associated amino acids. Additionally, we interactively visualize the graphs in the context of the molecular surface. We apply our method to the analysis of MD simulations of Proteinase 3, where we verify the previously described cavities and suggest a new potential cavity to be studied.
Assuntos
Aminoácidos/química , Biologia Computacional/métodos , Modelos Moleculares , Simulação de Acoplamento Molecular , Algoritmos , Sítios de Ligação , Gráficos por Computador , Humanos , Ligantes , Mieloblastina/química , Estrutura Terciária de ProteínaRESUMO
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/enzimologiaRESUMO
T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Leucemia Mieloide Aguda/terapia , Manitol/análogos & derivados , Mieloma Múltiplo/terapia , Ácidos Oleicos , Oligodesoxirribonucleotídeos , Peptídeos/uso terapêutico , Adolescente , Antígenos de Neoplasias/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Feminino , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Masculino , Manitol/efeitos adversos , Mucina-1/efeitos adversos , Mucina-1/química , Mucina-1/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Mieloblastina/efeitos adversos , Mieloblastina/química , Mieloblastina/imunologia , Estadiamento de Neoplasias , Neoplasia Residual/imunologia , Neoplasia Residual/patologia , Neoplasia Residual/terapia , Ácidos Oleicos/efeitos adversos , Oligodesoxirribonucleotídeos/efeitos adversos , Oligodesoxirribonucleotídeos/imunologia , Peptídeos/efeitos adversos , Peptídeos/imunologia , Projetos Piloto , Resultado do Tratamento , Proteínas WT1/efeitos adversos , Proteínas WT1/química , Proteínas WT1/imunologiaRESUMO
Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating "pro(C)"-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.
Assuntos
Elastase de Leucócito/metabolismo , Monócitos/enzimologia , Mieloblastina/metabolismo , Neutrófilos/enzimologia , Complexo 3 de Proteínas Adaptadoras/metabolismo , Animais , Antígenos CD/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/ultraestrutura , Endocitose , Precursores Enzimáticos/metabolismo , Humanos , Espaço Intracelular/enzimologia , Elastase de Leucócito/química , Monócitos/patologia , Mieloblastina/química , Neutrófilos/patologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Ratos , Proteínas de Ligação a Tacrolimo/metabolismo , Tetraspanina 30 , Tirosina/metabolismoRESUMO
PURPOSE OF REVIEW: This review focuses on proteinase 3 (PR3), the preferred target of antineutrophil cytoplasmic antibodies (ANCAs) in Wegener's granulomatosis. Deciphering the molecular associations that PR3 can make with its cognate partners might help to understand its pathophysiological significance in Wegener's granulomatosis and the potential role of ANCA as modulator of PR3 functions. RECENT FINDINGS: In neutrophils, PR3 is mainly localized within azurophilic granules but is also detected at the plasma membrane. Among PR3 partners (CD16, CD11b/CD18), CD177, a glycosylphosphatidylinositol (GPI)-anchored membrane protein is a potential receptor for PR3. In addition, PR3 can be externalized at the plasma membrane at a very early stage of neutrophil apoptosis, in the absence of degranulation. In these conditions, PR3 is associated with specific partners including phospholipidscramblase-1 and calreticulin. Interestingly, apoptosis-induced PR3 membrane expression significantly impaired macrophage phagocytosis. This new role of PR3 acting as a 'don't eat me signal' that delays neutrophil clearance might potentiate inflammation and autoimmunity. SUMMARY: Since PR3 membrane expression seems to represent a key element in the inflammatory and autoimmunity process, elucidation of the molecular basis of PR3 interaction with the plasma membrane or with receptor proteins led to the possibility of targeted therapy.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Autoimunidade/fisiologia , Granulomatose com Poliangiite/enzimologia , Proteínas de Membrana/metabolismo , Mieloblastina/metabolismo , Animais , Apoptose/imunologia , Proteínas Ligadas por GPI , Granulomatose com Poliangiite/genética , Granulomatose com Poliangiite/fisiopatologia , Humanos , Isoantígenos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Mieloblastina/química , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Receptores de Superfície Celular/metabolismoRESUMO
Many patients with Wegener's granulomatosis (WG) have anti-neutrophil cytoplasmic antibodies (c-ANCA). Aside from being a diagnostic marker, these autoantibodies may play roles in disease pathogenesis. Proteinase 3 (PR3) is the primary target of c-ANCA in WG patient sera. Of 60 c-ANCA-positive patients, 10 patients were selected for detailed humoral epitope analysis, contingent upon serum availability, using samples with positive levels of anti-PR3 by enzyme-linked immunosorbent assay (ELISA). Sequential epitope specificities of anti-PR3 antibodies detected by screening the maximally overlapping solid-phase octapeptides of PR3 showed seven major common antigenic targets bound by WG patient sera. These include novel and previously identified sequential PR3 epitopes bound by c-ANCA. B cell epitope prediction algorithms identified all or part of the seven defined epitopes. Several epitopes share sequence and structural proximity with functional sites, including the catalytic triad and proposed binding sites of other potential proteins [PR3 complementary peptide and soluble endothelial protein C receptor (sEPCR)]. Epitope 4 (VVLGAHNVRTQ) had the highest binding prevalence (90%) and epitope 2 (AQPHSRPYMAS) has the highest average reactivity of the antigenic regions. Epitope 4 includes the interaction site between sEPCR and PR3 which may serve as an important interaction to down-regulate inflammation. Epitopes 3, 5 and 7 are in direct proximity to amino acids that form the catalytic triad of the protein. c-ANCA targets both unique and previously known sequential PR3 peptides. This information may prove useful in understanding anti-PR3-mediated disease pathogenesis in systemic vasculitides.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Epitopos de Linfócito B/imunologia , Granulomatose com Poliangiite/imunologia , Mieloblastina/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Sequência de Aminoácidos , Anticorpos Anticitoplasma de Neutrófilos/sangue , Epitopos de Linfócito B/química , Feminino , Humanos , Ponto Isoelétrico , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mieloblastina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Propriedades de Superfície , Adulto JovemRESUMO
The S' subsites of human neutrophil proteinase 3 (Pr 3) were probed by constructing diverse libraries of compounds based on the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide using combinational and click chemistry methods. The multiple points of diversity embodied in the heterocyclic scaffold render it well-suited to the exploration of the S' subsites of Pr 3. Molecular modeling studies suggest that further exploration of the S' subsites of Pr 3 using the aforementioned heterocyclic scaffold may lead to the identification of highly selective, reversible competitive inhibitors of Pr 3.
Assuntos
Mieloblastina/antagonistas & inibidores , Mieloblastina/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Triazóis/química , Triazóis/farmacologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mieloblastina/química , Ligação ProteicaRESUMO
Proteinase 3 (PR3) is a serine protease of the neutrophils whose membrane expression is relevant in a number of inflammatory pathologies. It has been shown to strongly interact with reconstituted bilayers containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) or mixtures of both phospholipids. Here we present the results of molecular dynamics simulations of PR3 anchored at three different phospholipid bilayers: DMPC, DMPG and an equimolar mixture of DMPC/DMPG. We present for the first time a detailed model of membrane-bound PR3. A thorough inventory of the interaction between the lipids and the enzyme reveals three types of interactions contributing to the anchorage of PR3. Basic residues (R177, R186A, R186B, K187 and R222) interact via hydrogen bonds with the lipid headgroups to stabilize PR3 at the interfacial membrane region. Hydrophobic amino acids (V163, F165, F166, I217, L223, and F224) insert into the hydrophobic core below the carbonyl groups of the bilayers and six aromatic amino acids (F165, F192, F215, W218, F224, and F227) contribute electrostatic interaction via cation-pi interactions with the choline groups of DMPC. PR3 presents all the characteristics of a peripheral membrane protein with an ability to bind negative phospholipids. Although the catalytic triad remains unperturbed by the presence of the membrane, the ligand binding sites are located in close proximity to the membrane and amino acids K99 and I217 interact significantly with the lipids. We expect the binding of long ligands to be modified by the presence of the lipids.
Assuntos
Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Mieloblastina/metabolismo , Sítios de Ligação , Cátions , Membrana Celular/química , Membrana Celular/metabolismo , Colina/química , Colina/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Mieloblastina/química , Fenilalanina/química , Fenilalanina/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Eletricidade Estática , Triptofano/química , Triptofano/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1 : 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDS-free disc electrophoresis. CP affected the activities of NE, CG, and PR3 as a competitive inhibitor with K(i) ~ 1 µM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards p-phenylenediamine. CAP37 displayed the strongest inhibitory effect (K(i) ~ 20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (5-13, 61-63 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among serprocidins CAP37 can be regarded as the specific partner of CP.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Ceruloplasmina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Catepsina G/metabolismo , Ceruloplasmina/química , Cães , Golfinhos , Ensaios Enzimáticos , Cavalos , Humanos , Técnicas In Vitro , Cinética , Elastase de Leucócito/química , Elastase de Leucócito/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Mieloblastina/química , Mieloblastina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Coelhos , RatosRESUMO
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 α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-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 α1-PI and MNEI but not by SLPI. α2-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 α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105 m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-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 α2-M (39pep-α2-M). Since the 39pep-α2-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 α2-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.