RESUMEN
PURPOSE: Neutrophil serine proteases (NSPs) are implicated in numerous inflammatory diseases. Thus, a robust methodology to monitor and quantify NSPs is important to study disease progression and evaluate the effect of pharmacological interventions. A comparison of the various methods used to extract NSPs from neutrophil granulocytes has not been published, providing the impetus to conduct this method optimization and comparison study. METHODS: Two NSP recovery methodologies were evaluated on samples from five human donors: zymosan stimulation and cell pellet extraction. For the zymosan stimulation method, 1 mL donor blood was added to zymosan and samples were incubated at 37°C for 30 min while shaking. Samples were then centrifuged, and the plasma was collected for quantitation of NSP activity. For the cell pellet extraction procedure, 2 mL whole blood samples were centrifuged into white blood cell pellets following red blood cell lysis. To each pellet, three sequential lysis steps were performed using either 0.05% Nonidet P-40 Substitute (NP40) or 0.02% Triton X-100 lysis buffers under agitation followed by centrifugation. NSP activities were quantified using an exogenous peptide substrate specific to each of the three NSPs being analyzed: neutrophil elastase, cathepsin G, and proteinase 3. RESULTS AND DISCUSSION: The zymosan stimulation method resulted in lower recovery of active NSPs and was unable to stimulate significant release of active cathepsin G. In contrast, the NP40 pellet extraction method showed consistent inter-donor NSP release with greater recoveries of active NSPs than the Triton method or the zymosan stimulation method. Overall, the pellet extraction procedure provided 13.3-fold greater recovery of active neutrophil elastase, 283-fold greater recovery of active cathepsin G, and 2.9-fold greater recovery of active proteinase 3 than the zymosan method. CONCLUSION: The NP40 cell pellet extraction method resulted in greater extraction of active NSPs compared to the other methods investigated here, which may allow for a more accurate and complete biomarker profile when evaluating human clinical samples.
Asunto(s)
Métodos Analíticos de la Preparación de la Muestra , Serina Proteasas , Células Sanguíneas/química , Células Sanguíneas/enzimología , Catepsina G/química , Catepsina G/metabolismo , Humanos , Elastasa de Leucocito/química , Elastasa de Leucocito/metabolismo , Mieloblastina , Neutrófilos/química , Neutrófilos/metabolismo , Serina Proteasas/química , Serina Proteasas/metabolismo , Zimosan/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Catepsina G/química , Elastasa de Leucocito/química , Mieloblastina/química , Neutrófilos/enzimología , Inhibidores de Serina Proteinasa/química , Staphylococcus aureus/química , Cristalografía por Rayos X , Humanos , Estructura Cuaternaria de ProteínaRESUMEN
The oncogenic fusion protein RUNX1-ETO is a product of the t(8;21) translocation and consists of the hematopoietic transcriptional master regulator RUNX1 and the repressor ETO. RUNX1-ETO is found in 10-15% of acute myeloid leukemia and interferes with the expression of genes that are essential for myeloid differentiation. The neutrophil serine protease Cathepsin G is one of the genes suppressed by RUNX1-ETO, but little is known about its impact on the regulation of other lysosomal proteases. By lentiviral transduction of the t(8;21) positive cell line Kasumi-1 with an RUNX1-ETO specific shRNA, we analyzed long-term effects of stable RUNX1-ETO silencing on cellular phenotypes and target gene expression. Stable anti RUNX1-ETO RNAi reduces both proliferation and apoptosis in Kasumi-1 cells. In addition, long-term knockdown of RUNX1-ETO leads to an upregulation of proteolytic activity in Kasumi-1 cells, which may be released in vitro upon cell lysis leading to massive degradation of cellular proteins. We therefore propose that protein expression data of RUNX1-ETO-silenced Kasumi-1 cells must be analyzed with caution, as cell lysis conditions can heavily influence the results of studies on protein expression. Next, a mass spectrometry-based approach was used to identify protease cleavage patterns in RUNX1-ETO-depleted Kasumi-1 cells and Neutrophil Elastase has been identified as a RUNX1-ETO candidate target. Finally, proteolytic activity of Neutrophil Elastase and Cathepsin G was functionally confirmed by si/shRNA-mediated knockdown in Kasumi-1 cells.
Asunto(s)
Catepsina G/metabolismo , Expresión Génica , Elastasa de Leucocito/metabolismo , Proteínas de Fusión Oncogénica/genética , Catepsina G/química , Línea Celular Tumoral , Cromatografía Liquida , Silenciador del Gen , Humanos , Elastasa de Leucocito/química , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/metabolismo , Proteolisis , ARN Largo no Codificante , Espectrometría de Masas en TándemRESUMEN
Psoriasis therapy remains an extremely relevant area of modern drug design, due to necessity of adverse reaction reduction, inherent for actual methods of therapy. It was established that two serine proteases-neutrophil elastase 1 (HNE1) and cathepsin G (CatG)-are the key agents in psoriasis development. The collected molecular data for the presented targets form the basis for the molecular modeling strategy for the search for and identification of new target-specific inhibitors. The result of this work is a group of high-priority small-molecule compounds with double-targeted affinity, which are able to suppress the pro-psoriatic processes induced by the considered serine proteases at the initial stage of the disease.
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Catepsina G/antagonistas & inhibidores , Elastasa de Leucocito/antagonistas & inhibidores , Terapia Molecular Dirigida , Psoriasis/tratamiento farmacológico , Inhibidores de Serina Proteinasa/farmacología , Catepsina G/química , Descubrimiento de Drogas , Elastasa de Leucocito/química , Modelos Moleculares , Conformación Proteica , Psoriasis/enzimología , Inhibidores de Serina Proteinasa/uso terapéuticoRESUMEN
Cathepsin G is one of four members of the neutrophil serine protease family and constitutes an important biological target in various human inflammatory diseases, such as chronic obstructive pulmonary disease, acute respiratory distress syndrome and cystic fibrosis. Many studies have been focused on determining its biological roles, the latest ones concerning its involvement in acute myeloid leukemia, and as such, multiple chemical and biochemical tools were developed to investigate cathepsin G. Nevertheless, most of them lack selectivity or sensitivity and therefore cannot be used in complex systems. Here we present the development of an optimal cathepsin G Internally Quenched Fluorescence (IQF) substrate that incorporates unnatural amino acids causing the increase of its selectivity toward neutrophil elastase and potency in in vitro studies.
Asunto(s)
Catepsina G/química , Colorantes Fluorescentes/química , Elastasa de Leucocito/química , Humanos , Cinética , Neutrófilos/metabolismo , Especificidad por SustratoRESUMEN
Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km =â¯15⯱â¯2.9⯵M, kcat/Km =â¯930,000â¯s-1 M-1 and Km =â¯25⯱â¯5.4⯵M, kcat/Km =â¯640,000â¯s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1â¯nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.
Asunto(s)
Elastina/química , Elastasa de Leucocito/química , Pulmón/enzimología , Neumonía/enzimología , Animales , Líquido del Lavado Bronquioalveolar/química , Catepsina G/química , Elastina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Elastasa de Leucocito/aislamiento & purificación , Pulmón/efectos de los fármacos , Pulmón/patología , Imagen por Resonancia Magnética , Ratones , Mieloblastina/química , Neutrófilos/enzimología , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacología , Neumonía/metabolismo , Neumonía/patología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Especificidad por SustratoRESUMEN
Human neutrophils express at least four active serine proteases, cathepsin G, N-elastase, proteinase 3 and neutrophil serine protease 4 (NSP4). They have all been extensively studied due to their importance in neutrophil biology and immunity. However, their extended cleavage specificities have never been determined in detail. Here we present a detailed cleavage specificity analysis of human cathepsin G (hCG). The specificity was determined by phage display analysis and the importance of individual amino acids in and around the cleavage site was then validated using novel recombinant substrates. To provide a broader context to this serine protease, a comparison was made to the related mast cell protease, human chymase (HC). hCG showed similar characteristics to HC including both the primary and extended specificities. As expected, Phe, Tyr, Trp and Leu were preferred in the P1 position. In addition, both proteases showed a preference for negatively charged amino acids in the P2´ position of substrates and a preference for aliphatic amino acids both upstream and downstream of the cleavage site. However, overall the catalytic activity of hCG was ~10-fold lower than HC. hCG has previously been reported to have a dual specificity consisting of chymase and tryptase-type activities. In our analysis, tryptase activity against substrates with Lys in P1 cleavage position was indeed only 2-fold less efficient as compared to optimal chymase substrates supporting strong dual-type specificity. We hope the information presented here on extended cleavage specificities of hCG and HC will assist in the search for novel in vivo substrates for these proteases as well as aid in the efforts to better understand the role of hCG in immunity and bacterial defence.
Asunto(s)
Catepsina G/metabolismo , Neutrófilos/metabolismo , Secuencia de Aminoácidos , Catepsina G/química , Quimasas/metabolismo , Secuencia Conservada , Activación Enzimática , Humanos , Cinética , Mastocitos/metabolismo , Proteolisis , Especificidad por Sustrato , Triptasas/metabolismoRESUMEN
Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcß: 55.2%; Fucα1,6GlcNAcß: 22.7%), paucimannosidic N-glycans (Manß1,4GlcNAcß1,4GlcNAcß: 10.6%; Manß1,4GlcNAcß1,4(Fucα1,6)GlcNAcß: 7.9%; Manα1,6Manß1,4GlcNAcß1,4GlcNAcß: 3.7%, trace level of Manα1,6Manß1,4GlcNAcß1,4(Fucα1,6)GlcNAcß), and trace levels of monoantennary α2,6- and α2,3-sialylated complex N-glycans. High-resolution/mass accuracy LC-MS profiling of intact nCG confirmed the Asn71-glycoprofile and identified two C-terminal truncation variants at Arg243 (57.8%) and Ser244 (42.2%), both displaying oxidation of solvent-accessible Met152. Asn71 appeared proximal (~19 Å) to the active site of nCG, but due to the truncated nature of Asn71-glycans (~5-17 Å) we questioned their direct modulation of the proteolytic activity of the protein. This work highlights the continued requirement of using complementary technologies to accurately profile even relatively simple glycoproteins and illustrates important challenges associated with the analysis of unconventional protein N-glycosylation. Importantly, this study now facilitates investigation of the functional role of nCG Asn71-glycosylation.
Asunto(s)
Asparagina/metabolismo , Catepsina G/química , Catepsina G/metabolismo , Glicopéptidos/química , Glicoproteínas/química , Neutrófilos/enzimología , Polisacáridos/química , Secuencia de Aminoácidos , Dominio Catalítico , Catepsina G/aislamiento & purificación , Cromatografía Liquida , Glicopéptidos/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espacio Intracelular/metabolismo , Modelos Moleculares , Neutrófilos/citología , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Extracción en Fase Sólida , Espectrometría de Masas en TándemRESUMEN
Extracellular superoxide dismutase (EC-SOD) is expressed by both macrophages and neutrophils and is known to influence the inflammatory response. Upon activation, neutrophils generate hypochlorous acid (HOCl) and secrete proteases to combat invading microorganisms. This produces a hostile environment in which enzymatic activity in general is challenged. In this study, we show that EC-SOD exposed to physiologically relevant concentrations of HOCl remains enzymatically active and retains the heparin-binding capacity, although HOCl exposure established oxidative modification of the N-terminal region (Met32) and the formation of an intermolecular cross-link in a fraction of the molecules. The cross-linking was also induced by activated neutrophils. Moreover, we show that the neutrophil-derived proteases human neutrophil elastase and cathepsin G cleaved the N-terminal region of EC-SOD irrespective of HOCl oxidation. Although the cleavage by elastase did not affect the quaternary structure, the cleavage by cathepsin G dissociated the molecule to produce EC-SOD monomers. The present data suggest that EC-SOD is stable and active at the site of inflammation and that neutrophils have the capacity to modulate the biodistribution of the protein by generating EC-SOD monomers that can diffuse into tissue.
Asunto(s)
Catepsina G/química , Ácido Hipocloroso/farmacología , Elastasa de Leucocito/química , Macrófagos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Superóxido Dismutasa/química , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/enzimología , Catepsina G/metabolismo , Bovinos , Espacio Extracelular/química , Espacio Extracelular/enzimología , Heparina/química , Humanos , Ácido Hipocloroso/metabolismo , Elastasa de Leucocito/metabolismo , Macrófagos/citología , Macrófagos/enzimología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/enzimología , Oxidación-Reducción , Cultivo Primario de Células , Unión Proteica , Estructura Cuaternaria de Proteína/efectos de los fármacos , Superóxido Dismutasa/metabolismoRESUMEN
Cysteine protease is known to degrade amyloid beta peptide which is a causative agent of Alzheimer's disease. This cleavage mechanism has not been studied in detail at the atomic level. Hence, a three-dimensional structure of cysteine protease from Xanthomonas campestris was constructed by homology modeling using Geno3D, SWISS-MODEL, and MODELLER 9v7. All the predicted models were analyzed by PROCHECK and PROSA. Three-dimensional model of cysteine protease built by MODELLER 9v7 shows similarity with human cathepsin B crystal structure. This model was then used further for docking and simulation studies. The molecular docking study revealed that Cys17, His87, and Gln88 residues of cysteine protease form an active site pocket similar to human cathepsin B. Then the docked complex was refined by molecular dynamic simulation to confirm its stable behavior over the entire simulation period. The molecular docking and MD simulation studies showed that the sulfhydryl hydrogen atom of Cys17 of cysteine protease interacts with carboxylic oxygen of Lys16 of Aß peptide indicating the cleavage site. Thus, the cysteine protease model from X. campestris having similarity with human cathepsin B crystal structure may be used as an alternate approach to cleave Aß peptide a causative agent of Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/química , Proteínas Bacterianas/química , Proteasas de Cisteína/química , Simulación del Acoplamiento Molecular , Homología Estructural de Proteína , Xanthomonas campestris/enzimología , Catepsina G/química , HumanosRESUMEN
PURPOSE: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. EXPERIMENTAL DESIGN: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients. RESULTS: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL-mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. CONCLUSION: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.
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Catepsina G/inmunología , Antígeno HLA-A2/inmunología , Leucemia Mieloide Aguda/inmunología , Péptidos/inmunología , ADP-Ribosil Ciclasa 1/metabolismo , Antígenos CD34/metabolismo , Catepsina G/química , Catepsina G/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica , Epítopos/inmunología , Epítopos/metabolismo , Antígeno HLA-A2/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunoterapia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Péptidos/metabolismo , Unión Proteica/inmunología , Transporte de Proteínas , Linfocitos T Citotóxicos/inmunología , Trasplante HomólogoRESUMEN
Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. In addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. The Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by ß2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and ß2-tryptase. The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1)·s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.
Asunto(s)
Catepsina G/metabolismo , Quimasas/metabolismo , Sitios de Unión , Catepsina G/química , Línea Celular , Quimasas/química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/metabolismo , Humanos , Mastocitos/enzimología , Modelos Moleculares , Oligopéptidos/metabolismo , Especificidad por SustratoRESUMEN
Telechelic water-soluble HPMA copolymers and HPMA copolymer-doxorubicin (DOX) conjugates have been synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA) that contains an enzymatically degradable oligopeptide sequence. Postpolymerization aminolysis followed by chain extension with a bis-maleimide resulted in linear high molecular weight multiblock HPMA copolymer conjugates. These polymers are enzymatically degradable; in addition to releasing the drug (DOX), the degradation of the polymer backbone resulted in products with molecular weights similar to the starting material and below the renal threshold. The new multiblock HPMA copolymers hold potential as new carriers of anticancer drugs.
Asunto(s)
Acrilamidas/química , Acrilamidas/síntesis química , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/síntesis química , Doxorrubicina/química , Doxorrubicina/síntesis química , Portadores de Fármacos/química , Portadores de Fármacos/síntesis química , Acrilamidas/uso terapéutico , Antibióticos Antineoplásicos/uso terapéutico , Catepsina G/química , Preparaciones de Acción Retardada/síntesis química , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/uso terapéutico , Doxorrubicina/uso terapéutico , Portadores de Fármacos/uso terapéutico , Maleimidas/química , Oligopéptidos/química , Papaína/químicaRESUMEN
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.
Asunto(s)
Catepsina G/química , Catepsina G/fisiología , Elastasa de Leucocito/fisiología , Terapia Molecular Dirigida , Mieloblastina/fisiología , Animales , Dominio Catalítico , Catepsina G/antagonistas & inhibidores , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/química , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/enzimología , Mieloblastina/antagonistas & inhibidores , Mieloblastina/química , Neutropenia/tratamiento farmacológico , Neutropenia/enzimología , Enfermedad de Papillon-Lefevre/tratamiento farmacológico , Enfermedad de Papillon-Lefevre/enzimologíaRESUMEN
SUMMARY: The expression of major histocompatibility complex class II (MHC II) molecules is post-translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II-degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent-solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)-DR isolated from both HLA-DM-expressing and DM-null cells. Even following CatG cleavage, peptide binding was retained by pre-loaded, soluble recombinant HLA-DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane-proximal beta2 domain. All allelic variants of HLA-DR tested and murine I-A(g7) class II molecules were susceptible, whereas murine I-E(k) and HLA-DM were not, consistent with their altered sequence at the P1' position of the CatG cleavage site. CatG effects were reduced on HLA-DR molecules with DRB mutations in the region implicated in interaction with HLA-DM. In contrast, addition of CatG to intact B-lymphoblastoid cell lines (B-LCLs) did not cause degradation of membrane-bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen-presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context-dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis.
Asunto(s)
Catepsina G/química , Catepsina G/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Linfocitos B/metabolismo , Catepsina G/antagonistas & inhibidores , Catepsina G/genética , Catepsinas/metabolismo , Línea Celular , Células Dendríticas/metabolismo , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Antígeno HLA-DR1/genética , Antígeno HLA-DR1/metabolismo , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/farmacología , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Polimorfismo Genético/genética , Unión Proteica/fisiología , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Serine proteases released from neutrophils are central to the pathogenesis of cystic fibrosis lung disease and are considered to be obvious therapeutic targets. Neutrophil elastase digests key opsonins present in the lung and disrupts phagocytosis, allowing bacteria to persist despite established pulmonary inflammation. We have found that cathepsin G, an abundant serine protease found in human and murine neutrophils, has other roles in the development of suppurative lung diseases. Murine models of endobronchial inflammation indicate that cathepsin G inhibits airway defences and interferes with the host's ability to clear Pseudomonas aeruginosa from the lung with effects distinct from neutrophil elastase. We hypothesise that differences in bacterial killing are due to defects in innate defences created by proteolysis. Protein profiles of bronchoalveolar lavage of infected wild-type and cathepsin G-deficient mice were compared using two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry. Four proteins in bronchoalveolar lavage were cleaved by cathepsin G. Serum amyloid P component leaked into the lung during acute infection and was digested by cathepsin G. Its cleavage products had greater binding to lipopolysaccharide and interfered with phagocytosis. These results indicate that cleaved serum amyloid P component acts as an anti-opsonin and interferes with bacterial clearance from the lung.
Asunto(s)
Catepsina G/química , Animales , Bronquios/microbiología , Lavado Broncoalveolar , Catepsina G/metabolismo , Electroforesis en Gel Bidimensional/métodos , Células HL-60 , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Transgénicos , Neutrófilos/metabolismo , Proteínas Opsoninas/química , Fagocitosis , Componente Amiloide P Sérico/biosíntesis , Espectrometría de Masas en Tándem/métodosRESUMEN
Contributions from multiple cathepsins within endosomal antigen processing compartments are necessary to process antigenic proteins into antigenic peptides. Cysteine and aspartyl cathepsins have been known to digest antigenic proteins. A role for the serine protease, cathepsin G (CatG), in this process has been described only recently, although CatG has long been known to be a granule-associated proteolytic enzyme of neutrophils. In line with a role for this enzyme in antigen presentation, CatG is found in endocytic compartments of a variety of antigen presenting cells. CatG is found in primary human monocytes, B cells, myeloid dendritic cells 1 (mDC1), mDC2, plasmacytoid DC (pDC), and murine microglia, but is not expressed in B cell lines or monocyte-derived DC. Purified CatG can be internalized into endocytic compartments in CatG non-expressing cells, widening the range of cells where this enzyme may play a role in antigen processing. Functional assays have implicated CatG as a critical enzyme in processing of several antigens and autoantigens. In this review, historical and recent data on CatG expression, distribution, function and involvement in disease will be summarized and discussed, with a focus on its role in antigen presentation and immune-related events.