RESUMEN
Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S' specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO2)-NH2, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.
Asunto(s)
Catepsina C/química , Microscopía Fluorescente/métodos , Dominio Catalítico , Catepsina L/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Inflamación , Cinética , Mastocitos/citología , Conformación Molecular , Simulación del Acoplamiento Molecular , Neutrófilos/metabolismo , Péptidos/química , Unión Proteica , Proteínas Recombinantes/química , Especificidad por Sustrato , Linfocitos T Citotóxicos/citologíaRESUMEN
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 â¼80% 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.
Asunto(s)
Catepsina C/metabolismo , Pulmón/enzimología , Neutrófilos/enzimología , Neumonía/enzimología , Animales , Catepsina C/genética , Modelos Animales de Enfermedad , Células HL-60 , Humanos , Pulmón/patología , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Neutrófilos/patología , Neumonía/inducido químicamente , Neumonía/patología , Ratas Sprague-Dawley , Esputo/metabolismoRESUMEN
Papillon-Lefèvre syndrome (PLS) (OMIM: 245000) is a rare disease characterized by severe periodontitis and palmoplantar keratoderma. It is caused by mutations in both alleles of the cathepsin C (CatC) gene CTSC that completely abrogate the proteolytic activity of this cysteine proteinase. Most often, a genetic analysis to enable early and rapid diagnosis of PLS is unaffordable or unavailable. In this study, we tested the hypothesis that active CatC is constitutively excreted and can be easily traced in the urine of normal subjects. If this is true, determining its absence in the urine of patients would be an early, simple, reliable, low-cost and easy diagnostic technique. All 75 urine samples from healthy control subjects (aged 3 months to 80 years) contained proteolytically active CatC and its proform, as revealed by kinetic analysis and immunochemical detection. Of the urine samples of 31 patients with a PLS phenotype, 29 contained neither proteolytically active CatC nor the CatC antigen, so that the PLS diagnosis was confirmed. CatC was detected in the urine of the other two patients, and genetic analysis revealed no loss-of-function mutation in CTSC, indicating that they suffer from a PLS-like condition but not from PLS. Screening for the absence of urinary CatC activity soon after birth and early treatment before the onset of PLS manifestations will help to prevent aggressive periodontitis and loss of many teeth, and should considerably improve the quality of life of PLS patients.
Asunto(s)
Catepsina C/orina , Enfermedad de Papillon-Lefevre/diagnóstico , Enfermedad de Papillon-Lefevre/orina , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Catepsina C/genética , Catepsina C/metabolismo , Niño , Preescolar , Femenino , Voluntarios Sanos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Fenotipo , Adulto JovenRESUMEN
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.
Asunto(s)
Ésteres/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Mieloblastina/antagonistas & inhibidores , Mieloblastina/química , Oligopéptidos/química , Organofosfonatos/química , Animales , Apoptosis , Biotinilación , Línea Celular , Membrana Celular/metabolismo , Humanos , Hidrólisis , Inflamación , Insectos , Espectrometría de Masas , Modelos Químicos , Mutación , Activación Neutrófila , Neutrófilos/efectos de los fármacos , Péptidos/química , Prolina/química , Inhibidores de Proteasas/química , SolventesRESUMEN
Internally quenched cathepsin L (Cat L) substrate ABZ-Bip-Arg-Ala-Gln-Tyr(3-NO2)-NH2 with high specificity constant (kcat/KM=2.6×10(7)M(-1)s(-1)) was synthesized. The resultant compound displayed high selectivity over other members of the cathepsin family (B, S, X, V, C, K, H, F, D, and A). Activity of Cat L at picomolar (pM) concentrations was found using this substrate. Moreover, it was established that the presence of the selective Cat L inhibitor suppressed the proteolysis of the substrate to a non-detectable level. Incubation of the synthesized compound with a cell lysate of healthy and cancer cell lines indicated significant differences in Cat L activity. Based on the obtained results, it is proposed that this substrate could be used for selective monitoring of Cat L activity in biological systems.
Asunto(s)
Catepsina L/metabolismo , Péptidos/síntesis química , Bioensayo , Catepsinas/metabolismo , Línea Celular , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Límite de Detección , Péptidos/química , Péptidos/metabolismo , Especificidad por Sustrato , Tirosina/análogos & derivados , Tirosina/metabolismo , ortoaminobenzoatos/metabolismoRESUMEN
This article describes the synthesis and enzymatic study of newly synthesized analogs of trypsin inhibitors SFTI-1 that were fluorescent labeled on their N-terminal amino groups. Two fluorescent derivatives of benzoxazole (3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-L-alanine-[(4NPh2 )Ph]Box-Ala and 3-[2-(2',4',5'-trimethoxyphenyl)benzoxazol-5-yl]-L-alanine-[2,4,5-(OMe)3Ph]Box-Ala) were used as efficient fluorescent labels. The compounds obtained preserved their inhibitory activity and were efficient inhibitors of bovine trypsin or chymotrypsin. Nevertheless, their association inhibition constants were one or two orders of magnitude lower than those determined for unlabeled monocyclic SFTI-1 or [Phe(5)]SFTI-1, respectively. The conjugates obtained were found to be proteolytically stable in the presence of cognate enzymes. Applying such fluorescent peptides, we were able to investigate enzyme-inhibitor complex formation using fluorescent techniques. We found that such compounds were rapidly internalized by the fibroblast or cancer cells with no cytotoxic effects.
Asunto(s)
Helianthus/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/aislamiento & purificación , Semillas/química , Inhibidores de Tripsina/síntesis química , Inhibidores de Tripsina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Benzoxazoles/química , Bovinos , Línea Celular , Permeabilidad de la Membrana Celular , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Citometría de Flujo , Fluorescencia , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Péptidos Cíclicos/química , Factores de Tiempo , Inhibidores de Tripsina/químicaRESUMEN
In this work the efficient and simple method of improvement specificity and solubility of low molecular weight proteinase substrates is described. The series of fluorescent substrates of selected proteolytic enzymes (neutrophil elastase, cathepsin G and proteinase 3 along with human airway trypsin like protease) were synthesized and modified by selective pegylation by the attachment of 2-(2-(2-aminoethoxy)ethoxy)acetic acid. Modification of the C-terminal carboxyl group resulted in the decrease in the specificity constants (k(cat)/K(M)) for all obtained analogues. The covalent attachment of PEG to N-terminal amino group has the opposite effect, as the increase in specificity constant was observed for all studied compounds. This outcome was pronounced the most for proteinase 3 substrate PEG-ABZ-Tyr-Tyr-Abu-ANB-NH2, whose catalytic constant (k(cat)) increased over three fold. The introduction of PEG moieties at both C- and N-terminal yielded the substrates with lower specificity constants. For substrate (ABZ-Arg-Gln-Asp-Arg-ANB-NH2) the influence of the PEG chain length on its kinetic parameters was investigated. Elongation of the PEG chain at N-terminal of this peptide decreased the specificity constant. In addition to the effect of pegylation on the kinetic parameters of the studied substrates, the introduced modifications significantly improved their solubility in buffer solutions applied for enzymatic investigations.