RESUMEN
INTRODUCTION: Osteoporosis is a global health issue. Bisphosphonates that are commonly used to treat osteoporosis suppress both bone resorption and subsequent bone formation. Inhibition of cathepsin K, a cysteine proteinase secreted by osteoclasts, was reported to suppress bone resorption while preserving or increasing bone formation. Analyses of the different effects of antiresorptive reagents such as bisphosphonates and cysteine proteinase inhibitors will contribute to the understanding of the mechanisms underlying bone remodeling. MATERIALS AND METHODS: Our team has developed an in vitro system in which bone remodeling can be temporally observed at the cellular level by 2-photon microscopy. We used this system in the present study to examine the effects of the cysteine proteinase inhibitor E-64 and those of zoledronic acid on bone remodeling. RESULTS: In the control group, the amount of the reduction and the increase in the matrix were correlated in each region of interest, indicating the topological and quantitative coordination of bone resorption and formation. Parameters for osteoblasts, osteoclasts, and matrix resorption/formation were also correlated. E-64 disrupted the correlation between resorption and formation by potentially inhibiting the emergence of spherical osteoblasts, which are speculated to be reversal cells in the resorption sites. CONCLUSION: These new findings help clarify coupling mechanisms and will contribute to the development of new drugs for osteoporosis.
Asunto(s)
Resorción Ósea , Proteasas de Cisteína , Osteoporosis , Humanos , Proteasas de Cisteína/farmacología , Proteasas de Cisteína/uso terapéutico , Resorción Ósea/tratamiento farmacológico , Osteoclastos , Catepsina K , Osteoporosis/tratamiento farmacológico , Difosfonatos/farmacología , Difosfonatos/uso terapéuticoRESUMEN
The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L-KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L-mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.
Asunto(s)
Proteasas de Cisteína , Enfermedad de Parkinson , Humanos , Animales , Ratones , Glucosilceramidasa/genética , Catepsina L/genética , Catepsina L/metabolismo , Catepsinas/metabolismo , Catepsinas/uso terapéutico , Proteasas de Cisteína/metabolismo , Proteasas de Cisteína/uso terapéutico , Enfermedad de Parkinson/metabolismo , Lisosomas/metabolismoRESUMEN
Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bß, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague-Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.
Asunto(s)
Proteasas de Cisteína , Trombosis , Animales , Anticoagulantes/farmacología , Carragenina , Proteasas de Cisteína/uso terapéutico , Estrona/análogos & derivados , Fibrina/uso terapéutico , Fibrinógeno , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Ficaína , Ratas , Ratas Sprague-Dawley , Trombosis/tratamiento farmacológicoRESUMEN
BACKGROUND: Biomarkers of disease progression and treatment response are urgently needed for patients with lymphangioleiomyomatosis (LAM). Activity-based nanosensors, an emerging biosensor class, detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease. Because proteases are dysregulated in LAM and may directly contribute to lung function decline, activity-based nanosensors may enable quantitative, real-time monitoring of LAM progression and treatment response. We aimed to assess the diagnostic utility of activity-based nanosensors in a pre-clinical model of pulmonary LAM. METHODS: Tsc2-null cells were injected intravenously into female nude mice to establish a mouse model of pulmonary LAM. A library of 14 activity-based nanosensors, designed to detect proteases across multiple catalytic classes, was administered into the lungs of LAM mice and healthy controls, urine was collected, and mass spectrometry was performed to measure nanosensor cleavage products. Mice were then treated with rapamycin and monitored with activity-based nanosensors. Machine learning was performed to distinguish diseased from healthy and treated from untreated mice. RESULTS: Multiple activity-based nanosensors (PP03 (cleaved by metallo, aspartic and cysteine proteases), padjusted<0.0001; PP10 (cleaved by serine, aspartic and cysteine proteases), padjusted=0.017)) were differentially cleaved in diseased and healthy lungs, enabling strong classification with a machine learning model (area under the curve (AUC) 0.95 from healthy). Within 2â days after rapamycin initiation, we observed normalisation of PP03 and PP10 cleavage, and machine learning enabled accurate classification of treatment response (AUC 0.94 from untreated). CONCLUSIONS: Activity-based nanosensors enable noninvasive, real-time monitoring of disease burden and treatment response in a pre-clinical model of LAM.
Asunto(s)
Proteasas de Cisteína , Linfangioleiomiomatosis , Animales , Proteasas de Cisteína/uso terapéutico , Femenino , Humanos , Linfangioleiomiomatosis/tratamiento farmacológico , Ratones , Ratones Desnudos , Péptido Hidrolasas/uso terapéutico , Sirolimus/uso terapéuticoRESUMEN
Previous studies showed that P1G10, a proteolytic fraction from Vasconcellea cundinamarcensis latex, reduced the tumor mass in animals bearing melanoma, increased in vitro DNA fragmentation and decreased cell adhesion. Here, we present some molecular and cellular events related to the antimetastatic effect induced by the CMS-2 fraction derived from P1G10 in metastatic melanoma B16-F10 and melanocyte Melan-a. Using difference gel electrophoresis and mass spectrometry, we identified four proteins overexpressed in tumor cells, all of them related to proliferation, survival, migration and cell invasion, that had their expression normalized upon treatment with CMS-2: nucleophosmin 1, heat shock protein 65, calcyclin binding protein and eukaryotic translation initiation factor 4H. In addition, some antioxidant and glycolytic enzymes show increased expression after exposure to CMS-2, along with an induction of melanogenesis (differentiation marker). The down regulation of cofilin 1, a protein involved in cell motility, may explain the inhibition of cell migration and dendritic-like outgrowth in B16-F10 and Melan-a, observed after CMS-2 treatment. Taken together, it is argued that CMS-2 modulates the expression of proteins related to metastatic development, driving the cell to a more differentiated-like state. These effects support the CMS-2 antimetastatic activity and place this fraction in the category of anticancer agent.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Caricaceae/enzimología , Proteasas de Cisteína/farmacología , Melanoma/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Proteínas de Plantas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteasas de Cisteína/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Melanoma/patología , Ratones , Metástasis de la Neoplasia/patología , NucleofosminaRESUMEN
INTRODUCTION: Recently, the population of individuals with non-celiac gluten sensitivity (NCGS) who do not have celiac disease but show improved symptoms with a gluten-free diet, has increased. Enzyme replacement therapy using digestive enzymes is expected to improve the symptoms of NCGS and be sustainable, since gluten-related proteins that are indigestible by the digestive system have been considered triggers of NCGS. METHODS: We selected patients with NCGS by screening demographic interviews, as well as performing medical evaluations, anti-gluten antibody tests, and gluten challenge tests. We performed a single-blind and crossover clinical trial with these subjects using a gluten challenge with the enzyme mixture or a placebo. Our designed enzyme mixture contained peptidase, semi alkaline protease, deuterolysin, and cysteine protease derived from Aspergillus oryzae, Aspergillus melleus, Penicillium citrinum, and Carica papaya L., respectively. RESULTS: Administration of the enzyme mixture significantly decreased the change in the score of the symptom questionnaire before and after the gluten challenge compared with administration of the placebo in patients with NCGS without adverse events. In particular, the changes in the score of the gluten-induced incomplete evacuation feeling and headaches were significantly improved. The serum levels of interleukin (IL)-8, tumor necrosis factor (TNF)-α, andregulated on activation, normal T cell expressed and secreted (RANTES) in subjects were not significantly changed by gluten, as expected from previous studies, and the enzyme mixture did not affect these inflammatory markers. CONCLUSION: In this human clinical study, we demonstrated the efficacy of the enzyme mixture derived from microorganisms and papaya in improving the symptoms of NCGS.
Asunto(s)
Terapia de Reemplazo Enzimático , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/terapia , Glútenes/efectos adversos , Adulto , Anciano , Aspergillus/enzimología , Proteínas Bacterianas/uso terapéutico , Carica/enzimología , Estudios Cruzados , Proteasas de Cisteína/uso terapéutico , Citocinas/sangre , Dieta Sin Gluten , Digestión , Endopeptidasas/uso terapéutico , Femenino , Hipersensibilidad a los Alimentos/sangre , Humanos , Masculino , Persona de Mediana Edad , Penicillium/enzimología , Péptido Hidrolasas/uso terapéutico , Método Simple Ciego , Adulto JovenRESUMEN
Recombinant cysteine peptidase vaccine can induce protective immunity against cutaneous leishmaniasis. However, the antigenic diversity and variable immunogenicity prevents them from being approved for general vaccination. Different approaches like adjuvant application and antigen delivery systems are studied to increase their efficacy. Nanoparticles can both stimulated antigen uptakes and affect direction of immune response. In this study the effect of PLGA nanoparticles were considered to enhance the immune response against recombinant CPA (CPA) and CPB (CPB). For this purpose, L. major CPA and CPB were prepared. PLGA were conjugated to the proteins using Aldehyde/Hydrazine Reaction. Conjugation efficacy and created nanoparticle morphology were determined by FTIR and SEM methods, respectively. BALB/c mice were received intraperitoneally three boosts of 7⯵g/mouse of each antigen alone (CPA/CPB/CPAâ¯+â¯CPB) or as PLGA conjugated form in different Study groups, at 3â¯weeks interval. After vaccination, mice were challenged with 106L. major, subcutaneously. Time course study of lesion development demonstrated nanoparticle efficacy in parasite dissemination control that confirmed by spleen parasite burden assay. Significant induction of nitric oxide production by peritoneal macrophages and increase in splenocyte IFN-γ production showed the protective effect of PLGA-CPA/CPB vaccination in comparison to CPA and CPB alone. Current study demonstrated that the conjugation of the antigen with the PLGA can activate immune responses against L. major. However, further study is necessary to assess the long-term effect and other aspects of immune response.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Proteasas de Cisteína/uso terapéutico , Ácido Láctico/uso terapéutico , Vacunas contra la Leishmaniasis , Leishmaniasis Cutánea/tratamiento farmacológico , Nanopartículas/uso terapéutico , Ácido Poliglicólico/uso terapéutico , Adyuvantes Inmunológicos/química , Animales , Proteasas de Cisteína/química , Femenino , Interferón gamma/inmunología , Interleucina-4/inmunología , Ácido Láctico/química , Leishmania , Leishmaniasis Cutánea/parasitología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos BALB C , Nanopartículas/química , Óxido Nítrico/metabolismo , Carga de Parásitos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/parasitología , Células TH1/inmunologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The latex of Ervatamia heyneana (Wall.) T. Cooke plant has been used for wound healing and various skin diseases by Indian tribes and folklore. AIM OF THE STUDY: To validate the scientific basis of heynein - a key protease of Ervatamia heyneana, in hemostasis and wound healing process. MATERIALS AND METHODS: The latex from E. heyneana was processed and subjected to two step purification. The purified heynein was assayed for proteolytic activity using casein as substrate and also attested by zymography. The inhibition studies confirmed the nature of heynein. Pure fibrinogen was used for fibrinogenolytic activity and citrated plasma was used for coagulant and fibrinolytic activities. The edema inducing action and hemorrhagic activity of heynein were assessed on mice model. RESULTS: The purified heynein exhibited proteolytic activity, which was confirmed by caseinolytic assay and zymography. The inhibition studies confirmed heynein to be a cysteine protease. Heynein showed complete hydrolysis of all the three subunits of human fibrinogen (Aα, Bß, γ). It exhibited strong pro-coagulant activity by reducing plasma clotting time from 248 to 39s at 40µg concentration. Heynein cleaved α polymer subunit in fibrin clot and did not induce edema and hemorrhage in mice models. The non-hemorrhagic nature was supported with histopathological studies of skin samples. CONCLUSION: Heynein displays strong pro-coagulant action associated with fibrin(ogen)olytic activity. This provides basis for the observed pharmacological action of Ervatamia heyneana and thereby justifies its use in folk medicine.
Asunto(s)
Apocynaceae , Proteasas de Cisteína/farmacología , Fibrinolíticos/farmacología , Hemostáticos/farmacología , Látex/farmacología , Extractos Vegetales/farmacología , Adulto , Animales , Proteasas de Cisteína/aislamiento & purificación , Proteasas de Cisteína/uso terapéutico , Fibrinógeno/metabolismo , Fibrinolíticos/aislamiento & purificación , Fibrinolíticos/uso terapéutico , Hemorragia/tratamiento farmacológico , Hemorragia/metabolismo , Hemostasis/efectos de los fármacos , Hemostasis/fisiología , Hemostáticos/aislamiento & purificación , Hemostáticos/uso terapéutico , Humanos , Látex/aislamiento & purificación , Látex/uso terapéutico , Masculino , Ratones , Corteza de la Planta , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Adulto JovenRESUMEN
Bromelain is a cysteine protease found in pineapple tissue. Because of its anti-inflammatory and anti-cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid-liquid extractions by aqueous two-phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up-to-date compilation of the data reported on this enzyme.
Asunto(s)
Ananas/química , Bromelaínas/química , Proteasas de Cisteína/química , Secuencia de Aminoácidos/genética , Apoptosis/efectos de los fármacos , Bromelaínas/genética , Bromelaínas/aislamiento & purificación , Bromelaínas/uso terapéutico , Proteasas de Cisteína/uso terapéutico , HumanosRESUMEN
Insufficient and/or improper protein degradation is associated with the development of various human pathologies. Enzymatic therapy with proteolytic enzymes aimed to improve insufficient proteolytic activity was suggested as a treatment of protease deficiency-induced disorders. Since in many cases human degradome is incapable of degrading the entire target protein(s), other organisms can be used as a source of proteases exhibiting activities distinct from human enzymes, and plants are perspective candidates for this source. In this study recombinant wheat cysteine protease Triticain-α was shown to refold in vitro into an autocatalytically activated proteolytic enzyme possessing glutenase and collagenase activities at acidic (or close to neutral) pH levels at the temperature of human body. Mass-spectrometry analysis of the products of Triticain-α-catalyzed gluten hydrolysis revealed multiple cleavage sites within the sequences of gliadin toxic peptides, in particular, in the major toxic 33-mer α-gliadin-derived peptide initiating inflammatory responses to gluten in celiac disease (CD) patients. Triticain-α was found to be relatively stable in the conditions simulating stomach environment. We conclude that Triticain-α can be exploited as a basic compound for development of (i) pharmaceuticals for oral administration aimed at release of the active enzyme into the gastric lumen for CD treatment, and (ii) topically active pharmaceuticals for wound debridement applications.
Asunto(s)
Colagenasas/metabolismo , Proteasas de Cisteína/metabolismo , Terapia de Reemplazo Enzimático , Glútenes/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Triticum/enzimología , Secuencia de Aminoácidos , Enfermedad Celíaca/tratamiento farmacológico , Colagenasas/genética , Colagenasas/aislamiento & purificación , Colagenasas/uso terapéutico , Proteasas de Cisteína/genética , Proteasas de Cisteína/aislamiento & purificación , Proteasas de Cisteína/uso terapéutico , Desbridamiento/métodos , Estudios de Factibilidad , Glútenes/genética , Glútenes/aislamiento & purificación , Glútenes/uso terapéutico , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/uso terapéutico , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Triticum/genéticaRESUMEN
BACKGROUND: Cysteine proteinases (CPs) from papaya (Carica papaya) possess anthelmintic properties against human soil-transmitted helminths (STH, Ascaris lumbricoides, Trichuris trichiura and hookworm), but there is a lack of supportive and up-to-date efficacy data. We therefore conducted two randomized controlled trials in pigs to assess the efficacy of papaya CPs against experimental infections with T. suis. METHODS: First, we assessed efficacy by means of egg (ERR) and adult worm reduction rate (WRR) of a single-oral dose of 450 µmol active CPs (CP450) against low (inoculum of 300 eggs) and high (inoculum of 3,000 eggs) intensity T. suis infections and compared the efficacy with those obtained after a single-oral dose of 400 mg albendazole (ALB). In the second trial, we determined and compared the efficacy of a series of CP doses (45 [CP45], 115 [CP115], 225 [CP225], and 450 [CP450] µmol) against high intensity infections. RESULTS: CP450 was highly efficacious against both levels of infection intensity, resulting in ERR and WRR of more than 97%. For both levels of infection intensity, CP450 was significantly more efficacious compared to ALB by means of WRR (low infection intensity: 99.0% vs. 39.0%; high infection intensity; 97.4% vs. 23.2%). When the efficacy was assessed by ERR, a significant difference was only observed for high intensity infections, CP450 being more efficacious than ALB (98.9% vs. 59.0%). For low infection intensities, there was no significant difference in ERR between CP450 (98.3%) and ALB (64.4%). The efficacy of CPs increased as a function of increasing dose. When determined by ERR, the efficacy ranged from 2.1% for CP45 to 99.2% for CP450. For WRR the results varied from -14.0% to 99.0%, respectively. Pairwise comparison revealed a significant difference in ERR and WRR only between CP45 and CP450, the latter being more efficacious. CONCLUSIONS: A single dose of 450 µmol CPs provided greater efficacy against T. suis infections in pigs than a single-oral dose of 400 mg ALB. Although these results highlight the possibility of papaya CPs for controlling human STH, further development is needed in order to obtain and validate an oral formulation for human application.
Asunto(s)
Antihelmínticos/uso terapéutico , Carica/enzimología , Proteasas de Cisteína/uso terapéutico , Enfermedades de los Porcinos/parasitología , Tricuriasis/veterinaria , Trichuris/fisiología , Albendazol/administración & dosificación , Albendazol/uso terapéutico , Animales , Antihelmínticos/administración & dosificación , Proteasas de Cisteína/administración & dosificación , Relación Dosis-Respuesta a Droga , Proteínas de Plantas/uso terapéutico , Porcinos , Enfermedades de los Porcinos/tratamiento farmacológico , Tricuriasis/tratamiento farmacológico , Tricuriasis/parasitologíaRESUMEN
BACKGROUND: A recombinant cysteine proteinase from Leishmania (Leishmania) infantum chagasi (rLdccys1) was previously shown to induce protective immune responses against murine and canine visceral leishmaniasis. These findings encouraged us to use rLdccys1 in the immunotherapy of naturally infected dogs from Teresina, Piauí, a region of high incidence of visceral leishmaniasis in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Thirty naturally infected mongrel dogs displaying clinical signs of visceral leishmaniasis were randomly divided in three groups: one group received three doses of rLdccys1 in combination with the adjuvant Propionibacterium acnes at one month interval between each dose; a second group received three doses of P. acnes alone; a third group received saline. The main findings were: 1) dogs that received rLdccys1 with P. acnes did not display increase of the following clinical signs: weight loss, alopecia, onychogryphosis, cachexia, anorexia, apathy, skin lesions, hyperkeratosis, ocular secretion, and enlarged lymph nodes; they also exhibited a significant reduction in the spleen parasite load in comparison to the control dogs; 2) rLdccys1-treated dogs exhibited a significant delayed type cutaneous hypersensitivity elicited by the recombinant antigen, as well as high IgG2 serum titers and low IgG1 serum titers; sera from rLdccys1-treated dogs also contained high IFN-γ and low IL-10 concentrations; 3) control dogs exhibited all of the clinical signs of visceral leishmaniasis and had low serum IgG2 and IFN-γ levels and high concentrations of IgG1 and IL-10; 4) all of the dogs treated with rLdccys1 were alive 12 months after treatment, whereas dogs which received either saline or P. acnes alone died within 3 to 7 months. CONCLUSIONS/SIGNIFICANCE: These findings illustrate the potential use of rLdccys1 as an additional tool for the immunotherapy of canine visceral leishmaniasis and support further studies designed to improve the efficacy of this recombinant antigen for the treatment of this neglected disease.