RESUMO
L-Asparaginase (ASNase) is a biopharmaceutical used as an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Yet, some cases of ALL are naturally resistant to ASNase treatment, which results in poor prognosis. The REH ALL cell line, used as a model for studying the most common subtype of ALL, is considered resistant to treatment with ASNase. Cathepsin B (CTSB) is one of the proteases involved in the regulation of in vivo ASNase serum half-life and it has also been associated with the progression and resistance to treatment of several solid tumors. Previous works have shown that, in vitro, ASNase is degraded when incubated with REH cell lysate, which is prevented by a specific CTSB inhibitor, suggesting a function of this protease in the ASNase resistance of REH cells. In this work, we utilized a combination of CRISPR/Cas9 gene targeting and enzymatic measurements to investigate the relevance of CTSB on ASNase treatment resistance in the ALL model cell line. We found that deletion of CTSB in REH ALL cells did not confer ASNase treatment sensitivity, thus suggesting that intrinsic expression of CTSB is not a mechanism that drives the resistant nature of these ALL cells to enzymes used as the first-line treatment against leukemia.
Assuntos
Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Asparaginase/farmacologia , Asparaginase/metabolismo , Fator Intrínseco/uso terapêutico , Catepsina B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linhagem Celular , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Hepatitis C is considered a major public health problem caused by the hepatitis C virus (HCV). Viral infections are known to induce production of IL1ß through the signaling pathway of inflammasomes. Emerging evidences suggest that Inflammasome genes may influence the immune response against HCV as the host genetic background may contribute to the balance between acute and chronic inflammation. We investigated in 151 patients with chronic hepatitis C and 206 healthy blood donors' individuals (HD). Polymorphisms in the IL1B and IL18 genes were genotyped by PCR-RFLP, while NLRP3, CARD8, CTSB and AIM2 by RT- PCR. Serum assay of IL-1ß cytokine was performed by ELISA. 84 patients presented mild fibrosis (Assuntos
Predisposição Genética para Doença
, Hepatite C/genética
, Inflamassomos/genética
, Polimorfismo de Nucleotídeo Único
, Adulto
, Idoso
, Alelos
, Brasil
, Proteínas Adaptadoras de Sinalização CARD/genética
, Catepsina B/genética
, Proteínas de Ligação a DNA/genética
, Feminino
, Frequência do Gene
, Genótipo
, Humanos
, Interleucina-18/genética
, Interleucina-1beta/sangue
, Interleucina-1beta/genética
, Masculino
, Pessoa de Meia-Idade
, Proteína 3 que Contém Domínio de Pirina da Família NLR/genética
, Proteínas de Neoplasias/genética
, Adulto Jovem
RESUMO
Cysteine peptidases (CP) play a role as digestive enzymes in hemipterans similar to serine peptidases in most other insects. There are two major CPs: cathepsin L (CAL), which is an endopeptidase and cathepsin B (CAB) that is both an exopeptidase and a minor endopeptidase. There are thirteen putative CALs in Dysdercus peruvianus, which in some cases were confirmed by cloning their encoding genes. RNA-seq data showed that DpCAL5 is mainly expressed in the anterior midgut (AM), DpCAL10 in carcass (whole body less midgut), suggesting it is a lysosomal enzyme, and the other DpCALs are expressed in middle (MM) and posterior (PM) midgut. The expression data were confirmed by qPCR and enzyme secretion to midgut lumen by a proteomic approach. Two CAL activities were isolated by chromatography from midgut samples with similar kinetic properties toward small substrates. Docking analysis of a long peptide with several DpCALs modeled with digestive Tenebrio molitor CAL (TmCAL3) as template showed that on adapting to luminal digestion DpCALs (chiefly DpCAL5) changed in relation to their ancestral lysosomal enzyme (DpCAL10) mainly at its S2 subsite. A similar conclusion arrived from structure alignment-based clustering of DpCALs based on structural similarity of the modeled structures. Changes mostly on S2 subsite could mean the enzymes turn out less peptide-bond selective, as described in TmCALs. R. prolixus CALs changed on adapting to luminal digestion, although less than DpCALs. Both D. peruvianus and R. prolixus have two digestive CABs which are expressed in the same extension as CALs, in the first digestive section of the midgut, but less than in the other midgut sections. Mahanarva fimbriolata does not seem to have digestive CALs and their digestive CABs are mainly expressed in the first digestive section of the midgut and do not diverge much from their lysosomal counterparts. The data suggest that CABs are necessary at the initial stage of digestion in CP-dependent Hemipterans, which action is completed by CALs with low peptide-bond selectivity in Heteroptera species. In M. fimbriolata protein digestion is supposed to be associated with the inactivation of sap noxious proteins, making CAB sufficient as digestive CP. Hemipteran genomes and transcriptome data showed that CALs have been recruited as digestive enzymes only in heteropterans, whereas digestive CABs occur in all hemipterans.
Assuntos
Catepsina B/genética , Catepsina L/genética , Hemípteros/fisiologia , Proteínas de Insetos/genética , Sequência de Aminoácidos , Fenômenos Fisiológicos da Nutrição Animal , Animais , Sequência de Bases , Catepsina B/química , Catepsina B/metabolismo , Catepsina L/química , Catepsina L/metabolismo , Digestão , Hemípteros/enzimologia , Hemípteros/genética , Heterópteros/enzimologia , Heterópteros/genética , Heterópteros/fisiologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Rhodnius/enzimologia , Rhodnius/genética , Rhodnius/fisiologiaRESUMO
Mucopolysaccharidosis type I (MPS I) is caused by genetic deficiency of α-l-iduronidase and impairment of lysosomal catabolism of heparan sulfate and dermatan sulfate. In the brain, these substrates accumulate in the lysosomes of neurons and glial cells, leading to neuroinflammation and neurodegeneration. Their storage also affects lysosomal homeostasis-inducing activity of several lysosomal proteases including cathepsin B (CATB). In the central nervous system, increased CATB activity has been associated with the deposition of amyloid plaques due to an alternative pro-amyloidogenic processing of the amyloid precursor protein (APP), suggesting a potential role of this enzyme in the neuropathology of MPS I. In this study, we report elevated levels of protein expression and activity of CATB in cortex tissues of 6-month-old MPS I (Idua -/- mice. Besides, increased CATB leakage from lysosomes to the cytoplasm of Idua -/- cortical pyramidal neurons was indicative of damaged lysosomal membranes. The increased CATB activity coincided with an elevated level of the 16-kDa C-terminal APP fragment, which together with unchanged levels of ß-secretase 1 was suggestive for the role of this enzyme in the amyloidogenic APP processing. Neuronal accumulation of Thioflavin-S-positive misfolded protein aggregates and drastically increased levels of neuroinflammatory glial fibrillary acidic protein (GFAP)-positive astrocytes and CD11b-positive activated microglia were observed in Idua -/- cortex by confocal fluorescent microscopy. Together, our results point to the existence of a novel CATB-associated alternative amyloidogenic pathway in MPS I brain induced by lysosomal storage and potentially leading to neurodegeneration.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Catepsina B/metabolismo , Córtex Cerebral/metabolismo , Mucopolissacaridose I/metabolismo , Células Piramidais/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Catepsina B/genética , Córtex Cerebral/patologia , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Camundongos Knockout , Mucopolissacaridose I/genética , Mucopolissacaridose I/patologia , Células Piramidais/patologiaRESUMO
Giardia lamblia is one of the most common protozoan infectious agents in the world and is responsible for diarrheal disease and chronic postinfectious illness. During the host-parasite interaction, proteases are important molecules related to virulence, invasion, and colonization, not only for Giardia but also for other parasites. We aimed to characterize the cysteine protease activity detected in trophozoite lysates. This proteolytic activity showed the ability to cleave NH-terminal sequences with either a recognition sequence for a viral protease or a recognition sequence for thrombin. This cleavage activity was detected in nonencysting trophozoites and increased with the progression of encystation. This activity was also detected in excretion/secretion products of axenic trophozoites and in trophozoites cocultured with differentiated Caco-2 cells. Based on size exclusion chromatography, we obtained a fraction enriched in low- to medium-molecular-weight proteins that was capable of exerting this cleavage activity and aggregating human platelets. Finally, our results suggest that this proteolytic activity is shared with other protozoan parasites.
Assuntos
Cisteína Proteases/metabolismo , Giardia lamblia/enzimologia , Proteínas de Protozoários/metabolismo , Células CACO-2 , Catepsina B/química , Catepsina B/genética , Catepsina B/metabolismo , Cisteína Proteases/química , Cisteína Proteases/genética , Giardia lamblia/química , Giardia lamblia/genética , Giardíase , Humanos , Proteólise , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Especificidade por Substrato , Trofozoítos/química , Trofozoítos/enzimologia , Trofozoítos/genéticaRESUMO
The potential role of Blastocystis as a pathogen is controversial because it is found in both symptomatic and asymptomatic carriers. Since Cathepsin B has been identified as a main virulence factor that contributes to the pathogenesis of this parasite, the purpose of this study was to analyze the genetic polymorphisms of cathepsin B from Blastocystis from patients with irritable bowel syndrome and from asymptomatic carriers. DNA from fecal samples of both groups, which were previously genotyped by 18S sequencing, was used to amplify a fragment of the cathepsin B gene. Phylogenetic reconstructions were performed and some genetic population indexes were obtained. Amplicons of 27 samples (15 cases, 10 controls, and two commercial ATCC strains) were obtained and analyzed. Phylogenetic reconstructions using nucleotides or inferred amino acid sequences did not separate between cases or controls or among subtypes. Regarding the values of genetic variability, we found that the haplotype and nucleotide diversity indexes of cathepsin B from cases and controls were similar to the values of 18S from controls. By contrast, 18S from cases showed low variability, suggesting that the genetic variability of cathepsin B was not related to the symptomatology of Blastocystis carriers. However, since no polymorphisms related to cases or controls were found, it is logical to assume that the potential damage caused by Blastocystis in situ may be due to unclear mechanisms of Cathepsin B regulation and expression that should be studied in future studies.
Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis/genética , Blastocystis/patogenicidade , Catepsina B/genética , Síndrome do Intestino Irritável/parasitologia , Adulto , Sequência de Aminoácidos/genética , Blastocystis/classificação , Fezes/parasitologia , Feminino , Genética Populacional , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Polimorfismo Genético , Fatores de Virulência/genéticaRESUMO
Tuberculosis (TB) continues to be a major public health problem. An estimated one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb) but remains asymptomatic (latent TB) and only 5% to 10% of these latent individuals will develop active pulmonary TB. Factors affecting the balance between latent and active TB are mostly unknown, even if host genome has been shown to contribute to the outcome of Mtb response. Acute inflammation and Th1 response are important in the early clearance of the bacteria as it was emphasized by the association between immune genes (i.e.: HLA, IFNG, TNF, NRPAM1, IL10) variants and the development of active pulmonary TB. Recently, the role of the inflammasome in experimental TB has been demonstrated, however, to our knowledge, no data still exist about the contribution of inflammasome genetics to Mtb susceptibility and/or to the development of active TB. For this reason, selected polymorphisms in inflammasome genes were analysed in a case/control cohort of individuals with active pulmonary TB from an endemic area of Brazil Amazon. Our data evidence the novel association between polymorphisms in NLRP3-inflammasome encoding genes and active pulmonary TB, and replicated the association between P2X7 and TB observed in other populations. These results emphasize the role of NLRP3-inflammasome also in human TB, and contribute to our knowledge about pathways involved in the development of active TB, even if deeper investigation are needed to fully elucidate the role of the complex in Mtb infection.
Assuntos
Catepsina B/genética , Resistência à Doença/genética , Inflamassomos/genética , Tuberculose Latente/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Receptores Purinérgicos P2X7/genética , Tuberculose Pulmonar/genética , Adulto , Brasil , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Estudos de Casos e Controles , Catepsina B/imunologia , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Genótipo , Humanos , Inflamassomos/imunologia , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Tuberculose Latente/patologia , Pulmão/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Polimorfismo de Nucleotídeo Único , Receptores Purinérgicos P2X7/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 µM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 µM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 µM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.
Assuntos
Catepsina B/metabolismo , Citrus/parasitologia , Hemípteros/enzimologia , Doenças das Plantas/prevenção & controle , Doenças das Plantas/parasitologia , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise/efeitos dos fármacos , Catepsina B/química , Catepsina B/genética , Catepsina B/isolamento & purificação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemípteros/efeitos dos fármacos , Hemípteros/genética , Hemípteros/crescimento & desenvolvimento , Larva/genética , Dados de Sequência Molecular , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de Proteína , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
Cysteine proteinases (cathepsins) from Leishmania spp. are promising molecular targets against leishmaniasis. Leishmania mexicana cathepsin L is essential in the parasite life cycle and a pivotal in virulence factor in mammals. Natural products that have been shown to display antileishmanial activity were screened as part of our ongoing efforts to design inhibitors against the L. mexicana cathepsin L-like rCPB2.8. Among them, agathisflavone (1), tetrahydrorobustaflavone (2), 3-oxo-urs-12-en-28-oic acid (3), and quercetin (4) showed significant inhibitory activity on rCPB2.8 with IC50 values ranging from 0.43 to 18.03 µM. The mechanisms of inhibition for compounds 1-3, which showed Ki values in the low micromolar range (Ki = 0.14-1.26 µM), were determined. The biflavone 1 and the triterpene 3 are partially noncompetitive inhibitors, whereas biflavanone 2 is an uncompetitive inhibitor. The mechanism of action established for these leishmanicidal natural products provides a new outlook in the search for drugs against Leishmania.
Assuntos
Produtos Biológicos/farmacologia , Catepsina L/antagonistas & inibidores , Leishmania mexicana/enzimologia , Biflavonoides/farmacologia , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsina L/genética , Humanos , Concentração Inibidora 50 , Cinética , Leishmania mexicana/genética , Quercetina/farmacologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genéticaRESUMO
Despite the established role of Culex quinquefasciatus as a vector of various neurotropic viruses, such as the Rift Valley and West Nile viruses, as well as lymphatic filariasis, little is known regarding the organism's reproductive physiology. As in other oviparous animals, vitellogenin, the most important source of nutrients for the embryo development, is digested by intracellular proteases. Using mass spectrometry, we have identified two cathepsin B homologues partially purified by self-proteolysis of Cx. quinquefasciatus total egg extract. The transcriptional profile of these two cathepsin B homologues was determined by quantitative RT-PCR, and the enzymatic activity associated with the peptidase was determined in ovaries after female engorgement. According to the VectorBase (vectorbase.org) annotation, both cathepsin B homologues shared approximately 66% identity in their amino acid sequences. The two cathepsin B genes are expressed simultaneously in the fat body of the vitellogenic females, and enzymatic activity was detected within the ovaries, suggesting an extra-ovarian origin. Similar to the transcriptional profile of vitellogenin, cathepsin B transcripts were shown to accumulate post-blood meal and reached their highest expression at 36 h PBM. However, while vitellogenin expression decreased drastically at 48 h PBM, the expression of the cathepsins increased until 84 h PBM, at which time the females of our colony were ready for oviposition. The similarity between their transcriptional profiles strongly suggests a role for the cathepsin B homologues in vitellin degradation.