RESUMO
Fish processing generates plenty of waste that is directly discarded in open-air dumps and water sources, or treated in the same way as urban solid waste, causing serious pollution problems. The waste represents a significant source of high-value bioproducts with potential applications in different industrial processes such as the production of feed, fertilizers, biodiesel and biogas, detergent additives and cosmetics. The objective of this study was to characterize and compare specific activities under different pH values and temperature conditions of acid and alkaline proteinases and viscera yield from the following fish species: Argentine hake Merluccius hubbsi, Brazilian flathead Percophis brasiliensis, Brazilian codling Urophycis brasiliensis and Stripped weakfish Cynoscion guatucupa. Individuals were fished off the coast of Mar del Plata (Argentina) by a commercial fleet and the viscera were immediately extracted and kept on ice until use. Stomach proteinases from four species had the highest activity at pH 2, with stability in the range of pH 2-4. The optimum pH was 11.5 from intestinal enzymes of C. guatucupa, M. hubbsi and P. brasiliensis and 9.5 from intestinal enzymes of U. brasiliensis. Alkaline proteinases from all species were highly stable in the range of 7-11.5. The optimum temperature of stomach proteinases from the four species studied were 30 and 50°C, with stability at 10 and 30°C during 150 min. The optimum temperature of intestinal enzymes from the tested species were 50°C with high stability at 10 and 30°C during 150 min. Alkaline proteinase from all species and acid proteinases from C. guatucupa were inactive at 70°C after 150 min, while there was a residual activity lower than 5% at 80°C on pre-incubated stomach enzymes of M.hubbsi, P. brasiliensis and U. brasiliensis after 5, 10 and 20 min, respectively. Digestive proteinases recovered in this study could be appropriate for technological usage, reducing manufacturing costs, obtaining revenue from fishery wastes, and contributing to the reduction of environmental pollution.
Assuntos
Gadiformes , Peptídeo Hidrolases , Animais , Argentina , Oceano Atlântico , PesqueirosRESUMO
A proteomic approach was used to identify the digestive enzymes secreted by exocytosis and by microapocrine vesicles and enzyme midgut compartmentalization in Spodoptera frugiperda larvae. For this, proteomic analyses were performed in isolated midgut enterocyte microvillar membrane, in a fraction enriched in microapocrine vesicles (separated in soluble and membrane fractions), in the washings of the peritrophic membrane to isolate its loosely- and tightly-bound proteins, and in the peritrophic membrane contents. PM washings correspond to proteins extracted from the mucus layer surrounding PM. Serine endopeptidases (trypsins, chymotrypsins and serine endopeptidase homologs that have substitutions in the catalytic residues) and lipases are mainly secreted by exocytosis. Aminopeptidases are mainly microvillar enzymes and some are secreted membrane-bound to microapocrine vesicles, whereas carboxypeptidase isoforms follow different secretory routes. The results also showed that most polymer hydrolases (such as amylase and endopeptidases) are not retained in the ectoperitrophic fluid (found in PM washings but absent from PM contents). On the contrary, most enzymes involved in intermediate digestion (exemplified by carboxypeptidase and aminopeptidase) do not pass through the peritrophic membrane. Finally, the data revealed that the protein composition of PM includes peritrophins classified as peritrophic membrane proteins, PMP, and chitin deacetylase.
Assuntos
Proteínas de Insetos , Proteômica , Animais , Sistema Digestório , Proteínas de Insetos/genética , Larva , SpodopteraRESUMO
A proteomic approach was used to identify the digestive enzymes secreted by exocytosis and by microapocrine vesicles and enzyme midgut compartmentalization in Spodoptera frugiperda larvae. For this, proteomic analyses were performed in isolated midgut enterocyte microvillar membrane, in a fraction enriched in microapocrine vesicles (separated in soluble and membrane fractions), in the washings of the peritrophic membrane to isolate its loosely- and tightly-bound proteins, and in the peritrophic membrane contents. PM washings correspond to proteins extracted from the mucus layer surrounding PM. Serine endopeptidases (trypsins, chymotrypsins and serine endopeptidase homologs that have substitutions in the catalytic residues) and lipases are mainly secreted by exocytosis. Aminopeptidases are mainly microvillar enzymes and some are secreted membrane-bound to microapocrine vesicles, whereas carboxypeptidase isoforms follow different secretory routes. The results also showed that most polymer hydrolases (such as amylase and endopeptidases) are not retained in the ectoperitrophic fluid (found in PM washings but absent from PM contents). On the contrary, most enzymes involved in intermediate digestion (exemplified by carboxypeptidase and aminopeptidase) do not pass through the peritrophic membrane. Finally, the data revealed that the protein composition of PM includes peritrophins classified as peritrophic membrane proteins, PMP, and chitin deacetylase.
RESUMO
Babesia, Theileria and Cytauxzoon are tick-borne apicomplexan protozoans of the order Piroplasmida, notorious for the diseases they cause in livestock, pets and humans. Host cell invasion is their Achilles heel, allowing for the development of drug or vaccine-based therapies. In other apicomplexans, cleavage of the transmembrane domain of adhesins by the serine rhomboid proteinase ROM4 is required for successful completion of invasion. In this study, we record and classify the rhomboid repertoire encoded in the genomes of 10 piroplasmid species pertaining to the lineages Babesia sensu stricto (s.s., Clade VI), Theileria sensu stricto (Clade IV), Theileria equi (Clade IV), Cytauxzoon felis (Clade IIIb) and Babesia microti (Clade I), as defined by Schnittger et al. (2012). Fifty-six piroplasmid rhomboid-like proteins were assigned by phylogenetic analysis and bidirectional best hit to the ROM4, ROM6, ROM7 or ROM8 groups, and their crucial motifs for conformation and function were identified. Forty-four of these rhomboids had either been incorrectly classified or misannotated. Babesia s.s. encode five or three ROM4 proteinase paralogs, whereas the remaining piroplasmids encode two ROM4 paralogs. All piroplasmids encode a single ROM6, ROM7 and ROM8. Thus, an increased paralog number of ROM4 is the only feature distinguishing Babesia s.s. from other piroplasmid lineages. Piroplasmid ROM6 is related to the mammalian mitochondrial rhomboid and, accordingly, N-terminal mitochondrial targeting signal sequences was found in some cases. ROM6 is the only rhomboid encoded by piroplasmids that is ubiquitous in other organisms. ROM8 represents a pseudoproteinase that is highly conserved between studied piroplasmids, suggesting that it is important in regulatory functions. ROM4, ROM6, ROM7 and ROM8 are exclusively present in Aconoidasida, which comprises piroplasmids and Plasmodium, suggesting a relevant functional role in erythrocyte invasion. The correct classification and designation of piroplasmid rhomboids presented in this study facilitates an informed choice for future in-depth study of their functions.
Assuntos
Babesia , Babesiose , Animais , Babesia/genética , Humanos , Filogenia , RNA Ribossômico 18S , Serina , Serina Proteases/genéticaRESUMO
Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (-) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (-) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.
Assuntos
Cisteína Proteases/metabolismo , Ferro/administração & dosagem , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/efeitos dos fármacos , Vagina/parasitologia , Secreções Corporais/parasitologia , Feminino , Humanos , Trichomonas vaginalis/enzimologiaRESUMO
Among the ophidians that inhabit the Northeast of Argentina, the genus Bothrops such as B. alternatus and B. diporus species (also known as yararás) and Crotalus durisus terrificus (named cascabel), represent the most studied snake venom for more than thirty years. These two genera of venomous snakes account for the majority of poisonous snake envenomations and therefore, constitute a medical emergency in this region. This review presents a broad description of the compiled knowledge about venomous snakebite: its pathophysiological action, protein composition, isolated toxins, toxin synergism, toxin-antitoxin cross-reaction assays. Properties of some isolated toxins support a potential pharmacological application.
Assuntos
Venenos de Serpentes/farmacologia , Toxinas Biológicas/farmacologia , Animais , Argentina , Bothrops , Crotalus , Humanos , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Toxinas Biológicas/química , Toxinas Biológicas/isolamento & purificaçãoRESUMO
Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (pâ¯<â¯0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (pâ¯<â¯0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.
Assuntos
Antígenos de Diferenciação/biossíntese , Citrus/química , Cistatinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Animais , Cistatinas/química , Humanos , Macrófagos/patologia , Masculino , Camundongos , Proteínas de Plantas/química , Células RAW 264.7 , Ratos , Ratos WistarRESUMO
Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 252 ± 20 µmol.min-1 mg of protein-1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min-1 mg of protein-1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.
Assuntos
Membrana Celular/metabolismo , Citosol/metabolismo , Leishmania braziliensis/enzimologia , Serina Proteases/genética , Serina Proteases/metabolismo , Cromatografia de Afinidade , Simulação por Computador , Regulação da Expressão Gênica , Leishmania braziliensis/genética , Peso Molecular , Peroxidases/genética , Peroxidases/metabolismo , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sensibilidade e Especificidade , Subtilisina/genética , Subtilisina/metabolismoRESUMO
Trichomonas vaginalis induces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose on T. vaginalis cytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface of T. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.
Assuntos
Apoptose , Cisteína Endopeptidases/metabolismo , Glucose/metabolismo , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/patogenicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Glucose/farmacologia , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Fenômenos Fisiológicos da Nutrição , Proteólise , Proteínas de Protozoários/metabolismoRESUMO
Blood clotting, long considered dependent only on platelets and enzymes called coagulation factors, is now seen as a highly regulated, balanced and multifaceted system consisting of both molecular and cellular components and is divided into three overlapping phases : initiation (plasma exposure in an endothelial lesion), amplification (adhesion and activation of more platelets and activation of factors V, VIII and XI) and propagation (formation of tenase complexes and prothrombinase). After understanding the mechanism of coagulation factors, several studies are being developed in order to produce drugs with anticoagulant functions. It is already known that inhibitors of proteinases from salivary glands of hematopoietic and plant origin, mainly of the genus Bauhinia – object of study of the present work – have been widely studied and constitute an important class of biologically active molecules with promising results. Since there is still no ideal anticoagulant and the inhibitors of proteinases isolated from the Bauhinia genus can be used in enzyme- substrate interaction studies and as potential new drugs, the present work aimed to the development of peptidic inhibitors for serine proteinases human blood coagulation. Design, synthesis, purification and identification of peptides 1A and 1B were based on the reactive factor Xa inhibitor site (BuXI) and trypsin inhibitor (BvTI), respectively. In the enzyme inhibition assays, peptides 1A and 1B were evaluated for efficacy in inhibiting human factor Xa, only peptide 1A inhibited 20% activity of that central serine proteinase from the human blood coagulation cascade. Modifications in the peptide sequence provided an improvement in the association of these peptides with the target enzyme, effectively affecting its catalytic activity. And the program was also used for the chemical characteristics of the synthetic peptides.
A coagulação sanguínea, considerada por muito tempo dependente somente de plaquetas e enzimas denominadas fatores de coagulação, atualmente é vista como um sistema altamente regulado, equilibrado e multifacetado, constituído tanto por componentes moleculares quanto celulares e é dividida em três fases que se sobrepõem entre si: iniciação (exposição do plasma em uma lesão endotelial), amplificação (adesão e ativação de mais plaquetas e ativação dos fatores V, VIII e XI) e propagação (formação dos complexos tenases e protrombinase). Após entendimento do mecanismo dos fatores de coagulação, diversos estudos estão sendo desenvolvidos a fim de, produzir fármacos com funções anticoagulantes. Já se é sabido que inibidores de proteinases provenientes de glândulas salivares de animais hemátofogos e de origem vegetal, principalmente do gênero Bauhinia – objeto de estudo do presente trabalho – têm sido amplamente estudados e constituem uma importante classe de moléculas biologicamente ativas com resultados promissores. Visto que ainda não há um anticoagulante ideal e que os inibidores de proteinases isolados do gênero Bauhinia, podem ser usados em estudos de interação enzima-substrato e como potenciais novos fármacos, o presente trabalho teve como objetivo o desenvolvimento de inibidores peptídicos para serino proteinases da coagulação sanguínea humana. Desenho, síntese, purificação e identificação dos peptídeos 1A e 1B foram baseados no sítio reativo do inibidor de fator Xa (BuXI) e do inibidor de tripsina (BvTI), respectivamente. Nos ensaios de inibição enzimática, os peptídeos 1A e 1B foram avaliados quanto à eficácia na inibição do fator Xa humano, apenas o peptídeo 1A inibiu 20% a atividade dessa serino proteinase central da cascata da coagulação sanguínea humana. Modificações na sequência peptídica proporcionaram melhoria na associação desses peptídeos à enzima alvo, afetando de maneira efetiva a atividade catalítica da mesma. E também foi utilizado o programa protparam para características químicas dos peptídeos sintéticos.
RESUMO
The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500?µL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.
RESUMO
Ontogenetic changes in venom composition have important ecological implications due the relevance of venom in prey acquisition and defense. Additionally, intraspecific venom variation has direct medical consequences for the treatment of snakebite. However, ontogenetic changes are not well documented in most species. The Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) is large-bodied and broadly distributed in Mexico. To document venom variation and test for ontogenetic changes in venom composition, we obtained venom samples from twenty-seven C. m. nigrescens with different total body lengths (TBL) from eight states in Mexico. The primary components in the venom were detected by reverse-phase HPLC, western blot, and mass spectrometry. In addition, we evaluated the biochemical (proteolytic, coagulant and fibrinogenolytic activities) and biological (LD50 and hemorrhagic activity) activities of the venoms. Finally, we tested for recognition and neutralization of Mexican antivenoms against venoms of juvenile and adult snakes. We detected clear ontogenetic venom variation in C. m. nigrescens. Venoms from younger snakes contained more crotamine-like myotoxins and snake venom serine proteinases than venoms from older snakes; however, an increase of snake venom metalloproteinases was detected in venoms of larger snakes. Venoms from juvenile snakes were, in general, more toxic and procoagulant than venoms from adults; however, adult venoms were more proteolytic. Most of the venoms analyzed were hemorrhagic. Importantly, Mexican antivenoms had difficulties recognizing low molecular mass proteins (<12 kDa) of venoms from both juvenile and adult snakes. The antivenoms did not neutralize the crotamine effect caused by the venom of juveniles. Thus, we suggest that Mexican antivenoms would have difficulty neutralizing some human envenomations and, therefore, it may be necessary improve the immunization mixture in Mexican antivenoms to account for low molecular mass proteins, like myotoxins.
Assuntos
Venenos de Serpentes/química , Animais , Antivenenos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Caseínas/química , Crotalus , Feminino , Gelatina/química , Humanos , Dose Letal Mediana , Masculino , México , Camundongos Endogâmicos ICR , Neurotoxinas/análise , Neurotoxinas/farmacologia , Proteínas de Répteis/análise , Proteínas de Répteis/farmacologia , Venenos de Serpentes/farmacologiaRESUMO
Phage display is a protein engineering approach that involves construction of libraries of variant proteins displayed on the surface of bacteriophage as capsid fusion proteins and their screening for binding and inhibitory function through the use of bait proteins. Recently, we adapted a commercially available T7 phage display system to create phage-displayed serpin libraries hypervariable in up to five positions in their reactive center loop (RCL). The RCL is a key determinant in serpin specificity, the relationship between the structure of a given serpin and which target proteinase(s) it inhibits. In this chapter, we describe protocols to assess the feasibility of this method for different serpin/proteinase combinations and share experience with this technology gathered in the course of studying two serpins and multiple proteinases with this powerful iterative screening approach.
Assuntos
Bacteriófago T7 , Biblioteca de Peptídeos , Serina Proteases , Serpinas , Animais , Bacteriófago T7/química , Bacteriófago T7/genética , Humanos , Estrutura Secundária de Proteína , Serina Proteases/química , Serina Proteases/genética , Serpinas/química , Serpinas/genéticaRESUMO
Piroplasmid parasites comprising of Babesia, Theileria, and Cytauxzoon are transmitted by ticks to farm and pet animals and have a significant impact on livestock industries and animal health in tropical and subtropical regions worldwide. In addition, diverse Babesia spp. infect humans as opportunistic hosts. Molecular phylogeny has demonstrated at least six piroplasmid lineages exemplified by B. microti, B. duncani, C. felis, T. equi, Theileria sensu stricto (T. annulata, T. parva, and T. orientalis) and Babesia sensu stricto (B. bovis, B. bigemina, and B. ovis). C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. An expansion of C1A-Cp of T. annulata and T. parva with respect to B. bovis and B. ovis was previously described. In the present work, C1A-Cp paralogs were identified in available genomes of species pertaining to each piroplasmid lineage. Phylogenetic analysis revealed eight C1A-Cp groups. The profile of C1A-Cp paralogs across these groups corroborates and defines the existence of six piroplasmid lineages. C. felis, T. equi and Theileria s.s. each showed characteristic expansions into extensive families of C1A-Cp paralogs in two of the eight groups. Underlying gene duplications have occurred as independent unique evolutionary events that allow distinguishing these three piroplasmid lineages. We hypothesize that C1A-Cp paralog families may be associated with the advent of the schizont stage. Differences in the invertebrate tick host specificity and/or mode of transmission in piroplasmid lineages might also be associated with the observed C1A-Cp paralog profiles.
RESUMO
The present study demonstrates that the Leishmania (Viannia) braziliensis strain MCAN/BR/1998/R619 is composed of multiple subpopulations with measurable distinctions. Single parasites were separated from a culture of promastigotes in stationary phase by cell sorting and then cultivated as subpopulations. Subsequently, these subpopulations were evaluated for features of in vitro growth, infectivity to murine macrophages and proteinase gene expression. The first evidence of distinct characteristics was observed during the in vitro cultivation of isolated subpopulations, as distinct clusters of patterns were formed among the cultures, indicating the existence of quantifiable fluctuations in metrics. Further, when infecting murine macrophages, the subpopulations induced distinct patterns of production of immune response mediators. While some subpopulations mainly induced the production of IL-1ß, IL-6 and TNF-α, others induced the production of IL-12p70 and nitric oxide. Finally, amastigotes of these subpopulations had higher expression of proteinase genes than promastigotes. Additionally, cysteine proteinase, serine proteinase, metalloproteinase and aspartic proteinases were differentially expressed in promastigote and amastigote forms. These data suggest the existence of distinct profiles for the L. (V.) braziliensis MCAN/BR/1998/R619 strain and subpopulations that could drive the success of parasite adaptation to the environments that they inhabit.
Assuntos
Leishmania braziliensis/crescimento & desenvolvimento , Animais , Humanos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leishmania braziliensis/classificação , Leishmania braziliensis/genética , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Trichomonas vaginalis genome encodes â¼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a â¼35â¯kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a â¼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis.
Assuntos
Ácido Aspártico Endopeptidases/química , Glucose/química , Hemoglobinas/química , Proteínas de Protozoários/química , Trichomonas vaginalis/enzimologia , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Glucose/metabolismo , Hemoglobinas/metabolismo , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/fisiologia , Trichomonas vaginalis/genéticaRESUMO
Proteinases secreted by the prostate gland have a reproductive function in cleaving proteins in the ejaculate and in the female reproductive tract, but some may have a fundamental role in disease and pathological processes including cancer. The purpose of this study was to determine if there were differences in proteinase activities in urine samples collected following prostate massage of men positive (CaP) or negative (no evidence of malignancy, NEM) for biopsy determined prostate cancer. Matrix metalloproteinase (MMP) and serine proteinase activities were detected using protein substrate zymography. There were no differences in activities of MMP-2, proMMP-9, and MMP-9/NGAL (neutrophil gelatinase associated lipocalin) complex (gelatin substrate) in men with detected prostate cancer, although the latter two were somewhat diminished. A caseinolytic activity of about 75kDa inhibited by calcium did not differ between the NEM and CaP groups. Heparin stimulated calcium sensitive gelatinolytic activities of approximately 22, 42, and 60kDa, but did not affect activities of MMP-2, MMP-9, or the 75kDa caseinolytic activity. The 22, 42, and 60kDa activities appear to be serine proteinases since they were inhibited by benzamidine. There was a significant decrease in the 22kDa heparin-stimulated serine proteinase activity in urines of men with cancer. Proteinase expression and activities, perhaps in combination with other potential markers, may prove useful in urine for detection and evaluation of prostate cancer.
Assuntos
Biomarcadores Tumorais/urina , Metaloproteinase 2 da Matriz/urina , Metaloproteinase 9 da Matriz/urina , Neoplasias da Próstata/urina , Serina Proteases/urina , Idoso , Benzamidinas/administração & dosagem , Cálcio/metabolismo , Heparina/química , Humanos , Lipocalina-2/urina , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologiaRESUMO
Background: Fascioliasis is an infectious disease caused by parasites Fasciola hepatica and F. gigantica. Humans are infected by the consumption of vegetables and water contaminated with the infective form of the parasite. Materials and Methods: In this study, an IgM-ELISA with the cysteine proteinase Fas2 antigen was evaluated with sera from 76 patients infected with F. hepatica, 24 patients with other parasite infections and 84 healthy volunteers. Results: IgM-ELISA resulted in 43% positives in F. hepatica patients with positive serology to Fas2-ELISA, but no positives resulted from testing healthy volunteers and individuals infected with other parasites. The IgM-ELISA diagnostic parameters showed a sensitivity of 43.4% (95% CI 0.321-0.553), specificity of 100% (95% CI 0.957-1), and no cross-reactivity with other parasitic infection. Interference by rheumatoid factor in the IgM immunoassay was controlled by treating sera with rheumatoid factor absorbent before testing. Conclusions: Fas2 antigen is detected by circulating IgM in patients infected with F. hepatica and IgM-ELISA using Fas2 appears as a specific immunoassay to detect the acute phase of the acute phase of F. hepatica infection in humans.
Assuntos
Anticorpos Anti-Idiotípicos/sangue , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/sangue , Cisteína Endopeptidases/sangue , Fasciola hepatica/imunologia , Fasciolíase/sangue , Imunoglobulina G/sangue , Imunoglobulina M/imunologia , Animais , Área Sob a Curva , Ensaio de Imunoadsorção Enzimática , Fasciola hepatica/enzimologia , Fasciolíase/imunologia , Humanos , Fatores Imunológicos/farmacologia , Peru , Valor Preditivo dos Testes , Curva ROC , Fator Reumatoide/farmacologia , Sensibilidade e Especificidade , Estudos SoroepidemiológicosRESUMO
The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 °C. The enzyme activity in the medium was higher when the induction was carried out at 16 °C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation.
Assuntos
Cisteína Proteases , Expressão Gênica , Pichia/metabolismo , Proteínas de Protozoários , Proteínas Recombinantes de Fusão , Trichomonas vaginalis/genética , Aspergillus niger/enzimologia , Aspergillus niger/genética , Cisteína Proteases/biossíntese , Cisteína Proteases/química , Cisteína Proteases/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Pichia/genética , Sinais Direcionadores de Proteínas/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Trichomonas vaginalis/enzimologia , alfa-Amilases/biossíntese , alfa-Amilases/química , alfa-Amilases/genéticaRESUMO
Leishmania (Viannia) braziliensis presents adaptive protease-dependent mechanisms, as cysteine proteinases B (CPB). This study investigates the expression of three cpb gene isoforms and CPB enzymatic activity during the parasite differentiation. Relative expression levels of LbrM.08.0810 gene were assessed, exhibiting a higher quantity of transcripts in the logarithmic promastigotes phase than in the stationary promastigotes phase (>1.5 times). The cbp gene tends to decrease during acid pH shock and increases when the temperature rises (>1.3 times). LbrM.08.0820 and LbrM.08.0830 genes exhibited similar expression profiles to LbrM.08.0810 gene, with lower levels being observed overall. The proteolytic activity exhibits a gradual increase during the parasite's differentiation with low levels in samples of logarithmic promastigotes phase (3.2 ± 0.08 mmol min-1 mg protein-1) to a peak of activity after 72 h of incubation at 32 °C (4.2 ± 0.026 mmol min-1 mg protein-1) followed by a subsequent decrease of 68 % of peak activity levels after 96 h of incubation at 32 °C (2.8 ± 0.37 mmol min-1 mg protein-1). These activities were also measured in the presence of selective inhibitors for cysteine proteinases, such as Z-Phe-Phe-fluoromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, demonstrating their source as cathepsin-like proteinases. To the best of our knowledge, this report presents the first description of a modulation of cathepsin L-like expression during the L. (V.) braziliensis in vitro differentiation induced by acid pH and high temperature.