RESUMO
This study investigated the inhibitory activity of serine protease, as well as antibacterial and antibiotic modifying activities of the crude extract and fractions of A. cearensis seeds. Microdilution assay was used to evaluate the antibacterial activity and the antibiotic resistance-modulating effects of samples against multiresistant bacteria Staphylococcus aureus (SA10) and Escherichia coli (EC06). In the inhibition test for serine protease, all the samples showed inhibition of enzymatic activity. Crude extract and fractions of A. cearensis seeds showed a Minimum Inhibitory Concentration ≥1024⯵g/mL for all microorganisms tested. However, the samples acted as resistance modifying agent, presenting synergism when associated with gentamicin, norfloxacin and penicillin. The present study provides data indicating a possible use of the seeds extract of A. cearensis in association with antibiotics in the fight against bacterial infections.
Assuntos
Antibacterianos/farmacologia , Fabaceae/química , Extratos Vegetais/farmacologia , Inibidores de Serina Proteinase/farmacologia , Antibacterianos/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Extratos Vegetais/isolamento & purificação , Sementes/química , Inibidores de Serina Proteinase/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacosRESUMO
Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin.
Assuntos
Elastase de Leucócito/isolamento & purificação , Sementes/química , Inibidores de Serina Proteinase/isolamento & purificação , Vigna/química , Animais , Bioensaio , Bovinos , Extratos Vegetais/químicaRESUMO
The immobilization of Enterolobium contortisiliquum protease inhibitor, EcTI-Sepharose, as an affinity chromatography matrix is a powerful biotechnological tool to purify targets from Nasutitermes corniger in the investigation of insecticidal properties of natural compounds.
Assuntos
Fabaceae/química , Isópteros/efeitos dos fármacos , Isópteros/enzimologia , Peptídeo Hidrolases/química , Inibidores de Serina Proteinase/farmacologia , Animais , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/enzimologia , Concentração Inibidora 50 , Estrutura Secundária de Proteína , Sementes/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/isolamento & purificaçãoRESUMO
Two new iridoids (1-2) and a new decomposition product of valepotriates (3), together with fifteen known compounds (4-18) were isolated from the roots and rhizomes of Valeriana polystachya Smith, a native species from the Pampa Biome. Their structures were elucidated by means of NMR spectroscopy, mass spectrometry and optical rotation. The structures of 3 and 18 were further confirmed by single crystal X-ray diffraction analysis. In the group of the isolated compounds, 6ß-hydroxysitostenone, hydroxymaltol and isovillosol were isolated from the Valeriana genus for the first time. The extracts and isolated compounds were evaluated for their in vitro activities against acetylcholinesterase (AChE) and prolyloligopeptidase (POP). Compounds 7, 9 and 11 showed weak inhibitory activity against AChE, while 3 and 5 displayed exceptional POP inhibitory activity, with IC50 values of 5.3⯱â¯0.07 and 7.9⯱â¯0.4⯵M, respectively.
Assuntos
Inibidores da Colinesterase/isolamento & purificação , Iridoides/isolamento & purificação , Inibidores de Serina Proteinase/isolamento & purificação , Valeriana/química , Acetilcolinesterase , Brasil , Inibidores da Colinesterase/farmacologia , Iridoides/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Raízes de Plantas/química , Prolil Oligopeptidases , Rizoma/química , Serina Endopeptidases , Inibidores de Serina Proteinase/farmacologiaRESUMO
Serine peptidase inhibitor (serpin) is the name given to the superfamily of proteins with wide range of biological functions, and that the main feature is the inhibition of serine proteases. Here we describe the inhibitory characterization of a serpin from Gloeobacter violaceus that we named vioserpin. The serpin presented a high specificity to inhibit trypsin-like enzymes with a rapid inhibition rate constant (2.1 × 10(6) M(-1) s(-1)). We also demonstrated that the inhibitory activity of the vioserpin is influenced by the concentration of heparin, and this finding may throw a new light on understanding the molecular evolution of serpins.
Assuntos
Cianobactérias , Heparina/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Cinética , Camundongos , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/metabolismo , Tripsina/metabolismoRESUMO
The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.
Assuntos
Biflavonoides/farmacologia , Produtos Biológicos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Calicreínas Teciduais/antagonistas & inibidores , Biflavonoides/química , Biflavonoides/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Relação Dose-Resposta a Droga , Garcinia/química , Humanos , Modelos Moleculares , Conformação Molecular , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/isolamento & purificação , Relação Estrutura-Atividade , Calicreínas Teciduais/metabolismoRESUMO
Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.
Assuntos
Arabidopsis/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiologia , Botrytis/patogenicidade , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/metabolismoRESUMO
A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.
Assuntos
Antifibrinolíticos/farmacologia , Venenos Elapídicos/farmacologia , Fibrinolisina/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Animais , Antifibrinolíticos/isolamento & purificação , Venenos Elapídicos/isolamento & purificação , Elapidae , Fibrinolisina/metabolismo , Humanos , Inibidores de Serina Proteinase/isolamento & purificaçãoRESUMO
BACKGROUND: The genus Hypericum (family Clusiaceae) comprises various species that are used in traditional medicine, such as wound healing, antidepressant, and anticancer agents. OBJECTIVE: The aim of this study was to evaluate the inhibitory capacity of extracts and fractions from two Hypericum species used in the Brazilian folk medicine (H. brasiliense and H. connatum) against the enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase-IV (DPP-IV), and acetylcholinesterase (AChE), as well as to identify their main active constituents. METHODS: Dried aerial parts of H. connatum and H. brasiliense were subjected to extraction with 8:2 methanol-H2O. Each hydroalcoholic extract was fractioned resulting in ethyl acetate and aqueous fractions. The activity of POP, DPP-IV and AChE was determined in vitro in 96-well microplates. RESULTS: The main components identified in the plant extracts were chlorogenic acid (1), quercitrin (2), rutin (3), quercetin (4), and isoquercitrin (5). Hydroalcoholic extracts, ethyl acetate and aqueous fractions showed high POP inhibitory activity with IC50 values of 2.6 to 3.7 µg/mL. AChE and DPP-IV inhibitory effects were very low for all extracts and substances. CONCLUSION: Chlorogenic acid (1) and quercetin (4) were the main constituent responsible for the activity observed against POP. Parallel artificial membrane permeability assay of ethyl acetate fractions of both species showed that the metabolite that can effectively pass through the lipid membrane is 4, the aglycone form of 2, 3 and 5.
Assuntos
Acetilcolinesterase/química , Inibidores da Colinesterase/química , Extratos Vegetais/química , Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Ácido Clorogênico/química , Ácido Clorogênico/isolamento & purificação , Inibidores da Colinesterase/isolamento & purificação , Difusão , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/isolamento & purificação , Ensaios Enzimáticos , Hypericum , Membranas Artificiais , Permeabilidade , Fosfolipídeos/química , Extratos Vegetais/isolamento & purificação , Prolil Oligopeptidases , Quercetina/química , Quercetina/isolamento & purificação , Inibidores de Serina Proteinase/isolamento & purificaçãoRESUMO
Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.(AU)
Assuntos
Animais , Animais Peçonhentos , Venenos de Anfíbios , Inibidores de Serina Proteinase/isolamento & purificaçãoRESUMO
Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.
Assuntos
Antifúngicos/farmacologia , Capsicum/química , Fragmentos de Peptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antifúngicos/isolamento & purificação , Capsicum/genética , Quimera , Besouros/enzimologia , Eletroforese em Gel de Poliacrilamida , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , Saliva/enzimologia , Sementes/química , Sementes/genética , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/isolamento & purificaçãoRESUMO
Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.
Assuntos
Animais , Animais Peçonhentos , Inibidores de Serina Proteinase/isolamento & purificação , Venenos de AnfíbiosRESUMO
Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.
Assuntos
Rhipicephalus/química , Inibidores de Serina Proteinase/isolamento & purificação , Animais , Larva/química , ProteínasRESUMO
Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.
Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.
Assuntos
Animais , Rhipicephalus/química , Inibidores de Serina Proteinase/isolamento & purificação , Larva/química , ProteínasRESUMO
Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.
Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.
Assuntos
Animais , Rhipicephalus/química , Inibidores de Serina Proteinase/isolamento & purificação , Larva/química , ProteínasRESUMO
Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.
Assuntos
Trato Gastrointestinal/enzimologia , Insetos Vetores/parasitologia , Phlebotomus/enzimologia , Inibidores de Serina Proteinase/isolamento & purificação , Animais , Células CHO , Quimotripsina/metabolismo , Cricetulus , Dípteros/genética , Feminino , Expressão Gênica , Leishmaniose/prevenção & controle , Estágios do Ciclo de Vida/genética , Masculino , Psychodidae/parasitologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/metabolismo , Trombina/metabolismo , Tripsina/metabolismoRESUMO
Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.
Assuntos
Animais , Feminino , Masculino , Trato Gastrointestinal/enzimologia , Insetos Vetores/parasitologia , Phlebotomus/enzimologia , Inibidores de Serina Proteinase/isolamento & purificação , Células CHO , Cricetulus , Quimotripsina/metabolismo , Dípteros/genética , Expressão Gênica , Leishmaniose/prevenção & controle , Estágios do Ciclo de Vida/genética , Psychodidae/parasitologia , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/metabolismo , Trombina/metabolismo , Tripsina/metabolismoRESUMO
Kallikrein-related peptidases (KLKs) are trypsin-like and chymotrypsin-like serine proteases which are expressed in several tissues. Their activity is tightly controlled by inhibitors including members of the serine protease Kazal-type (SPINK) family. These enzymes are promising targets for the treatment of skin desquamation, inflammation and cancer. Spink3 or caltrin I is expressed in mouse pancreas and males accessory glands and the resulting mature protein has been associated with different activities such as an inhibitor of trypsin and acrosin activity, calcium transport inhibitor in sperm and inhibitor of cell proliferation during embryogenesis. In this study, we produced a soluble recombinant Spink3 from mouse seminal vesicle (rmSpink3) that inhibited the activity of human KLKs. Using FRET substrates, rmSpink3 exhibited a potent inhibitory activity against human KLK2, KLK3, KLK5 (Ki ranging from 260 to 1500 nM), and to a lesser extent against KLK6, KLK1 and KLK7 (Ki around 3000 nM). As shown by mass spectrometry analysis of rmSpink3 incubated with trypsin, the inhibitor was not truncated by the target enzyme. Based on the in silico analysis of the expression of Spink3/SPINK1 and KLKs it is speculated that some KLKs may be natural targets of Spink3/SPINK1, however experimental confirmation using both proteins from mouse or human origin is needed. This work shows that rmSpink3 is a potent inhibitor of various human KLK members suggesting the potential of this molecule in the diagnosis/prevention of several human diseases.
Assuntos
Glicoproteínas/farmacologia , Calicreínas/antagonistas & inibidores , Proteínas Secretadas pela Próstata/farmacologia , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Humanos , Calicreínas/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Secretadas pela Próstata/química , Proteínas Secretadas pela Próstata/genética , Proteínas Secretadas pela Próstata/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/isolamento & purificação , Inibidor da Tripsina Pancreática de KazalRESUMO
Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serine-protease inhibitor was isolated from Acacia polyphylla seeds (AcKI).Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of â¼20 kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of â¼50% with the lower concentration of AcKI used in the study (0.5%). Although additionalassays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.
Assuntos
Acacia/química , Inseticidas/isolamento & purificação , Mariposas/efeitos dos fármacos , Peptídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Animais , Digestão/efeitos dos fármacos , Inseticidas/química , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Dados de Sequência Molecular , Mariposas/crescimento & desenvolvimento , Mariposas/fisiologia , Peptídeos/química , Peptídeos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Alinhamento de Sequência , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/farmacologiaRESUMO
The phenylpropanoid glycoside verbascoside [2-(3,4-dihydroxyphenylethyl)-1-O-α-L-rhamnopyranosyl-(1â3)-ß-D-(4-O-caffeyl)-glucopyranoside] (1) has been isolated as the main constituent of the crude extract of Buddleja brasiliensis Jacq. ex Spreng from Southern Brazil. The crude extract, main fractions and the compound 1 were evaluated for inhibition of the enzymes acetylcholinesterase (AChE), dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). Compound 1 showed weak activity against DPP-IV with an IC(50) >> 150 µM and was inactive against AChE, with a pMIQ determined by bioautography of 9.6. In contrast, 1 displayed significant inhibition of POP in a dose-dependent manner with an IC(50) value of 1.3 ± 0.2 µM, similar to the positive control, baicalin, with a POP IC(50) of 12 ± 3 µM.