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1.
J Agric Food Chem ; 67(38): 10604-10613, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31466448

RESUMO

The aim of this study was to investigate the dipeptidyl peptidase-IV (DPP-IV) inhibition and metabolic stability of a casein-derived peptide Val-Pro-Tyr-Pro-Gln (VPYPQ) and its fragments as well as their release from casein following hydrolysis. Results showed that VPYPQ was the most potent DPP-IV inhibitory peptide among them with an IC50 value of 41.45 µM. This might be due to its two internal Pro residues at positions 2 and 4. Moreover, VPYPQ was resistant to hydrolysis by gastrointestinal enzymes and was relatively more stable to hydrolysis by DPP-IV and peptidases in plasma compared with its fragments. Additionally, oral administration of VPYPQ at a dose of 90 µmol/kg body weight could reduce the postprandial blood glucose levels in mice. More importantly, VPYPQ could be released efficiently from casein following hydrolysis by a combination of papain and in vitro digestion, reaching up to 3211.15 µg/g. Therefore, VPYPQ was a promising casein-derived DPP-IV inhibitor.


Assuntos
Caseínas/química , Preparações de Ação Retardada/química , Inibidores da Dipeptidil Peptidase IV/química , Peptídeos/química , Animais , Biocatálise , Glicemia/metabolismo , Preparações de Ação Retardada/administração & dosagem , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Teste de Tolerância a Glucose , Humanos , Hidrólise , Camundongos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Peptídeos/administração & dosagem , Ratos , Ratos Sprague-Dawley
2.
Adv Exp Med Biol ; 1152: 401-411, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456196

RESUMO

Understanding breast cancer cell proteolysis and migration is crucial for developing novel therapies to prevent local and distant metastases. Human cancer cells utilize many biological functions comparable to those observed during embryogenesis conferring the cancer cells with survival advantages. One such advantage is the ability to secrete proteases into the tumor microenvironment in order to remodel the extracellular matrix to facilitate migration. These proteases degrade the extracellular matrix, which initially functions as a barrier to cancer cell escape from their site of origin. The extracellular matrix also functions as a reservoir for growth factors that can be released by the secreted proteases and thereby further aid tumor growth and progression. Other survival advantages of tumor cells include: the ability to utilize multiple modes of motility, thrive in acidic microenvironments, and the tumor cell's ability to hijack stromal and immune cells to foster their own migration and survival. In order to reduce metastasis, we must focus our efforts on addressing the survival advantages that tumor cells have acquired.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Proteólise , Matriz Extracelular , Feminino , Humanos , Peptídeo Hidrolases/metabolismo , Microambiente Tumoral
3.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
4.
World J Microbiol Biotechnol ; 35(8): 122, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346836

RESUMO

To promote enzymatic unhairing for leather production, a new unhairing enzyme is developed. The Keratinase (kerT) gene, which is amplified from B. amyloliquefaciens TCCC11319 by PCR, is expressed in B. subtilis WB600. The recombinant KerT reduces the collagenolytic protease content as well as improving the keratinase content effectively. Therefore, the improved keratinase leads to the obviously unhairing effect, whereas the low collagenolytic protease ensures the integrity of collagen fibers in hide. It represents, the leather grain surface isn't destroyed thereby the value of finished leather can be maintained. In addition, by analyzing the properties of KerT, tits activity isn't inhibited with Na+, K+ and Ca2+ which are commonly used in leather production. The freeze-dried fermentation broth can be used directly as unhairing enzyme without addition of traditional sulfide chemicals. By evaluating the properties of unhaired hide, the results show that the collagen degradation ability of this new unhairing enzyme is slightly and it does not cause any adverse effects on the leather quality. Besides, this unhairing enzyme doesn't further degrade collagen in the time range of 8 h to 24 h, thus it is safely and easy-control in actual production. In conclusion, the enzymatic unhairing method with recombinant KerT has the potential to be more sustainable and efficient alternative than current sulphur-lime method, and it does not require the further purification thereby saving the cost.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Bacteriano/isolamento & purificação , Peptídeo Hidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Fragmentação do DNA , DNA Bacteriano/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Nat Commun ; 10(1): 2853, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253808

RESUMO

Plant innate immunity restricts growth of bacterial pathogens that threaten global food security. However, the mechanisms by which plant immunity suppresses bacterial growth remain enigmatic. Here we show that Arabidopsis thaliana secreted aspartic protease 1 and 2 (SAP1 and SAP2) cleave the evolutionarily conserved bacterial protein MucD to redundantly inhibit the growth of the bacterial pathogen Pseudomonas syringae. Antibacterial activity of SAP1 requires its protease activity in planta and in vitro. Plants overexpressing SAP1 exhibit enhanced MucD cleavage and resistance but incur no penalties in growth and reproduction, while sap1 sap2 double mutant plants exhibit compromised MucD cleavage and resistance against P. syringae. P. syringae lacking mucD shows compromised growth in planta and in vitro. Notably, growth of ΔmucD complemented with the non-cleavable MucDF106Y is not affected by SAP activity in planta and in vitro. Our findings identify the genetic factors and biochemical process underlying an antibacterial mechanism in plants.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Serina Endopeptidases/metabolismo , Arabidopsis/imunologia , Proteínas de Bactérias/genética , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidases/genética
6.
Nat Commun ; 10(1): 2876, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253842

RESUMO

Osteoarthritis (OA) is a common, painful disease. Currently OA is incurable, and its etiology largely unknown, partly due to limited understanding of OA as a whole-joint disease. Here we report that two homologous microRNAs, miR-204 and miR-211, maintain joint homeostasis to suppress OA pathogenesis. Specific knockout of miR-204/-211 in mesenchymal progenitor cells (MPCs) results in Runx2 accumulation in multi-type joint cells, causing whole-joint degeneration. Specifically, miR-204/-211 loss-of-function induces matrix-degrading proteases in articular chondrocytes and synoviocytes, stimulating articular cartilage destruction. Moreover, miR-204/-211 ablation enhances NGF expression in a Runx2-dependent manner, and thus hyper-activates Akt signaling and MPC proliferation, underlying multiplex non-cartilaginous OA conditions including synovial hyperplasia, osteophyte outgrowth and subchondral sclerosis. Importantly, miR-204/-211-deficiency-induced OA is largely rescued by Runx2 insufficiency, confirming the miR-204/-211-Runx2 axis. Further, intraarticular administration of miR-204-expressing adeno-associated virus significantly decelerates OA progression. Collectively, miR-204/-211 are essential in maintaining healthy homeostasis of mesenchymal joint cells to counteract OA pathogenesis.


Assuntos
Células-Tronco Mesenquimais/fisiologia , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Animais , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Osteoartrite/etiologia , Osteoartrite/patologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Sinoviócitos/metabolismo
7.
Food Chem ; 297: 124931, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253328

RESUMO

Enzyme hydrolysis of corn gluten meal (CGM) is a promising process to prepare oligopeptides with high Fischer ratios (HFOPs). However, the relationship between Fischer ratios and enzyme hydrolysis approaches remains poorly understood. In this study, peptidomes of varying corn protein hydrolysates (CPHs) before and after activated carbon adsorption were profiled and analyzed according to sequence composition and chain length. Fischer ratios of HFOPs depended on sequences in CPHs by differing enzyme hydrolysis approaches, especially branched-chain amino acid (BCAA)-aromatic amino acid (AAA)-containing-oligopeptides. Activated carbon adsorption increased BCAA-containing-oligopeptide contents and decreased oligopeptide contents including AAAs, preferring BCAA-AAA-containing-oligopeptides with long chain length. Employing a three-enzyme hydrolysis approach, HFOPs were obtained with a yield of 49%, comprising 90% of dipeptides and tripeptides and possessing additional bioactivities. This work revealed the mechanism of HFOP production depending on the release and selective removal of oligopeptides and confirmed CGM was a promising alternative for value-added HFOP production.


Assuntos
Glutens/metabolismo , Oligopeptídeos/química , Zea mays/metabolismo , Adsorção , Aminoácidos Aromáticos/química , Aminoácidos de Cadeia Ramificada/química , Carbono/química , Cromatografia Líquida de Alta Pressão , Hidrólise , Peso Molecular , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Espectrometria de Massas em Tandem
8.
Food Chem ; 295: 599-606, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174801

RESUMO

The impact of Aqualysin 1 (Aq1), the thermo-active peptidase of Thermus aquaticus, on wheat albumin, globulin, gliadin and glutenin proteins during heat treatment of wheat dough and bread baking was examined. The level of protein extractable in sodium dodecyl sulfate containing medium under non-reducing conditions (SDS-EP-NR) from wheat dough decreases upon heating to a lesser extent when Aq1 is used than in control experiments. The higher SDS-EP-NR level is caused by the release by Aq1 of peptides from the repetitive gluten protein domains during baking. These peptides are also extractable from bread crumb with salt solution. The resultant thermoset gluten network in bread crumb is mainly made up by protein from non-repetitive gluten domains.


Assuntos
Pão/análise , Glutens/química , Peptídeo Hidrolases/metabolismo , Thermus/enzimologia , Triticum/metabolismo , Culinária , Farinha/análise , Glutens/metabolismo , Peso Molecular , Dodecilsulfato de Sódio/química , Temperatura Ambiente
9.
Plant Sci ; 285: 132-140, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203877

RESUMO

Xanthomonas campestris pv. campestris (Xcc)- responsive soluble and cell wall-bound hydroxycinnamic acids (HAs) and flavonoids accumulation in relation to hormonal changes in two Brassica napus cultivars contrasting disease susceptibility were interpreted with regard to the disease resistance. At 14-day post inoculation with Xcc, disease resistance in cv. Capitol was distinguished by an accumulation of specific (HAs) and flavonoids particularly in cell-wall bound form, and was characterized by higher endogenous jasmonic acid (JA) resulting in a decrease of JA-based balance with other hormones, as well as enhanced expression of JA signaling that was concurrently based on upregulation of PAP1 (production of anthocyanin pigment 1), MYB transcription factor, and phenylpropanoid biosynthetic genes. Fourier transform infrared spectra confirmed higher amounts of esterified phenolic acids in cv. Capitol. These results indicate that enhanced JA levels and signaling in resistant cultivar was associated with a higher accumulation of HAs and flavonoids, particularly in the cell wall-bound form, and vice versa in the susceptible cultivar (cv. Mosa) with enhanced SA-, ABA-, and CK- levels and signaling. Thus the JA-mediated phenolic metabolites accumulation is an important feature for the management and breeding program to develop disease-resistant B. napus cultivar.


Assuntos
Brassica napus/imunologia , Parede Celular/metabolismo , Ácidos Cumáricos/metabolismo , Ciclopentanos/metabolismo , Resistência à Doença , Oxilipinas/metabolismo , Fenóis/metabolismo , Reguladores de Crescimento de Planta/fisiologia , Xanthomonas campestris , Brassica napus/metabolismo , Brassica napus/microbiologia , Brassica napus/fisiologia , Parede Celular/fisiologia , Resistência à Doença/fisiologia , Suscetibilidade a Doenças/microbiologia , Suscetibilidade a Doenças/fisiopatologia , Flavonoides/metabolismo , Peroxidação de Lipídeos , Microscopia Eletrônica de Varredura , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo
10.
Biochemistry (Mosc) ; 84(Suppl 1): S1-S18, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213192

RESUMO

Various sensors designed for optical and photo(opto)acoustic imaging in living systems are becoming essential components of basic and applied biomedical research. Some of them including those developed for determining enzyme activity in vivo are becoming commercially available. These sensors can be used for various fluorescent signal detection methods: from whole body tomography to endoscopy with miniature cameras. Sensor molecules including enzyme-cleavable macromolecules carrying multiple quenched near-infrared fluorophores are able to deliver their payload in vivo and have long circulation time in bloodstream enabling detection of enzyme activity for extended periods of time at low doses of these sensors. In the future, more effective "activated" probes are expected to become available with optimized sensitivity to enzymatic activity, spectral characteristics suitable for intraoperative imaging of surgical field, biocompatibility and lack of immunogenicity and toxicity. New in vivo optical imaging methods such as the fluorescence lifetime and photo(opto)acoustic imaging will contribute to early diagnosis of human diseases. The use of sensors for in vivo optical imaging will include more extensive preclinical applications of experimental therapies. At the same time, the ongoing development and improvement of optical signal detectors as well as the availability of biologically inert and highly specific fluorescent probes will further contribute to the introduction of fluorescence imaging into the clinic.


Assuntos
Técnicas Biossensoriais/métodos , Diagnóstico Precoce , Corantes Fluorescentes/química , Imagem Óptica/métodos , Peptídeo Hidrolases/metabolismo , Animais , Modelos Animais de Doenças , Humanos
11.
Lett Appl Microbiol ; 69(2): 88-95, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102470

RESUMO

Natural enzyme inhibitors have been widely described in literature because of its pharmacological and cosmetic applications. Fungi found in caves represent a promising source of bioactive substances that are still little explored scientifically. Thus, the present work evaluated the presence of enzymatic modulators in a filtrate obtained from the cultivation of the cave fungus Lecanicillium aphanocladii (Family: Cordycipitaceae). Snake venoms from Bothrops alternatus and Bothrops atrox were used as an enzymatic source for the induction of the phospholipase, proteolytic, thrombolytic, cytotoxic and coagulant activities. Compounds present in the fungal filtrate inhibited 50, 23·8, 26·6, 50·9 and 52·5% of the proteolytic, phospholipase, haemolytic, thrombolytic and coagulant activities respectively. The filtrate was not cytotoxic on erythrocytes, but induced partial dissolution of thrombi. Fungal enzyme inhibitors that have low or no toxicity and can be obtained on a large scale and at low cost are relevant in the medical-scientific context. Therefore, the inhibition of phospholipases A2 and proteases observed in the present work highlights the potential of fungal metabolites for the development of drugs that can be used in the treatment of haemostasis and inflammation-related disorders. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, secondary metabolites synthesized by Lecanicillium aphanocladii, a fungus isolated from caves, demonstrated modulating action on proteases and phospholipases A2 present in snake venoms of the Bothrops genus, widely used as tools for the study of pathophysiology processes related to haemostasis and inflammation. The results suggest the possibility of future applications for these metabolites in the development of pharmaceuticals of medical-scientific interest.


Assuntos
Ascomicetos/química , Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Peptídeo Hidrolases/metabolismo , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Inibidores de Proteases/farmacologia , Animais , Ascomicetos/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemostasia/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Proteólise/efeitos dos fármacos
12.
Plant Physiol Biochem ; 141: 40-50, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128562

RESUMO

Agrobacterium-mediated soybean transformation has been greatly improved in recent years, however the transformation efficiency is still low and highly genotype-dependent when compared to other species. Here, we characterized seventeen soybean genotypes based on their genetic transformation efficiencies, i.e., high and low, during Agrobacterium-mediated transformation. To reveal the molecular basis of this transformation difference, we constructed a highly efficient transient transgene expression system using soybean cotyledon protoplasts and then assess the methylation levels of promoter and coding regions of an EYFP (enhanced yellow fluorescent protein) gene introduced into the protoplast cultures of various soybean genotypes using BSP (bisulfite sequencing PCR). Increased methylation was found to be associated with the considerably decreased transfection efficiency (as percentage of EYFP fluorescent protoplasts) in low-efficacy genotypes as compared with those in high-efficacy on three DAT (day after transfection). 5-Azacytidine (5-Azac), a demethylating reagent commonly applied in epigenetic researches, significantly improved the transient transfection efficiency and transgene expression level in low-efficiency genotypes. Furthermore, the shoot regeneration efficiency in low-efficiency genotypes was substantially increased by 5-Azac treatment in an Agrobacterium-mediated soybean transformation system. Taken together, we concluded that lower methylation level in transgene contributed to enhanced shoot regeneration in Agrobacterium-mediated soybean transformation.


Assuntos
Agrobacterium tumefaciens/genética , Azacitidina/farmacologia , Técnicas de Transferência de Genes , Brotos de Planta/efeitos dos fármacos , Soja/efeitos dos fármacos , Cotilédone/genética , Metilação de DNA , Fabaceae/genética , Genes de Plantas , Genótipo , Metilação , Peptídeo Hidrolases/metabolismo , Plantas Geneticamente Modificadas/genética , Protoplastos , Regeneração , Análise de Sequência de DNA , Soja/genética , Transfecção , Transformação Genética , Transgenes
13.
Biochimie ; 163: 50-57, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078584

RESUMO

Acylpeptidyl-oligopeptidase (AOP), which has been recently identified as a novel enzyme in a periodontopathic bacterium, Porphyromonas gingivalis, removes di- and tri-peptides from N-terminally acylated polypeptides, with a preference for hydrophobic P1-position amino acid residues. To validate enzymatic properties of AOP, characteristics of two bacterial orthologues from Bacteroides dorei (BdAOP), a Gram-negative intestinal rod, and Lysinibacillus sphaericus (LsAOP), a Gram-positive soil rod, were determined. Like P. gingivalis AOP (PgAOP), two orthologues more efficiently hydrolyzed N-terminal acylated peptidyl substrates than non-acylated ones. Optimal pH was shifted from 7.0 to 8.9 for N-acylated to 8.5-9.5 for non-acylated substrates, indicating preference for non-charged hydrophobic N-terminal residues. Hydrophobic P1- and P2-position preferences were common in the three AOPs, although PgAOP preferred Leu and the others preferred Phe at the P1 position. In vitro mutagenesis demonstrated that Phe647 in PgAOP was responsible for the P1 Leu preference. In addition, bacterial AOPs commonly liberated acetyl-Ser1-Tyr2 from α-melanocyte-stimulating hormone. Taken together, although these three bacterial AOPs exhibited some variations in biochemical properties, the present study demonstrated the existence of a group of exopeptidases that preferentially liberates mainly dipeptides from N-terminally acylated polypeptides with a preference for hydrophobic P1 and P2-position residues.


Assuntos
Peptídeo Hidrolases/metabolismo , Porphyromonas gingivalis/enzimologia , Bacillaceae/enzimologia , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Hidrólise , Cinética , Peptídeo Hidrolases/efeitos dos fármacos , Especificidade por Substrato
14.
Artigo em Inglês | MEDLINE | ID: mdl-31129293

RESUMO

Adaptive mechanisms underlying the long-term existence of intestinal parasites in their enzymatically hostile environment are still poorly understood, particularly with regard to fish cestodes. The study describes the activity distribution of proteolytic enzymes along the gut of the bream Abramis brama infected with intestinal cestodes Caryophyllaeus laticeps and characterizes the capacity of these worms to inhibit host proteinase activity. Mucosal proteolytic activity was mainly presented by serine proteinases. The research revealed an insignificant increase in total proteolytic activity from anterior and middle to posterior part of the gut accompanied with changes in proportions of various proteinase subclasses along the intestine. The trypsin (but not chymotrypsin) activity in the posterior section was significantly higher than in the mid-section. Both the incubation medium of the worms and their extract had a significant inhibitory effect on mucosal proteolytic activity and commercial trypsin samples. In both instances, the effect was comparable with that of a synthetic serine protease inhibitor, PMSF. SDS-PAGE electrophoregrams of the incubation medium of C. laticeps and its extract revealed three common protein bands, with apparent molecular masses from 19 to 47 kDa, possibly responsible for the worms' inhibitory capacities. According to casein-zymography performed, the target host proteinases for a putative cestode inhibitor (inhibitors) have an approximate molecular weight of 28-53 kDa. A comparative test with the extracts from three other cestodes showed that each of them can suppress the proteolytic activity of the bream mucosa. The level of inhibitory activity was found to increase with protein content in the extracts of these tapeworms.


Assuntos
Infecções por Cestoides/veterinária , Cyprinidae/metabolismo , Cyprinidae/parasitologia , Doenças dos Peixes/enzimologia , Doenças dos Peixes/parasitologia , Peptídeo Hidrolases/metabolismo , Animais , Cestoides/metabolismo , Cestoides/patogenicidade , Infecções por Cestoides/enzimologia , Infecções por Cestoides/parasitologia , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/metabolismo , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Mucosa Intestinal/enzimologia , Mucosa Intestinal/parasitologia , Peso Molecular , Peptídeo Hidrolases/isolamento & purificação , Perciformes , Inibidores de Proteases/metabolismo
15.
Prep Biochem Biotechnol ; 49(7): 718-726, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050583

RESUMO

A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655 U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29 kDa. The optimal temperature and pH of the protease were 45 °C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg2+, Zn2+, K+, Na+ and Tween-80 could improve its activity. The calculated Km and Vmax values of the protease towards α-casein were 12.74 mg/mL and 28.57 µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.


Assuntos
1-Propanol/metabolismo , Bacillus/enzimologia , Peptídeo Hidrolases/metabolismo , Bacillus/química , Bacillus/metabolismo , Detergentes/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Íons/metabolismo , Metais/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Solventes/metabolismo , Especificidade por Substrato , Tensoativos/metabolismo , Temperatura Ambiente
16.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035313

RESUMO

Seed storage proteins must be hydrolyzed by proteases to deliver the amino acids essential for embryo growth and development. Several groups of proteases involved in this process have been identified in both the monocot and the dicot species. This review focuses on the implication of proteases during germination in two cereal species, barley and wheat, where proteolytic control during the germination process has considerable economic importance. Formerly, the participation of proteases during grain germination was inferred from reports of proteolytic activities, the expression of individual genes, or the presence of individual proteins and showed a prominent role for papain-like and legumain-like cysteine proteases and for serine carboxypeptidases. Nowadays, the development of new technologies and the release of the genomic sequences of wheat and barley have permitted the application of genome-scale approaches, such as those used in functional genomics and proteomics. Using these approaches, the repertoire of proteases known to be involved in germination has increased and includes members of distinct protease families. The development of novel techniques based on shotgun proteomics, activity-based protein profiling, and comparative and structural genomics will help to achieve a general view of the proteolytic process during germination.


Assuntos
Germinação/fisiologia , Hordeum/enzimologia , Hordeum/fisiologia , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Triticum/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Germinação/genética , Peptídeo Hidrolases/genética , Proteínas de Plantas/genética
17.
MBio ; 10(2)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040245

RESUMO

Staphylococcus aureus has the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (ΔpckA) and glutamate dehydrogenase (ΔgudB), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for S. aureus growth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can support S. aureus growth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting that S. aureus has the ability to detect peptides in the environment.IMPORTANCE Staphylococcus aureus has the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients for S. aureus Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin support S. aureus growth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by which S. aureus senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Animais , Análise Mutacional de DNA , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL
18.
J Microbiol ; 57(6): 498-508, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054137

RESUMO

Outer membrane vesicles (OMVs) are nanostructures of 20-200 nm diameter deriving from the surface of several Gram-negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical "fingerprint", suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Vesículas Secretórias/química , Vesículas Secretórias/enzimologia , Sphingomonadaceae/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Exocitose , Ácidos Graxos/análise , Humanos , Queratinócitos/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Nanopartículas , Peptídeo Hidrolases/metabolismo , Proteômica/métodos , Sphingomonadaceae/citologia
19.
mSphere ; 4(3)2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068431

RESUMO

The human malaria parasite Plasmodium falciparum causes disease as it replicates within the host's erythrocytes. We have found that an erythrocyte serine hydrolase, acylpeptide hydrolase (APEH), accumulates within developing asexual parasites. Internalization of APEH was associated with a proteolytic event that reduced the size of the catalytic polypeptide from 80 to 55 kDa. A triazole urea APEH inhibitor, termed AA74-1, was employed to characterize the role of parasite-internalized APEH. In cell lysates, AA74-1 was a potent and highly selective inhibitor of both host erythrocyte and parasite-internalized APEH. When added to cultures of ring-stage parasites, AA74-1 was a poor inhibitor of replication over one asexual replication cycle; however, its potency increased dramatically after a second cycle. This enhancement of potency was not abrogated by the addition of exogenous isopentenyl pyrophosphate, the sole essential product of apicoplast metabolism. High-potency inhibition of parasite growth could be effected by adding AA74-1 to schizont-stage parasites, which resulted in parasite death at the early trophozoite stage of the ensuing replication cycle. Analysis of APEH inhibition in intact cultured cells revealed that host erythrocyte APEH, but not the parasite-internalized APEH pool, was inhibited by exogenous AA74-1. Our data support a model for the mode of parasiticidal activity of AA74-1 whereby sustained inactivation of host erythrocyte APEH is required prior to merozoite invasion and during parasite asexual development. Together, these findings provide evidence for an essential catalytic role for parasite-internalized APEH.IMPORTANCE Nearly half a million deaths were attributed to malaria in 2017. Protozoan parasites of the genus Plasmodium cause disease in humans while replicating asexually within the host's erythrocytes, with P. falciparum responsible for most of the mortality. Understanding how Plasmodium spp. have adapted to their unique host erythrocyte environment is important for developing malaria control strategies. Here, we demonstrate that P. falciparum coopts a host erythrocyte serine hydrolase termed acylpeptide hydrolase. By showing that the parasite requires acylpeptide hydrolase activity for replication, we expand our knowledge of host cell factors that contribute to robust parasite growth.


Assuntos
Eritrócitos/enzimologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Peptídeo Hidrolases/metabolismo , Plasmodium falciparum/fisiologia , Reprodução Assexuada , Células Cultivadas , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo
20.
Talanta ; 200: 236-241, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036179

RESUMO

We report herein a rationally designed pyrene linked substrate for quantitative protease activity assay via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this proof-of-concept study, a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe, Py-GGGGRG. In the presence of trypsin, the Py-GGGGRG probe can be specifically hydrolyzed into Py-GGGGR. The introduction of pyrene greatly increased ionization efficiency of Py-peptides, and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene coated MALDI plate through hydrophobic and π-π stacking interactions. As a result, trypsin activity can be directly quantified by relative intensity ratio of product and substrate via MALDI-TOF-MS without the use of external internal standard. A linear range of 0.1-10 µg/mL and a relatively low detection limit of 29 ng/mL were obtained. This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors of trypsin. Besides, the proposed strategy was also validated for another protease, chymotrypsin, by using the probe Py-GGGGGGYG. Therefore, owing to simplicity, high-throughput capacity and quantificational accuracy, the proposed method shows great potential for activity assay of various proteases and screening their inhibitors via application of specific peptide sequences.


Assuntos
Sondas Moleculares/química , Peptídeo Hidrolases/análise , Peptídeos/química , Pirenos/química , Peptídeo Hidrolases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/análise , Tripsina/metabolismo
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