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1.
Cell Mol Life Sci ; 76(16): 3193-3206, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31201463

RESUMO

Alzheimer's Disease (AD) is the sixth-leading cause of death in industrialized countries. Neurotoxic amyloid-ß (Aß) plaques are one of the pathological hallmarks in AD patient brains. Aß accumulates in the brain upon sequential, proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretases. However, so far disease-modifying drugs targeting ß- and γ-secretase pathways seeking a decrease in the production of toxic Aß peptides have failed in clinics. It has been demonstrated that the metalloproteinase meprin ß acts as an alternative ß-secretase, capable of generating truncated Aß2-x peptides that have been described to be increased in AD patients. This indicates an important ß-site cleaving enzyme 1 (BACE-1)-independent contribution of the metalloprotease meprin ß within the amyloidogenic pathway and may lead to novel drug targeting avenues. However, meprin ß itself is embedded in a complex regulatory network. Remarkably, the anti-amyloidogenic α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a direct competitor for APP at the cell surface, but also a sheddase of inactive pro-meprin ß. Overall, we highlight the current cellular, molecular and structural understanding of meprin ß as alternative ß-secretase within the complex protease web, regulating APP processing in health and disease.


Assuntos
Proteína ADAM10/metabolismo , Metaloendopeptidases/metabolismo , Proteína ADAM10/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Metaloendopeptidases/química , Presenilina-1/metabolismo , Proteólise , Serina Endopeptidases/metabolismo
2.
J Agric Food Chem ; 67(27): 7650-7659, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31241944

RESUMO

Neutrase-hydrolysates hydrolyzed from mulberry leaf proteins were separated by ion exchange chromatography, gel filtration chromatography, and semipreparative reverse-phase HPLC. Purified fractions were analyzed for their radical scavenging activity, hemolysis inhibition ability, and cellular antioxidant activity (CAA). Three new antioxidant peptides, P1 (SVL, 317 Da), P2 (EAVQ, 445 Da), and P3 (RDY, 452 Da), were obtained from the most active HPLC fraction (R1) and identified using UPLC-QTOF-MS. These three peptides were then synthesized, and their antioxidant activities were analyzed. P1 and P2 had no ability to inhibit hemolysis of erythrocytes but did show antioxidant activity on HepG2 cells. P3 showed the highest hemolysis inhibition ability (92%) and CAA value (2204 µM QE/100 g peptide). The Tyr residues at the C-terminal region play an important role in the antioxidant activity in P3. Thus, the natural peptide R1 and synthesized P3 could be used as antioxidants and might be promising components of functional foods.


Assuntos
Antioxidantes/farmacologia , Hemólise/efeitos dos fármacos , Morus/química , Peptídeos/farmacologia , Folhas de Planta/química , Proteínas de Plantas/metabolismo , Cromatografia Líquida de Alta Pressão , Células Hep G2 , Humanos , Hidrólise , Fígado/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação
3.
Microb Pathog ; 132: 230-242, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31082528

RESUMO

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation to disrupt quorum-sensing in these bacteria could pave the way for the new development in decreasing resistance strains and are of significant interest for clinical, agricultural, and industrial applications. Isolated endophytic Bacillus thuringiensis strain KMCL07 showing quorum quenching activity on Pseudomonas aeruginosa PAO1 has been studied. AiiA lactonase KMMI17 identified belongs to metallo- ß-lactamase superfamily preserving conserved regions of 106HXDH-59 amino acids-H169-21 amino acids-D191 motif, significantly inhibits the biofilm formation and attenuates virulence factor pyocyanin production of PAO1. Insilico molecular docking analysis of lactonase KMMI17 using alternative catalytic site (PDB entry: 3DHA) with the AHL-based QS system regulators of PAO-1, C4 AHL, C6 AHL and 3-oxo-C12 AHL molecules showed good binding affinity between the protein and ligands, Phe111 and Tyr198 residues plays an important role in binding them. Crude enzyme extract was found to have Km value for C6-HSL: 134.2702 ±â€¯34.83 µM-1, C4-HSL: 308.217 ±â€¯139.9 µM-1 and 3-oxo-C12-HSL: 760.463 ±â€¯251.3 µM-1. LCMS analysis confirms the degradation activity of lactonase KMMI17 on AHL molecules and its hydrolytic process, which indicates the potential application of lactonase KMMI17 as a biocontrol agent or an anti-pathogenic drug.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Lactonas/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/metabolismo , Índia , Madhuca/microbiologia , Metaloendopeptidases/metabolismo , Simulação de Acoplamento Molecular , Fenótipo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Piocianina/metabolismo , Fatores de Virulência
4.
Comput Biol Chem ; 80: 292-306, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054542

RESUMO

Human meprin beta metalloprotease, a small subgroup of the astacin family, is a potent drug target for the treatment of several disorders such as fibrosis, neurodegenerative disease in particular Alzheimer and inflammatory bowel diseases. In this study, a ligand-based pharmacophore approach has been used for the selection of potentially active compounds to understand the inhibitory activities of meprin-ß by using the sulfonamide scaffold based inhibitors. Using this dataset, a pharmacophore model (Hypo1) was selected on the basis of a highest correlation coefficient (0.959), lowest total cost (105.89) and lowest root mean square deviation (1.31 Å) values. All the pharmacophore hypotheses generated from the candidate inhibitors comprised four features: two hydrogen-bond acceptor, one hydrogen-bond donor and one zinc binder feature. The best validated pharmacophore model (Hypo1) was used for virtual screening of compounds from several databases. The selective hit compounds were filtered by drug likeness property, acceptable ADMET profile, molecular docking and DFT study. Molecular dynamic simulations with the final 10 hit compounds revealed that a large number of non-covalent interactions were formed with the active site and specificity sub-pockets of the meprin beta metalloprotease. This study assists in the development of the new lead molecules as well as gives a better understanding of their interaction with meprin-ß.


Assuntos
Metaloendopeptidases/química , Inibidores de Proteases/química , Sulfonamidas/química , Domínio Catalítico , Conjuntos de Dados como Assunto , Teoria da Densidade Funcional , Desenho de Drogas , Humanos , Ligantes , Metaloendopeptidases/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Ligação Proteica , Sulfonamidas/metabolismo
5.
Molecules ; 24(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897764

RESUMO

To investigate methods for improving the processing of porcine waste, porcine skin was hydrolyzed using different commercially available proteases (Alcalase, Flavorzyme, Neutrase, Bromeline, Protamex, and Papain) under several optimal conditions. Following enzymatic hydrolysis, the collagen hydrolysates (CHs) were fractionated by molecular weight (3 kDa) via membrane ultrafiltration. The CHs were analyzed for physical properties (pH, protein recovery, free amino group content, molecular weight distribution, and amino composition) as well as for functional properties (antioxidant activities and anti-aging activities). Among the CHs, CHs hydrolyzed by Alcalase (CH-Alcalase) exhibited the highest degree of hydrolysis compared to other CHs. Both "CH-Alcalase" and "CH-Alcalase < 3 kDa" fractions showed a considerably high antioxidant activity and collagenase inhibition activity. Therefore, resulting bioactives have potential for development as antioxidants and anti-aging ingredients in the food, cosmetics, and pharmaceuticals, from animal by-products.


Assuntos
Colágeno/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Antioxidantes , Eletroforese em Gel de Poliacrilamida , Hidrólise , Metaloendopeptidases/metabolismo , Papaína/metabolismo , Subtilisinas/metabolismo , Suínos
6.
Nat Commun ; 10(1): 1198, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867416

RESUMO

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.


Assuntos
Proteínas de Bactérias/metabolismo , Doença Celíaca/imunologia , Proteínas na Dieta/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Metaloendopeptidases/metabolismo , Receptor PAR-2/imunologia , Adulto , Idoso , Animais , Antígenos/imunologia , Antígenos/metabolismo , Proteínas de Bactérias/genética , Biópsia , Estudos de Casos e Controles , Doença Celíaca/diagnóstico por imagem , Doença Celíaca/microbiologia , Doença Celíaca/patologia , Estudos de Coortes , Colonoscopia , Proteínas na Dieta/metabolismo , Modelos Animais de Doenças , Duodeno/imunologia , Duodeno/metabolismo , Duodeno/microbiologia , Duodeno/patologia , Feminino , Microbioma Gastrointestinal/imunologia , Vida Livre de Germes , Glutens/imunologia , Glutens/metabolismo , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteólise , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Receptor PAR-2/metabolismo , Regulação para Cima , Adulto Jovem
7.
Food Chem ; 275: 696-702, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30724251

RESUMO

The RAW264.7 cell model was employed to screen immunomodulatory selenium-containing peptides from selenium-enriched rice protein hydrolysates (SPHs). Moreover, the selenium-containing peptides of high-activity protein hydrolysates were purified by Sephadex G-25, and identified by reversed phase ultra performance liquid chromatography coupled with triple quadrupole time-of-flight mass spectrometry. The results showed that 25 peptide sequences containing selenomethionine (SeMet) information above 90% of probability confidence were found in a fraction of alcalase hydrolysates. SeMDPGQQ and TSeMMM of 100% probability confidence were speculated as two novel selenium-containing peptide sequences. The artificially synthesized peptide TSeMMM was subsequently verified by an excellent immunomodulatory activity at a concentration of 80 µg/mL. In conclusion, the immunomodulatory activity of SPHs was correlated to SeMet sequence in the structure of selenium-containing peptides, and TSeMMM with a stronger immunomodulatory activity demonstrated potential as functional food additives for improving human health.


Assuntos
Oryza/metabolismo , Peptídeos/análise , Hidrolisados de Proteína/química , Selênio/química , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fatores Imunológicos/análise , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Oryza/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Células RAW 264.7 , Selenometionina/química , Sonicação
8.
Mol Cell ; 73(5): 1028-1043.e5, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733118

RESUMO

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.


Assuntos
Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/enzimologia , Proteínas Quinases/metabolismo , Motivos de Aminoácidos , Antiparkinsonianos/farmacologia , Transporte Biológico , Desenho de Drogas , Ativação Enzimática , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Development ; 146(2)2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30635283

RESUMO

The timing of Drosophila egg chamber development is controlled by a germline Delta signal that activates Notch in the follicle cells to induce them to cease proliferation and differentiate. Here, we report that follicle cells lacking the RNA-binding protein IMP go through one extra division owing to a delay in the Delta-dependent S2 cleavage of Notch. The timing of Notch activation has previously been shown to be controlled by cis-inhibition by Delta in the follicle cells, which is relieved when the miRNA pathway represses Delta expression. i mp mutants are epistatic to Delta mutants and give an additive phenotype with belle and Dicer-1 mutants, indicating that IMP functions independently of both cis-inhibition and the miRNA pathway. We find that the i mp phenotype is rescued by overexpression of Kuzbanian, the metalloprotease that mediates the Notch S2 cleavage. Furthermore, Kuzbanian is not enriched at the apical membrane in i mp mutants, accumulating instead in late endosomes. Thus, IMP regulates Notch signalling by controlling the localisation of Kuzbanian to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.


Assuntos
Desintegrinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Metaloendopeptidases/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Divisão Celular , Polaridade Celular , Epistasia Genética , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Mutação/genética , Fatores de Tempo
10.
Mol Plant Microbe Interact ; 32(7): 841-852, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30694091

RESUMO

Bacterial panicle blight caused by Burkholderia glumae is a major bacterial disease of rice. Our preliminary RNA-seq study showed that a serine metalloprotease gene, prtA, is regulated in a similar manner to the genes for the biosynthesis and transport of toxoflavin, which is a known major virulence factor of B. glumae. prtA null mutants of the virulent strain B. glumae 336gr-1 did not show a detectable extracellular protease activity, indicating that prtA is the solely responsible gene for the extracellular protease activity detected from this bacterium. In addition, inoculation of rice panicles with the prtA mutants resulted in a significant reduction of disease severity compared with the wild-type parent strain, suggesting the requirement of prtA for the full virulence of B. glumae. A double mutant deficient in both serine metalloprotease and toxoflavin (ΔtoxA/prtA-) exhibited a further numeric but not statistically significant decrease of disease development compared with the ΔtoxA strain. Both the prtA-driven extracellular protease activity and the toxoflavin production were dependent on both the tofI/tofR quorum-sensing and the global regulatory gene qsmR, indicating the important roles of the two global regulatory factors for the bacterial pathogenesis by this pathogen.


Assuntos
Burkholderia , Regulação Bacteriana da Expressão Gênica , Metaloendopeptidases , Virulência , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Burkholderia/genética , Burkholderia/patogenicidade , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Virulência/genética
11.
J Med Microbiol ; 68(3): 355-367, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30628885

RESUMO

PURPOSE: Candida albicans and Staphylococcus aureus can be co-isolated in biofilm-associated infections. However, treatments have not been well established due to a lack of antibiofilm strategies. Hence, this study aims to characterize the mechanism and impact of Staphylokinase (Sak) on fungal-bacterial polymicrobial biofilms. METHODOLOGY: Sak generation levels were obtained via chromogenic analysis. C. albicans and S. aureus polymicrobial biofilm formation and integrity were analysed using a bright-field microscope and scanning electron microscopy (SEM). Metabolic mitochondrial activity, growth rate and adhesive capacity were also measured. Quantification real-time RT-PCR (qRT-PCR) was carried out to evaluate the expression levels of biofilm-related genes. Furthermore, the biofilm inhibitory potential of Sak alone or combined with antimicrobials was investigated. RESULTS: Sak production levels varied, ranging from 0.130 to 0.648. A strong decrease of biomass, metabolic activity andearly stage growth rate was demonstrated in the Sak-treated group. SEM showed S. aureus attached on hyphae of C. albicans in sporadic small microcolonies after Sak treatment. Moreover, the gene expression levels of HWP1, EFG1 and NRG1 were significantly altered, while no obvious difference was observed in ALS3. Finally, Sak had a notable impact on mature polymicrobial biofilms alone or when combined with vancomycin and fluconazole. CONCLUSION: The effect induced by Sak to C. albicans and S. aureus polymicrobial biofilms is caused by decreased biomass, biofilm integrity, metabolic activity and early stage growth rate. Alterations of gene expression levels were consistent with Sak-induced phenotypic change. Combined treatment strategies are essential for optimal activities against fungal-bacterial polymicrobial biofilms.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Metaloendopeptidases/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Biomassa , Candida albicans/genética , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real , Staphylococcus aureus/genética
12.
Dev Comp Immunol ; 90: 176-185, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30261235

RESUMO

Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 ΔlasB mutant and found that PO activity in hemolymph of the PAO1 ΔlasB-infected larvae was significantly higher than that in the wild type-infected. The mutant-inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 ΔlasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the ΔlasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Bombyx/imunologia , Metaloendopeptidases/metabolismo , Pseudomonas aeruginosa/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/genética , Bombyx/microbiologia , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Hemolinfa/metabolismo , Sistema Imunitário , Imunidade Inata , Larva , Metaloproteinase 12 da Matriz , Metaloendopeptidases/genética , Microrganismos Geneticamente Modificados , Mutação/genética , Proteínas/metabolismo , Pseudomonas aeruginosa/patogenicidade , Virulência
13.
J Dairy Sci ; 102(1): 54-67, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30527978

RESUMO

Oxidative stress caused by free radicals has been implicated in several human disorders. Dietary antioxidants can help the body to counteract those reactive species and reduce oxidative stress. Antioxidant activity is one of the multiple health-promoting attributes assigned to bovine whey products. The present study investigated whether this activity was retained during upper gut transit using a static simulated in vitro gastrointestinal digestion (SGID) model. The capacity to scavenge free radicals and reduce ferric ion of whey protein isolate (WPI), individual whey proteins, and hydrolysates pre- and post-SGID were measured and compared using various antioxidant assays. In addition, the free AA released from individual protein fractions in physiological gut conditions were characterized. Our results indicated that the antioxidant activity of WPI after exposure to the harsh conditions of the upper gut significantly increased compared with intact WPI. From an antioxidant bioactivity viewpoint, this exposure negates the need for prior hydrolysis of WPI. The whey protein α-lactalbumin showed the highest antioxidant properties post-SGID (oxygen radical absorbance capacity = 1,825.94 ± 50.21 µmol of Trolox equivalents/g of powder) of the 4 major whey proteins tested with the release of the highest amount of the antioxidant AA tryptophan, 6.955 µmol of tryptophan/g of protein. Therefore, α-lactalbumin should be the preferred whey protein in food formulations to boost antioxidant defenses.


Assuntos
Antioxidantes/metabolismo , Trato Gastrointestinal/metabolismo , Proteínas do Soro do Leite/metabolismo , Animais , Antioxidantes/administração & dosagem , Bromelaínas/metabolismo , Bovinos , Cromanos/administração & dosagem , Cromanos/metabolismo , Digestão , Depuradores de Radicais Livres/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Lactalbumina/metabolismo , Metaloendopeptidases/metabolismo , Proteínas do Leite/metabolismo , Estresse Oxidativo , Subtilisinas/metabolismo , Soro do Leite/química , Proteínas do Soro do Leite/administração & dosagem
14.
Int J Biol Macromol ; 121: 1037-1045, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30342946

RESUMO

Thrombolytic agents are routinely used to dissolve blood clot by activating fibrinolytic system. Among different thrombolytic agents available, staphylokinase (SAK) is gaining much attention because of their fibrin specificity and reduced inhibition by α2 antiplasmin. Though SAK had exhibited less circulatory half life, they are equipotent to tissue plasminogen activator and streptokinase and had shown more potency for clot dissolution during retracted thrombi. In this study, SAK was lipid modified at the N-terminal by a protein engineering approach to enhance its stability and activity. Native SAK as well as the gene encoding SAK with lipobox was cloned into E. coli GJ1158 using pRSET-B expression vector for higher expression. The lipid modification of SAK was confirmed by a mobility shift of 1.3 kDa against the 15.5 kDa of native SAK using tricine SDS-PAGE. Lipid modification of SAK was confirmed by LC MS/MS. The secondary structure analysis was carried out using circular dichroism and deconvoluted fourier transform infrared spectroscopy. LMSAK was found to have a slightly higher denaturation temperature compared to SAK. The improved stablility and activity of lipid modified SAK was studied by heated plasma agar plate assay and mouse tail bleeding test.


Assuntos
Metabolismo dos Lipídeos , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Estabilidade Enzimática , Meia-Vida , Proteômica
15.
Biomed Pharmacother ; 109: 1586-1592, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551412

RESUMO

Osteoarthritis (OA) is a joint disease characterized by inflammation and cartilage degradation. Accumulating evidence has demonstrated that luteolin, a natural flavonoid, has anti-inflammatory and anticatabolic effects. The present study aimed to assess the protective effect of luteolin on interleukin (IL)-1ß-stimulated rat chondrocytes and a monosodium iodoacetate (MIA)-induced model of OA. Rat chondrocytes were pretreated with luteolin (0, 25, 50, and 100 µM for 12 h) prior to stimulation with IL-1ß (10 ng/ml for 24 h). Nitric oxide (NO) production was determined using the Griess method. Production of prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-2, -8, and -9 (MMP-2, MMP-8 and MMP-9) was measured by an enzyme-linked immunosorbent assay (ELISA). Protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), MMP-1, MMP-3, MMP-13, p65, p-p65, IκB, and p-IκB were determined by Western blotting. The OA rats received luteolin (10 mg/kg/day) by gavage in vivo. Morphological and ultrastructural scanning electron microscopy (SEM) observations were performed to assess the severity of OA at 45 days following MIA injection. Collagen II protein expression was determined by immunohistochemistry. In this study, luteolin considerably reduced the IL-1ß-induced production of NO, PGE2, TNF-α, MMP-2, MMP-8 and MMP-9 and the expression of COX-2, iNOS, MMP-1, MMP-3 and MMP-13. Luteolin reversed the degradation of collagen II induced by IL-1ß. Luteolin also significantly inhibited IL-1ß-induced phosphorylation of NF-κB in vitro. Luteolin treatment prevented cartilage destruction and enhanced collagen II expression in OA rats in vivo. Overall, our findings suggest that luteolin may be a useful therapeutic agent for patients with OA.


Assuntos
Condrócitos/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Interleucina-1beta/farmacologia , Luteolina/farmacologia , Osteoartrite/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Condrócitos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Flavonoides/farmacologia , Inflamação/metabolismo , Masculino , Metaloendopeptidases/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
16.
J Mol Microbiol Biotechnol ; 28(4): 169-178, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30566956

RESUMO

The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for Chlamydomonas reinhardtii using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of C. reinhardtii. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of C. reinhardtii, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans-formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.


Assuntos
Parede Celular/metabolismo , Chlamydomonas reinhardtii/citologia , Subtilisina/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Chlamydomonas reinhardtii/genética , Vidro , Metaloendopeptidases/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Subtilisina/farmacologia , Transformação Genética
17.
Int J Mol Sci ; 19(12)2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30544562

RESUMO

Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.


Assuntos
Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Western Blotting , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células HEK293 , Humanos , Metaloendopeptidases/genética , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética
18.
Vet Res ; 49(1): 109, 2018 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-30373658

RESUMO

Streptococcus suis is a swine pathogen and zoonotic agent responsible for meningitis and septic shock. Although several putative virulence factors have been described, the initial steps of the S. suis pathogenesis remain poorly understood. While controversial results have been reported for a S. suis serotype 2 zinc metalloprotease (Zmp) regarding its IgA protease activity, recent phylogenetic analyses suggested that this protein is homologous to the ZmpC of Streptococcus pneumoniae, which is not an IgA protease. Based on the previously described functions of metalloproteases (including IgA protease and ZmpC), different experiments were carried out to study the activities of that of S. suis serotype 2. First, results showed that S. suis, as well as the recombinant Zmp, were unable to cleave human IgA1, confirming lack of IgA protease activity. Similarly, S. suis was unable to cleave P-selectin glycoprotein ligand-1 and to activate matrix metalloprotease 9, at least under the conditions tested. However, S. suis was able to partially cleave mucin 16 and syndecan-1 ectodomains. Experiments carried out with an isogenic Δzmp mutant showed that the Zmp protein was partially involved in such activities. The absence of a functional Zmp protein did not affect the ability of S. suis to adhere to porcine bronchial epithelial cells in vitro, or to colonize the upper respiratory tract of pigs in vivo. Taken together, our results show that S. suis serotype 2 Zmp is not a critical virulence factor and highlight the importance of independently confirming results on S. suis virulence by different teams.


Assuntos
Metaloendopeptidases/metabolismo , Streptococcus suis/enzimologia , Animais , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases/genética , Camundongos , Domínios Proteicos , Serina Endopeptidases/metabolismo , Sorogrupo , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/patogenicidade , Virulência
19.
Mar Drugs ; 16(8)2018 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-30126169

RESUMO

Ultraviolet (UV) B exposure induces DNA damage and production of reactive oxygen species (ROS), which causes skin photoaging through signaling pathways of inflammation and modulation of extracellular matrix remodeling proteins, collagens, and matrix metalloproteinase (MMP). As low molecular-weight fucoidan (LMF) has potential antioxidant and anti-inflammatory properties, we examined the protective effects of LMF against UVB-induced photoaging. A UVB-irradiated mouse model was topically treated with myricetin or LMF at 2.0, 1.0 and 0.2 mg/cm² (LMF2.0, LMF1.0 and LMF0.2, respectively) once a day for 15 weeks. Wrinkle formation, inflammation, oxidative stress, MMP expression, and apoptosis in the treated regions were compared with those in a distilled water-treated photoaging model (UVB control). LMF treatments, particularly LMF2.0 and LMF1.0, significantly inhibited the wrinkle formation, skin edema, and neutrophil recruitment into the photo-damaged lesions, compared with those in the UVB control. While LMF decreased interleukin (IL)-1ß release, it increased IL-10. The LMF treatment inhibited the oxidative stresses (malondialdehyde and superoxide anion) and enhanced endogenous antioxidants (glutathione). Additionally, LMF reduced the mRNA expression of MMP-1, 9, and 13. The histopathological analyses revealed the anti-photoaging effects of LMF exerted via its antioxidant, anti-apoptotic, and MMP-9-inhibiting effects. These suggest that LMF can be used as a skin-protective remedy for photoaging.


Assuntos
Polissacarídeos/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Colágeno/metabolismo , Feminino , Interleucina-10/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Pelados , Peso Molecular , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo
20.
Appl Microbiol Biotechnol ; 102(15): 6525-6536, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29948116

RESUMO

In this work, we describe the preparation and characterization of a biopreparate for efficient and rapid animal glue removal. The biopreparate is based on the extracellular proteolytic enzymes of an Exiguobacterium undae environmental isolate. Liquid chromatography-mass spectrometry analysis showed that the biopreparate is predominantly composed of hydrolytic enzymes-proteases and peptidases, nucleases, peptide ABC transporter substrate-binding proteins, and a phosphatase. The two main proteins present are bacillolysin and a peptide ABC transporter substrate-binding protein. Inhibition and proteomic analyses of the biopreparate revealed that bacillolysin, a neutral metalloendopeptidase, is mainly responsible for its proteolytic activity. This biopreparate was able to satisfactorily remove two types of animal glue from different kinds of material surfaces. These results suggest that this biopreparate could serve as a potential new tool for the restoration of historical objects rather than living microorganisms.


Assuntos
Adesivos/metabolismo , Antropologia Cultural/métodos , Bacillaceae/enzimologia , Animais , Bacillaceae/química , Bacillaceae/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida , Metaloendopeptidases/metabolismo , Proteoma , Proteômica , Espectrometria de Massas em Tandem
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