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1.
Sheng Wu Gong Cheng Xue Bao ; 36(11): 2435-2442, 2020 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-33244937

RESUMO

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.


Assuntos
Quimotripsina , Proteínas de Membrana , Sequência de Aminoácidos , Cromatografia Líquida , Quimotripsina/metabolismo , Digestão , Humanos , Espectrometria de Massas em Tandem , Tripsina , Vitamina K Epóxido Redutases
2.
PLoS One ; 15(9): e0238406, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886667

RESUMO

INTRODUCTION: In cancer treatment an attempt has been made to pharmacologically regulate the proteasome functions, thus the aim was to test whether 20S proteasome chymotrypsin-like (ChT-L) activity has a role in glial brain tumors. Furthermore, we analyzed the correlation between proteasome activity and IL-8, CCL2, NF-κB1 and NF-κB2 concentrations, which impact on brain tumors has already been indicated. METHODS: Plasma 20S proteasome ChT-L activity was assayed using the fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence of SDS. IL-8, CCL2, NF-κB1 and NF-κB2 concentration was analyzed with the use of ELISA method. Immunohistochemistry for IDH1-R132H was done on 5-microns-thick formalin-fixed, paraffin-embedded tumor sections with the use of antibody specific for the mutant IDH1-R132H protein. Labelled streptavidin biotin kit was used as a detection system. RESULTS: Brain tumor patients had statistically higher 20S proteasome ChT-L activity (0.649 U/mg) compared to non-tumoral individuals (0.430 U/mg). IDH1 wild-type patients had statistically higher 20S proteasome ChT-L activity (1.025 U/mg) compared to IDH1 mutants (0.549 U/mg). 20S proteasome ChT-L activity in brain tumor patients who died as the consequence of a tumor (0.649) in the following 2 years was statistically higher compared to brain tumor patients who lived (0.430 U/mg). In brain tumor patients the 20S proteasome ChT-L activity positively correlated with IL-8 concentration. CONCLUSIONS: Elevated 20S proteasome ChT-L activity was related to the increased risk of death in glial brain tumor patients. A positive correlation between 20S proteasome ChT-L activity and IL-8 concentration may indicate the molecular mechanisms regulating glial tumor biology. Thus research on proteasomes may be important and should be carried out to verify if this protein complexes may represent a potential therapeutic target to limit brain tumor invasion.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Serina Endopeptidases/metabolismo , Adulto , Idoso , Biometria , Quimiocina CCL2/metabolismo , Quimotripsina/metabolismo , Cisteína Endopeptidases/metabolismo , Feminino , Glioma/patologia , Humanos , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/sangue , Proteólise , Serina Endopeptidases/sangue
3.
Sci Rep ; 10(1): 11680, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669617

RESUMO

Bioactive plant peptides have received considerable interest as potential antihypertensive agents with potentially fewer side effects than antihypertensive drugs. Here, the blood pressure-lowering effects of the Bowman-Birk protease inhibitor, BTCI, and its derived peptides, PepChy and PepTry, were investigated using normotensive (Wistar-WR) and spontaneously hypertensive rats (SHR). BTCI inhibited the proteases trypsin and chymotrypsin, respectively, at 6 µM and 40 µM, a 10-fold greater inhibition than observed with PepTry (60 µM) and PepChy (400 µM). These molecules also inhibited angiotensin converting enzyme (ACE) with IC50 values of 54.6 ± 2.9; 24.7 ± 1.1; and 24.4 ± 1.1 µM, respectively, occluding its catalytic site, as indicated by molecular docking simulation, mainly for PepChy and PepTry. Gavage administration of BTCI and the peptides promoted a decrease of systolic and diastolic blood pressure and an increase of renal and aortic vascular conductance. These effects were more expressive in SHR than in WR. Additionally, BTCI, PepChy and PepTry promoted coronary vasodilation and negative inotropic effects in isolated perfused hearts. The nitric oxide synthase inhibitor blunted the BTCI and PepChy, with no cardiac effects on PepTry. The findings of this study indicate a therapeutic potential of BTCI and its related peptides in the treatment of hypertension.


Assuntos
Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Contração Miocárdica/efeitos dos fármacos , Peptídeos/farmacologia , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Animais , Anti-Hipertensivos/química , Sítios de Ligação , Quimotripsina/química , Quimotripsina/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Simulação de Acoplamento Molecular , NG-Nitroarginina Metil Éster/química , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Peptídeos/síntese química , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Tripsina/química , Tripsina/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/química , Vasodilatação/efeitos dos fármacos
4.
Sci Rep ; 10(1): 11731, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678161

RESUMO

The digestive enzyme chymotrypsin protects the pancreas against pancreatitis by reducing harmful trypsin activity. Genetic deficiency in chymotrypsin increases pancreatitis risk in humans and pancreatitis severity in mice. Pancreatic chymotrypsin is produced in multiple isoforms including chymotrypsin B1, B2, C and chymotrypsin-like protease (CTRL). Here we investigated the role of CTRL in cerulein-induced pancreatitis in mice. Biochemical experiments with recombinant mouse enzymes demonstrated that CTRL cleaved trypsinogens and suppressed trypsin activation. We generated a novel CTRL-deficient strain (Ctrl-KO) using CRISPR-Cas9 genome engineering. Homozygous Ctrl-KO mice expressed no detectable CTRL protein in the pancreas. Remarkably, the total chymotrypsinogen content in Ctrl-KO mice was barely reduced indicating that CTRL is a low-abundance isoform. When given cerulein, Ctrl-KO mice exhibited lower intrapancreatic chymotrypsin activation and a trend for higher trypsin activation, compared with C57BL/6N mice. Despite the altered protease activation, severity of cerulein-induced acute pancreatitis was similar in Ctrl-KO and C57BL/6N mice. We conclude that CTRL is a minor chymotrypsin isoform that plays no significant role in cerulein-induced pancreatitis in mice.


Assuntos
Pâncreas/enzimologia , Pancreatite/etiologia , Pancreatite/metabolismo , Serina Endopeptidases/deficiência , Doença Aguda , Animais , Biópsia , Linhagem Celular , Quimotripsina/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Pancreatite/patologia , Peroxidase/genética , Peroxidase/metabolismo , Índice de Gravidade de Doença , Tripsina/metabolismo
5.
Phys Chem Chem Phys ; 22(28): 16325-16333, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32648563

RESUMO

The use of cosolvents and high hydrostatic pressure (HHP) has been described as an efficient means to modulate the stability of enzymes and their catalytic activity. Cosolvents and pressure can lead to increased reaction rates without affecting the stability of the enzyme. Here, we studied the combined effects of one of the most used organic cosolvents, dimethyl sulfoxide (DMSO), and HHP to reveal their combined effect on the kinetic constants of an α-chymotrypsin-catalyzed peptide hydrolysis reaction. The Michaelis constant and the turnover number of the reaction respond differently to the two variables, and we observed an opposite effect of hydrostatic pressure and the dipolar cosolvent DMSO on the kinetic parameters. The results could be rationalized by determining the volume diagram of the reaction at the different solution conditions. In our case, the use of high hydrostatic pressure in concert with DMSO does not lead to an improvement of the enzymatic activity. However, the advantages of DMSO and HHP to increase the temperature stability of the enzyme and to increase the solubility of more hydrophobic substrates could still be useful.


Assuntos
Quimotripsina/metabolismo , Dimetil Sulfóxido/metabolismo , Animais , Biocatálise , Bovinos , Quimotripsina/química , Dimetil Sulfóxido/química , Hidrólise , Pressão Hidrostática , Cinética , Estrutura Molecular , Pâncreas/enzimologia , Termodinâmica
6.
Anim Sci J ; 91(1): e13409, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32524726

RESUMO

This study was aimed to explore the comparative acidifying properties of 2-hydroxy-4-(methylthio) butanoic acid (HMTBA) and a combination of DL-methionine (DLM) and acidifier in male broiler production. A total of 480 1-day-old broiler chicks were randomly divided into four treatments: A (low HMTBA, 0.057% HMTBA); B (low acidifier, 0.05% DLM + 0.057% acidifier); C (high HMTBA, 0.284% HMTBA); and D (high acidifier, 0.25% DLM + 0.284% acidifier). At 21 d, growth performance, chyme pH, digestive enzyme activities, and intestinal microflora were measured. The pH of crop, gizzard, and ileum contents was higher in the HMTBA treatment group than in DLM + acidifier treatment group. Furthermore, acidifier supplementation promoted growth of butyrate-producing bacteria such as Faecalibacterium, whereas high HMTBA (0.284%) inhibited the proliferation of acid-producing bacteria including Roseburia and Collinsella. The chymotrypsin activity was lower in the HMTBA group than in the DLM + acidifier group. In contrast, high-level HMTBA group showed higher average daily gain and average daily feed intake than the DLM + acidifier group. These results suggested that HMTBA work through different pathways with DLM plus acidifier.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Galinhas/crescimento & desenvolvimento , Galinhas/microbiologia , Galinhas/fisiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Metionina/análogos & derivados , Metionina/farmacologia , Animais , Quimotripsina/metabolismo , Papo das Aves , Ingestão de Alimentos/efeitos dos fármacos , Conteúdo Gastrointestinal , Moela das Aves , Concentração de Íons de Hidrogênio , Íleo , Masculino , Ganho de Peso/efeitos dos fármacos
7.
J Colloid Interface Sci ; 571: 174-184, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32199270

RESUMO

The preferred spatial orientation of single-wall carbon nanotubes (SWCNTs) in their interaction with enzymes determines their behavior either as nano-supports or as inhibitors. α -chymotrypsin (α-CT) is considered a serine protease model for studying nanomaterial/proteases interactions. The interaction of α-CT with pristine single-wall carbon nanotubes is still unknown. Here α-CT/SWCNT hybrids are synthesized and characterized. Spectroscopic, microscopic and kinetic measurements, coupled to molecular dynamics simulations, provide a detailed description of the interaction between α-CT and SWCNTs. The SWCNT binding pocket was unambiguously identified. A perfect match is observed with the crevice structure of the α-CT substrate binding pocket. The activity of α-CT, upon SWCNT binding, is dramatically reduced, as expected by the interaction of the SWCNT in the active site of the protein. π-π stacking between aromatic residues and the conjugated surface of SWCNT governs α-CT/SWCNT interactions. An important role in the bonding appears also for purely hydrophobic residues and with residues able to establish surfactant-like interactions. The secondary structure of α-CT and the catalytic triad structure are not perturbed by the complex formation, on the contrary the volume of the substrate binding pocket is strongly reduced by SWCNT binding because SWCNT occupies the α-CT substrate binding site, clogging the active site.


Assuntos
Quimotripsina/antagonistas & inibidores , Fulerenos/farmacologia , Nanotubos de Carbono/química , Inibidores de Serino Proteinase/farmacologia , Sítios de Ligação/efeitos dos fármacos , Quimotripsina/metabolismo , Fulerenos/química , Simulação de Dinâmica Molecular , Tamanho da Partícula , Inibidores de Serino Proteinase/química , Propriedades de Superfície
8.
ACS Appl Mater Interfaces ; 12(13): 15615-15621, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32134235

RESUMO

Construction of large-scale single-layer two-dimensional (2D) frameworks in water is significant due to their utilities in various fields. Utilizing macrocycle-mediated supramolecular self-assembly represents a promising approach; however, challenges still remain in their practical preparation. Here, we exploited a two-step supramolecular strategy to build 2D organic-inorganic hybrid frameworks at a micrometer scale in water. Taking advantage of the high binding affinity to cucurbit[7]uril (CB[7]), mono-quaternary ammonium tetraphenylethene (MQATPE) derivatives were first included with CB[7] to form a 1:1 complex (MQATPE@CB[7]). Then, just mixing the complex with anionic polyoxometalate Na9[EuW10O36]·32H2O (denoted as Eu-POM) in a 3:1 molar ratio leads to the formation of single-layer 2D films with tens of micrometers via electrostatic and π-π stacking interactions. The most unique feature of this strategy is that the steric effect imposed by CB[7] would not only lead the modules to adopt a periodic hexagonal assembly but also forbid stacking between layers through comparison with the merely multilayered 2D nanosheets self-assembled by MQATPE/Eu-POM. Interestingly, the charge interactions between MQATPE and Eu-POM would lead to the aggregation-induced emission (AIE) fluorescence of MQATPE, and white light emission could be obtained through the simple regulation of the contents of Eu-POM and MQATPE. Furthermore, due to the high surface areas and more accessible active sites, the single-layer films can act as an effective enzyme inhibitor to modulate the activity of α-chymotrypsin (ChT). These findings suggest a simple but universal approach for single-layer hybrid materials, which may hold promise for practical applications in photophysical and biomedical fields.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Quimotripsina/antagonistas & inibidores , Inibidores Enzimáticos/química , Imidazóis/química , Estilbenos/química , Compostos de Tungstênio/química , Quimotripsina/metabolismo , Inibidores Enzimáticos/metabolismo , Európio/química , Íons/química , Teoria Quântica , Eletricidade Estática
9.
Int J Mol Sci ; 21(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033479

RESUMO

The aim of this study was to isolate and identify angiotensin I-converting enzyme (ACE) inhibitory peptides from sesame protein through simulated gastrointestinal digestion in vitro, and to explore the underlying mechanisms by molecular docking. The sesame protein was enzymatically hydrolyzed by pepsin, trypsin, and α-chymotrypsin. The degree of hydrolysis (DH) and peptide yield increased with the increase of digest time. Moreover, ACE inhibitory activity was enhanced after digestion. The sesame protein digestive solution (SPDS) was purified by ultrafiltration through different molecular weight cut-off (MWCO) membranes and SPDS-VII (< 3 kDa) had the strongest ACE inhibition. SPDS-VII was further purified by NGC Quest™ 10 Plus Chromatography System and finally 11 peptides were identified by Nano UHPLC-ESI-MS/MS (nano ultra-high performance liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry) from peak 4. The peptide GHIITVAR from 11S globulin displayed the strongest ACE inhibitory activity (IC50 = 3.60 ± 0.10 µM). Furthermore, the docking analysis revealed that the ACE inhibition of GHIITVAR was mainly attributed to forming very strong hydrogen bonds with the active sites of ACE. These results identify sesame protein as a rich source of ACE inhibitory peptides and further indicate that GHIITVAR has the potential for development of new functional foods.


Assuntos
Digestão/fisiologia , Trato Gastrointestinal/metabolismo , Peptidil Dipeptidase A/metabolismo , Sesamum/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Quimotripsina/metabolismo , Digestão/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Simulação de Acoplamento Molecular/métodos , Pepsina A/metabolismo , Peptídeos/metabolismo , Hidrolisados de Proteína/metabolismo , Coelhos , Tripsina/metabolismo
10.
Protein Expr Purif ; 170: 105595, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32044416

RESUMO

Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation.


Assuntos
Quimotripsina/metabolismo , Cisteína/metabolismo , Dissulfetos/metabolismo , Elastase Pancreática/metabolismo , Saccharomyces cerevisiae/genética , Serpinas/genética , Proliferação de Células , Quimotripsina/antagonistas & inibidores , Quimotripsina/genética , Clonagem Molecular , Dissulfetos/química , Ensaios Enzimáticos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Peso Molecular , Oxirredução , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Serpinas/isolamento & purificação , Serpinas/metabolismo
11.
J Biol Chem ; 295(11): 3447-3455, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32014997

RESUMO

Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested ß-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.


Assuntos
Quimotripsina/metabolismo , Proteólise , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo , Biocatálise , Caseínas/metabolismo , Células HEK293 , Humanos , Cinética , Mutação/genética , Inibidor da Tripsina Pancreática de Kazal/metabolismo
12.
Chem Commun (Camb) ; 56(15): 2348-2351, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31993621

RESUMO

A miniaturized mimic of the active site of a protease, chymotrypsin, was linked to a target recognition unit to generate "Miniature Artificial Proteases" (mAPs). Time-resolved MALDI-TOF data analyses indicated that mAPs cleaved every amide bond between Lys16-Phe20 of the amyloid ß fragment (Aß12-21) and Aß1-40, resulting in inhibition of fibrillization and disruption of the preformed amyloid. Such a platform may offer not only new therapeutic options against various amyloidoses but also novel routes for the selective knockdown of specific proteins.


Assuntos
Amiloide/metabolismo , Quimotripsina/metabolismo , Amiloide/química , Domínio Catalítico , Quimotripsina/química , Humanos , Modelos Moleculares , Estrutura Molecular
13.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 1): 8-13, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929180

RESUMO

PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size (∼100 kDa), flexibility and complicated folding of PitA, obtaining diffraction-quality crystals has been a challenge. However, by the use of limited proteolysis with α-chymotrypsin, the diffraction quality of the PitA crystals was considerably enhanced to 2.16 Šresolution. These crystals belonged to space group P1, with unit-cell parameters a = 61.48, b = 70.87, c = 82.46 Å, α = 80.08, ß = 87.02, γ = 87.70°. The anomalous signal from the terbium derivative of α-chymotrypsin-treated PitA crystals prepared with terbium crystallophore (Tb-Xo4) was sufficient to obtain an interpretable electron-density map via terbium SAD phasing.


Assuntos
Adesinas Bacterianas/química , Placa Dentária/química , Fímbrias Bacterianas/química , Streptococcus oralis/química , Actinomyces , Adesinas Bacterianas/genética , Adesinas Bacterianas/isolamento & purificação , Adesinas Bacterianas/metabolismo , Biofilmes , Quimotripsina/metabolismo , Cristalização , Cristalografia por Raios X , Placa Dentária/metabolismo , Placa Dentária/microbiologia , Escherichia coli , Fímbrias Bacterianas/genética , Expressão Gênica/genética , Humanos , Streptococcus oralis/patogenicidade , Difração de Raios X
14.
Res Vet Sci ; 128: 1-8, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31706217

RESUMO

The aim of this work was to identify the molecular characteristics of a chymotrypsin-like enzyme from Trichinella spiralis (Tschy) and its facilitation of larval penetration into enteral epithelial cells (EECs). The complete Tschy cDNA sequence was cloned and expressed in Escherichia coli BL21. RT-PCR, IIFA and western blotting showed that Tschy was expressed at the T. spiralis muscle larvae (ML), intestinal infective L1 larvae (IL1), adult worms (AW) and embryo stages and was primarily located in the stichosome of this parasite. The results of ELISA, IIFA and Far-western assays showed that there was a specific binding between rTschy and EECs, and the binding was dependent on the dose of both rTschy and EEC proteins. Confocal microscopy demonstrated that the binding was located in the EEC cytoplasm. rTschy facilitated T. spiralis larval penetration of EECs, and anti-rTschy antibodies impeded the larval intrusion of EECs. These results demonstrate that Tschy facilitated the larval intrusion of the host's enteral epithelium and could be a candidate molecular target for vaccine against the enteral invasive phase of T. spiralis.


Assuntos
Quimotripsina/genética , Expressão Gênica , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita/fisiologia , Trichinella spiralis/fisiologia , Animais , Quimotripsina/metabolismo , Embrião não Mamífero/enzimologia , Embrião não Mamífero/fisiologia , Células Epiteliais/parasitologia , Escherichia coli/genética , Proteínas de Helminto/metabolismo , Intestino Delgado/parasitologia , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Microrganismos Geneticamente Modificados/genética , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Vacinas/análise
15.
J Dairy Sci ; 103(2): 1141-1150, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31785876

RESUMO

Food protein allergies are a major global concern. Hydrolysis of food proteins reduces their allergenicity, but another novel approach is the covalent attachment of polysaccharides to proteins via the Maillard reaction (i.e., glycation), which blocks some IgE binding epitopes on the protein allergen. We wanted to examine whether enzymatic hydrolysis, combined with glycation, could further reduce IgE binding for people with a cow milk protein allergy. Whey protein isolate (WPI) was hydrolyzed by immobilized trypsin and chymotrypsin to degree of hydrolysis (DH) values of 17 to 27%. Immobilized enzymes were used to avoid heat-treating the hydrolysate (to inactivate the enzymes, because heating could also affect the IgE binding ability of the protein). The resultant whey protein isolate hydrolysates (WPIH) were then glycated with 10-kDa dextran (DX) in aqueous solutions held at 62°C for 24 h. We analyzed the molar mass (MW) of WPIH samples and their corresponding glycates (WPIH-DX) using size-exclusion chromatography with multi-angle laser light scattering. We obtained blood sera from 8 patients who had been diagnosed with a cow milk protein allergy, and we used a composite serum for IgE binding analysis. The average MW values of samples WPIH-1 to WPIH-3 decreased from 11.15, 9.46, and 7.57 kDa with increasing DH values of 18.7, 22.5, and 27.1%. Glycation significantly reduced the high bitterness of the WPIH samples, as assessed by a trained sensory panel. The WPIH-DX glycates had significantly reduced WPI-specific IgE binding capacity compared to WPI or unglycated WPIH; we found an almost 99% reduction in IgE binding for the WPIH-DX glycate made from WPIH with a DH value of 27.1%. Hydrolysis of WPI followed by glycation with DX via the Maillard reaction significantly decreased the allergenicity of whey proteins.


Assuntos
Dextranos/metabolismo , Hipersensibilidade Alimentar/imunologia , Imunoglobulina E/sangue , Hipersensibilidade a Leite/imunologia , Proteínas do Soro do Leite/metabolismo , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Bovinos , Criança , Quimotripsina/metabolismo , Epitopos/metabolismo , Feminino , Hipersensibilidade Alimentar/sangue , Glicosilação , Humanos , Hidrólise , Reação de Maillard , Hidrolisados de Proteína/metabolismo , Tripsina/metabolismo , Proteínas do Soro do Leite/imunologia
16.
Biomed Res Int ; 2019: 7521715, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31737677

RESUMO

This study aimed to investigate the effects of leucine (Leu) on the synthesis and secretion of digestive enzymes in cultured pancreatic tissue of dairy goats and on the signaling molecules. Fresh pancreatic tissue from dairy goats was cut into approximately 2 mm × 2 mm pieces and incubated in oxygenated Krebs-Ringer bicarbonate buffer containing 0 (the control), 0.40, 0.80, or 1.60 mM Leu at 39°C in a CO2 incubator for 180 min. The results showed that Leu increased the release of α-amylase, trypsin, and chymotrypsin in the buffer and tissue, as well as the total activity (P < 0.05), especially at 0.40 and 0.80 mM. Compared with the control, 1.60 mM Leu increased the release of α-amylase and the total activity of trypsin and chymotrypsin (P < 0.05) but had no effect on the tissue concentration of α-amylase, trypsin, and chymotrypsin or the total activity of α-amylase (P > 0.05). Leu improved the mRNA expression of α-amylase, trypsin, and chymotrypsin (P < 0.05), especially at 0.80 and 1.60 mM. The activity and mRNA expression of lipase were not affected (P > 0.05). Compared with the control, 0.40 and 0.80 mM Leu increased the expression of the γ isoform of 4EBP1 (P < 0.05), implying increased phosphorylation of 4EBP1. Leu increased the phosphorylation of S6K1 (P < 0.05). Compared with the control, 0.40 and 0.80 mM Leu decreased the eEF2 phosphorylation level (P < 0.05). Conclusively, these results suggested that Leu could regulate the synthesis of pancreatic enzymes by increasing the mRNA expression and phosphorylation level of protein factors in the mammalian target of rapamycin pathway and the optimal Leu level in this experiment was 0.80 mM.


Assuntos
Cabras/metabolismo , Leucina/metabolismo , Pâncreas Exócrino/metabolismo , Pâncreas/metabolismo , Animais , Quimotripsina/metabolismo , Lipase/metabolismo , Fosforilação/fisiologia , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Tripsina/metabolismo , alfa-Amilases/metabolismo
17.
Int J Mol Sci ; 20(22)2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31717952

RESUMO

Earlier this year we published a method article aimed at optimising protein extraction from mature buds of medicinal cannabis for trypsin-based shotgun proteomics (Vincent, D., et al. Molecules 2019, 24, 659). We then developed a top-down proteomics (TDP) method (Vincent, D., et al. Proteomes 2019, 7, 33). This follow-up study aims at optimising the digestion of medicinal cannabis proteins for identification purposes by bottom-up and middle-down proteomics (BUP and MDP). Four proteases, namely a mixture of trypsin/LysC, GluC, and chymotrypsin, which target different amino acids (AAs) and therefore are orthogonal and cleave proteins more or less frequently, were tested both on their own as well as sequentially or pooled, followed by nLC-MS/MS analyses of the peptide digests. Bovine serum albumin (BSA, 66 kDa) was used as a control of digestion efficiency. With this multiple protease strategy, BSA was reproducibly 97% sequenced, with peptides ranging from 0.7 to 6.4 kD containing 5 to 54 AA residues with 0 to 6 miscleavages. The proteome of mature apical buds from medicinal cannabis was explored more in depth with the identification of 27,123 peptides matching 494 unique accessions corresponding to 229 unique proteins from Cannabis sativa and close relatives, including 130 (57%) additional annotations when the list is compared to that of our previous BUP study (Vincent, D., et al. Molecules 2019, 24, 659). Almost half of the medicinal cannabis proteins were identified with 100% sequence coverage, with peptides composed of 7 to 91 AA residues with up to 9 miscleavages and ranging from 0.6 to 10 kDa, thus falling into the MDP domain. Many post-translational modifications (PTMs) were identified, such as oxidation, phosphorylations, and N-terminus acetylations. This method will pave the way for deeper proteome exploration of the reproductive organs of medicinal cannabis, and therefore for molecular phenotyping within breeding programs.


Assuntos
Cannabis/química , Maconha Medicinal/química , Proteínas de Plantas/química , Proteômica/métodos , Quimotripsina/metabolismo , Flores/química , Espectrometria de Massas/métodos , Proteólise
18.
Pediatr Ann ; 48(11): e441-e447, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710363

RESUMO

Exocrine pancreatic insufficiency in children can lead to lifelong complications related to malnutrition and poor growth. The clinical presentation can be subtle in the early stages of insufficiency as the large functional capacity of the pancreas is gradually lost. The pediatrician plays a crucial role in the early identification of these children to ensure a timely referral so that a diagnosis can be made and therapy initiated. Early nutritional therapy allows for prevention and correction of deficiencies, which leads to improved outcomes and survival. When insufficiency is suspected, the workup should start with an indirect test of exocrine pancreatic function, such as fecal elastase, to establish the diagnosis. Once a diagnosis is established, further testing to delineate the etiology should be pursued, with cystic fibrosis being high on the differential list and assessed for with a sweat test. Assessment of anthropometry at every visit is key, as is monitoring of laboratory parameters and physical examination findings that are suggestive of malabsorption and malnutrition. The mainstay of management is administration of exogenous pancreatic enzymes to facilitate digestion and absorption. [Pediatr Ann. 2019;48(11):e441-e447.].


Assuntos
Transtornos da Nutrição Infantil/etiologia , Insuficiência Pancreática Exócrina/diagnóstico , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Anus Imperfurado/complicações , Criança , Transtornos da Nutrição Infantil/diagnóstico , Transtornos da Nutrição Infantil/terapia , Quimotripsina/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea/complicações , Fibrose Cística/complicações , Gorduras na Dieta/metabolismo , Displasia Ectodérmica/complicações , Terapia de Reposição de Enzimas , Insuficiência Pancreática Exócrina/etiologia , Insuficiência Pancreática Exócrina/terapia , Fezes/enzimologia , Transtornos do Crescimento/complicações , Perda Auditiva Neurossensorial/complicações , Humanos , Hipotireoidismo/complicações , Deficiência Intelectual/complicações , Erros Inatos do Metabolismo Lipídico/complicações , Doenças Mitocondriais/complicações , Doenças Musculares/complicações , Nariz/anormalidades , Avaliação Nutricional , Pâncreas/diagnóstico por imagem , Pâncreas/fisiologia , Pancreatopatias/complicações , Elastase Pancreática/metabolismo , Testes de Função Pancreática , Pancreatite Crônica/complicações , Pancreatite Crônica/etiologia , Síndrome de Shwachman-Diamond/complicações , Esteatorreia/etiologia , Tripsinogênio/sangue
19.
ACS Chem Biol ; 14(12): 2616-2628, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31710461

RESUMO

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.


Assuntos
Quimotripsina/metabolismo , Espectrometria de Massas/métodos , Quimotripsina/genética , Escherichia coli/genética , Glicosilação , Ensaios de Triagem em Larga Escala , Humanos , Especificidade por Substrato
20.
Nutrients ; 11(10)2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31640215

RESUMO

Diabetes mellitus is a non-communicable disease entity currently constituting one of the most significant health problems. The development of effective therapeutic strategies for the prevention and/or treatment of diabetes mellitus based on the selection of methods to restore and maintain blood glucose homeostasis is still in progress. Among the different courses of action, inhibition of dipeptidyl peptidase IV (DPP-IV) can improve blood glucose control in diabetic patients. Pharmacological therapy offering synthetic drugs is commonly used. In addition to medication, dietary intervention may be effective in combating metabolic disturbances caused by diabetes mellitus. Food proteins as a source of biologically active sequences are a potential source of anti-diabetic peptides (DPP-IV inhibitors and glucose uptake stimulating peptides). This study showed that in silico pork meat proteins digested with gastrointestinal enzymes are a potential source of bioactive peptides with a high potential to control blood glucose levels in patients with type 2 diabetes mellitus. Analysis revealed that the sequences released during in silico digestion were small dipeptides (with an average weight of 270.07 g mol-1), and most were poorly soluble in water. The selected electron properties of the peptides with the highest bioactivity index (i.e., GF, MW, MF, PF, PW) were described using the DFT method. The contribution of hydrophobic amino acids, in particular Phe and Trp, in forming the anti-diabetic properties of peptides released from pork meat was emphasized.


Assuntos
Simulação por Computador , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/farmacologia , Glucose/metabolismo , Hipoglicemiantes , Proteínas de Carne/farmacologia , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Fenômenos Químicos , Quimotripsina/metabolismo , Diabetes Mellitus Tipo 2/sangue , Dipeptídeos/química , Dipeptídeos/farmacologia , Inibidores da Dipeptidil Peptidase IV/química , Humanos , Proteínas de Carne/química , Proteínas de Carne/metabolismo , Pepsina A/metabolismo , Sus scrofa , Tripsina/metabolismo
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