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1.
BMC Plant Biol ; 21(1): 113, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627080

RESUMO

BACKGROUND: Recent studies indicate that amylase-trypsin inhibitors (ATIs) and certain carbohydrates referred to as FODMAPs (fermentable oligo-, di-, monosaccharides and polyols) play an important role in promoting wheat sensitivity. Hitherto, no study has investigated the accumulation of ATIs during the development of the wheat caryopsis. We collected caryopses of common wheat cv. 'Arnold' at eight different grain developmental stages to study compositional changes in ATI and FODMAP content. RESULTS: The harvested caryopses were analysed for their size, protein and carbohydrate concentrations. ATIs were further characterized by MALDI-TOF MS, and their trypsin inhibition was evaluated by an enzymatic assay. The results showed that ATI accumulation started about 1 week after anthesis and subsequently increased steadily until physiological maturity. However, the biological activity of ATIs in terms of enzyme inhibition was not detectable before about 4 weeks after anthesis. Carbohydrate analysis revealed the abundance of short-chain fructans in early stages of grain development, whereas non-water-soluble carbohydrates increased during later developmental stages. CONCLUSIONS: The results provide new insights into the complex metabolisms during grain filling and maturation, with particular emphasis on the ATI content as well as the inhibitory potential towards trypsin. The time lag between ATI accumulation and development of their biological activity is possibly attributed to the assembling of ATIs to dimers and tetramers, which seems to be crucial for their inhibitory potential.


Assuntos
Amilases/metabolismo , Grão Comestível/crescimento & desenvolvimento , Inibidores Enzimáticos/metabolismo , Sementes/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimento , Inibidores da Tripsina/metabolismo , Tripsina/metabolismo , Áustria , Metabolismo dos Carboidratos
2.
J Vis Exp ; (167)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33554961

RESUMO

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (λem = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.


Assuntos
Eletroforese/métodos , Ouro/metabolismo , Luminescência , Proteólise , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Animais , Sítios de Ligação , Cátions , Bovinos , Cristalografia por Raios X , Fluorescência , Géis , Concentração de Íons de Hidrogênio , Domínios Proteicos , Tripsina/metabolismo
3.
Microbiome ; 9(1): 12, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436102

RESUMO

BACKGROUND: Proteolysis regulation allows gut microbes to respond rapidly to dynamic intestinal environments by fast degradation of misfolded proteins and activation of regulatory proteins. However, alterations of gut microbial proteolytic signatures under complex disease status such as inflammatory bowel disease (IBD, including Crohn's disease (CD) and ulcerative colitis (UC)), have not been investigated. Metaproteomics holds the potential to investigate gut microbial proteolysis because semi-tryptic peptides mainly derive from endogenous proteolysis. RESULTS: We have developed a semi-tryptic peptide centric metaproteomic mining approach to obtain a snapshot of human gut microbial proteolysis signatures. This approach employed a comprehensive meta-database, two-step multiengine database search, and datasets with high-resolution fragmentation spectra to increase the confidence of semi-tryptic peptide identification. The approach was validated by discovering altered proteolysis signatures of Escherichia coli heat shock response. Utilizing two published large-scale metaproteomics datasets containing 623 metaproteomes from 447 fecal and 176 mucosal luminal interface (MLI) samples from IBD patients and healthy individuals, we obtain potential signatures of altered gut microbial proteolysis at taxonomic, functional, and cleavage site motif levels. The functional alterations mainly involved microbial carbohydrate transport and metabolism, oxidative stress, cell motility, protein synthesis, and maturation. Altered microbial proteolysis signatures of CD and UC mainly occurred in terminal ileum and descending colon, respectively. Microbial proteolysis patterns exhibited low correlations with ß-diversity and moderate correlations with microbial protease and chaperones levels, respectively. Human protease inhibitors and immunoglobulins were mainly negatively associated with microbial proteolysis patterns, probably because of the inhibitory effects of these host factors on gut microbial proteolysis events. CONCLUSIONS: This semi-tryptic peptide centric mining strategy offers a label-free approach to discover signatures of in vivo gut microbial proteolysis events if experimental conditions are well controlled. It can also capture in vitro proteolysis signatures to facilitate the evaluation and optimization of experimental conditions. Our findings highlight the complex and diverse proteolytic events of gut microbiome, providing a unique layer of information beyond taxonomic and proteomic abundance. Video abstract.


Assuntos
Microbioma Gastrointestinal , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Proteólise , Proteômica , Tripsina/metabolismo , Adolescente , Adulto , Criança , Escherichia coli/metabolismo , Humanos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Adulto Jovem
4.
Nucleic Acids Res ; 49(2): 791-804, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398338

RESUMO

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.


Assuntos
Enterócitos/metabolismo , Histonas/metabolismo , Intestino Delgado/citologia , Celulas de Paneth/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Células-Tronco/metabolismo , Animais , Catepsina L/metabolismo , Diferenciação Celular , Homeostase , Mucosa Intestinal/citologia , Camundongos , Microvilosidades/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Organoides , Domínios Proteicos , Tripsina/metabolismo
5.
J Dairy Sci ; 104(2): 1351-1363, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33309364

RESUMO

During the thermal processing of milk, Maillard reactions occur between proteins and lactose to generate glycated proteins. In this study, a lactose-glycated caseinate was hydrolyzed by trypsin. The obtained glycated caseinate (GCN) hydrolysate had a lactose content of 10.8 g/kg of protein. We identified its glycation sites and then assessed it for its protective effect against lipopolysaccharide-induced barrier injury using a rat intestinal epithelial cell line (IEC-6 cells) as a cell model and unglycated caseinate (CN) hydrolysate as a reference. Results from our liquid chromatography-mass spectrometry analysis of the GCN hydrolysate verified that lactose glycation occurred at the Lys residues in 3 casein components (αS1-casein, ß-casein, and κ-casein), and this resulted in the formation of 5 peptides with the following amino acid sequences: EMPFPKYPKYPVEPF, HIQKEDVPSE, GSENSEKTTMPL, NQDKTEIPT, and EGIHAQQKEPM. The results from cell experiments showed that the 2 hydrolysates could promote cell growth and decrease lactate dehydrogenase release in the lipopolysaccharide-injured cells; more importantly, they could partially protect the damaged barrier function of the cells by increasing trans-epithelial electrical resistance, decreasing epithelial permeability, and upregulating the expression of the 3 tight junction proteins zonula occludens-1, occludin, and claudin-1. However, compared with CN hydrolysate, GCN hydrolysate showed lower efficacy in protecting against cellular barrier dysfunction. We propose that the different chemical characteristics of the CN hydrolysate and the GCN hydrolysate (i.e., amino acid loss and lactose conjugation) contributed to the lower barrier-protective efficacy of the GCN hydrolysate. During dairy processing, protein glycation of the Maillard type might have a non-negligible, unfavorable effect on dairy proteins, in view of the resulting protein glycation we found and the critical function of proteins for maintaining the integrity of the intestinal barrier.


Assuntos
Caseínas/metabolismo , Células Epiteliais/efeitos dos fármacos , Lactose/metabolismo , Lipopolissacarídeos/farmacologia , Peptídeos/farmacologia , Animais , Sítios de Ligação , Caseínas/química , Linhagem Celular , Claudina-1/metabolismo , Células Epiteliais/fisiologia , Glicosilação , Hidrólise , Intestinos/citologia , Intestinos/efeitos dos fármacos , Reação de Maillard , Peptídeos/química , Permeabilidade , Ratos , Tripsina/metabolismo
6.
Food Chem ; 337: 127759, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777568

RESUMO

High-resolution ultrasonic spectroscopy (HR-US) was applied for real-time monitoring of ß-casein hydrolysis by trypsin at various conditions for the first time. The technique is based on the precision measurement of hydration changes proportional to the number of peptide bond hydrolyzed. As HR-US exhibits ultrasonic transparency for most solution, the analysis did not require optical transparency like for 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay. Appropriate enzymatic models were fitted with degree of hydrolysis (dh) profiles to provide kinetic and mechanistic description of proteolysis in terms of initial hydrolysis rate, r0, and rate constant of hydrolysis, kh, and enzyme inactivation, kd. Maximal r0 and dh were obtained at 45 °C and pH 8. The exponential dependence of kinetic parameters allowed determination of the activation (EA = 50.3 ± 7 kJ/mol) and deactivation (ED = 62.23 ± 3 kJ/mol) energies of hydrolysis. The ultrasonic assay provided rapid detection of trypsin activity even at sub-nanomolar concentration.


Assuntos
Caseínas/metabolismo , Ensaios Enzimáticos/métodos , Análise Espectral , Tripsina/metabolismo , Ondas Ultrassônicas , Ativação Enzimática , Hidrólise , Cinética , Proteólise , Soluções
7.
Methods Mol Biol ; 2213: 147-161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33270200

RESUMO

Interdisciplinary chemical proteomics approaches have been widely applied to the identification of specific targets of bioactive small molecules or drugs. In this chapter, we describe the application of a cell-permeable activity-based curcumin probe (Cur-P) with an alkyne moiety to detect and identify specific binding targets of curcumin in HCT116 colon cancer cells. Through click chemistry, a fluorescent tag or a biotin tag is attached to the probe-modified curcumin targets for visualization or affinity purification followed by mass spectrometric identification. A quantitative proteomics approach of isobaric tags for relative and absolute quantification (iTRAQ)™ is applied to distinguish specific curcumin targets from nonspecific binding proteins.


Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Proteômica/métodos , Cromatografia por Troca Iônica , Cromatografia Líquida , Química Click , Eletroforese em Gel de Poliacrilamida , Fluorescência , Células HCT116 , Humanos , Marcação por Isótopo , Nanotecnologia , Peptídeos/metabolismo , Rodaminas , Estreptavidina/química , Espectrometria de Massas em Tandem , Tripsina/metabolismo
8.
Ann Surg ; 272(5): 863-870, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32833754

RESUMO

OBJECTIVE: We investigated the activation of pancreatic proenzymes and signs of peripancreatic inflammation in patients with clinically relevant postoperative pancreatic fistulas (POPFs). SUMMARY BACKGROUND DATA: An increase of systemic amylase concentration was associated with POPFs. This suggested parallels in the pathomechanisms between the development of POPFs and pancreatitis. METHODS: Trypsinogen, procathepsin B, and IL-6 concentrations as well as cathepsin B, myeloperoxidase and trypsin activities were determined throughout the first 7 postoperative days in drain fluids of 128 consecutive patients after pancreas resection. Histology and immunohistochemistry were performed in pancreatic specimens after total pancreatectomy due to complications and after placing experimental pancreatic sutures in the pancreatic tail of C57/Bl6 mice. RESULTS: Trypsin activity, cathepsin B activity and myeloperoxidase activity on the first postoperative day were elevated and predictive for clinically relevant pancreatic fistulas. Drain fluid stabilized trypsin activity and prevented the activation of the cascade of digestive enzymes. Leukocytes were the source of cathepsin B in drain fluid. Findings differed between fistulas after distal pancreatectomy and pancreatoduodenectomy. Immunohistochemistry of the pancreatic remnant revealed an inflammatory infiltrate expressing cathepsin B, independent of the presence of pancreatic fistulas. The infiltrate could be reproduced experimentally by sutures placed in the pancreatic tail of C57/Bl6 mice. CONCLUSIONS: Trypsinogen activation, increased cathepsin B activity and inflammation around the pancreato-enteric anastomosis on post operative day 1 are associated with subsequent clinically relevant POPFs after pancreatoduodenectomy. The parenchymal damage seems to be induced by placing sutures in the pancreatic parenchyma during pancreatic surgery.


Assuntos
Pancreatectomia , Fístula Pancreática/enzimologia , Complicações Pós-Operatórias/enzimologia , Amilases/metabolismo , Animais , Catepsina B/metabolismo , Precursores Enzimáticos/metabolismo , Feminino , Humanos , Inflamação/enzimologia , Interleucina-6/metabolismo , Masculino , Camundongos , Peroxidase/metabolismo , Estudos Prospectivos , Tripsina/metabolismo , Tripsinogênio/metabolismo
9.
J Biosci Bioeng ; 130(5): 514-519, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32782194

RESUMO

We have investigated the potential of bile acid (BA)-binding short-chain peptides for suppressing cholesterol absorption in the intestine. In our previous report, we have revealed the physicochemical characteristics of high binding peptides using principal component analysis. In this study, we investigated the characteristics of amino acid residues of BA-binding short-chain peptides. We found that short-chain peptides containing lysine (K) and arginine (R) had a higher BA-binding ability than peptides containing other amino acids. Since short-chain tryptic peptides contain K or R residues, we focused on 4-mer, 5-mer, and 6-mer peptides, which were expectedly released from the edible proteins by trypsin. Forty-four short-chain peptides from lactoproteins (Bos taurus) and glutelin (Oryza sativa subsp. Japonica) were synthesized, and their BA micelle disruption activity was evaluated. We could observe such activities in nearly all tested peptides. We found that CEVFR, NGLK, and NSVFR had particularly high disruption activities. We determined that the 50% cholesterol concentration decrease value (DC50) of the micellar solution upon peptide addition was almost the same in case of the aforementioned peptides as that of the VAWWMY as a positive control. In addition, 4-mer and 5-mer peptides had higher BA micelle disruption activity than 6-mer peptides. Our results confirmed that the BA binding and micelle disruption activities were significantly higher if the short-chain peptides contained K and R residues.


Assuntos
Ácidos e Sais Biliares/química , Proteínas na Dieta/metabolismo , Micelas , Fragmentos de Peptídeos/química , Tripsina/metabolismo , Animais , Bovinos , Hidrólise
10.
Nat Commun ; 11(1): 3974, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769995

RESUMO

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Tripsina/metabolismo
11.
PLoS One ; 15(7): e0236740, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722706

RESUMO

Tryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and biopharmaceutical product characterization, owing to the high level of cleavage fidelity produced with this technique. However, nonspecific trypsin cleavages have been frequently reported and shown to be related to a number of digestion conditions and predigestion sample treatments. In this work, we reveal that, for a number of commercial trypsins, reconstitution and storage conditions can have a significant impact on the occurrence of trypsin nonspecific cleavages. We analyzed the tryptic digestion of a variety of biotherapeutics, using trypsins reconstituted under different conditions. The results indicate that, for many commercial trypsins, commonly recommended reconstitution/storage conditions (mildly acidic, e.g., 50 mM acetic acid, 1 mM HCl) can actually promote nonspecific trypsin activities, which are time dependent and can be as high as 20% in total relative abundance. In contrast, using water for reconstitution and storage can effectively limit nonspecific cleavages to 1%. Interestingly, the performances of different commercial trypsins were found to be quite distinct in their levels of nonspecific cleavages and responses to the two reconstitution conditions. Our findings demonstrate the importance of choosing the appropriate trypsin for tryptic digestion and the necessity of assessing the impact of trypsin reconstitution and storage on nonspecific cleavages. We advocate for manufacturers of commercial trypsins to reevaluate manufacturing processes and reconstitution/storage conditions to provide good cleavage specificity.


Assuntos
Ácidos/química , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Proteólise , Proteômica/métodos , Tripsina/metabolismo , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/isolamento & purificação
12.
Chem Biol Interact ; 328: 109201, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32717190

RESUMO

The caseinate and glycated caseinate generated from the transglutaminase-catalyzed reaction of caseinate and oligochitosan were digested using pepsin and trypsin, and the activity of the resultant digests was measured in rat intestinal epithelial cell line (IEC-6) using several biological responses as indicators. Compared with the caseinate digest, the glycated caseinate digest had similar contents in 17 amino acids but less reactable -NH2 contents, and 6.57 g glucosamine per kg protein; moreover, it showed higher activity in the cells (P < 0.05) to promote cell growth, accumulate the cell-cycle progression at the S-phase, and prevent the camptothecin-induced cell apoptosis. The glycated caseinate digest also showed higher differentiation activity in the cells than the caseinate digest, resulting in enhanced activities of the three brush-border membrane enzymes (P < 0.05) and increased microvilli on the cell surfaces. The real-time reverse transcription-polymerase chain reaction, Western-blot assay, and Dickkopf-1 (a receptor inhibitor of the Wnt/ß-catenin signaling pathway) were used to determine both gene and protein expression changes in the cells. A Wnt/ß-catenin signaling pathway responsible for these enhanced effects was proposed because the five genes (glycogen synthase kinase 3ß, Wnt3a, ß-catenin, c-Myc, and cyclin D1) and three proteins (nuclear and cytosolic ß-catenin, cyclin D1, and c-Myc) as part of this signaling pathway were regulated in the treated cells. The oligochitosan glycation of caseinate induced by transglutaminase is thus suggested endowing the peptic-tryptic caseinate digest with higher activity in the cells through its effects on the Wnt/ß-catenin signaling pathway.


Assuntos
Caseínas/metabolismo , Quitina/análogos & derivados , Enterócitos/metabolismo , Pepsina A/metabolismo , Tripsina/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Bovinos , Pontos de Checagem do Ciclo Celular , Diferenciação Celular/genética , Proliferação de Células , Sobrevivência Celular , Quitina/metabolismo , Enterócitos/citologia , Enterócitos/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
13.
Arch Biochem Biophys ; 690: 108460, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32603715

RESUMO

BACKGROUND: Our previous research revealed that trypsin is abundantly expressed in atherosclerotic plaques and its distribution overlaps with that of matrix metalloproteinase-9 (MMP-9). This study was performed to explore the possible roles of trypsin in vulnerable atherosclerotic plaque formation. METHODS AND RESULTS: Twenty-four rabbits were randomly assigned to a normal (control) group, an atherosclerosis (experimental) group and a trypsin inhibitor (aprotinin) group. In the 13th feeding week, the aprotinin group was treated with 5 mg/kg/day aprotinin via ear vein for 4 weeks. At the end of the 16th week, coronary arterial and aortic expression of trypsin, proteinase-activated receptor-2 (PAR-2), activated MMP-9, and pro-inflammatory cytokines were significantly greater in the experimental group than in the control group. Aprotinin decreased trypsin expression and activation in plaques, blocked PAR-2 and MMP-9 activation, and decreased cytokine expression; it also increased fibrous cap thickness, decreased the intima-media thickness and intimal/medial ratio, thus significantly ameliorating plaque vulnerability. Upregulated trypsin, MMP-9 and PAR-2 were also found in coronary intimal atherosclerotic plaques of patients undergoing coronary artery bypass grafting. CONCLUSIONS: Ectopic trypsin was significantly upregulated in atherosclerotic plaques, which increased pro-inflammatory cytokine levels by activating PAR-2 and promoted plaque instability by activating proMMP-9, thereby promoting atherosclerosis and plaque vulnerability. In addition, the high trypsin expression in human coronary intimal atherosclerotic plaques suggests that targeting trypsin may be a new strategy for acute coronary syndrome prevention.


Assuntos
Aterosclerose/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Placa Aterosclerótica/química , Tripsina/metabolismo , Animais , Aorta/química , Aprotinina/administração & dosagem , Aprotinina/metabolismo , Espessura Intima-Media Carotídea , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Coelhos , Receptor PAR-2/metabolismo , Tripsina/genética , Inibidores da Tripsina/administração & dosagem , Inibidores da Tripsina/metabolismo
14.
J Biol Chem ; 295(36): 12686-12696, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675285

RESUMO

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.


Assuntos
Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Serina Proteases/metabolismo , Células A549 , Células Cultivadas , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Domínios Proteicos , Transporte Proteico , Mucosa Respiratória/citologia , Serina Proteases/química , Serina Proteases/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tripsina/metabolismo
15.
J Vis Exp ; (160)2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32597868

RESUMO

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.


Assuntos
Separação Celular/métodos , Implantação do Embrião , Embrião de Mamíferos/citologia , Trofoblastos/citologia , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Forma Celular , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Feminino , Humanos , Gravidez , Imagem com Lapso de Tempo , Fixação de Tecidos , Tripsina/metabolismo , Vitrificação
16.
PLoS One ; 15(6): e0233745, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32542029

RESUMO

The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (ß-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/µg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.


Assuntos
Alérgenos/química , Análise de Alimentos/métodos , Peptídeos/química , Proteólise , Alérgenos/metabolismo , Animais , Cromatografia Líquida/métodos , Inocuidade dos Alimentos , Hemoglobinas/química , Hemoglobinas/metabolismo , Lactalbumina/química , Lactalbumina/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Peptídeos/metabolismo , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Suínos , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo
18.
PLoS One ; 15(5): e0232253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365084

RESUMO

Proteases have been implicated in the tumorigenesis and aggressiveness of a variety of cancer types. In fact, proteases have proven to be very clinically useful as tumor biomarkers in the blood of patients. Proteases are typically involved in complex systems of substrates, activators, and inhibitors, thus making our ability to establish their exact function in cancer more difficult. Trypsin, perhaps the most famous of proteases, has been shown to play a role in cancer progression, but its functional role in ovarian cancer has not been much studied. PAR2, a transmembrane receptor that is known to be activated by trypsin, has been reported to be associated with ovarian cancer. Here, we found that stimulation of ovarian cancer cell lines with trypsin or PAR2 activating peptide markedly increased MAPK signaling and cell proliferation. Additionally, HE4, a WAP-family glycoprotein and ovarian cancer biomarker, was found to inhibit trypsin degradation, thereby retaining its activity. Patient data seemed to support this phenomenon, as the serum of ovarian cancer patients with high HE4 expression, revealed significantly elevated trypsin levels. These data support the hypothesis that trypsin plays a tumorigenic role in ovarian cancer, which can be mediated by its receptor PAR2, and potentiated by HE4.


Assuntos
Carcinoma Epitelial do Ovário/genética , Neoplasias Ovarianas/genética , Receptor PAR-2/metabolismo , Tripsina/genética , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/metabolismo , Carcinoma Epitelial do Ovário/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Ovarianas/metabolismo , Proteólise , Receptor PAR-2/genética , Tripsina/química , Tripsina/metabolismo , Regulação para Cima
19.
J Pharmacol Exp Ther ; 374(2): 233-240, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32423989

RESUMO

CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low-therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaska Native people, ultrarare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862), and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus-infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in Escherichia coli toward (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I, and P279T variants, but as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wild-type protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 oxidoreductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen to the expected monohydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wild-type CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen, and (S)-naproxen. We conclude that carriers of the variant N218I and, especially, the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance. SIGNIFICANCE STATEMENT: Novel gene variants of CYP2C9-M1L, and N218I, along with P279T (CYP2C9*29)-are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency toward several substrates, including the low-therapeutic index drugs (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this understudied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities.


Assuntos
Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Povos Indígenas/genética , Alaska/etnologia , Escherichia coli/genética , Expressão Gênica , Células HEK293 , Humanos , Proteólise , Tripsina/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-32361468

RESUMO

The present study aimed to evaluate the effect of the immobilization method of trypsin on biochar on the hydrolysis of casein from different sources, when compared to the process using trypsin in native form, to obtain bioactive peptides. The modification of the surface of biochar with glutaraldehyde was effective, as shown by the results of FTIR assay and the texture profile of the materials. Both activated and functionalized biochar showed high immobilization efficiency (greater than 87%) and high binding capacity (greater than 91 mg/g). During hydrolysis, the biocatalyst obtained by enzyme immobilization on the functionalized biochar presented a higher hydrolysis capacity for the different caseins when compared to the enzyme immobilized by adsorption, with values of 3.05 and 2.73 U/mg for goat casein, 2.36 and 1.85 U/mg for bovine casein, and 2.60 and 2.37 U/mg for buffalo, casein, respectively, with 60 min of reaction. The results of inhibitory activity in this study ranged from 93.5% and 25.5% for trypsin in its free form and immobilized on functionalized activated carbon, respectively, under the same reaction conditions. The immobilization methods were efficient, presenting high immobilization capacity. The proteolytic activity of trypsin immobilized via covalent binding was higher when compared the immobilization by adsorption. Thus, the functionalized biochar has proven to be potential support for enzyme immobilization, and the biocatalyst can be reused for more than 4 cycles. Despite lower ACE inhibition values of hydrolyzed obtained with the immobilized enzymes compared to free enzymes, biocatalysts present advantage due to the possibility of reuse.


Assuntos
Caseínas/química , Carvão Vegetal/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Tripsina/química , Tripsina/metabolismo , Adsorção , Animais , Biocatálise , Bovinos , Estabilidade Enzimática , Glutaral/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Ácidos Fosfóricos/química , Proteólise , Propriedades de Superfície , Temperatura
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