Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38.916
Filtrar
1.
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
2.
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
4.
Nat Commun ; 11(1): 2918, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522984

RESUMO

Coarse-graining of fully atomistic molecular dynamics simulations is a long-standing goal in order to allow the description of processes occurring on biologically relevant timescales. For example, the prediction of pathways, rates and rate-limiting steps in protein-ligand unbinding is crucial for modern drug discovery. To achieve the enhanced sampling, we perform dissipation-corrected targeted molecular dynamics simulations, which yield free energy and friction profiles of molecular processes under consideration. Subsequently, we use these fields to perform temperature-boosted Langevin simulations which account for the desired kinetics occurring on multisecond timescales and beyond. Adopting the dissociation of solvated sodium chloride, trypsin-benzamidine and Hsp90-inhibitor protein-ligand complexes as test problems, we reproduce rates from molecular dynamics simulation and experiments within a factor of 2-20, and dissociation constants within a factor of 1-4. Analysis of friction profiles reveals that binding and unbinding dynamics are mediated by changes of the surrounding hydration shells in all investigated systems.


Assuntos
Modelos Teóricos , Benzamidinas/química , Sítios de Ligação , Simulação de Dinâmica Molecular , Ligação Proteica , Cloreto de Sódio/química , Termodinâmica , Tripsina/química , Água/química
5.
Clin Imaging ; 65: 113-118, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32387800

RESUMO

OBJECTIVES: To review the imaging of patients with Genetically-Mediated Pancreatitis (GMP), identify common imaging findings in this cohort and assess phenotypical characteristics of specific genotypes. MATERIALS AND METHODS: Retrospective review of the databases of the Irish National Surgical Centre for Pancreatic Cancer (NSCPC) and Cystic Fibrosis (CF) from November 2010 to January 2018. Retrospective imaging and chart review for the patients with positive genetics for GMP. RESULTS: The NSCPC database contained 699 patients; the CF database included 352 patients. Of these 1051, 14 were identified as having GMP (age range: 20-65, M:F ratio of 1:1). 14 of 1051 patients from the database had positive genetics for GMP. 10 had imaging to support a diagnosis of hereditary pancreatitis or familial recurrent pancreatitis (1.3%) and 4 had imaging to support a diagnosis of CF-related pancreatitis. Imaging findings were considered in 3 categories, determined by genotype - PRSS1 hereditary pancreatitis, SPINK 1 autosomal recessive pancreatitis and those for CFTR - cystic fibrosis related pancreatitis. Imaging findings in PRSS1 hereditary pancreatitis patients included: pancreatic atrophy, calcification and main pancreatic duct (MPD) dilatation, referred to as the PRSS1 imaging triad. Patients with the SPINK1 gene mutation had less severe imaging manifestations (pancreatic atrophy 33%, MPD dilatation 33%, pancreatic calcification 33%). CFTR patients with imaging findings had pancreatic atrophy (100%). CONCLUSION: GMP should be suspected when the features of 'chronic pancreatitis' are seen in young adults with no history of excess alcohol intake. Genetic testing, endocrinology review and long-term imaging follow-up for pancreatic carcinoma are indicated.


Assuntos
Pancreatite/diagnóstico por imagem , Adulto , Idoso , Proteínas de Transporte/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Pancreáticas , Pancreatite/terapia , Pancreatite Crônica/genética , Pancreatite Crônica/cirurgia , Estudos Retrospectivos , Tripsina/genética , Inibidor da Tripsina Pancreática de Kazal/genética , Adulto Jovem
6.
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
7.
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
8.
Arch Insect Biochem Physiol ; 104(3): e21687, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32342573

RESUMO

The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant Ki and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 Å2 . The kinetic analysis revealed a noncompetitive tight-binding mechanism. The survival curves obtained using Kaplan-Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect.


Assuntos
Mariposas/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Inibidores da Tripsina/farmacologia , Animais , Fabaceae/química , Larva/efeitos dos fármacos , Larva/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mariposas/enzimologia , Mariposas/crescimento & desenvolvimento , Tripsina/metabolismo
9.
BMC Bioinformatics ; 21(1): 143, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293241

RESUMO

BACKGROUND: Protein-protein interactions (PPIs) are fundamental in many biological processes and understanding these interactions is key for a myriad of applications including drug development, peptide design and identification of drug targets. The biological data deluge demands efficient and scalable methods to characterize and understand protein-protein interfaces. In this paper, we present ppiGReMLIN, a graph based strategy to infer interaction patterns in a set of protein-protein complexes. Our method combines an unsupervised learning strategy with frequent subgraph mining in order to detect conserved structural arrangements (patterns) based on the physicochemical properties of atoms on protein interfaces. To assess the ability of ppiGReMLIN to point out relevant conserved substructures on protein-protein interfaces, we compared our results to experimentally determined patterns that are key for protein-protein interactions in 2 datasets of complexes, Serine-protease and BCL-2. RESULTS: ppiGReMLIN was able to detect, in an automatic fashion, conserved structural arrangements that represent highly conserved interactions at the specificity binding pocket of trypsin and trypsin-like proteins from Serine-protease dataset. Also, for the BCL-2 dataset, our method pointed out conserved arrangements that include critical residue interactions within the conserved motif LXXXXD, pivotal to the binding specificity of BH3 domains of pro-apoptotic BCL-2 proteins towards apoptotic suppressors. Quantitatively, ppiGReMLIN was able to find all of the most relevant residues described in literature for our datasets, showing precision of at least 69% up to 100% and recall of 100%. CONCLUSIONS: ppiGReMLIN was able to find highly conserved structures on the interfaces of protein-protein complexes, with minimum support value of 60%, in datasets of similar proteins. We showed that the patterns automatically detected on protein interfaces by our method are in agreement with interaction patterns described in the literature.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Animais , Gráficos por Computador , Mineração de Dados , Complexos Multiproteicos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tripsina/química , Tripsina/metabolismo
10.
Nat Commun ; 11(1): 1620, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: covidwho-17830

RESUMO

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/fisiologia , Anticorpos Amplamente Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Betacoronavirus/química , Betacoronavirus/imunologia , Canais de Cálcio/metabolismo , Catepsina L/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Fusão Celular , Infecções por Coronavirus/imunologia , Reações Cruzadas , Endocitose , Células Gigantes/fisiologia , Células HEK293 , Humanos , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Pneumonia Viral/imunologia , Domínios Proteicos , Multimerização Proteica , Receptores Virais/metabolismo , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/química , Tripsina/metabolismo
11.
Nat Commun ; 11(1): 1620, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221306

RESUMO

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/fisiologia , Anticorpos Amplamente Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Betacoronavirus/química , Betacoronavirus/imunologia , Canais de Cálcio/metabolismo , Catepsina L/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Fusão Celular , Infecções por Coronavirus/imunologia , Reações Cruzadas , Endocitose , Células Gigantes/fisiologia , Células HEK293 , Humanos , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Pneumonia Viral/imunologia , Domínios Proteicos , Multimerização Proteica , Receptores Virais/metabolismo , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/química , Tripsina/metabolismo
12.
Food Chem ; 318: 126333, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32151919

RESUMO

Dipeptidyl peptidase-IV (DPP-IV) is an enzyme that break down the antidiabetic hormone glucagon-like peptide-1. Therefore, inhibition of DPP-IV could be an effective strategy to treat Type 2 diabetes (T2D). The α-lactalbumin-rich whey protein concentrate was hydrolyzed by trypsin, and the hydrolysates were then fractionated at a semi-preparative scale using a Superdex Gel filtration Chromatography. The peptides were analyzed by using HPLC coupled with tandem mass spectrometry (RP-HPLC-MS/MS), and their Dipeptidyl peptidase-IV inhibitory activity was determined by the enzymatic assay. Among tested fragments, a potent fragment (LDQWLCEKL), with the half-maximal inhibitory concentration (IC50) of 131 µM was obtained. Further analysis shows that the LDQWLCEKL peptide corresponds to the amino acid sequence of f(115-123) in α-lactalbumin. Furthermore, LDQWLCEKL exhibited a typical non-competitive mode of inhibition. The results indicate that α-lactalbumin contains active peptides with DPP-IV inhibitory activity that may be used to prevent and treat T2D.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Lactalbumina/metabolismo , Peptídeos/química , Proteínas do Soro do Leite/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/metabolismo , Hidrólise , Concentração Inibidora 50 , Cinética , Lactalbumina/química , Peptídeos/análise , Peptídeos/metabolismo , Espectrometria de Massas em Tandem , Tripsina/metabolismo
13.
PLoS One ; 15(3): e0228461, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160194

RESUMO

Simulating drug binding and unbinding is a challenge, as the rugged energy landscapes that separate bound and unbound states require extensive sampling that consumes significant computational resources. Here, we describe the use of interactive molecular dynamics in virtual reality (iMD-VR) as an accurate low-cost strategy for flexible protein-ligand docking. We outline an experimental protocol which enables expert iMD-VR users to guide ligands into and out of the binding pockets of trypsin, neuraminidase, and HIV-1 protease, and recreate their respective crystallographic protein-ligand binding poses within 5-10 minutes. Following a brief training phase, our studies shown that iMD-VR novices were able to generate unbinding and rebinding pathways on similar timescales as iMD-VR experts, with the majority able to recover binding poses within 2.15 Å RMSD of the crystallographic binding pose. These results indicate that iMD-VR affords sufficient control for users to carry out the detailed atomic manipulations required to dock flexible ligands into dynamic enzyme active sites and recover crystallographic poses, offering an interesting new approach for simulating drug docking and generating binding hypotheses.


Assuntos
Protease de HIV/metabolismo , Simulação de Dinâmica Molecular , Neuraminidase/metabolismo , Tripsina/metabolismo , Realidade Virtual , Benzamidinas/metabolismo , Sítios de Ligação , Carbamatos/metabolismo , Ligantes , Oseltamivir/metabolismo , Ligação Proteica , Sulfonamidas/metabolismo , Zanamivir/metabolismo
14.
Food Chem ; 319: 126472, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32163839

RESUMO

Whey protein is one of the most relevant co-products manufactured by the dairy industry and it is a powerful environmental pollutant. Therefore, the enzymatic hydrolysis of whey protein concentrate (WPC 35) to produce antioxidant peptides is an innovative approach which can provide added value to whey. The WPC 35 hydrolysis with trypsin was carried out for 4.31 h at 41.1 °C with an enzyme/substrate ratio of 0.017. Under such hydrolysis conditions, the peptides produced have the highest radical scavenging activity and cytoprotector effect. The WPC hydrolysate and a permeate ≤3 kDa were characterized by SDS-page, RP-HPLC and MALDI-TOF-MS. Furthermore, O2•- and HO• scavenging activity and the cytoprotective effect against a stress agent in epithelial cells of the rat ileum (IEC-18) were determined. In this study, strong antioxidant and cytoprotective peptides were obtained from a low-cost dairy industry product, which could improve consumers' health when used as functional ingredients.


Assuntos
Peptídeos/metabolismo , Tripsina/metabolismo , Proteínas do Soro do Leite/metabolismo , Animais , Antioxidantes/metabolismo , Hidrólise , Ratos , Soro do Leite/química
15.
Curr Diab Rep ; 20(6): 16, 2020 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-32221727

RESUMO

PURPOSE OF REVIEW: The aim was to review evidence about diabetes secondary to hereditary pancreatitis, seeking novel diagnostic and treatment features. RECENT FINDINGS: Hereditary pancreatitis (HP) is an autosomal dominant condition, characterized by recurrent episodes of acute pancreatitis, progression to fibrosis, and chronic pancreatitis. Clinical presentation includes diabetes of the exocrine pancreas (DEP). HP prevalence ranges from 0.3 to 0.57 per 100,000 people, with up to 80% of these develop DEP. This condition often requires specific interventions: with regard to metabolic control, metformin is the first choice for those with mild DEP, and for those in advanced disease, insulin is considered the first-line therapy. Insulin analogues and insulin pump therapy are preferred due to the brittle glycemic pattern and risk of hypoglycemia. In case of exocrine insufficiency, pancreatic enzyme replacement therapy is recommended. Pancreatic polypeptide administration is a promising novel treatment feature. DEP due to HP appears to be a misdiagnosed condition. The requirement of specific management demonstrates the importance of this matter; therefore, appropriate recognition and classification are important.


Assuntos
Diabetes Mellitus/genética , Pâncreas Exócrino/patologia , Pancreatite Crônica/genética , Tripsina/genética , Doença Aguda , Carcinoma Ductal Pancreático/etiologia , Quimotripsina/genética , Complicações do Diabetes/complicações , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/fisiopatologia , Diabetes Mellitus/terapia , Insuficiência Pancreática Exócrina/genética , Insuficiência Pancreática Exócrina/fisiopatologia , Insuficiência Pancreática Exócrina/terapia , Fibrose/etiologia , Humanos , Pâncreas Exócrino/fisiopatologia , Neoplasias Pancreáticas/etiologia , Pancreatite Crônica/complicações , Pancreatite Crônica/diagnóstico , Pancreatite Crônica/fisiopatologia , Recidiva , Fatores de Risco , Inibidor da Tripsina Pancreática de Kazal/genética
16.
Nat Commun ; 11(1): 1318, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165630

RESUMO

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.


Assuntos
Replicação do DNA , Receptores Virais/metabolismo , Tripsina/química , Camptotecina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genética
17.
Emerg Microbes Infect ; 9(1): 457-468, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-124862

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.


Assuntos
Fusão Celular , Coronavirus/fisiologia , Fusão de Membrana , Tripsina/metabolismo , Animais , Linhagem Celular , Humanos , Suínos , Internalização do Vírus , Replicação Viral
18.
Nat Microbiol ; 5(4): 562-569, 2020 04.
Artigo em Inglês | MEDLINE | ID: covidwho-1966

RESUMO

Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geographic regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.


Assuntos
Betacoronavirus/fisiologia , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Animais , Betacoronavirus/química , Betacoronavirus/classificação , Antígenos CD13/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/metabolismo , Humanos , Mutação , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da SARS/química , Vírus da SARS/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Tripsina/metabolismo
19.
Nat Commun ; 11(1): 1049, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103000

RESUMO

Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL-1 trypsin is electrosonically sprayed (-3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development.


Assuntos
Hormônio Adrenocorticotrópico/análise , Mioglobina/análise , Análise de Sequência de Proteína/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Trastuzumab/análise , Hormônio Adrenocorticotrópico/metabolismo , Enzimas Imobilizadas/química , Humanos , Mioglobina/metabolismo , Trastuzumab/metabolismo , Tripsina/metabolismo
20.
Emerg Microbes Infect ; 9(1): 457-468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32090689

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.


Assuntos
Fusão Celular , Coronavirus/fisiologia , Fusão de Membrana , Tripsina/metabolismo , Animais , Linhagem Celular , Humanos , Suínos , Internalização do Vírus , Replicação Viral
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA