RESUMO
Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of â¼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.
Assuntos
Echinococcus granulosus , Proteínas de Helminto , Echinococcus granulosus/enzimologia , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Animais , Cães , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/química , Bovinos , Sequência de Aminoácidos , Tripsina/química , Tripsina/metabolismoRESUMO
Recent advancements in enzyme research have unveiled a new proteoform of bovine trypsin, expanding our understanding of this well-characterized enzyme. While generally similar to other trypsins, this novel proteoform comprises three polypeptide chains, marking a significant difference in activity, kinetic properties, and conformational stability. Compared with the already known bovine trypsin proteoforms, the results showed a lower: activity, kcat and kcat.KM-1 and protein 'foldedness' ratio for the new proteoform. Molecular autolysis, a common feature in trypsin and chymotrypsin, has been explored through comparative physical chemistry properties with other proteoforms. This new proteoform of trypsin not only enriches the existing enzyme repertoire but also promises to shed light on the intricate physiological pathway for enzyme inactivation. Our results suggest that the new trypsin proteoform is one of the likely final pathways for enzyme inactivation in a physiological environment. This discovery opens up new avenues for further research into the functional implications of this new trypsin proteoform.
Assuntos
Tripsina , Tripsina/química , Tripsina/metabolismo , Animais , Bovinos , Cinética , Estabilidade Enzimática , Conformação ProteicaRESUMO
This study presents an innovative method for synthesizing ß-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti-isomer with 97 % conversion and 16 : 84 (syn : anti) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn-isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.
Assuntos
Quimotripsina , Enzimas Imobilizadas , Nanotubos , Titânio , Tripsina , Titânio/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Quimotripsina/química , Quimotripsina/metabolismo , Tripsina/metabolismo , Tripsina/química , Nanotubos/química , Estereoisomerismo , Biocatálise , Cicloexanonas/químicaRESUMO
INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.
Assuntos
Bauhinia , Inibidores da Tripsina , Animais , Bovinos , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Bauhinia/metabolismo , Tripsina/análise , Tripsina/química , Tripsina/metabolismo , Espectrometria de Massas em Tandem , Sementes/química , Anticoagulantes/farmacologia , Anticoagulantes/análise , Anticoagulantes/químicaRESUMO
Proteases are the main enzymes traded worldwide-comprising 60% of the total enzyme market-and are fundamental to the degradation and processing of proteins and peptides. Due to their high commercial demand and biological importance, there is a search for alternative sources of these enzymes. Crotalaria stipularia is highlighted for its agroecological applications, including organic fertilizers, nematode combat, and revegetation of areas contaminated with toxic substances. Considering the pronounced biotechnological functionality of the studied species and the necessity to discover alternative sources of proteases, we investigated the extraction, purification, and characterization of a protease from seeds of the C. stipularia plant. Protease isolation was achieved by three-phase partitioning and single-step molecular exclusion chromatography in Sephacryl S-100, with a final recovery of 47% of tryptic activity. The molecular mass of the isolated enzyme was 40 kDa, demonstrating optimal activities at pH 8.0 and 50 °C. Enzymatic characterization demonstrated that the protease can hydrolyze the specific trypsin substrate, BApNA. This trypsin-like protease had a Km, Vmax, Kcat, and catalytic efficiency constant of 0.01775 mg/mL, 0.1082 mM/min, 3.86 s-1, and 217.46, respectively.
Assuntos
Crotalaria , Sementes , Crotalaria/química , Sementes/química , Sementes/enzimologia , Concentração de Íons de Hidrogênio , Tripsina/metabolismo , Tripsina/química , Cinética , Especificidade por Substrato , Temperatura , Peso MolecularRESUMO
This study aimed to immobilize trypsin on activated carbon submitted to different surface modifications and its application in casein hydrolysis. With the aim of determining which support can promote better maintenance of the immobilized enzyme. Results showed that pH 5.0 was obtained as optimal for immobilization and pH 9.0 for the casein hydrolysis reaction for activated carbon and glutaraldehyde functionalized carbon. Among the supports used, activated carbon modified with iron ions in the presence of a chelating agent was the one that showed best results, under the conditions evaluated in this study. Presenting an immobilization yield of 95.15% and a hydrolytic activity of 4.11 U, same as soluble enzyme (3.76 U). This derivative kept its activity stable at temperatures above 40 °C for1 h and when stored for 30 days at 5 °C. Furthermore, it was effective for more than 6 reuse cycles (under the same conditions as the 1st cycle). In general, immobilization of trypsin on metallized activated carbon can be an alternative to biocatalysis, highlighting the advantages of protease immobilization.
Assuntos
Caseínas , Carvão Vegetal , Hidrólise , Estabilidade Enzimática , Tripsina/metabolismo , Concentração de Íons de Hidrogênio , Enzimas Imobilizadas/metabolismo , TemperaturaRESUMO
Proteases such as trypsins in the gut of Spodoptera frugiperda are responsible for breaking down dietary proteins into amino acids necessary for insect growth and development. In this study, we characterized the insecticidal potential of dioscorin, the storage protein of yam (Dioscorea alata), using molecular docking and molecular dynamics simulations to determine the interactions between trypsin enzymes and the protein inhibitor dioscorin. To achieve this, we used the three-dimensional structures of the trypsin-like digestive enzymes of S. frugiperda, a pest of corn and cotton, as receptors or target molecules. We performed protein-protein docking using Cluspro software, estimation of the binding free energy, and information on the dynamic and time-dependent behavior of dioscorin-trypsin complexes using the NAMD package. Our computational analysis showed that dioscorin can bind to the digestive trypsins of S. frugiperda, as confirmed by the affinity energy values (-1022.4 to -1236.9), stability of the complexes during the simulation trajectory, and binding free energy values between -57.3 and -66.9 kcal/mol. Additionally, dioscorin uses two reactive sites to bind trypsin, but the largest contribution to the interaction energy is made by amino acid residues between amino acid backbone positions 8-14 by hydrogen bonds, hydrophobic, and Van der Waals (VdW) interactions. VdW is the energy that makes the greatest contribution to the binding energy. Collectively, our findings demonstrate, for the first time, the binding capacity of the yam protein dioscorin to the digestive trypsin of S. frugiperda. These promising results suggest a possible bioinsecticide action of dioscorin.
Assuntos
Dioscorea , Animais , Dioscorea/química , Dioscorea/metabolismo , Proteínas de Plantas/metabolismo , Simulação de Acoplamento Molecular , Tripsina/metabolismo , Aminoácidos/metabolismo , Simulação de Dinâmica MolecularRESUMO
Skin aging represents a health and aesthetic problem that could result in infections and skin diseases. Bioactive peptides can potentially be used in skin aging regulation. Chickpea (Cicer arietinum L.) selenoproteins were obtained from germination with 2 mg Na2SeO3/100 g of seeds for 2 days. Alcalase, pepsin, and trypsin were used as hydrolyzers, and a membrane < 10 kDa was used to fractionate the hydrolysate. Se content, antioxidant capacity, elastase and collagen inhibition, functional stability, and preventative capacity were analyzed. Significant increases in Se content were found in germinated chickpea flour and protein related to the control. An increase of 38% in protein was observed in the selenized flour related to the control. A band (600-550 cm-1) observed in the selenized hydrolysates suggested the insertion of Se into the protein. Hydrolysates from pepsin and trypsin had the highest antioxidant potential. Se enhanced the stability of total protein and protein hydrolysates through time and increased their antioxidant capacity. Hydrolysates > 10 kDa had higher elastase and collagenase inhibition than the total protein and hydrolysates < 10 kDa. Protein hydrolysates < 10 kDa 6 h before UVA radiation had the highest inhibition of collagen degradation. Selenized protein hydrolysates showed promising antioxidant effects that could be related to skin anti-aging effects.
Assuntos
Antioxidantes , Cicer , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Cicer/química , Hidrolisados de Proteína/química , Pepsina A/metabolismo , Tripsina/metabolismo , Elastase Pancreática/metabolismoRESUMO
Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of Helicoverpa armigera larvae to soybean peptidase inhibitors (SPIs) is associated with large-scale gene expression changes including the upregulation of genes encoding serine peptidases in the digestive system. Furthermore, approximately 60% of the gene expression changes induced by SPIs persisted in the next generation of larvae fed on SPI-free diets including genes encoding regulatory, oxidoreductase, and protease functions. To investigate the role of epigenetic mechanisms in regulating SPI adaptation, the methylome of the digestive system of first-generation larvae (fed on a diet with and without SPIs) and of the progeny of larvae exposed to SPIs were characterized. A comparative analysis between RNA-seq and Methyl-seq data did not show a direct relationship between differentially methylated and differentially expressed genes, while trypsin and chymotrypsin genes were unmethylated in all treatments. Rather, DNA methylation potential epialleles were associated with transcriptional and translational controls; these may play a regulatory role in the adaptation of H. armigera to SPIs. Altogether, our findings provided insight into the mechanisms of insect adaptation to plant antiherbivore defense proteins and illustrated how large-scale transcriptional reprograming of insect genes can be transmitted across generations.
Assuntos
Glycine max , Mariposas , Animais , Glycine max/genética , Glycine max/metabolismo , Inibidores de Proteases/farmacologia , Regulação para Cima , Serina Proteases/metabolismo , Mariposas/genética , Mariposas/metabolismo , Quimotripsina/genética , Quimotripsina/metabolismo , Tripsina/metabolismo , Larva/genética , Larva/metabolismo , Serina/metabolismoRESUMO
Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino- or carboxy-terminal end, and they allowed us to identify those proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348. Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP33 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication. IMPORTANCE Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and VP38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.