Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.030
Filtrar
1.
Int J Mol Sci ; 25(11)2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38892254

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.


Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais , Ipomoea batatas , SARS-CoV-2 , Serina Endopeptidases , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Animais , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Ipomoea batatas/virologia , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , COVID-19/virologia , COVID-19/metabolismo , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/metabolismo , Internalização do Vírus/efeitos dos fármacos , Chlorocebus aethiops , Células Vero , Regulação para Baixo/efeitos dos fármacos , Camundongos
2.
J Chem Inf Model ; 64(13): 5194-5206, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38870039

RESUMO

The serine protease trypsin forms a tightly bound inhibitor complex with the bovine pancreatic trypsin inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to α-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores the inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown, and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before, and unrestrained umbrella sampling simulations of these complexes suggest additional energetic minima. Here, we use random acceleration molecular dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the prebound state. The prebound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the BPTI variants, and changes in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between the fully bound state and prebound state and also lowers the energy minimum of the prebound state. While the effect of fluorination is in general difficult to quantify, here, it is in part caused by favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the prebound state offers insights into the inhibitory mechanism of BPTI and might add valuable information for the design of serine protease inhibitors.


Assuntos
Aprotinina , Simulação de Dinâmica Molecular , Ligação Proteica , Tripsina , Tripsina/metabolismo , Tripsina/química , Aprotinina/química , Aprotinina/metabolismo , Animais , Bovinos , Halogenação , Ligação de Hidrogênio , Conformação Proteica , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
3.
Int J Mol Sci ; 25(11)2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38891766

RESUMO

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman-Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.


Assuntos
Edição de Genes , Glycine max , Inibidor da Tripsina de Soja de Bowman-Birk , Quimotripsina/metabolismo , Quimotripsina/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Glycine max/genética , Glycine max/metabolismo , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Sementes/genética , Sementes/metabolismo , Tripsina/metabolismo , Tripsina/genética , Tripsina/química , Inibidor da Tripsina de Soja de Bowman-Birk/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/genética , Inibidores da Tripsina/metabolismo
4.
Biochem J ; 481(11): 717-739, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38752933

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 ∼20 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/metabolismo
5.
Pestic Biochem Physiol ; 201: 105899, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38685208

RESUMO

This study investigated the function of the MDR49 gene in Aedes aegypti. MDR49 mutants were constructed using CRISPR/Cas9 technology; the mutation led to increased sensitivity to ivermectin (LC50: from 1.3090 mg L-1 to 0.5904 mg L-1), and a reduction in midgut trypsin activity. These findings suggest that the P-gp encoded by MDR49 confers resistance to ivermectin and impacts the reproductive function in Ae. aegypti. RNA interference technology showed that knockdown of MDR49 gene resulted in a significant decrease in the expression of VGA1 after a blood meal, as well as a decrease in the number of eggs laid and their hatching rate. LC-MS revealed that following ivermectin treatment, the MDR493d+2s/3d+2s strain larvae exhibited significantly higher drug concentrations in the head and fat body compared to the wild type. Modeling of inward-facing P-gp and molecular docking found almost no difference in the affinity of P-gp for ivermectin before and after the mutation. However, modeling of the outward-facing conformation demonstrated that the flexible linker loop between TM5 and TM6 of P-gp undergoes changes after the mutation, resulting in a decrease in trypsin activity and an increase in sensitivity to ivermectin. These results provide useful insights into ivermectin resistance and the other roles played by the MDR49 gene.


Assuntos
Aedes , Proteínas de Insetos , Ivermectina , Animais , Aedes/efeitos dos fármacos , Aedes/genética , Aedes/metabolismo , Ivermectina/farmacologia , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Tripsina/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Fertilidade/efeitos dos fármacos , Resistência a Inseticidas/genética , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia , Simulação de Acoplamento Molecular , Inseticidas/farmacologia
6.
Plant J ; 116(5): 1342-1354, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37614094

RESUMO

Miraculin-like proteins (MLPs), members of the Kunitz trypsin inhibitor (KTI) family that are present in various plants, have been discovered to have a role in defending plants against pathogens. In this study, we identified a gene StMLP1 in potato that belongs to the KTI family. We found that the expression of StMLP1 gradually increases during Ralstonia solanacearum (R. solanacearum) infection. We characterized the promoter of StMLP1 as an inducible promoter that can be triggered by R. solanacearum and as a tissue-specific promoter with specificity for vascular bundle expression. Our findings demonstrate that StMLP1 exhibits trypsin inhibitor activity, and that its signal peptide is essential for proper localization and function. Overexpression of StMLP1 in potato can enhance the resistance to R. solanacearum. Inhibiting the expression of StMLP1 during infection accelerated the infection by R. solanacearum to a certain extent. In addition, the RNA-seq results of the overexpression-StMLP1 lines indicated that StMLP1 was involved in potato immunity. All these findings in our study reveal that StMLP1 functions as a positive regulator that is induced and specifically expressed in vascular bundles in response to R. solanacearum infection.


Assuntos
Ralstonia solanacearum , Solanum tuberosum , Solanum tuberosum/genética , Ralstonia solanacearum/fisiologia , Inibidores da Tripsina/metabolismo , Feixe Vascular de Plantas , Plantas , Doenças das Plantas
7.
Food Res Int ; 169: 112825, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37254400

RESUMO

The presence of so-called anti-nutritional factors can reduce the bioavailability of nutrients following consumption of seeds which are otherwise an excellent source of proteins, carbohydrates and micronutrients. Among the proteins associated with negative effects on quality in pea (Pisum sativum L.) seeds are lectin, pea albumin 2 (PA2) and trypsin inhibitors (TI). Here we have investigated the impact of these proteins on protein digestibility and amino acid availability, using naturally occurring and derived mutant lines of pea lacking these proteins. The mutations were stacked to generate a triple mutant which was compared with a wild-type progenitor and a line lacking the major seed trypsin inhibitors alone. In vitro digestions following the INFOGEST protocol revealed significant differences in the degree of hydrolysis, protein profile and apparent amino acid availability among the pea variants. Proteins resistant to digestion were identified by MALDI-TOF mass spectrometry and amino acid profiles of digested samples determined. The results indicate that pea seeds lacking certain proteins can be used in the development of novel foods which have improved protein digestibility, and without negative impact on seed protein concentration or yield.


Assuntos
Pisum sativum , Proteínas de Plantas , Proteínas de Plantas/análise , Pisum sativum/genética , Pisum sativum/química , Inibidores da Tripsina/metabolismo , Mutação com Perda de Função , Aminoácidos/metabolismo
8.
Plant Cell Rep ; 42(4): 723-734, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36740647

RESUMO

KEY MESSAGE: We identified a miraculin-like protein (NaMLP) who is a new Kunitz trypsin inhibitor regulated synergistically by JA and ethylene signals and confers Spodoptera litura resistance in wild tobacco Nicotiana attenuata. The findings revealed a new source of trypsin inhibitor activities after herbivory, and provide new insights into the complexity of the regulation of trypsin inhibitor-based defense after insect herbivore attack. Upon insect herbivore attack, wild tobacco Nicotiana attenuata accumulates trypsin protease inhibitor (TPI) activities as a defense response from different protease inhibitor (PI) coding genes, including WRKY3-regulated NaKTI2, and JA-dependent NaPI. However, whether any other TPI gene exists in N. attenuata is still unclear. A miraculin-like protein gene (NaMLP) was highly up-regulated in N. attenuata after Alternaria alternata infection. However, silencing or overexpression of NaMLP had no effect on the lesion diameter developed on N. attenuata leaves after A. alternata inoculation. Meanwhile, the transcripts of NaMLP could be induced by wounding and amplified by Spodoptera litura oral secretions (OS). S. litura larvae gained significantly more biomass on NaMLP-silenced plants but less on NaMLP overexpressed plants. Although NaMLP showed low sequence similarity to NaKTI2, it had conserved reaction sites of Kunitz trypsin inhibitors, and exhibited TPI activities when its coding gene was overexpressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP overexpressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficient AOC silencing and ethylene-reduced ACO-silencing plants, and the expression of NaMLP could be significantly induced by methyl jasmonate or ethephon alone, but dramatically amplified by co-treatment of both methyl jasmonate and ethephon. Thus, our results demonstrate that in addition to JA-regulated NaPI, and WRKY3/6-dependent NaKTI2, N. attenuata plants also up-regulates TPI activities via NaMLP, which confers S. litura resistance through JA and ethylene signaling pathways in a synergistic way.


Assuntos
Nicotiana , Inibidores da Tripsina , Animais , Nicotiana/genética , Nicotiana/metabolismo , Inibidores da Tripsina/metabolismo , Spodoptera/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oxilipinas/farmacologia , Oxilipinas/metabolismo , Ciclopentanos/farmacologia , Ciclopentanos/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/metabolismo , Etilenos/metabolismo , Herbivoria
9.
J Anim Sci ; 1012023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36444860

RESUMO

Two experiments were conducted to determine the digestible energy and metabolizable energy contents, as well as the apparent ileal digestibility and standardized ileal digestibility of amino acids in full-fat soybean fed to growing pigs. Ten full-fat soybean samples were collected from different areas in China and used in two experiments in this study. In Exp. 1, 66 growing pigs (initial body weight = 18.48 ± 1.2 kg) were randomly allotted to 1 of 11 diets (n = 6) including a corn basal diet and 10 experimental diets formulated by replacing the corn with 30% full-fat soybean. In Exp. 2, 11 growing pigs (initial body weight = 50.45 ± 3.2 kg) were surgically equipped with a T-cannula in the distal ileum and arranged in a 6 × 11 Youden square design with 11 diets and 6 periods. The diets included an N-free diet based on cornstarch and sucrose and 10 experimental diets formulated with full-fat soybeans as the sole source of amino acids. Chromic oxide was added into the diets as an indigestible maker to calculate the digestibility of the amino acids. Results showed that there was considerable variation in neutral detergent fiber, acid detergent fiber, and trypsin inhibitor contents in the 10 full-fat soybean samples with a coefficient of variation greater than 10%. On a dry matter basis, the averaged digestible energy and metabolizable energy values in the 10 full-fat soybean samples were 4,855 and 4,555 kcal/kg, respectively, both were positively correlated with the ether extract content. The best-fitted prediction equations for digestible energy and metabolizable energy of full-fat soybean were: digestible energy, kcal/kg = 3,472 + 94.87 × ether extract - 97.63 × ash (R2 = 0.91); metabolizable energy, kcal/kg = 3,443 + 65.11 × ether extract - 36.84 × trypsin inhibitor (R2 = 0.91). In addition, all full-fat soybean samples showed high apparent ileal digestibility and standardized ileal digestibility values in amino acids and were all within the range of previously published values. Those values significantly varied among different samples (P < 0.05) for most amino acids, except for glycine and proline. In conclusion, full-fat soybean is a high-quality protein ingredient with high ileal digestibility of amino acids when fed to growing pigs, and the metabolizable energy value of full-fat soybean could be predicted based on its ether extract and trypsin inhibitor contents.


Full-fat soybean is an excellent protein source supplied in swine diets, especially in weaned and growing stages. However, the high price limits its utilization in practice, so it is vital to accurately evaluate the available energy and digestible amino acids contents in full-fat soybean to better formulate a least-cost diet. Ten full-fat soybean samples were collected from different areas in China, and two experiments were conducted to evaluate the energy concentration and amino acids digestibility of full-fat soybean and to establish the corresponding prediction equations. The averaged digestible and metabolizable energy of FFSB were 4,855 and 4,555 kcal/kg (dry matter basis), and the best-fitted prediction equations for digestible energy and metabolizable energy of full-fat soybean were: digestible energy, kcal/kg = 3,472 + 94.87 × ether extract − 97.63 × ash; metabolizable energy, kcal/kg = 3,443 + 65.11 × ether extract − 36.84 × trypsin inhibitor. Except for glycine and proline, the digestibility of other amino acids significantly varied among 10 full-fat soybean samples but all were within the range of previously published values. In addition, all the amino acids exhibited high digestibility, indicating that full-fat soybean is a protein ingredient with high quality.


Assuntos
Aminoácidos , Glycine max , Suínos , Animais , Glycine max/química , Aminoácidos/metabolismo , Digestão , Detergentes/metabolismo , Inibidores da Tripsina/metabolismo , Ração Animal/análise , Dieta , Íleo/metabolismo , Éteres/metabolismo , Extratos Vegetais , Fenômenos Fisiológicos da Nutrição Animal , Zea mays/metabolismo
10.
Proteins ; 91(1): 22-31, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35927030

RESUMO

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.


Assuntos
Phaseolus , Inibidor da Tripsina de Soja de Bowman-Birk , Bovinos , Animais , Inibidor da Tripsina de Soja de Bowman-Birk/química , Inibidor da Tripsina de Soja de Bowman-Birk/metabolismo , Tripsina/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Quimotripsina
11.
Trop Anim Health Prod ; 54(5): 330, 2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36173493

RESUMO

In this study, the in vitro apparent rumen degradability of organic matter (ARDOM) and plant secondary metabolites (ARDPSM) of three tropical legumes (Mucuna pruriens, Canavalia ensiformis, and Leucaena leucocephala) were assessed. For this, 3 experiments were set up, i.e., single end-point incubations (24 h) with ruminal inoculum from either Belgian or Cuban sheep, as well as kinetic assessments (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h) inoculum from Belgian sheep. L-mimosine, L-canavanine, Concanavalin A (Con A), and trypsin inhibitor (TI) were the plant secondary metabolites (PSM) targeted in this study. In all three experiments, both beans, as well as forage/bean meals of M. pruriens and C. ensiformis and their PSM, were extensively degraded during 24 h incubation, irrespective of the inoculum source (0.44 to 0.70 and 0.43 to 0.78 g/g of organic matter (OM) for ARDOM, respectively, and > 0.80 g/g for L-canavanine, > 0.76 TIU/TIU for TI, and > 0.95 g/g for Con A, for both legumes). Forage meal of L. leucocephala was considerably less degraded, with apparent ruminal degradabilities of 0.20 g/g OM and 0.35 g/g OM after 24 h incubation with Belgian or Cuban sheep inoculum, respectively. This could - at least partially - be related to L-mimosine, present in L. leucocephala, which was hardly degraded in the Belgian incubation, while a more extensive ruminal breakdown was observed under the Cuban conditions (0.05 g/g PSM vs. 0.78 g/g PSM, respectively). The negative effect of L-mimosine on OM degradability was supported in an additional in vitro experiment with straw and inoculum from Belgian sheep, as ruminal degradation of straw was 31% lower when pure L-mimosine was supplemented.


Assuntos
Fabaceae , Rúmen , Ração Animal/análise , Animais , Canavanina/metabolismo , Concanavalina A/metabolismo , Digestão , Fabaceae/metabolismo , Fermentação , Mimosina/metabolismo , Rúmen/metabolismo , Ovinos , Inibidores da Tripsina/metabolismo , Verduras/metabolismo
12.
Int J Mol Sci ; 23(14)2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35887084

RESUMO

Cysteine-rich trypsin inhibitor-like domain (TIL)-harboring proteins are broadly distributed in nature but remain understudied in vector mosquitoes. Here we have explored the biology of a TIL domain-containing protein of the arbovirus vector Aedes aegypti, cysteine-rich venom protein 379 (CRVP379). CRVP379 was previously shown to be essential for dengue virus infection in Ae. aegypti mosquitoes. Gene expression analysis showed CRVP379 to be highly expressed in pupal stages, male testes, and female ovaries. CRVP379 expression is also increased in the ovaries at 48 h post-blood feeding. We used CRISPR-Cas9 genome editing to generate two mutant lines of CRVP379 with mutations inside or outside the TIL domain. Female mosquitoes from both mutant lines showed severe defects in their reproductive capability; mutant females also showed differences in their follicular cell morphology. However, the CRVP379 line with a mutation outside the TIL domain did not affect male reproductive performance, suggesting that some CRVP379 residues may have sexually dimorphic functions. In contrast to previous reports, we did not observe a noticeable difference in dengue virus infection between the wild-type and any of the mutant lines. The importance of CRVP379 in Ae. aegypti reproductive biology makes it an interesting candidate for the development of Ae. aegypti population control methods.


Assuntos
Aedes , Dengue , Viroses , Animais , Cisteína/metabolismo , Feminino , Masculino , Mosquitos Vetores/genética , Reprodução/genética , Tripsina/metabolismo , Inibidores da Tripsina/metabolismo
13.
Plant Foods Hum Nutr ; 77(1): 20-29, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35000105

RESUMO

Plant-based diets are a great source of protease inhibitors (PIs). Two of the most well-known families of PIs are Bowman-Birk inhibitors (BBI) and Kunitz-type inhibitors (KTI). The first group acts mainly on trypsin, chymotrypsin, and elastase; the second is on serine, cysteine, and aspartic proteases. PIs can retard or inhibit the catalytic action of enzymes; therefore, they are considered non-nutritional compounds; nevertheless, animal studies and cell line experiments showed promising results of PIs in treating human illnesses such as obesity, cardiovascular diseases, autoimmune diseases, inflammatory processes, and different types of cancer (gastric, colorectal, breast, and lung cancer). Anticarcinogenic activity's proposed mechanisms of action comprise several inhibitory effects at different molecular levels, i.e., transcription, post-transcription, translation, post-translation, and secretion of cancer cells. This work reviews the potential therapeutic applications of PIs as anticarcinogenic and anti-inflammatory agents in human diseases and the mechanisms by which they exert these effects.


Assuntos
Ácido Aspártico Proteases , Inibidor da Tripsina de Soja de Bowman-Birk , Animais , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Tripsina , Inibidor da Tripsina de Soja de Bowman-Birk/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Inibidor da Tripsina de Soja de Bowman-Birk/uso terapêutico , Inibidores da Tripsina/metabolismo
14.
Sci Rep ; 12(1): 698, 2022 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-35027639

RESUMO

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10-7 mol L-1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70-80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and ß-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL-1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.


Assuntos
Cacau/química , Cacau/parasitologia , Inibidores da Tripsina/isolamento & purificação , Inibidores da Tripsina/farmacologia , Agaricales/efeitos dos fármacos , Agaricales/crescimento & desenvolvimento , Animais , Cacau/metabolismo , Estabilidade de Medicamentos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Isoformas de Proteínas , Temperatura , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo
15.
In Vivo ; 35(5): 2551-2558, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410942

RESUMO

BACKGROUND/AIM: We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath system. However, secretion of trypsin, another pancreatic enzyme, interferes with insulin production in such systems. We aimed to ascertain the minimum trypsin inhibitor (TI), dose for obtaining a sustained, stable rate of insulin secretion. MATERIALS AND METHODS: The action of TI (1-10 µg/ml) on pancreatic preparations of male Wistar-Imamichi rats in organ bath experiments was assessed by measuring insulin, amylase, and trypsin activity. RESULTS: The level of insulin outflow remained steady in the TI-treated samples, in contrast to that in the untreated control, where insulin secretion decreased over time. The level of amylase outflow did not change significantly. Trypsin activity was significantly lower in the TI-treated samples than in the control. CONCLUSION: Even low concentrations of TI can maintain insulin secretion by inhibiting trypsin activity in organ bath experiments.


Assuntos
Amilases , Inibidores da Tripsina , Animais , Insulina/metabolismo , Secreção de Insulina , Masculino , Pâncreas/metabolismo , Ratos , Ratos Wistar , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
16.
J Basic Microbiol ; 61(8): 709-720, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34228389

RESUMO

Trypsin is a protein-digesting enzyme that is essential for the growth and regeneration of bone, muscle, cartilage, skin, and blood. The trypsin inhibitors have various role in diseases such as inflammation, Alzheimer's disease, pancreatitis, rheumatoid arthritis, cancer prognosis, metastasis and so forth. From 10 endophytic fungi isolated, we were able to screen only one strain with the required activity. The fungus with activity was obtained as an endophyte from Dendrophthoe falcata and was later identified as Nigrospora sphaerica. The activity was checked by enzyme assays using trypsin. The fungus was fermented and the metabolites were extracted and further purified by bioassay-guided chromatographic methods and the compound isolated was identified using gas chromatography-mass spectrometry. The compound was identified as quercetin. Docking studies were employed to study the interaction. The absorption, distribution, metabolism, and excretion analysis showed satisfactory results and the compound has no AMES and hepatotoxicity. This study reveals the ability of N. sphaerica to produce bioactive compound quercetin has been identified as a potential candidate for trypsin inhibition. The present communication describes the first report claiming that N. sphaerica strain AVA-1 can produce quercetin and it can be considered as a sustainable source of trypsin active-site inhibitors.


Assuntos
Ascomicetos/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Tripsina/metabolismo , Antioxidantes , Endófitos/metabolismo , Fermentação , Loranthaceae , Simulação de Acoplamento Molecular , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
17.
Theor Appl Genet ; 134(10): 3427-3441, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34245321

RESUMO

KEY MESSAGE: Wheat cultivars largely differ in the content and composition of ATI proteins, but heritability was quite low for six out of eight ATIs. The genetic architecture of ATI proteins is built up of few major and numerous small effect QTL. Amylase trypsin inhibitors (ATIs) are important allergens in baker's asthma and suspected triggers of non-celiac wheat sensitivity (NCWS) inducing intestinal and extra-intestinal inflammation. As studies on the expression and genetic architecture of ATI proteins in wheat are lacking, we evaluated 149 European old and modern bread wheat cultivars grown at three different field locations for their content of eight ATI proteins. Large differences in the content and composition of ATIs in the different cultivars were identified ranging from 3.76 pmol for ATI CM2 to 80.4 pmol for ATI 0.19, with up to 2.5-fold variation in CM-type and up to sixfold variation in mono/dimeric ATIs. Generally, heritability estimates were low except for ATI 0.28 and ATI CM2. ATI protein content showed a low correlation with quality traits commonly analyzed in wheat breeding. Similarly, no trends were found regarding ATI content in wheat cultivars originating from numerous countries and decades of breeding history. Genome-wide association mapping revealed a complex genetic architecture built of many small, few medium and two major quantitative trait loci (QTL). The major QTL were located on chromosomes 3B for ATI 0.19-like and 6B for ATI 0.28, explaining 70.6 and 68.7% of the genotypic variance, respectively. Within close physical proximity to the medium and major QTL, we identified eight potential candidate genes on the wheat reference genome encoding structurally related lipid transfer proteins. Consequently, selection and breeding of wheat cultivars with low ATI protein amounts appear difficult requiring other strategies to reduce ATI content in wheat products.


Assuntos
Cromossomos de Plantas/genética , Fenótipo , Proteínas de Plantas/metabolismo , Locos de Características Quantitativas , Triticum/metabolismo , Inibidores da Tripsina/metabolismo , alfa-Amilases/antagonistas & inibidores , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , Melhoramento Vegetal , Proteínas de Plantas/genética
18.
Food Chem ; 360: 130057, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34029924

RESUMO

In this study, the effect of ultrasonic frequency and power on the inactivation of soy trypsin inhibitors (TIs) was investigated to explore the ultrasound-induced inactivation mechanism. It was observed that 20 kHz and 355 kHz ultrasound have better inactivation efficiency than 1056 kHz. First-order rate constants for the inactivation process were obtained, which increased with increasing ultrasonic power at both 20 kHz and 355 kHz. For 20 kHz ultrasound, the formation of TI aggregates resulting from the physical effects of acoustic cavitation decreased the interactions between the active sites of TIs and trypsin, thus reducing the TI activity. For 355 kHz ultrasound, most of the methionine in the TIs was oxidised within 5 mins, resulting in a faster reduction of TI activity. Subsequent aggregation of TIs resulted in further TI inactivation. SDS-PAGE showed that neither disulphide bonds nor CC coupling were involved in the formation of aggregates.


Assuntos
Glycine max/metabolismo , Proteínas de Plantas/química , Sonicação , Inibidores da Tripsina/química , Domínio Catalítico , Cinética , Tamanho da Partícula , Proteínas de Plantas/metabolismo , Inibidores da Tripsina/metabolismo
19.
ACS Chem Biol ; 16(5): 829-837, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33881318

RESUMO

Angiogenesis is important for tumor growth, and accordingly, targeting angiogenesis has become an important pathway for antitumor therapy. A novel proapoptotic peptide, CIGB-300 (P15-Tat), has been shown to be involved in the casein kinase II phosphorylation pathway, conferring it with antiangiogenic activity. Cyclic peptides have been widely used as scaffolds in drug design studies due to their high stability and favorable biopharmaceutical properties. Here, we chose two very stable cyclic trypsin inhibitors, MCoTI-II and SFTI-1, as frameworks to incorporate the bioactive epitope P15 into various backbone loops. NMR studies revealed that all re-engineered analogs had similar secondary structures to their native cyclic frameworks. One key analog, MCoP15, displayed significant improvement for inhibiting human umbilical vein endothelial cell migration, was nontoxic, and had higher stability than the P15 epitope alone. Overall, the results show the value of P15 being engineered into cyclic trypsin inhibitor scaffolds for improving antiangiogenic activity and stability. More broadly, the study highlights the versatility of cyclic peptide frameworks in drug design for antiangiogenic therapies.


Assuntos
Inibidores da Angiogênese/química , Colágeno/química , Ciclotídeos/química , Neovascularização Patológica/tratamento farmacológico , Fragmentos de Peptídeos/química , Peptídeos Cíclicos/química , Inibidores da Tripsina/química , Sequência de Aminoácidos , Inibidores da Angiogênese/metabolismo , Caseína Quinase II/metabolismo , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Desenho de Fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Inibidores da Tripsina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Methods Mol Biol ; 2263: 161-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877597

RESUMO

Microscale thermophoresis (MST) has become a widely used technique to determine the KD or EC50 of protein-ligand interactions. The method exploits the tendency of macromolecules to migrate along a thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield KD. In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily available, inexpensive materials to plan, execute, and analyze an MST experiment.


Assuntos
Quimotripsina/metabolismo , Glycine max/metabolismo , Inibidores da Tripsina/metabolismo , Animais , Humanos , Cinética , Proteínas de Plantas/metabolismo , Ligação Proteica , Termodinâmica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...