RESUMO
We advance the quality of first-principles calculations of protein electronic circular dichroism (CD) through an amelioration of a key deficiency of a previous procedure that involved diabatization of electronic states on the amide chromophore (to obtain interamide couplings) in a ß-strand conformation of a diamide. This yields substantially improved calculated far-ultraviolet (far-UV) electronic circular dichroism (CD) spectra for ß-sheet conformations. The interamide couplings from the diabatization procedure for 13 secondary structural elements (13 diamide structures) are applied to compute the CD spectra for seven example proteins: myoglobin (α helix), jacalin (ß strand), concanavalin A (ß type I), elastase (ß type II), papain (α + ß), 310-helix bundle (310-helix) and snow flea antifreeze protein (polyproline). In all cases, except concanavalin A and papain, the CD spectra computed using the interamide couplings from the diabatization procedure yield improved agreement with experiment with respect to previous first-principles calculations.
Assuntos
Dicroísmo Circular , Concanavalina A , Concanavalina A/química , Mioglobina/química , Proteínas/química , Papaína/química , Papaína/metabolismo , Peptídeos/química , Elétrons , Elastase Pancreática/química , Elastase Pancreática/metabolismo , Estrutura Secundária de ProteínaRESUMO
Enzymatic peptide synthesis is a powerful alternative to solid-phase methods, as enzymes can have high regio- and stereoselectivity and high yield and require mild reaction conditions. This is beneficial in formulation research due to the rise of nucleic acid therapies. Peptide nucleic acids (PNAs) have a high affinity toward DNA and RNA, and their solubility and cellular delivery can be improved via conjugation to peptides. Here, we designed and assessed the viability of the papain enzyme to conjugate four PNA-peptide models in water and an organic solvent using QM/MM metadynamics. We found that the reactions in water yield better results, where three conjugates could potentially be synthesized by the enzyme, with the first transition state as the rate-limiting step, with an associated energy of 14.53 kcal mol-1, although with a slight endergonic profile. The results highlight the importance of considering the enzyme pockets and different substrate acceptivities and contribute to developing greener, direct, and precise synthetic routes for nucleic acid-based therapies. By exploring the enzyme's potential in conjunction with chemical synthesis, current protocols can be simplified for the synthesis of longer nucleic acids and peptide sequences (and, by extension, proteins) from smaller oligo or peptide blocks.
Assuntos
Teoria da Densidade Funcional , Papaína , Ácidos Nucleicos Peptídicos , Peptídeos , Solventes , Água , Ácidos Nucleicos Peptídicos/química , Papaína/química , Papaína/metabolismo , Água/química , Solventes/química , Peptídeos/química , Simulação de Dinâmica MolecularRESUMO
Obesity is becoming a worldwide pandemic. Interfacial engineering of food lipid is expected to inhibit diet-induced obesity without damage to the eating enjoyment brought by high-fat diets. Unfortunately, this strategy has not been achieved yet. After screening different plant proteins, bromelain and papain were found to form wormlike and long-straight protein fibrils, respectively. The conversion of long-straight amyloid-like fibrils to wormlike fibrils was demonstrated in the fibrillation of bromelain. Using oil-in-water high internal phase emulsions (HIPEs) as a proof of concept, bromelain fibrils showed dramatically stronger interfacial stabilization capabilities than papain fibrils with high application potentials in the real-world formulation of high-fat food products such as mayonnaise. Compared with papain fibrils, oral administration of HIPEs stabilized by bromelain fibrils resulted in substantially higher fecal lipid contents and significantly decreased expression levels of the genes related to lipid absorption and transport in the intestine, including CD36, FATP-2, FATP-4, and APOA-4, without a difference in intervening gut microbiota. Consequently, dramatically less lipid absorption in the small intestine, markedly smaller chylomicron particles in the plasma, lower serum triglycerides, and controlled energy and lipid metabolism, as well as the inhibition of adipose expansion and overweight, were observed in the group with gavage of HIPEs stabilized by the bromelain fibrils rather than the papain fibrils. Furthermore, with the same calorie, substitution of all the fat in the standard high-fat feed of mice with the HIPEs emulsified by the bromelain fibrils showed a significantly stronger effect than the ones prepared by the papain fibrils on preventing high-fat-diet (HFD)-induced obesity including alleviation of adipose expansion and inflammation as well as fatty liver, also via inhibiting the absorption and transport of lipid in the intestine. The effect is ascribed to the suppressed lipolysis caused by a more compact and elastic interfacial layer formed by the wormlike fibrils than that of the long-straight fibrils, which are resistant to gastric environments and replacement by bile acids in digestion. Therefore, we provide an appealing and general strategy for controlling obesity by reducing the supply of free fatty acids (FAs) for absorption in the enteric lumen through protein fibril polymorphisms at the interface.
Assuntos
Obesidade , Papaína , Animais , Obesidade/metabolismo , Camundongos , Papaína/metabolismo , Papaína/química , Bromelaínas/farmacologia , Bromelaínas/química , Bromelaínas/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Dieta Hiperlipídica , Emulsões/química , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacosRESUMO
The environmental benefits of utilizing protease as a biocatalyst for wool shrink-resist finishing have been widely recognized. However, the efficacy of individual protease treatment is unsatisfactory due to its incapability towards the outermost cuticle layer of wool fibers that contains hydrophobic fatty acids. In order to weaken the structural integrity of the highly cross-linked scales and promote the enzymatic anti-felting, sodium sulfite and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were employed in combination with papain, respectively, aiming at obtaining a low shrinkage without unacceptable fiber damages. Based on the synergistic effect of papain and TCEP, the edges of wool scales were slightly destroyed by the reduction of disulfide bonds, accompanied by enzymatic hydrolysis of the keratin component. Through the controlled reduction and hydrolysis of wool scales, satisfactory anti-felting result was achieved without causing severe damage to the fiber interiors. In the presence of 0.25 g/L TCEP and 25 U/mL papain, the area shrinkage of wool fabric decreased to approximately 6 %, with a low strength loss of less than 8 %. Meanwhile, the dyeing behavior of the wool fabric under low-temperature conditions was dramatically improved, leading to decreased energy consumption during production. The present work provides an alternative for eco-friendly finishing of wool fabrics, which can be applied commercially.
Assuntos
Dissulfetos , Papaína , Lã , Papaína/química , Animais , Lã/química , Dissulfetos/química , Substâncias Redutoras/química , Sulfitos/química , Sulfitos/farmacologia , Fosfinas/química , Fibra de Lã , Hidrólise , TêxteisRESUMO
Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol-1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.
Assuntos
Inibidores da Enzima Conversora de Angiotensina , Simulação de Acoplamento Molecular , Peptídeos , Hidrolisados de Proteína , Solubilidade , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Animais , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Água/química , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Papaína/metabolismo , Papaína/antagonistas & inibidores , Papaína/química , Proteínas de Peixes/química , Proteínas de Peixes/metabolismoRESUMO
The dual-frequency ultrasound-assisted enzymatic digestion (DUED) technique was developed for synchronous green extraction of five heavy metal ions in root vegetables. The combination of α-amylase, cellulase, and papain showed significant advantageous in extracting heavy metal ions. Under optimized dual-frequency ultrasonic conditions, the extraction rates of Cr, As, Cd, Pb, and Hg in carrots reached 99.04%, 105.88%, 104.65%, 104.10%, and 103.13% respectively. And the extraction process is highly efficient, completing in just 15 min. Compared to conventional microwave-assisted acid hydrolysis method, this technique eliminates the need for high-temperature concentrated acid, enhancing its environmental sustainability while maintaining mild reaction conditions, making it ideal for biosensors application. Additionally, simultaneous extraction and detection of four heavy metals in lotus roots were successfully achieved by using DUED and a fluorescent paper-based microfluidic chip. The obtained results are consistent with those obtained using conventional methods.
Assuntos
Metais Pesados , Raízes de Plantas , Verduras , Metais Pesados/isolamento & purificação , Metais Pesados/química , Verduras/química , Raízes de Plantas/química , alfa-Amilases/metabolismo , alfa-Amilases/química , Celulase/química , Celulase/metabolismo , Papaína/química , Papaína/metabolismo , Ultrassom , Contaminação de Alimentos/análise , Daucus carota/químicaRESUMO
Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5 h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of âCOOH and âNH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.
Assuntos
Cálcio , Galinhas , Animais , Feminino , Cálcio/metabolismo , Galinhas/metabolismo , Hidrolisados de Proteína/química , Peptídeos/química , Hidrólise , Papaína/química , Aminoácidos , Cálcio da Dieta/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Carne , EtanolRESUMO
The bitterness of soy protein isolate hydrolysates prepared using five proteases at varying degree of hydrolysis (DH) and its relation to physicochemical properties, i.e., surface hydrophobicity (H0), relative hydrophobicity (RH), and molecular weight (MW), were studied and developed for predictive modelling using machine learning. Bitter scores were collected from sensory analysis and assigned as the target, while the physicochemical properties were assigned as the features. The modelling involved data pre-processing with local outlier factor; model development with support vector machine, linear regression, adaptive boosting, and K-nearest neighbors algorithms; and performance evaluation by 10-fold stratified cross-validation. The results indicated that alcalase hydrolysates were the most bitter, followed by protamex, flavorzyme, papain, and bromelain. Distinctive correlation results were found among the physicochemical properties, influenced by the disparity of each protease. Among the features, the combination of RH-MW fitted various classification models and resulted in the best prediction performance.
Assuntos
Proteínas de Soja , Paladar , Hidrólise , Proteínas de Soja/química , Peptídeo Hidrolases/metabolismo , Papaína/química , Hidrolisados de Proteína/químicaRESUMO
The COVID-19 pandemic is caused by SARS-CoV-2, an RNA virus with high transmissibility and mutation rate. Given the paucity of orally bioavailable antiviral drugs to combat SARS-CoV-2 infection, there is a critical need for additional antivirals with alternative mechanisms of action. Papain-like protease (PLpro) is one of the two SARS-CoV-2 encoded viral cysteine proteases essential for viral replication. PLpro cleaves at three sites of the viral polyproteins. In addition, PLpro antagonizes the host immune response upon viral infection by cleaving ISG15 and ubiquitin from host proteins. Therefore, PLpro is a validated antiviral drug target. In this study, we report the X-ray crystal structures of papain-like protease (PLpro) with two potent inhibitors, Jun9722 and Jun9843. Subsequently, we designed and synthesized several series of analogs to explore the structure-activity relationship, which led to the discovery of PLpro inhibitors with potent enzymatic inhibitory activity and antiviral activity against SARS-CoV-2. Together, the lead compounds are promising drug candidates for further development.
Assuntos
COVID-19 , Papaína , Humanos , Papaína/química , Papaína/genética , Papaína/metabolismo , SARS-CoV-2/metabolismo , Pandemias , Antivirais/farmacologia , Antivirais/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/químicaRESUMO
The highly infectious respiratory illness 'COVID-19' was caused by SARS-CoV-2 and is responsible for millions of deaths. SARS-single-stranded viral RNA genome encodes several structural and nonstructural proteins, including papain-like protease (PLpro), which is essential for viral replication and immune evasion and serve as a potential therapeutic target. Multiple computational techniques were used to search the natural compounds that may block the protease and deubiquitinase activities of PLpro. Five compounds showed strong interactions and binding energy (ranges between -8.18 to -8.69 Kcal/mol) in our in-silico studies. Interestingly, those molecules strongly bind in the PLpro active site and form a stable complex, as shown by microscale molecular dynamic simulations (MD). The dynamic movements indicate that PLpro acquires closed conformation by the attachment of these molecules, thereby changing its normal function. In the in-vitro evaluation, compound COMP4 showed the most potent inhibitory potential for PLpro (protease activity: 2.24 ± 0.17 µM and deubiquitinase activity: 1.43 ± 0.14 µM), followed by COMP1, 2, 3, and 5. Furthermore, the cytotoxic effect of COMP1-COMP5 on a human BJ cell line revealed that these compounds demonstrate negligible cytotoxicity at a dosage of 30 µM. The results suggest that these entities bear therapeutic efficacy for SARS-CoV-2 PLpro.
Assuntos
Produtos Biológicos , COVID-19 , Humanos , Papaína/química , Peptídeo Hidrolases/metabolismo , SARS-CoV-2 , Produtos Biológicos/farmacologia , Enzimas Desubiquitinantes , Antivirais/farmacologiaRESUMO
Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.
Assuntos
Equartevirus , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Cavalos , Suínos , Humanos , Papaína/química , Papaína/genética , Papaína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Mutagênese , Peptídeo Hidrolases/genética , Replicação Viral , Interferons/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Cystatins encode a high functional variability not only because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils; however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Noncovalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The noncovalent dimeric form of AtCYT6 retained activity against its target proteases, papain and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to the AtCYT6's N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications for plant protease regulation.
Assuntos
Arabidopsis , Cistatinas , Animais , Papaína/química , Amiloide/química , Cistatinas/química , Cistatinas/farmacologia , Peptídeo Hidrolases , MamíferosRESUMO
Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 Mpro and PLpro protease activity was identified. In silico docking studies also predicted the binding of the phenothiazine urea to the active sites of structurally similar Mpro and PLpro proteases from distantly related alphacoronavirus, HCoV-229 E (229 E), and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cellulo. It was further demonstrated that the compound inhibited 229 E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles, and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative readily inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses.
Assuntos
Peptídeo Hidrolases , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Papaína/química , Proteases Virais , Fenotiazinas/farmacologia , Inibidores de Proteases/química , Antivirais/farmacologia , Antivirais/químicaRESUMO
To date, mechanistic insights into many clinical drugs against COVID-19 remain unexplored. Dexamethasone, a corticosteroid, is one of them. While treating the entire corticosteroid database, including vitamins D2 and D3, with cutting-edge computational techniques, several intriguing results are unfolded. From the top-notch candidates, dexamethasone is likely to inhibit the viral main protease (Mpro), with vitamin D3 exhibiting multitarget [Mpro, papain-like protease (PLpro), and nucleocapsid protein (N-pro)] roles and ciclesonide's dynamic flipping disinterring a cryptic allosteric site in the PLpro enzyme. The results rationalize why these drugs improve the health of COVID-19 patients. Understanding an enzyme's secret binding site is essential to understanding how the enzyme works and how to inhibit its function. Ciclesonide's allosteric inhibition could not only jeopardize PLpro's catalytic role in polyprotein processing but also make it less vulnerable to the host body's defense machinery. Hotspot residues in the identified allosteric site could be considered for effective therapeutic designs against PLpro.
Assuntos
COVID-19 , Papaína , Humanos , Papaína/química , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , Sítio Alostérico , SARS-CoV-2/metabolismo , Ubiquitina , Simulação de Dinâmica Molecular , Sítios de Ligação , Dexametasona , Antivirais/química , Inibidores de ProteasesRESUMO
The aim of this study is to explore differences in the peptidomics of Saccharomyces pastorianus protein hydrolysates treated with different enzymes. Briefly, differences in the peptide fingerprints and active peptides of neutral protease/papain-hydrolyzed S. pastorianus were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) combined with PEAKS Online 1.7 analysis software, Peptide Ranker, and the BIOPEP database. Compared to traditional databases, the PEAKS Online uses de novo sequencing for analysis to obtain oligopeptides smaller than pentapeptides. It provides more comprehensive data of the peptide sample. In this study, enzymatic hydrolysates of S. pastorianus protein were prepared under the optimum conditions of neutral protease and papain respectively. In total, 7221 and 7062 polypeptides were identified in the hydrolysates of neutral protease and papain, respectively; among these polypeptides, 980 were common to the two enzymes. The 6241 and 6082 unique peptides found in the hydrolysates of neutral protease and papain, respectively, indicated that the peptide fingerprints of the two hydrolysates are quite different. Peptide Ranker predicted that 3013 (41.73%) and 3095 (43.83%) peptides were potentially bioactive in the hydrolysates of neutral protease and papain, respectively. According to the BIOPEP database, neutral protease and papain contained 295 and 357 active peptides, respectively; these peptides were mainly composed of angiotensin converting enzyme (ACE) inhibitors and dipeptidyl peptidase IV inhibitors and antioxidant peptides. The number of active peptides in the hydrolysate of papain was higher than that in the hydrolysate of neutral protease, but the total ion intensity of active peptides in the former was lower than that in the latter. This study revealed the influence of protease type on the composition of enzymatic hydrolysates from S. pastorianus protein. The above results provide a reference for the development of functional products of S. pastorianus protein peptides and the high-value utilization of yeast resources.
Assuntos
Papaína , Hidrolisados de Proteína , Papaína/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/análise , Inibidores da Enzima Conversora de Angiotensina/metabolismo , HidróliseRESUMO
This study aimed to hydrolyze soy isolate protein (SPI) using five enzymes (alcalase, pepsin, trypsin, papain, and bromelain) in order to obtain five enzymatic hydrolysates and to elucidate the effect of enzymes on structural and biological activities of the resulting hydrolysates. The antioxidant and hypoglycemic activities of the soy protein isolate hydrolysates (SPIEHs) were evaluated through in silico analysis, revealing that the alcalase hydrolysate exhibited the highest potential, followed by the papain and bromelain hydrolysates. Subsequently, the degree of hydrolysis (DH), molecular weight distribution (MWD), amino acid composition, structure, antioxidant activities, and hypoglycemic activity in vitro of SPIEHs were analyzed. After enzymatic treatment, the particle size, polymer dispersity index (PDI), ζ-potentials, ß-sheet content and α-helix content of SPIEHs was decreased, and the maximum emission wavelength of all SPIEHs exhibited red-shifted, which all suggesting the structure of SPIEHs was unfolded. More total amino acids (TAAs), aromatic amino acids (AAAs), and hydrophobic amino acids (HAAs) were found in alcalase hydrolysate. For 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, metal ion chelating activity, α-glucosidase inhibitory activity and α-amylase inhibitory activity, alcalase hydrolysate had the lowest IC50; alcalase hydrolysate and papain hydrolysate had the lowest IC50 for hydroxyl radical scavenging activity. Physiological activity of SPIEHs was evaluated thoroughly by 5-Axe cobweb charts, and the results revealed that alcalase hydrolysate exhibited the greatest biological activities.
Assuntos
Antioxidantes , Bromelaínas , Antioxidantes/farmacologia , Antioxidantes/química , Glycine max/metabolismo , Papaína/química , Hidrolisados de Proteína/química , Proteínas de Soja , Aminoácidos , Subtilisinas/químicaRESUMO
Papain-like protease (PLpro) from severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is a prime target for the development of antivirals for Coronavirus disease 2019 (COVID-19). However, drugs that target the PLpro protein have not yet been approved. In order to gain insights into the development of a PLpro inhibitor, conformational dynamics of PLpro in complex with GRL0617, the most well-characterized PLpro inhibitor, were investigated using nuclear magnetic resonance (NMR) spectroscopy in solution. Although mutational analyses demonstrated that the L162 sidechain interaction is responsible for the affinity for GRL0617, NMR analyses revealed that L162 in the inhibitor-binding pocket underwent conformational exchange and was not fixed in the conformation in which it formed a contact with ortho-methyl group of GRL0617. The identified conformational dynamics would provide a rationale for the binding mechanism of a covalent inhibitor designed based on GRL0617.
Assuntos
COVID-19 , Papaína , Humanos , Papaína/química , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/metabolismo , Sítios de Ligação , Antivirais/farmacologia , Espectroscopia de Ressonância MagnéticaRESUMO
The study aimed to investigate the effects of alcalase, papain, flavourzyme, and neutrase on the structural characteristics and bioactivity stability of Cucumaria frondosa intestines and ovum hydrolysates (CFHs). The findings revealed that flavourzyme exhibited the highest hydrolysis rate (51.88% ± 1.87%). At pH 2.0, the solubility of hydrolysate was the lowest across all treatments, while the solubility at other pH levels was over 60%. The primary structures of hydrolysates of different proteases were similar, whereas the surface hydrophobicity of hydrolysates was influenced by the types of proteases used. The hydrolysates produced by different proteases were also analyzed for their absorption peaks and antioxidant activity. The hydrolysates of flavourzyme had ß-fold absorption peaks (1637 cm-1), while the neutrase and papain hydrolysates had N-H bending vibrations. The tertiary structure of CFHs was unfolded by different proteases, exposing the aromatic amino acids and red-shifting of the λ-peak of the hydrolysate. The alcalase hydrolysates showed better antioxidant activity in vitro and better surface hydrophobicity than the other hydrolysates. The flavourzyme hydrolysates displayed excellent antioxidant stability and pancreatic lipase inhibitory activity during gastrointestinal digestion, indicating their potential use as antioxidants in the food and pharmaceutical industries.
Assuntos
Cucumaria , Peptídeo Hidrolases , Animais , Peptídeo Hidrolases/metabolismo , Papaína/química , Antioxidantes/farmacologia , Hidrólise , Intestinos , Subtilisinas/química , Hidrolisados de Proteína/químicaRESUMO
Okara is a solid byproduct created during the processing of soy milk. The production of protein hydrolysates utilizing enzymatic tests such as papain can result in the production of bioactive peptides (BPs), which are amino acid sequences that can also be produced from the okara protein by hydrolysis. The objective of this study was to investigate the antioxidant activities of okara hydrolysates using papain, based on the in silico and in vitro assays using the papain enzyme. We found that using the in silico assessment, the antioxidant peptides can be found from the precursor (glycinin and conglycinin) in okara. When used as a protease, papain provides the maximum degree of hydrolysis for antioxidative peptides. The highest-peptide-rank peptide sequence was predicted using peptide ranks such as proline-histidine-phenylalanine (PHF), alanine-aspartic acid-phenylalanine (ADF), tyrosine-tyrosine-leucine (YYL), proline-histidine-histidine (PHH), isoleucine-arginine (IR), and serine-valine-leucine (SVL). Molecular docking studies revealed that all peptides generated from the parent protein impeded substrate access to the active site of xanthine oxidase (XO). They have antioxidative properties and are employed in the in silico approach to the XO enzyme. We also use papain to evaluate the antioxidant activity by using in vitro tests for protein hydrolysate following proteolysis. The antioxidant properties of okara protein hydrolysates have been shown in vitro, utilizing DPPH and FRAP experiments. This study suggests that okara hydrolysates generated by papain can be employed as natural antioxidants in food and for further applications, such as active ingredients for antioxidants in packaging.
Assuntos
Antioxidantes , Hidrolisados de Proteína , Antioxidantes/farmacologia , Antioxidantes/química , Hidrolisados de Proteína/química , Papaína/química , Simulação de Acoplamento Molecular , Histidina , Leucina , Hidrólise , Peptídeos/farmacologia , Peptídeos/químicaRESUMO
Coronaviruses that can infect humans can cause either common colds (HCoV-NL63, HCoV-229E, HCoV-HKU1, and HCoV-OC43) or severe respiratory symptoms (SARS-CoV-2, SARS-CoV, and MERS-CoV). The papain-like proteases (PLPs) of SARS-CoV, SARS-CoV-2, MERS-CoV, and HCoV-NL63 function in viral innate immune evasion and have deubiquitinating (DUB) and deISGylating activities. We identified the PLPs of HCoV-229E, HCoV-HKU1, and HCoV-OC43 and found that their enzymatic properties correlated with their ability to suppress innate immune responses. A conserved noncatalytic aspartic acid residue was critical for both DUB and deISGylating activities, but the PLPs had differing ubiquitin (Ub) chain cleavage selectivities and binding affinities for Ub, K48-linked diUb, and interferon-stimulated gene 15 (ISG15) substrates. The crystal structure of HKU1-PLP2 in complex with Ub revealed binding interfaces that accounted for the unusually high binding affinity between this PLP and Ub. In cellular assays, the PLPs from the severe disease-causing coronaviruses strongly suppressed innate immune IFN-I and NF-κB signaling and stimulated autophagy, whereas the PLPs from the mild disease-causing coronaviruses generally showed weaker effects on immune suppression and autophagy induction. In addition, a PLP from a SARS-CoV-2 variant of concern showed increased suppression of innate immune signaling pathways. Overall, these results demonstrated that the DUB and deISGylating activities and substrate selectivities of these PLPs differentially contribute to viral innate immune evasion and may affect viral pathogenicity.