RESUMEN
This study evaluated the effect of ultrasound using papain and sodium bicarbonate (SC) on meat tenderness to achieve the desired texture for elderly individuals. Meats were immersed in distilled water (DW) or papain (PI), ultrasonically treated with papain (UPI), or ultrasonically treated with papain and SC (UPIS). Response surface methodology was used to optimize the processing conditions with the lowest hardness, and the optimal conditions were determined as follows: 400 U/mL papain, ultrasonic for 30 min, and 4% SC. Hardness, color, and myofibrillar fragmentation index (MFI) were investigated. The hardness followed the order of DW (22.50 N), PI (18.62 N), UPI (12.08 N), and UPIS (7.16 N), and UPIS showed the highest MFI. Papain and SC affected the color of the meat. Overall, ultrasound-assisted treatment using papain and SC resulted in hardness levels of less than 7.8 N, which can be easily compressed by low tongue pressure.
RESUMEN
PURPOSE: Palmar or plantar fibromatosis is a benign fibroproliferative disorder affecting the fascia of the hands or feet. Management involves surgery, typically reserved for cases where progression limits function. Retrospective series demonstrate that radiation therapy (RT) can stabilize the disease course in many patients and improve symptoms in some cases. RT techniques vary between the use of electrons and superficial or orthovoltage photons and often require lead cutouts or custom boluses. We present a new approach demonstrating the implementation and effectiveness of three-dimensional (3D)-printed bolus material in patients receiving RT for fibromatosis. MATERIALS AND METHODS: A total of 3 patients, one with plantar and two with palmar fibromatosis, were treated with radiation using 3D-printed boluses over the past year. Bolus's design was based on computed tomography (CT) imaging data. Palmar patients were treated with a single en-face electron field, with a two-part accessory as a bolus and an immobilization device encasing the hand. The plantar case required 6MV photons delivered with a Volumetric Modulated Arc Therapy (VMAT) technique to cover the deeper target volume adequately. Dose and fractionation were based on guidelines from the Royal College of Radiologists in the United Kingdom. CT was used to assess printed shape and density accuracy. RESULTS: The mean deviations in shape between the printed bolus pieces and their designs were all less than 0.4 mm. The differences in mean Hounsefield units (HU) between the printed boluses and their expected values were between 7 and 44 HU. No significant issues were encountered when applying the bolus to patients. The thermoluminescent dosimeters (TLD) used demonstrated dose accuracy to within TLD precision (5 %). CONCLUSIONS: 3D printing bolus technology represents a novel approach to treating fibromatosis with radiation. It offers superior dosimetry through the reduction of air gaps and by permitting custom bolus thickness. Also, it simplifies clinical set-up by acting as an immobilization device and a visual aid for daily field placement.
RESUMEN
BACKGROUND: Soybean meal yogurt was prepared from soybean meal using papain and Bifidobacterium animalis subsp. lactis. A non-targeted metabolomics approach was employed to analyze the relevance of papain to the differences in volatile and non-volatile metabolites of soybean meal yogurt. RESULTS: The results showed that the main up-regulated metabolites and metabolic pathways after enzymatic digestion were dominated by amino acids and their derivatives. Conversely, the main down-regulated metabolites and pathways were predominantly associated with flavonoid metabolism. Amino acids and their derivatives, as well as flavonoids, were found to be highly correlated with the formation of sweet, umami, astringent, and bitterness. The addition of papain enriched the content of aromatic compounds in soybean meal yogurt. Aromatic components such as 2-heptanone, naphthalene, and p-xylene increased in concentration. Synthetic peptide of aspartate and serine, gramine, geissospermine, N-desmethyl vinblastine, and 3,7-dihydroxyflavone were the major non-volatile differential metabolites distinguishing the soybean meal yogurt. CONCLUSION: This study provided a comprehensive analysis of the metabolic traits of products co-fermented by papain and Bifidobacterium animalis subsp. lactis, offering insights for the application of papain in fermented goods. © 2024 Society of Chemical Industry.
RESUMEN
INTRODUCTION AND AIMS: Potential secondary or toxic effects of peroxide-based whitening gels have driven the search for alternative methods that use natural compounds with gentle action on tooth enamel that provide remineralizing benefits. METHODS: This study introduces four innovative experimental whitening gels (GC, G1, G3, G4) formulated with enzymes (Bromelaine and Papaine) and natural extracts, along with SiO2. The efficacy of these gels was tested on nanohybrid dental composite (EsCOM100, Spident Company) and dental enamel stained with coffee and natural juice (Tedi) over 10 days. The structural changes in samples before and after bleaching were examined using scanning electron microscopy and atomic force microscopy. Additionally, cytotoxicity tests were conducted on the gels using mesenchymal stem cells from human dental pulp (dMSC) and human keratinocytes (HaCaT). Antibacterial activity was assessed on five strains (Streptococcus mutans. Porphyromonas gingivalis; Enterococcus faecalis; Escherichia coli; Staphylococcus aureus). RESULTS: Coffee and natural juice stains significantly increase the roughness of composite and enamel surfaces by forming deposits. The enzymatic action of bromelain and papain effectively disorganizes and removes these clusters, significantly reducing surface roughness. CONCLUSION: Notably, the gel containing papain and nanostructured SiO2 proved to be the most effective in removing coffee stains from both composite surfaces and enamel. On the other hand, the gel with bromelain and nanostructured SiO2 was the most efficient in removing natural juice stains. The absence of SiO2 in the experimental gels slightly decreased the enzymes' effectiveness in stain removal. The antibacterial activity observed in the experimental gels is attributed solely to the enzymatic compounds.
RESUMEN
The present study focused on preparing rohu egg sauce using optimized conditions through enzymatic and fermentative methods. The enzymatic preparation of rohu egg sauce (ERS) involved homogenizing the eggs in water at a ratio of 1:0.9 (w/v), followed by the addition of salt (20% w/w) and papain (3% w/w). A mixture containing salt (25% w/w), sugar (7.5% w/w), and inoculum (10% (w/v)) of Pediococcus pentosaceus FSBP4-40 was utilized to prepare fermentatively produced rohu egg sauce (FRS). ERS and FRS were then stored at room temperature (25 ± 2 °C) and 37 °C for 180 days. After storage, both sauces were evaluated for their scavenging activity against DPPH, ABTS, and superoxide anion (SOA). The ERS demonstrated significantly higher DPPH, ABTS, and SOA scavenging activity compared to the FRS, with values of 61.61 ± 7.33%, 71.21 ± 2.14%, and 85.11 ± 4.92%, respectively, as opposed to 37.49 ± 5.34, 52.31 ± 1.76%, and 63.09 ± 2.31%. Significant changes were observed in the fatty acid profile of the sauces during 180 day storage. Furthermore, after 180 days, the bacterial counts in the FRS were much lower than in the ERS. Overall, this study highlights the importance of using enzymes and LAB in accelerating the hydrolytic process to produce biofunctional rohu egg sauce.
RESUMEN
The unique and complex structure of papain-like protease (PLpro) of the SARS-CoV-2 virus represents a difficult challenge for antiviral development, yet it offers a compelling validated target for effective therapy of COVID-19. The surge in scientific interest in inhibiting this cysteine protease emerged after its demonstrated connection to the cytokine storm in patients with COVID-19 disease. Furthermore, the development of new inhibitors against PLpro may also be beneficial for the treatment of respiratory infections caused by emerging coronavirus variants of concern. This review article provides a comprehensive overview of PLpro inhibitors, focusing on the structural framework of the known inhibitor GRL0617 and its analogs. We categorize PLpro inhibitors on the basis of their structures and binding site: Glu167 containing site, BL2 groove, Val70Ub site, and Cys111 containing catalytic site. We summarize and evaluate the majority of GRL0617-like inhibitors synthesized so far, highlighting their published biochemical parameters, which reflect their efficacy. Published research has shown that strategic modifications to GRL0617, such as decorating the naphthalene ring, extending the aromatic amino group or the orthomethyl group, can substantially decrease the IC50 from micromolar up to nanomolar concentration range. Some advantageous modifications significantly enhance inhibitory activity, paving the way for the development of new potent compounds. Our review places special emphasis on structures that involve direct modifications to the GRL0617 scaffold, including piperidine carboxamides and modified benzylmethylnaphthylethanamines (Jun9 scaffold). All these compounds are believed to inhibit the proteolytic, deubiquitination, and deISGylation activity of PLpro, biochemical processes linked to the severe progression of COVID-19. Finally, we summarize the development efforts for SARS-CoV-2 PLpro inhibitors, in detailed structure-activity relationships diagrams. This aims to inform and inspire future research in the search for potent antiviral agents against PLpro of current and emerging coronavirus threats.
RESUMEN
Epigenetic changes have long-lasting impacts, which influence the epigenome and are maintained during cell division. Thus, human genome changes have required a very long timescale to become a major contributor to the current obesity pandemic. Whereas bidirectional effects of coronavirus disease 2019 (COVID-19) and obesity pandemics have given the opportunity to explore, how the viral microribonucleic acids (miRNAs) use the human's transcriptional machinery that regulate gene expression at a posttranscriptional level. Obesity and its related comorbidity, type 2 diabetes (T2D), and new-onset diabetes due to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are additional risk factors, which increase the severity of COVID-19 and its related mortality. The higher mortality rate of these patients is dependent on severe cytokine storm, which is the sum of the additional cytokine production by concomitant comorbidities and own cytokine synthesis of COVID-19. Patients with obesity facilitate the SARS-CoV-2 entry to host cell via increasing the host's cell receptor expression and modifying the host cell proteases. After entering the host cells, the SARS-CoV-2 genome directly functions as a messenger ribonucleic acid (mRNA) and encodes a set of nonstructural proteins via processing by the own proteases, main protease (Mpro), and papain-like protease (PLpro) to initiate viral genome replication and transcription. Following viral invasion, SARS-CoV-2 infection reduces insulin secretion via either inducing ß-cell apoptosis or reducing intensity of angiotensin-converting enzyme 2 (ACE2) receptors and leads to new-onset diabetes. Since both T2D and severity of COVID-19 are associated with the increased serum levels of pro-inflammatory cytokines, high glucose levels in T2D aggravate SARS-CoV-2 infection. Elevated neopterin (NPT) value due to persistent interferon gamma (IFN-γ)-mediated monocyte-macrophage activation is an indicator of hyperactivated pro-inflammatory phenotype M1 macrophages. Thus, NPT could be a reliable biomarker for the simultaneously occurring COVID-19-, obesity- and T2D-induced cytokine storm. While host miRNAs attack viral RNAs, viral miRNAs target host transcripts. Eventually, the expression rate and type of miRNAs also are different in COVID-19 patients with different viral loads. It is concluded that specific miRNA signatures in macrophage activation phase may provide an opportunity to become aware of the severity of COVID-19 in patients with obesity and obesity-related T2D.
Asunto(s)
COVID-19 , Síndrome de Activación Macrofágica , Obesidad , SARS-CoV-2 , Humanos , COVID-19/virología , COVID-19/epidemiología , COVID-19/inmunología , COVID-19/complicaciones , Obesidad/complicaciones , Obesidad/metabolismo , Obesidad/epidemiología , Obesidad/virología , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , Síndrome de Activación Macrofágica/virología , Síndrome de Activación Macrofágica/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/virología , Diabetes Mellitus Tipo 2/metabolismo , Pandemias , MicroARNs/genética , MicroARNs/metabolismo , Citocinas/metabolismo , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/virologíaRESUMEN
Context: Dental bleaching, a common cosmetic treatment, typically uses hydrogen peroxide (H2O2). Yet, the search for natural options has prompted an investigation into fruit extract efficacies such as papain, bromelain, and actinidin for tooth whitening. Aim: The aim of this study was to evaluate the efficacy of 30% hydrogen peroxide and fruit extracts (papaya, pineapple, and kiwi) on human enamel using a spectrophotometer at different time intervals. Study Design: Eighty maxillary anterior teeth were stained with tea solution and evaluated for baseline color. They were then divided into four groups: hydrogen peroxide alone and hydrogen peroxide combined with papaya, pineapple, or kiwi extracts. Each group was further divided based on bleaching duration: 10 or 20 min. Materials and Methods: The color value of the bleached teeth was measured using a reflectance spectrophotometer. In order to analyze the data, one-way ANOVA, post hoc Tukey, and paired t-tests were used. The significance level was established at α =0.05. Results: Combining hydrogen peroxide with pineapple extract showed the highest efficacy, followed by papaya and kiwi extracts. Hydrogen peroxide alone also demonstrated significant bleaching efficacy, albeit lower than the combinations with fruit extracts. Conclusion: Combining hydrogen peroxide with pineapple and papaya extracts notably improves dental bleaching efficacy, as shown by reduced color difference (ΔE) values. This underscores the potential of natural enzymes in tooth whitening.
RESUMEN
PURPOSE: The aim of the present study is to assess the molluscicidal, larvicidal and genotoxicological activities of papain and how it can affect the host-parasite interactions. METHODS: Toxicity of papain on snails by making series of concentrations to calculate LC50, and then study its larvicide effect on the free larval stages of S. mansoni and infection rate of snails. RESULTS: Papain has a molluscicidal activity on adult snails of Biomphalaria alexandrina with a lethal concentration LC50 equals to 43.1 mg/L. In addition, it has activity on miracidia with half Lethal time (LT50) of 16.11 min., and on cercariae with 12.1 min. compared to control ones. The sub lethal concentration LC10 and LC25 (6.9 or 24.1 mg/L, respectively) decreased the survival rate of snails at the first cercarial shedding, the rate of infection, the average total number of cercariae per snail, the shedding period and the life span of snails, while the prepatent period was significantly increased than the control ones. The morphological alterations in cercariae after exposure to papain were occurred where the cercariae lacked motility and some had a dark tail with complete detachment of head and tail. Compared to the control group, the levels of cytochrome oxidase subunit I (COI) and (ND1) genes significantly decreased in snails after exposure to papain. CONCLUSIONS: Papain could be used as a potential molluscicide for elimination of schistosomiasis and decrease its transmission and deterioration of host-parasite interaction.
RESUMEN
Papain (PN) is a proteolytic enzyme derived from Carica Papaya L. While the pharmacological effects of PN have not been extensively studied compared to its enzymatic activity, PN also holds potential benefits beyond protein digestion. This study aimed to investigate the potential effects of PN against skin inflammation in house dust mite Dermatophagoides farinae body (Dfb)-exposed NC/Nga atopic dermatitis (AD) mice and human HaCaT keratinocytes and their underlying mechanisms. The effects of PN on the skin were assessed via histological examination, measurements of transepidermal water loss (TEWL), quantitative reverse transcription-polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Our findings indicated that the oral intake of PN decreased the severity scores of lesions resembling AD, TEWL, and the levels of inflammatory cytokines and serum immunoglobulin E in Dfb-induced AD mice, along with a reduction in epidermal thickness and mast cell infiltration. Additionally, PN inhibited the activation of the mitogen-activated protein kinases (MAPKs) and the signal transducer and activator of transcription (STAT) pathways in Dfb-induced AD mice and HaCaT keratinocytes. Moreover, PN improved survival and reduced ROS production in H2O2-damaged HaCaT keratinocytes and enhanced the expression of antioxidant enzymes in Dfb-induced AD mice. Concludingly, the oral administration of PN suppressed inflammatory mediators and downregulated the MAPKs/STAT pathway, suggesting its potential role in AD pathogenesis.
RESUMEN
Papain-like protease (PLpro) is an attractive anti-coronavirus target. The development of PLpro inhibitors, however, is hampered by the limitations of the existing PLpro assay and the scarcity of validated active compounds. We developed a novel in-cell PLpro assay based on BRET and used it to evaluate and discover SARS-CoV-2 PLpro inhibitors. The developed assay demonstrated remarkable sensitivity for detecting the reduction of intracellular PLpro activity while presenting high reliability and performance for inhibitor evaluation and high-throughput screening. Using this assay, three protease inhibitors were identified as novel PLpro inhibitors that are structurally disparate from those previously known. Subsequent enzymatic assays and ligand-protein interaction analysis based on molecular docking revealed that ceritinib directly inhibited PLpro, showing high geometric complementarity with the substrate-binding pocket in PLpro, whereas CA-074 methyl ester underwent intracellular hydrolysis, exposing a free carboxyhydroxyl group essential for hydrogen bonding with G266 in the BL2 groove, resulting in PLpro inhibition.
Asunto(s)
Simulación del Acoplamiento Molecular , Pirimidinas , SARS-CoV-2 , Sulfonas , Humanos , SARS-CoV-2/enzimología , SARS-CoV-2/efectos de los fármacos , Sulfonas/farmacología , Sulfonas/química , Pirimidinas/química , Pirimidinas/farmacología , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus/química , Transferencia de Energía por Resonancia de Bioluminiscencia , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Estructura Molecular , Relación Dosis-Respuesta a Droga , Relación Estructura-ActividadRESUMEN
Sous vide meat is an emerging food category, the consumption of which has increased owing to greater convenience, sensory traits, elderly consumers acceptance, and low-cost cuts use. However, required prolonged thermal treatment to achieve desired tenderness, impact energy-consumption besides triggering lipid oxidation, undesired off-flavors, and cooked meat profiles. Using a response surface methodology (RSM), this study evaluated the effects of the vegetal proteolytic papain (0 to 20 mg/kg) and low-temperature sous vide cooking (SVC) time (1 to 8 h at 65°C) in low-value marinated M. semitendinosus beefsteaks on technological characteristics associated with tenderness, and lipid oxidation. Additionally, the sensory profile traits of the pre-selected treatments were described using check-all-that-apply (CATA) and preference mapping. Shear force (WBsSF) was reduced with greater papain addition, whereas higher cooking losses (CL) were observed with longer SVC cooking times. Both the released total collagen and TBARS values increased with increasing papain concentrations and SVC times. Combining high levels of papain (>10 mg/kg) and SVC time (>6 h) resulted in lower WBsSF values (<20 N) but higher CL (>27%) and the CATA descriptors "aftertaste" and "mushy." The optimized conditions (14 mg/kg papain; 2 h SVC) also reduced WBsSF values (<26 N) with lower CL (<20%) and were most preferred and described as "juicy" and "tender" by consumers. Observed results suggest that combined mild SVC and papain may potentiate tenderness, conjointly favor juiciness and oxidation, further representing a promising tool for reducing SVC time without compromising valued sous vide sensory traits.
Asunto(s)
Culinaria , Papaína , Gusto , Culinaria/métodos , Animales , Bovinos , Humanos , Carne Roja/análisis , Masculino , Carne/análisis , FemeninoRESUMEN
The single-stranded RNA genome of SARS-CoV-2 encodes several structural and non-structural proteins, among which the papain-like protease (PLpro) is crucial for viral replication and immune evasion and has emerged as a promising therapeutic target. The current study aims to discover new inhibitors of PLpro that can simultaneously disrupt its protease and deubiquitinase activities. Using multiple computational approaches, six compounds (CP1-CP6) were selected from our in-house compounds database, with higher docking scores (-7.97 kcal/mol to -8.14 kcal/mol) and fitted well in the active pocket of PLpro. Furthermore, utilizing microscale molecular dynamics simulations (MD), the dynamic behavior of selected compounds was studied. Those molecules strongly binds at the PLpro active site and forms stable complexes. The dynamic motions suggest that the binding of CP1-CP6 brought the protein to a closed conformational state, thereby altering its normal function. In an in vitro evaluation, CP2 showed the most significant inhibitory potential for PLpro (protease activity = 2.71 ± 0.33 µM and deubiquitinase activity = 3.11 ± 0.75 µM), followed by CP1, CP5, CP4 and CP6. Additionally, CP1-CP6 showed no cytotoxicity at a concentration of 30 µM in the human BJ cell line.
Asunto(s)
Proteasas Similares a la Papaína de Coronavirus , Enzimas Desubicuitinizantes , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , SARS-CoV-2 , SARS-CoV-2/enzimología , SARS-CoV-2/efectos de los fármacos , Humanos , Enzimas Desubicuitinizantes/metabolismo , Enzimas Desubicuitinizantes/química , Proteasas Similares a la Papaína de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Dominio Catalítico , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Antivirales/farmacología , Antivirales/química , Productos Biológicos/farmacología , Productos Biológicos/química , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , Unión ProteicaRESUMEN
Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host immune proteins such as MDA5 and IRF3 in a process called ISGylation, thereby promoting type I IFN induction to limit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through deISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387, and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.IMPORTANCEThe role of protein ISGylation in regulating host cellular processes has been studied extensively; however, how ISG15 conjugation influences the activity of viral proteins, particularly coronaviral proteins, is largely unknown. Our study uncovered that the nucleocapsid (N) protein of SARS-CoV-2 is ISGylated by the HERC5 ISGylation machinery and that this modification impedes the functional assembly of N into oligomers ultimately inhibiting viral RNA synthesis. This antiviral restriction mechanism is antagonized by the PLpro deISGylation activity of SARS-CoV-2 NSP3. This study deepens our understanding of SARS-CoV-2 protein regulation by posttranslational modifications and may open new avenues for designing antiviral strategies for COVID-19.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , Proteasas Similares a la Papaína de Coronavirus , Citocinas , ARN Viral , SARS-CoV-2 , Ubiquitina-Proteína Ligasas , Ubiquitinas , Replicación Viral , Humanos , Ubiquitinas/metabolismo , Ubiquitinas/genética , SARS-CoV-2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Similares a la Papaína de Coronavirus/metabolismo , ARN Viral/metabolismo , ARN Viral/genética , Citocinas/metabolismo , Células HEK293 , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Fosfoproteínas/metabolismo , COVID-19/virología , COVID-19/metabolismo , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Péptidos y Proteínas de Señalización IntracelularRESUMEN
A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.
Asunto(s)
COVID-19 , Proteínas de la Nucleocápside de Coronavirus , Proteasas Similares a la Papaína de Coronavirus , Citocinas , SARS-CoV-2 , Ubiquitina-Proteína Ligasas , Ubiquitinas , Replicación Viral , Humanos , Ubiquitinas/metabolismo , Ubiquitinas/genética , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Citocinas/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , COVID-19/virología , COVID-19/inmunología , COVID-19/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Células HEK293 , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Evasión Inmune , Proteínas de la Nucleocápside/metabolismo , Proteasas 3C de Coronavirus/metabolismo , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host (immune) proteins such as MDA5 and IRF3 in a process called ISGylation, thereby limiting the replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through de-ISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387 and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.
RESUMEN
Papain is useful for the enzymatic digestion of various proteins to produce functional peptides or protein fragments. Immobilized papain being reactive toward proteins and easily removable from a reaction mixture is worth developed. In the present work, liposomes were applied as colloidal carriers of papain for the catalytic digestion of polyclonal immunoglobulin G (IgG). Papain was covalently conjugated at pH = 7.0 via tris-succinimidyl aminotriacetate (TSAT) to liposomes incorporated with 5 mol % poly(ethylene glycol)-tethered lipid with a reactive amino group. The papain-conjugated liposome (liposome-papain) catalyzed the hydrolysis of Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPNA) at pH = 5.0-7.0. The activity of liposome-papain significantly increased with increasing temperature from 25 to 50 °C. The Michaelis constant Km was determined with respect to the liposome-papain- and free papain-catalyzed reactions with BAPNA at 37 °C as Km = 1.11 ± 0.13 and 11.6 ± 2.9 mM, respectively. Liposome-papain was applied to the catalytic digestion of 10 mg·mL-1 IgG at 37 °C for 24 h at pH = 5.0-7.0. The reaction mixture could be analyzed without pretreatment by using the affinity columns immobilized with the protein A or protein L ligand because colloidal liposome-papain quickly flowed through the chromatographic stationary phase, exhibiting little proteolytic effect on the proteinaceous ligands. The analysis clearly demonstrated the catalytic production of antigen-binding fragments (Fab) from IgG in an enzyme concentration- and pH-dependent manner. Liposome-papain with 15 or 50 mol % anionic lipids also catalyzed the formation of Fab from IgG. The above results demonstrated that liposome-papain was useful to digest IgG and to purify Fab formed with the affinity chromatography.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas , Liposomas , Papaína , Liposomas/química , Papaína/química , Papaína/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Ensayo de Materiales , Inmunoglobulina G/química , Materiales Biocompatibles/química , Materiales Biocompatibles/síntesis química , Tamaño de la Partícula , Catálisis , Concentración de Iones de HidrógenoRESUMEN
The single-step synthesis of nitro-derivatized SG using dimethyldichlorosilane in an aprotic solvent dichloromethane at 300 K is efficient and straightforward. Reduction and diazotization effectively functionalize the material for enzyme coupling at the O-carbon of the enzyme's tyrosine. The high extraction efficiency of protonated dichromate ions with a breakthrough capacity of 480 µmol·g-1 is notable. Eco-friendly elution using distilled water achieves a significant enrichment factor of 23.2. Excellent reusability (up to 900 cycles) and stable sorption efficiency (ζ ≥ 0.9) highlight the material's potential for practical applications and future research.
Asunto(s)
Ensayo de Materiales , Papaína , Propiedades de Superficie , Papaína/química , Papaína/metabolismo , Aminas/química , Tamaño de la Partícula , Estructura Molecular , Materiales Biocompatibles/química , Materiales Biocompatibles/síntesis química , ProtonesRESUMEN
In the global fight against the COVID-19 pandemic caused by the highly transmissible SARS-CoV-2 virus, the search for potent medications is paramount. With a focused investigation on the SARS-CoV-2 papain-like protease (PLpro) as a promising therapeutic target due to its pivotal role in viral replication and immune modulation, the catalytic triad of PLpro comprising Cys111, His272, and Asp286, highlights Cys111 as an intriguing nucleophilic center for potential covalent bonds with ligands. The detailed analysis of the binding site unveils crucial interactions with both hydrophobic and polar residues, demonstrating the structural insights of the cavity and deepening our understanding of its molecular landscape. The sequence of PLpro among variants of concern (Alpha, Beta, Gamma, Delta and Omicron) and the recent variant of interest, JN.1, remains conserved with no mutations at the active site. Moreover, a thorough exploration of apo, non-covalently bound, and covalently bound PLpro conformations exposes significant conformational changes in loop regions, offering invaluable insights into the intricate dynamics of ligand-protein complex formation. Employing strategic in silico medication repurposing, this study swiftly identifies potential molecules for target inhibition. Within the domain of covalent docking studies and molecular dynamics, using reported inhibitors and clinically tested molecules elucidate the formation of stable covalent bonds with the cysteine residue, laying a robust foundation for potential therapeutic applications. These details not only deepen our comprehension of PLpro inhibition but also play a pivotal role in shaping the dynamic landscape of COVID-19 treatment strategies.
Asunto(s)
Antivirales , Dominio Catalítico , Simulación de Dinámica Molecular , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , Humanos , Antivirales/química , Antivirales/farmacología , Antivirales/metabolismo , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus/química , Sitios de Unión , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , Unión Proteica , Simulación del Acoplamiento MolecularRESUMEN
Kafirin in sorghum inhibits starch digestion and exhibits antioxidant properties, however its potential in food industry remains unexplored. Therefore, the study was aimed to explore and improve the potential of kafirin as natural carbohydrate blocker using papain (6 NFU/mL) and/or infrared treatment (220 °C/3 min). Results indicated that the combined treatment, PIR (infrared + papain) is the most efficient treatment to modify kafirin. PIR generated a new â¼37 kDa high molecular weight moiety in kafirin with a crystal size of 157.44 Å. All samples showed superior antioxidant activity post-treatments, with PIR exhibiting highest scavenging activity from 31.09 to 82.97%, 15.09 to 42.82%, and 25.92 to 38.58% for DPPH, FRAP, and ABTS, respectively. PIR-modified kafirin limited malondialdehyde production, and increased α-amylase and α-glucosidase inhibition. Incorporation of 7.5% kafirin in corn starch increased resistant starch from 5.09 to 21.04% after cooking, which suggests potential of kafirin in development of diabetic-friendly food formulations.