Effect of ultrasound-assisted treatment on meat tenderization for elderly individuals.

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.

Investigation of the impact of papain on the volatile and non-volatile metabolites of soybean meal yogurt.

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.

Use of 3D printing technology for custom bolus fabrication in the management of palmar or plantar fibromatosis with radiotherapy: A retrospective case series.

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.

Biofunctional components and bacterial dynamics of enzymatic and fermentatively prepared rohu (Labeo rohita) egg sauce.

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.

Macrophage Activation Syndrome in Coinciding Pandemics of Obesity and COVID-19: Worse than Bad.

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.

Design of inhibitors of SARS-CoV-2 papain-like protease deriving from GRL0617: Structure-activity relationships.

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.

Phytochemicals in vital tooth bleaching: Spectrophotometric evaluation of efficacy with papaya, pineapple, or kiwi extracts and 30% hydrogen peroxide.

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.

Effect of Experimental Bleaching Gels With Enzymes on Composite and Enamel.

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.

Identification of Novel Allosteric Sites of SARS-CoV-2 Papain-Like Protease (PLpro) for the Development of COVID-19 Antivirals.

Coronaviruses such as SARS-CoV-2 encode a conserved papain-like protease (PLpro) that is crucial for viral replication and immune evasion, making it a prime target for antiviral drug development. In this study, three surface pockets on SARS-CoV-2 PLpro that may function as sites for allosteric inhibition were computationally identified. To evaluate the effects of these pockets on proteolytic activity, 52 residues were separately mutated to alanine. In Pocket 1, located between the Ubl and thumb domains, the introduction of alanine at T10, D12, T54, Y72, or Y83 reduced PLpro activity to <12% of that of WT. In Pocket 2, situated at the interface of the thumb, fingers, and palm domains, Q237A, S239A, H275A, and S278A inactivated PLpro. Finally, introducing alanine at five residues in Pocket 3, between the fingers and palm domains, inactivated PLpro: S212, Y213, Y251, K254, and Y305. Pocket 1 has a higher druggability score than Pockets 2 and 3. MD simulations showed that interactions within and between domains play critical roles in PLpro activity and thermal stability. The essential residues in Pockets 1 and 2 participate in a combination of intra- and inter-domain interactions. By contrast, the essential residues in Pocket 3 predominantly participate in inter-domain interactions. The most promising targets for therapeutic development are Pockets 1 and 3, which have the highest druggability score and the largest number of essential residues, respectively. Non-competitive inhibitors targeting these pockets may be antiviral agents against COVID-19 and related coronaviruses.

Does enamel deproteinisation with 10% papain affect shear bond strength of orthodontic adhesives: a randomised controlled trial.

OBJECTIVE: To evaluate the effect of 10% papain as an enamel deproteinising agent on the shear bond strength (SBS) of three orthodontic adhesives: Transbond XT, resin-modified glass ionomer cement (RMGIC) and Biofix. DESIGN: Single-centre, double-blinded, split-mouth randomised controlled trial. SETTING: Department of Orthodontics and Dentofacial Orthopaedics, Nair Hospital Dental College, Mumbai, India. PARTICIPANTS: A total of 20 participants requiring bilateral premolar extraction for fixed orthodontic treatment in both the maxillary and mandibular arches were included in this study. METHODS: In total, 80 premolars from the above-mentioned participants were divided into four groups as follows: group A: Transbond XT deproteinised with 10% papain gel; group B: Biofix deproteinised with 10% papain gel; group C: RMGIC deproteinised with 10% papain gel; and group D: Transbond XT without enamel deproteinisation as a control group-bonded as instructed by the manufacturer. After deproteinisation, brackets were bonded and after a follow-up period of 28 days, the teeth were extracted. The SBS was then measured using the Universal Testing Machine. The force needed to shear the bracket was documented, and bond strengths were subsequently calculated in megapascals (MPa). The obtained results were subjected to statistical analysis and one-way ANOVA was performed to compare the mean SBS between the groups. Subsequently, pairwise comparisons were conducted using Tukey's post hoc test. RESULTS: There was a statistically significant difference in SBS among all groups (P = 0.002). The SBS of TransXT with deproteinisation increased significantly compared with TransXT without deproteinisation (P = 0.03). There was no statistically significant difference between the SBS of TransXT without deproteinisation and RMGIC (P = 0.47) and Biofix (P = 0.39), both with deproteinisation. CONCLUSION: The use of 10% papain for deproteinising enamel improved the SBS of all materials. Deproteinising improved the SBS of RMGIC and Biofix to the level of TransXT without deproteinisation.

Papain Injection Creates a Nucleotomy-like Cavity for Testing Gels in Intervertebral Discs.

Biomaterials, such as hydrogels, have an increasingly important role in the development of regenerative approaches for the intervertebral disc. Since animal models usually resist biomaterial injection due to high intradiscal pressure, preclinical testing of the biomechanical performance of biomaterials after implantation remains difficult. Papain reduces the intradiscal pressure, creates cavities within the disc, and allows for biomaterial injections. But papain digestion needs time, and cadaver experiments that are limited to 24 h for measuring range of motion (ROM) cannot not be combined with papain digestion just yet. In this study, we successfully demonstrate a new organ culture approach, facilitating papain digestion to create cavities in the disc and the testing of ROM, neutral zone (NZ), and disc height. Papain treatment increased the ROM by up to 109.5%, extended NZ by up to 210.9%, and decreased disc height by 1.96 ± 0.74 mm. A median volume of 0.73 mL hydrogel could be injected after papain treatment, and histology revealed a strong loss of proteoglycans in the remaining nucleus tissue. Papain has the same biomechanical effects as known from nucleotomies or herniations and thus creates a disc model to study such pathologies in vitro. This new model can now be used to test the performance of biomaterials.

Identification of New Angiotensin-Converting Enzyme Inhibitory Peptides Isolated from the Hydrolysate of the Venom of Nemopilema nomurai Jellyfish.

Recently, jellyfish venom has gained attention as a promising reservoir of pharmacologically active compounds, with potential applications in new drug development. In this investigation, novel peptides, isolated from the hydrolysates of Nemopilema nomurai jellyfish venom (NnV), demonstrate potent inhibitory activities against angiotensin-converting enzyme (ACE). Proteolytic enzymes-specifically, papain and protamex-were utilized for the hydrolysis under optimized enzymatic conditions, determined by assessing the degree of hydrolysis through the ninhydrin test. Comparative analyses revealed that papain treatment exhibited a notably higher degree of NnV hydrolysis compared to protamex treatment. ACE inhibitory activity was quantified using ACE kit-WST, indicating a substantial inhibitory effect of 76.31% for the papain-digested NnV crude hydrolysate, which was validated by captopril as a positive control. The separation of the NnV-hydrolysate using DEAE sepharose weak-anion-exchange chromatography revealed nine peaks under a 0-1 M NaCl stepwise gradient, with peak no. 3 displaying the highest ACE inhibition of 96%. The further purification of peak no. 3 through ODS-C18 column reverse-phase high-performance liquid chromatography resulted in five sub-peaks (3.1, 3.2, 3.3, 3.4, and 3.5), among which 3.2 exhibited the most significant inhibitory activity of 95.74%. The subsequent analysis of the active peak (3.2) using MALDI-TOF/MS identified two peptides with distinct molecular weights of 896.48 and 1227.651. The peptide sequence determined by MS/MS analysis revealed them as IVGRPLANG and IGDEPRHQYL. The docking studies of the two ACE-inhibitory peptides for ACE molecule demonstrated a binding affinity of -51.4 ± 2.5 and -62.3 ± 3.3 using the HADDOCK scoring function.

SARS-CoV-2 Genotyping Highlights the Challenges in Spike Protein Drift Independent of Other Essential Proteins.

As of 2024, SARS-CoV-2 continues to propagate and drift as an endemic virus, impacting healthcare for years. The largest sequencing initiative for any species was initiated to combat the virus, tracking changes over time at a full virus base-pair resolution. The SARS-CoV-2 sequencing represents a unique opportunity to understand selective pressures and viral evolution but requires cross-disciplinary approaches from epidemiology to functional protein biology. Within this work, we integrate a two-year genotyping window with structural biology to explore the selective pressures of SARS-CoV-2 on protein insights. Although genotype and the Spike (Surface Glycoprotein) protein continue to drift, most SARS-CoV-2 proteins have had few amino acid alterations. Within Spike, the high drift rate of amino acids involved in antibody evasion also corresponds to changes within the ACE2 binding pocket that have undergone multiple changes that maintain functional binding. The genotyping suggests selective pressure for receptor specificity that could also confer changes in viral risk. Mapping of amino acid changes to the structures of the SARS-CoV-2 co-transcriptional complex (nsp7-nsp14), nsp3 (papain-like protease), and nsp5 (cysteine protease) proteins suggest they remain critical factors for drug development that will be sustainable, unlike those strategies targeting Spike.

Green Nanotechnology Through Papain Nanoparticles: Preclinical in vitro and in vivo Evaluation of Imaging Triple-Negative Breast Tumors.

Background: Recent advancements in nanomedicine and nanotechnology have expanded the scope of multifunctional nanostructures, offering innovative solutions for targeted drug delivery and diagnostic agents in oncology and nuclear medicine. Nanoparticles, particularly those derived from natural sources, hold immense potential in overcoming biological barriers to enhance therapeutic efficacy and diagnostic accuracy. Papain, a natural plant protease derived from Carica papaya, emerges as a promising candidate for green nanotechnology-based applications due to its diverse medicinal properties, including anticancer properties. Purpose: This study presents a novel approach in nanomedicine and oncology, exploring the potential of green nanotechnology by developing and evaluating technetium-99m radiolabeled papain nanoparticles (99mTc-P-NPs) for imaging breast tumors. The study aimed to investigate the efficacy and specificity of these nanoparticles in breast cancer models through preclinical in vitro and in vivo assessments. Methods: Papain nanoparticles (P-NPs) were synthesized using a radiation-driven method and underwent thorough characterization, including size, surface morphology, surface charge, and cytotoxicity assessment. Subsequently, P-NPs were radiolabeled with technetium-99m (99mTc), and in vitro and in vivo studies were conducted to evaluate cellular uptake at tumor sites, along with biodistribution, SPECT/CT imaging, autoradiography, and immunohistochemistry assays, using breast cancer models. Results: The synthesized P-NPs exhibited a size mean diameter of 9.3 ± 1.9 nm and a spherical shape. The in vitro cytotoxic activity of native papain and P-NPs showed low cytotoxicity in HUVEC, MDA-MB231, and 4T1 cells. The achieved radiochemical yield was 94.2 ± 3.1% that were sufficiently stable (≥90%) for 6 h. The tumor uptake achieved in the 4T1 model was 2.49 ± 0.32% IA/g at 2 h and 1.51 ± 0.20% IA/g at 6 h. In the spontaneous breast cancer model, 1.19 ± 0.20% IA/g at 2 h and 0.86 ± 0.31% IA/g at 6 h. SPECT/CT imaging has shown substantial tumor uptake of the new nanoradiopharmaceutical and clear tumor visualization. 99mTc-P-NPs exhibited a high affinity to tumoral cells confirmed by ex vivo autoradiography and immunohistochemistry assays. Conclusion: The findings underscore the potential of green nanotechnology-driven papain nanoparticles as promising agents for molecular imaging of breast and other tumors through SPECT/CT imaging. The results represent a substantial step forward in the application of papain nanoparticles as carriers of diagnostic and therapeutic radionuclides to deliver diagnostic/therapeutic payloads site-specifically to tumor sites for the development of a new generation of nanoradiopharmaceuticals.

Papain Suppresses Atopic Skin Inflammation through Anti-Inflammatory Activities Using In Vitro and In Vivo Models.

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.

ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis.

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.

SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

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.

ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis.

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.

Disrupting protease and deubiquitinase activities of SARS-CoV-2 papain-like protease by natural and synthetic products discovered through multiple computational and biochemical approaches.

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.