c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 innate lymphoid cells.

Group-2 innate lymphoid cells (ILC2s), which are involved in type 2 inflammatory diseases such as allergy, can exhibit immunological memory, but the basis of this ILC2 "trained immunity" has remained unclear. Here, we found that stimulation with IL-33/IL-25 or exposure to the allergen papain induces the expression of the transcription factor c-Maf in mouse ILC2s. Chronic papain exposure results in high production of IL-5 and IL-13 cytokines and lung eosinophil recruitment, effects that are blocked by c-Maf deletion in ILCs. Transcriptomic analysis revealed that knockdown of c-Maf in ILC2s suppresses expression of type 2 cytokine genes, as well as of genes linked to a memory-like phenotype. Consistently, c-Maf was found highly expressed in human adult ILC2s but absent in cord blood and required for cytokine production in isolated human ILC2s. Furthermore, c-Maf-deficient mouse or human ILC2s failed to exhibit strengthened ("trained") responses upon repeated challenge. Thus, the expression of c-Maf is indispensable for optimal type 2 cytokine production and proper memory-like responses in group-2 innate lymphoid cells.

Deubiquitinating activity of SARS-CoV-2 papain-like protease does not influence virus replication or innate immune responses in vivo.

The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis.

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.

Phytocystatin 6 is a context-dependent, tight-binding inhibitor of Arabidopsis thaliana legumain isoform ß.

Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform ß (AtLEGß) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEGß and papain-like cysteine proteases through two separate cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEGß. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEGß suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEGß regulation and its broader physiological significance.

The Papain-Like Protease of Porcine Reproductive and Respiratory Syndrome Virus Impedes STING Translocation from the Endoplasmic Reticulum to the Golgi Apparatus by Deubiquitinating STIM1.

Stimulator of interferon (IFN) genes (STING) was recently pinpointed as an antiviral innate immune factor during the infection of RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), the swine arterivirus, is an enveloped RNA virus which has evolved many strategies to evade innate immunity. To date, the interactive network between PRRSV and STING remains to be fully established. Herein, we report that STING suppresses PRRSV replication through type I interferon signaling. However, PRRSV impedes STING trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, leading to the decreased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Furthermore, PRRSV nonstructural protein 2 (Nsp2) colocalizes with STING, blocks STING translocation, and disrupts the STING-TBK1-IRF3 complex. Mechanistically, PRRSV Nsp2 retains STING at the ER by increasing the level of Ca2+ sensor stromal interaction molecule 1 (STIM1) protein. Functional analysis reveals that PRRSV Nsp2 deubiquitinates STIM1 by virtue of its papain-like protease 2 (PLP2) deubiquitinating (DUB) activity. Finally, we demonstrate that loss of STIM1 is associated with an elevated IFN response and restricts PRRSV replication. This work delineates the relationship between PRRSV infection and STING signaling and the importance of papain-like proteases (PLPs) in interfering in this axis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae, is responsible for reproductive disorders in pregnant sows and respiratory problems in piglets, resulting in huge losses in the swine industry worldwide. Of note, PRRSV infection causes immunosuppression, of which the mechanism is not completely understood. Here, we demonstrate for the first time that STING, a protein typically associated with the antiviral response in DNA viruses, plays a critical role in controlling PRRSV infection. However, PRRSV utilizes its encoded protein Nsp2 to inhibit STING activity by blocking its translocation from the ER to the Golgi apparatus. In particular, Nsp2 retains STING at the ER by interacting with and further deubiquitinating STIM1. For this process, the activity of the viral PLP2 DUB enzyme is indispensable. The study describes a novel mechanism by which PLP2 plays a critical role in suppressing the innate immune response against arteriviruses and potentially other viruses that encode similar proteases.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Anti-biofilm effect of enzymatic hydrolysates of ovomucin in Listeria monocytogenes and Staphylococcus aureus.

Despite modern advances in food hygiene, food poisoning due to microbial contamination remains a global problem, and poses a great threat to human health. Especially, Listeria monocytogenes and Staphylococcus aureus are gram-positive bacteria found on food-contact surfaces with biofilms. These foodborne pathogens cause a considerable number of food poisoning and infections annually. Ovomucin (OM) is a water-insoluble gel-type glycoprotein in egg whites. Enzymatic hydrolysis can be used to improve the bioactive properties of OM. This study aimed to investigate whether ovomucin hydrolysates (OMHs) produced using five commercial enzymes (Alcalase®, Bromelain, α-Chymotrypsin, Papain, and Pancreatin) can inhibit the biofilm formation of L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7. Particularly, OMH prepared with papain (OMPP; 500 µg/mL) significantly inhibited biofilm formation in L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7 by 85.56 %, 80.28 %, 91.70 %, and 79.00 %, respectively. In addition, OMPP reduced the metabolic activity, exopolysaccharide production (EPS), adhesion ability, and gene expression associated with the biofilm formation of these bacterial strains. These results suggest that OMH, especially OMPP, exerts anti-biofilm effects against L. monocytogenes and S. aureus. Therefore, OMPP can be used as a natural anti-biofilm agent to control food poisoning in the food industry.

A novel ACE inhibitory peptide from Pelodiscus sinensis Wiegmann meat water-soluble protein hydrolysate.

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.

Effects of Leukocyte-rich platelet-rich plasma and Leukocyte-poor platelet-rich plasma on cartilage in a rabbit osteoarthritis model.

Osteoarthritis is a prevalent chronic disease. One of its primary pathological processes involves the degeneration of articular cartilage. Platelet-rich plasma (PRP) contains cytokines and growth factors that can stimulate the repair and regeneration of articular cartilage tissues. PRP may also slow the progression of osteoarthritis. The purpose of this experiment is to compare the efficacy of Leukocyte poor (LP) - PRP and Leukocyte rich (LR) - PRP in treating rabbit osteoarthritis and to investigate their mechanisms of action. Analyzing the impact of leukocytes on PRP therapeutic effectiveness will provide a valuable clinical reference for the choice of which PRP is better for the treatment of osteoarthritis. A rabbit osteoarthritis model was established by injecting papain into the knee joint cavity, and LP-PRP and LR-PRP were prepared through different centrifugation methods for injection into the knee joint cavity. Eight weeks after injection, rabbit knee cartilage specimens were observed for gross changes, HE staining, senna O-solid green staining, and immunohistochemistry of type II collagen and were quantitatively compared using Pelletier's score, Mankin's pathology score, and ImageJ image processing software. Injection of papain into the knee joint cavity successfully established a rabbit model of osteoarthritis. All three evaluation indexes differed significantly from those of the blank group (P<0.05). LP-PRP and LR-PRP exhibited therapeutic effects when compared with the model group. The two PRP groups had similar gross tissue appearance and pathology (P>0.05). The LR-PRP group had higher collagen type-II expression (P < 0.05) than the LP-PRP group. Both LP-PRP and LR-PRP proved therapeutic for the rabbit papain osteoarthritis model. The difference in leukocyte content between the two groups did not yield different cartilage morphology or other factors by 8 weeks posttreatment. LR-PRP displayed the ability to release more factors relevant to the metabolism of type II collagen than LP-PRP, enabling the preservation of into cartilage collagen content of type II collagen and delaying osteoarthritis progression.

Thermal Inactivation, Denaturation and Aggregation Processes of Papain-Like Proteases.

The investigation into the behavior of ficin, bromelain, papain under thermal conditions holds both theoretical and practical significance. The production processes of medicines and cosmetics often involve exposure to high temperatures, particularly during the final product sterilization phase. Hence, it's crucial to identify the "critical" temperatures for each component within the mixture for effective technological regulation. In light of this, the objective of this study was to examine the thermal inactivation, aggregation, and denaturation processes of three papain-like proteases: ficin, bromelain, papain. To achieve this goal, the following experiments were conducted: (1) determination of the quantity of inactivated proteases using enzyme kinetics with BAPNA as a substrate; (2) differential scanning calorimetry (DSC); (3) assessment of protein aggregation using dynamic light scattering (DLS) and spectrophotometric analysis at 280 nm. Our findings suggest that the inactivation of ficin and papain exhibits single decay step which characterized by a rapid decline, then preservation of the same residual activity by enzyme stabilization. Only bromelain shows two steps with different kinetics. The molecular sizes of the active and inactive forms are similar across ficin, bromelain, and papain. Furthermore, the denaturation of these forms occurs at approximately the same rate and is accompanied by protein aggregation.

Enzymatic Hydrolysis Optimization of Yak Whey Protein Concentrates and Bioactivity Evaluation of the Ultrafiltered Peptide Fractions.

Yak whey protein concentrates (YWPCs) have good functional properties, but there is still a gap in the study of their peptides. In this study, peptides were obtained by enzymatic hydrolysis, and the bioactivity of each ultrafiltration fraction was evaluated using an optimal process. YWPCs were isolated and purified from yak milk as the raw material. Alkaline protease, trypsin, and papain were used to hydrolyze YWPCs. The protease with the highest degree of hydrolysis (DH) and peptide concentration was selected as the most suitable enzyme. The effects of pH, temperature, time, and the enzyme-to-substrate ratio (E/S) on the DH and peptide concentration were investigated, and response surface methodology was utilized to optimize the hydrolysis process. The hydrolysate was separated using ultrafiltration membranes with molecular weight cut-offs of 10 kDa, 5 kDa, 3 kDa, and 1 kDa. The bioactivity of each ultrafiltration component was analyzed, including the inhibition rates of α-amylase and xanthine oxidase (XOD) activities and the scavenging rates of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radicals. The results indicated that alkaline protease was the best enzyme for hydrolyzing YWPCs. The peptide concentration in the YWPC hydrolysate was the highest (17.21 mg/mL) at a pH of 8 and a concentration of 7500 U/g, after 2.5 h at 62 °C. The enzymatic hydrolysate was ultrafiltered to yield four peptide fractions, of which the <1 kDa peptides exhibited the highest α-amylase inhibitory activity (22.06%), XOD inhibitory activity (17.15%), and ABTS cationic free radical scavenging rate (69.55%). This demonstrates the potential of YWPC hydrolyzed peptides for hypoglycemic, uric acid-lowering, and antioxidant applications, providing a theoretical basis for the high-value utilization of YWPCs.

Proteolytic Activity of Silkworm Thorn (Cudrania tricuspidata) Fruit for Enzymatic Hydrolysis of Food Proteins.

This study aimed to isolate the proteolytic fraction from the silkworm thorn fruit (Cudrania tricuspidata) through ethanol precipitation at different ratios, and to determine its proteolytic activity and optimal activity conditions. Furthermore, the hydrolysis characteristics and antioxidant activity of soy protein isolate (SPI) and whey protein concentrate (WPC) hydrolyzates obtained through the enzymatic hydrolysis of freeze-dried silkworm thorn fruit powder (SF) were evaluated. For isolation and partial purification of proteolytic fraction, the water-solubilized fraction of the silkworm thorn fruit was purified through ethanol precipitation at four different ratios of 1:1, 1:2, 1:4, and 1:6 (v/v). The protein recovery rate, caseinolytic activity, protein pattern, and optimal activity (pH, temperature, and inhibitors) of fractional ethanol precipitate obtained from the silkworm thorn fruit (ESF) were evaluated. The proteolytic fraction obtained from silkworm thorn fruit exhibited a major protein band around 65-70 kDa and showed the highest proteolytic activity at a 1:4 ratio of ethanol precipitation (p < 0.05). The optimal activity of the measured enzyme fraction was determined to be at pH 9.0 and 50 °C, and the proteolytic activity of ESF was almost inhibited by phenyl methyl sulphonyl fluoride (PMSF, 2 mM), a serine protease inhibitor. Compared to Alcalase and papain, extensively used as commercial enzymes, the silkworm thorn fruit powder was less effective in hydrolyzing SPI and WPC. Nevertheless, SPI and WPC hydrolyzates mediated with silkworm thorn fruit powder showed even better antioxidant activities than those mediated with Alcalase and papain. Thus, our results show the potential application of silkworm thorn fruit as a novel source of plant protease for producing human-grade protein hydrolyzates.

Bioactive Properties of Enzymatic Gelatin Hydrolysates Based on In Silico, In Vitro, and In Vivo Studies.

This current study aims to analyze the potential bioactivities possessed by the enzymatic hydrolysates of commercial bovine, porcine, and tilapia gelatins using bioinformatics in combination with in vitro and in vivo studies. The hydrolysate with superior inhibition of angiotensin converting enzyme (ACE) activity was used to treat the D-galactose (DG)-induced amnesic mice. In silico digestion of the gelatins led to the identification of peptide sequences with potential antioxidant, ACE-inhibitory, and anti-amnestic properties. The results of in vitro digestion revealed that the <1 kDa peptide fraction of porcine gelatin hydrolysate obtained after 1 h digestion with papain (PP) (PP1, <1 kDa) potently inhibited ACE, acetylcholinesterase, and prolyl endopeptidase activities at 87.42%, 21.24%, and 48.07%, respectively. Administering the PP1 to DG-induced amnesic mice ameliorated the spatial cognitive impairment and Morris water maze learning abilities. The dentate area morphology in the PP1-treated mice was relatively similar to the control group. In addition, PP1 enhanced the antioxidant capacity in the DG-induced amnesic mice. This study suggests that PP1 could serve as a potential treatment tool against oxidative stress, hypertension, and neurodegenerative diseases.

A Puccinia striiformis f. sp. tritici effector inhibits high-temperature seedling-plant resistance in wheat.

Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1)-induced protein kinase (RIPK) in Arabidopsis belongs to the receptor-like cytoplasmic kinase (RLCK) family and plays a vital role in immunity. However, the role of RLCKs in the high-temperature seedling-plant (HTSP) resistance of wheat (Triticum aestivum) to Puccinia striiformis f. sp. tritici (Pst), the stripe rust pathogen, remains unclear. Here, we identified a homologous gene of RIPK in wheat, namely TaRIPK. Expression of TaRIPK was induced by Pst inoculation and high temperatures. Silencing of TaRIPK reduced the expression level of TaRPM1, resulting in weaker HTSP resistance. Moreover, TaRIPK interacts with and phosphorylates papain-like cysteine protease 1 (TaPLCP1). Meanwhile, we found that the Pst-secreted protein PSTG_01766 targets TaPLCP1. Transient expression of PSTG_01766 inhibited basal immunity in tobacco (Nicotiana benthamiana) and wheat. The role of PSTG_01766 as an effector involved in HTSP resistance was further supported by host-induced gene silencing and bacterial type three secretion system-mediated delivery into wheat. PSTG_01766 inhibited the TaRIPK-induced phosphorylation of TaPLCP1. Furthermore, PSTG_01766 has the potential to influence the subcellular localization of TaPLCP1. Overall, we suggest that the TaRIPK-TaPLCP1-TaRPM1 module fits the guard model for disease resistance, participating in HTSP resistance. PSTG_01766 decreases HTSP resistance via targeting TaPLCP1. Guarded by wheat and attacked by Pst, TaPLCP1 may serve as a central hub of the defense response. Our findings improve the understanding of the molecular mechanism of wheat HTSP resistance, which may be an important strategy for controlling stripe rust in the face of global warming.

NMR Characterization of the Papain-like Protease from SARS-CoV-2 Identifies the Conformational Heterogeneity in Its Inhibitor-Binding Site.

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.

Mouse Abdominal Aortic Aneurysm Model Induced by Periarterial Incubation of Papain.

For decades, numerous experimental animal models have been developed to examine the pathophysiologic mechanisms and potential treatments for abdominal aortic aneurysms (AAAs) in diverse species with varying chemical or surgical approaches. This study aimed to create an AAA mouse model by the periarterial incubation with papain, which can mimic human AAA with advantages such as simplicity, convenience, and high efficiency. Eighty C57BL/6J male mice were randomly assigned to 1 of the 4 groups: papain (1.0 or 2.0 mg), porcine pancreatic elastase, and phosphate-buffered solution. The aortic segment was wrapped for 20 minutes, and the diameter was measured using ultrasound preoperatively and postoperative days 7 and 14. Then, the mice were killed for histomorphometric and immunohistochemical analyses. According to ultrasound measurements and histomorphometric analyses, on postoperative day 7, 65% of mice in the 1.0-mg papain group and 60% of mice in the 2.0-mg papain group developed AAA. In both papain groups, 100% of mice developed AAA, and 65% of mice in the porcine pancreatic elastase group developed AAA on postoperative day 14. Furthermore, hematoxylin/eosin, elastin van Gieson, and Masson staining of tissues from the papain group revealed thickened media and intimal hyperplasia, collagen sediments, and elastin destruction, indicating that AAA histochemical alteration was similar to that of humans. In addition, the immunohistochemical analysis was conducted to detect infiltrated inflammatory cells, such as macrophages and leukocytes, in the aortic wall and hyperplasic adventitia. The expression of matrix metalloproteinase 2 and 9 was significantly upregulated in papain and human AAA tissues. Periarterial incubation with 1.0 mg of papain for 20 minutes can successfully create an experimental AAA model in mice for 14 days, which can be used to explore the mechanism and treatment of human AAA.

Multipoint Immobilization at Inert Center of Papain on Homo-Functional Diazo-Activated Silica Support: A Way of Restoring "Above Room-Temperature" Bio-Catalytic Sustainability.

Although enzymes play a significant role in industrial applications, their potential usage at high-level efficiency, particularly above room temperature, has not yet been fully harnessed. It brings above room-temperature catalytic sustainability of an immobilized (imm.) bio-catalyst as a long pending issue to improve enzyme stability, activity, specificity, or selectivity, particularly the enantio-selectivity over the native-enzymes. At this juncture, in a robust methodology, a heterogeneous solid phase bio-catalyst, {Si(OSi)4(H2O)1.03}n=328{OSi(CH3)2-NH-C6H4-N═N}4{papain}(H2O)251, has efficiently been prepared by immobilizing papain on homo-functionalized SG (silica-gel) via multipoint covalent attachment. The bio-catalyst is easy to be recovered and reused multiple times. The homo-functional -N═N+, which appears on the SG-surface, makes the multipoint diazo-links with the inert center of the tyrosine-moiety to couple the enzyme where all the amino, thiol, phenol, and so forth, groups of the protein, including those that belong to the active-site, remain intact. The immobilized enzyme (13.9 µmol g-1) swims in pore-water within the pore-channel, remains stable up to 70 ± 5 °C, and exhibits wider temperature adaptability in performing its hydrolyzing activities. The relative activity, 78 ± 2% at 27 °C, remains quantitative for 60 days and can be reused for 60 cycles with 53% activity at room-temperature. The thermal (relative activity: 87%; incubated at 70 ± 5 °C for 24 h) and mechanical (relative activity: 92%; incubated at 2500 rpm for 2 h at 27 °C) stability was outstanding.

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Amino acid variants of SARS-CoV-2 papain-like protease have impact on drug binding.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused both a health and economic crisis around the world. Its papain-like protease (PLpro) is one of the protein targets utilized in designing new drugs that would aid vaccines in the fight against the virus. Although there are already several potential candidates for a good inhibitor of this protein, the degree of variability of the protein itself is not taken into account. As an RNA virus, SARS-CoV-2 can mutate to a high degree, but PLpro variability has not been studied to date. Based on sequence data available in databases, we analyzed the mutational potential of this protein. We focused on the effect of observed mutations on inhibitors' binding mode and their efficacy as well as protein's activity. Our analysis identifies five mutations that should be monitored and included in the drug design process: P247S, E263D-Y264H and T265A-Y268C.