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The Plant Cell Aug 2023Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is...
Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.
Topics: Root Nodules, Plant; Glycine max; Plant Proteins; Nitrogen Fixation; Cysteine Proteases; Symbiosis; Gene Expression Regulation, Plant
PubMed: 37177994
DOI: 10.1093/plcell/koad129 -
PloS One 2023Hypertrophic scars and keloids are characterized by an excessive collagen deposition. The available treatment options are invasive and can result in recurrence of scar...
Hypertrophic scars and keloids are characterized by an excessive collagen deposition. The available treatment options are invasive and can result in recurrence of scar formation. Using liposomes and transferosomes for the topical delivery of papain, a proteolytic enzyme, can be effective treatment. The objective of the study is to formulate papain-loaded liposomes and transferosomes, characterize the formulations, and study in vitro permeation using shed snake skin and Sprague-Dawley rat skin as models for stratum corneum and full thickness skin. Papain-loaded liposomes and transferosomes were formulated using the thin-film hydration method for the delivery of papain across the stratum corneum barrier. An in vitro permeation study carried out using shed-snake skin and Sprague-Dawley rat skin models showed that transferosomes were able to deliver papain across the stratum corneum barrier, while papain solution and papain liposomes were not able to cross the barrier. However, transferosomes were not able to deliver papain across the full thickness rat skin model suggesting the deposition of papain loaded transferosomes in the epidermal or dermal layer of skin. In addition, an ex-vivo model was used to analyze the effect of papain exposure on the morphology of the epidermis taken from rat skin exposed to papain solution, papain in transferosomes and papain in liposomes. Papain in solution resulted in a noticeable degradation of the epidermis, but when embedded in either transferosomes or liposomes there was no noticeable change when compared to control animals. The cytotoxicity study performed using HeLa cells showed that the cells were viable at papain concentrations lower than 0.01 mg/ml.
Topics: Humans; Rats; Animals; Liposomes; Keloid; Cicatrix, Hypertrophic; Papain; Rats, Sprague-Dawley; HeLa Cells; Administration, Cutaneous
PubMed: 38100466
DOI: 10.1371/journal.pone.0290224 -
MBio Aug 2023Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the global pandemic of COVID-19. SARS-CoV-2 genome encodes a main protease (nsp5, also...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the global pandemic of COVID-19. SARS-CoV-2 genome encodes a main protease (nsp5, also called Mpro) and a papain-like protease (nsp3, also called PLpro), which are responsible for processing viral polyproteins to assemble a functional replicase complex. In this study, we found that Mpro of SARS-CoV-2 can cleave human MAGED2 and other mammalian orthologs at Gln-263. Moreover, SARS-CoV and MERS-CoV Mpro can also cleave human MAGED2, suggesting MAGED2 cleavage by Mpro is an evolutionarily conserved mechanism of coronavirus infection in mammals. Intriguingly, Mpro from Beta variant cleaves MAGED2 more efficiently than wild type, but Omicron Mpro is opposite. Further studies show that MAGED2 inhibits SARS-CoV-2 infection at viral replication step. Mechanistically, MAGED2 is associated with SARS-CoV-2 nucleocapsid protein through its N-terminal region in an RNA-dependent manner, and this disrupts the interaction between SARS-CoV-2 nucleocapsid protein and viral genome, thus inhibiting viral replication. When MAGED2 is cleaved by Mpro, the N-terminal of MAGED2 will translocate into the nucleus, and the truncated MAGED2 is unable to suppress SARS-CoV-2 replication. This work not only discovers the antiviral function of MAGED2 but also provides new insights into how SARS-CoV-2 Mpro antagonizes host antiviral response. IMPORTANCE Host factors that restrict severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain elusive. Here, we found that MAGED2 can be cleaved by SARS-CoV-2 main protease (Mpro) at Gln-263. SARS-CoV and MERS-CoV Mpro can also cleave MAGED2, and MAGED2 from multiple species can be cleaved by SARS-CoV-2 Mpro. Mpro from Beta variant cleaves MAGED2 more efficiently efficiently than wild type, but Omicron is the opposite. MAGED2 depletion enhances SARS-CoV-2 infection, suggesting its inhibitory role in SARS-CoV-2 infection. Mechanistically, MAGED2 restricts SARS-CoV-2 replication by disrupting the interaction between nucleocapsid and viral genomes. When MAGED2 is cleaved, its N-terminal will translocate into the nucleus. In this way, Mpro relieves MAGED2' inhibition on viral replication. This study improves our understanding of complex viral-host interaction and provides novel targets to treat SARS-CoV-2 infection.
Topics: Animals; Humans; Antiviral Agents; SARS-CoV-2; COVID-19; Coronavirus 3C Proteases; Middle East Respiratory Syndrome Coronavirus; Nucleocapsid Proteins; Mammals; Antigens, Neoplasm; Adaptor Proteins, Signal Transducing
PubMed: 37439567
DOI: 10.1128/mbio.01373-23 -
Foods (Basel, Switzerland) Nov 2023Plant proteases, including actinidin, papain and bromelain, have been widely used in the food industry but with limited application in dairy systems. This research aimed...
Plant proteases, including actinidin, papain and bromelain, have been widely used in the food industry but with limited application in dairy systems. This research aimed to establish and compare operational parameters (kinetics, temperature, enzyme type, time and thermodynamics) relevant to the applications of these enzymes in the hydrolysis of whey protein isolates (WPI), whey protein concentrates (WPC) or milk protein concentrates (MPC). The degree of hydrolysis (DH) increased with the rise in temperature, and the maximum DH was achieved at 60 °C for all three dairy systems. The addition of papain resulted in a greater %DH of whey proteins in comparison to bromelain. The cleavage of proteins was clearly time-dependent ( < 0.05), while the pH did not change significantly ( > 0.05) during this time. PAGE analysis revealed that all three enzymes mainly acted on α-lactalbumin and α-casein in WPI and MPC, respectively. Kinetic parameters from the Lineweaver-Burk plot at 60 °C using WPC and MPC as a substrate varied widely, establishing that WPC hydrolysis was characterised by a lower KM, higher kcat, kcat/KM and Vmax compared to MPC in the case of all three enzymes. The difference in kcat/KM values amongst all enzymes (actinidin > papain > bromelain) indicated the difference in the strength of substrate binding sites. The thermodynamic parameters of these enzymes with MPC and WPC were also determined at a temperature range of 15-60 °C, and the results indicate the potential application of papain and actinidin in the dairy industry.
PubMed: 38231667
DOI: 10.3390/foods12234248 -
International Journal of Molecular... Jul 2023Peptidases of the papain family play a key role in protein degradation, regulated proteolysis, and the host-pathogen arms race. Although the papain family has been the...
Peptidases of the papain family play a key role in protein degradation, regulated proteolysis, and the host-pathogen arms race. Although the papain family has been the subject of many studies, knowledge about its diversity, origin, and evolution in Eukaryota, Bacteria, and Archaea is limited; thus, we aimed to address these long-standing knowledge gaps. We traced the origin and expansion of the papain family with a phylogenomic analysis, using sequence data from numerous prokaryotic and eukaryotic proteomes, transcriptomes, and genomes. We identified the full complement of the papain family in all prokaryotic and eukaryotic lineages. Analysis of the papain family provided strong evidence for its early diversification in the ancestor of eukaryotes. We found that the papain family has undergone complex and dynamic evolution through numerous gene duplications, which produced eight eukaryotic ancestral paralogous C1A lineages during eukaryogenesis. Different evolutionary forces operated on C1A peptidases, including gene duplication, horizontal gene transfer, and gene loss. This study challenges the current understanding of the origin and evolution of the papain family and provides valuable insights into their early diversification. The findings of this comprehensive study provide guidelines for future structural and functional studies of the papain family.
Topics: Papain; Cysteine; Evolution, Molecular; Phylogeny; Eukaryota; Archaea; Cysteine Proteases; Peptide Hydrolases
PubMed: 37511529
DOI: 10.3390/ijms241411761 -
Biophysical Reviews Oct 2023In the process of the development of structural biology, both the size and the complexity of the determined macromolecular structures have grown significantly. As a... (Review)
Review
In the process of the development of structural biology, both the size and the complexity of the determined macromolecular structures have grown significantly. As a result, the range of application areas for the results of structural studies of biological macromolecules has expanded. Significant progress in the development of structural biology methods has been largely achieved through the use of synchrotron radiation. Modern sources of synchrotron radiation allow to conduct high-performance structural studies with high temporal and spatial resolution. Thus, modern techniques make it possible to obtain not only static structures, but also to study dynamic processes, which play a key role in understanding biological mechanisms. One of the key directions in the development of structural research is the drug design based on the structures of biomolecules. Synchrotron radiation offers insights into the three-dimensional time-resolved structure of individual viral proteins and their complexes at atomic resolution. The rapid and accurate determination of protein structures is crucial for understanding viral pathogenicity and designing targeted therapeutics. Through the application of experimental techniques, including X-ray crystallography and small-angle X-ray scattering (SAXS), it is possible to elucidate the structural details of SARS-CoV-2 virion containing 4 structural, 16 nonstructural proteins (nsp), and several accessory proteins. The most studied potential targets for vaccines and drugs are the structural spike (S) protein, which is responsible for entering the host cell, as well as nonstructural proteins essential for replication and transcription, such as main protease (M), papain-like protease (PL), and RNA-dependent RNA polymerase (RdRp). This article provides a brief overview of structural analysis techniques, with focus on synchrotron radiation-based methods applied to the analysis of SARS-CoV-2 proteins.
PubMed: 37974992
DOI: 10.1007/s12551-023-01153-7 -
Frontiers in Nutrition 2023Chickpea ( L.), an annual plant of the family Fabaceae is mainly grown in semiarid and temperate regions. Among pulses, cultivated worldwide chickpeas are considered an... (Review)
Review
Chickpea ( L.), an annual plant of the family Fabaceae is mainly grown in semiarid and temperate regions. Among pulses, cultivated worldwide chickpeas are considered an inexpensive and rich source of protein. Chickpea is a good source of protein and carbohydrate, fiber, and important source of essential minerals and vitamins. The quality of protein is better among other pulses. Consumption of chickpeas is related to beneficial health outcomes. Dietary peptides from the protein of chickpeas gaining more attention. Peptides can be obtained through acid, alkali, and enzymatic hydrolysis. Among all these, enzymatic hydrolysis is considered safe. Various enzymes are used for the production of peptides, i.e., flavorzyme, chymotrypsin, pepsin, alcalase, papain, and trypsin either alone or in combinations. Chickpea hydrolysate and peptides have various bioactivity including angiotensin 1-converting enzyme inhibition, digestive diseases, hypocholesterolemic, CVD, antioxidant activity, type 2 diabetes, anti-inflammatory, antimicrobial, and anticarcinogenic activity. This review summarizes the nutritional composition and bioactivity of hydrolysate and peptides obtained from chickpea protein. The literature shows that chickpea peptides and hydrolysate have various functional activities. But due to the limited research and technology, the sequences of peptides are unknown, due to which it is difficult to conduct the mechanism studies that how these peptides interact. Therefore, emphasis must be given to the optimization of the production of chickpea bioactive peptides, studies of chickpea bioactivity, and conducting human study trials to check the bioactivity of these peptides and hydrolysate.
PubMed: 37854353
DOI: 10.3389/fnut.2023.1218468 -
Genes Jul 2023Salt and osmotic stress seriously restrict the growth, development, and productivity of horticultural crops in the greenhouse. The papain-like cysteine proteases (PLCPs)...
Salt and osmotic stress seriously restrict the growth, development, and productivity of horticultural crops in the greenhouse. The papain-like cysteine proteases (PLCPs) participate in multi-stress responses in plants. We previously demonstrated that salt and osmotic stress affect cysteine protease 15 of pepper ( L.) (); however, the role of in salt and osmotic stress responses is unknown. Here, the function of in regulating pepper salt and osmotic stress resistance was explored. Pepper plants were subjected to abiotic (sodium chloride, mannitol, salicylic acid, ethrel, methyl jasmonate, etc.) and biotic stress ( inoculation). The was silenced through the virus-induced gene silencing (VIGS) and transiently overexpressed in pepper plants. The full-length fragment is 1568 bp, with an open reading frame of 1032 bp, encoding a 343 amino acid protein. CaCP15 is a senescence-associated gene 12 (SAG12) subfamily member containing two highly conserved domains, Inhibitor 129 and Peptidase_C1. expression was the highest in the stems of pepper plants. The expression was induced by salicylic acid, ethrel, methyl jasmonate, and was infected by inoculation. Furthermore, was upregulated under salt and osmotic stress, and silencing in pepper enhanced salt and mannitol stress resistance. Conversely, transient overexpression of increased the sensitivity to salt and osmotic stress by reducing the antioxidant enzyme activities and negatively regulating the stress-related genes. This study indicates that negatively regulates salt and osmotic stress resistance in pepper via the ROS-scavenging.
Topics: Osmoregulation; Sodium Chloride; Capsicum; Antioxidants; Salicylic Acid; Mannitol
PubMed: 37510313
DOI: 10.3390/genes14071409