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Chembiochem : a European Journal of... Oct 2022Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets.... (Review)
Review
Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PL ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.
Topics: Aniline Compounds; Antiviral Agents; Benzamides; Coronavirus Papain-Like Proteases; Humans; Naphthalenes; Papain; Peptide Hydrolases; Protease Inhibitors; SARS-CoV-2; Ubiquitin; Viral Proteins; COVID-19 Drug Treatment
PubMed: 35993805
DOI: 10.1002/cbic.202200327 -
Biomolecular Concepts Jun 2013Papain-like cysteine peptidases are a diverse family of peptidases found in most known organisms. In eukaryotes, they are divided into multiple evolutionary groups,... (Review)
Review
Papain-like cysteine peptidases are a diverse family of peptidases found in most known organisms. In eukaryotes, they are divided into multiple evolutionary groups, which can be clearly distinguished on the basis of the structural characteristics of the proenzymes. Most of them are endopeptidases; some, however, evolved into exopeptidases by obtaining additional structural elements that restrict the binding of substrate into the active site. In humans, papain-like peptidases, also called cysteine cathepsins, act both as non-specific hydrolases and as specific processing enzymes. They are involved in numerous physiological processes, such as antigen presentation, extracellular matrix remodeling, and hormone processing. Their activity is tightly regulated and dysregulation of one or more cysteine cathepsins can result in severe pathological conditions, such as cardiovascular diseases and cancer. Other organisms can utilize papain-like peptidases for different purposes and they are often part of host-pathogen interactions. Numerous parasites, such as Plasmodium and flukes, utilize papain-like peptidases for host invasion, whereas plants, in contrast, use these enzymes for host defense. This review presents a state-of-the-art description of the structure and phylogeny of papain-like peptidases as well as an overview of their physiological and pathological functions in humans and in other organisms.
Topics: Animals; Catalytic Domain; Evolution, Molecular; Humans; Models, Molecular; Papain; Peptide Hydrolases; Phylogeny; Protein Structure, Secondary
PubMed: 25436581
DOI: 10.1515/bmc-2012-0054 -
The Journal of Biological Chemistry Jun 2023Papain-like cysteine peptidases form a big and highly diverse superfamily of proteins involved in many important biological functions, such as protein turnover,...
Papain-like cysteine peptidases form a big and highly diverse superfamily of proteins involved in many important biological functions, such as protein turnover, deubiquitination, tissue remodeling, blood clotting, virulence, defense, and cell wall remodeling. High sequence and structure diversity observed within these proteins hinders their comprehensive classification as well as the identification of new representatives. Moreover, in general protein databases, many families already classified as papain like lack details regarding their mechanism of action or biological function. Here, we use transitive remote homology searches and 3D modeling to newly classify 21 families to the papain-like cysteine peptidase superfamily. We attempt to predict their biological function and provide structural characterization of 89 protein clusters defined based on sequence similarity altogether spanning 106 papain-like families. Moreover, we systematically discuss observed diversity in sequences, structures, and catalytic sites. Eventually, we expand the list of human papain-related proteins by seven representatives, including dopamine receptor-interacting protein 1 as potential deubiquitinase, and centriole duplication regulating CEP76 as retaining catalytically active peptidase-like domain. The presented results not only provide structure-based rationales to already existing peptidase databases but also may inspire further experimental research focused on peptidase-related biological processes.
Topics: Humans; Catalytic Domain; Centrioles; Cysteine Proteases; Deubiquitinating Enzymes; Models, Molecular; Papain; Databases, Protein
PubMed: 37164157
DOI: 10.1016/j.jbc.2023.104801 -
Biochemistry Jul 2013Possible reaction pathways for papain-catalyzed hydrolysis of N-acetyl-Phe-Gly 4-nitroanilide (APGNA) have been studied by performing pseudobond first-principles quantum...
Possible reaction pathways for papain-catalyzed hydrolysis of N-acetyl-Phe-Gly 4-nitroanilide (APGNA) have been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy (QM/MM-FE) calculations. The whole hydrolysis process includes two stages: acylation and deacylation. For the acylation stage of the catalytic reaction, we have explored three possible paths (A, B, and C) and the corresponding free energy profiles along the reaction coordinates. It has been demonstrated that the most favorable reaction path in this stage is path B consisting of two reaction steps: the first step is a proton transfer to form a zwitterionic form (i.e., Cys-S⁻/His-H⁺ ion-pair), and the second step is the nucleophilic attack on the carboxyl carbon of the substrate accompanied by the dissociation of 4-nitroanilide. The deacylation stage includes the nucleophilic attack of a water molecule on the carboxyl carbon of the substrate and dissociation between the carboxyl carbon of the substrate and the sulfhydryl sulfur of Cys25 side chain. The free energy barriers calculated for the acylation and deacylation stages are 20.0 and 10.7 kcal/mol, respectively. Thus, the acylation is rate-limiting. The overall free energy barrier calculated for papain-catalyzed hydrolysis of APGNA is 20.0 kcal/mol, which is reasonably close to the experimentally derived activation free energy of 17.9 kcal/mol.
Topics: Acylation; Binding Sites; Biocatalysis; Dipeptides; Hydrolysis; Indicators and Reagents; Kinetics; Models, Molecular; Molecular Conformation; Molecular Dynamics Simulation; Papain; Plant Proteins; Protons; Quantum Theory
PubMed: 23862626
DOI: 10.1021/bi400629r -
Marine Drugs Mar 2021Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine... (Comparative Study)
Comparative Study
Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan) and specific activity (U mg protein), leading to the preservation of more than 90% of the initial total activity (U mL). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6-7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by and . As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded . Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.
Topics: Anti-Bacterial Agents; Biofilms; Carica; Chitosan; Drug Carriers; Drug Compounding; Enzyme Stability; Hydrogen-Ion Concentration; Molecular Weight; Papain; Staphylococcus aureus; Staphylococcus epidermidis; Temperature
PubMed: 33807362
DOI: 10.3390/md19040197 -
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 -
International Journal of Biological... Oct 2021Papain is a cysteine protease from papaya, with many applications due to its broad specificity. This paper reviews for first time the immobilization of papain on... (Review)
Review
Papain is a cysteine protease from papaya, with many applications due to its broad specificity. This paper reviews for first time the immobilization of papain on different supports (organic, inorganic or hybrid supports) presenting some of the features of the utilized immobilization strategies (e.g., epoxide, glutaraldehyde, genipin, glyoxyl for covalent immobilization). Special focus is placed on the preparation of magnetic biocatalysts, which will permit the simple recovery of the biocatalyst even if the medium is a suspension. Problems specific to the immobilization of proteases (e.g., steric problems when hydrolyzing large proteins) are also defined. The benefits of a proper immobilization (enzyme stabilization, widening of the operation window) are discussed, together with some artifacts that may suggest an enzyme stabilization that may be unrelated to enzyme rigidification.
Topics: Carica; Enzyme Stability; Enzymes; Enzymes, Immobilized; Papain
PubMed: 34375660
DOI: 10.1016/j.ijbiomac.2021.08.016 -
Meat Science Apr 2021The present study investigated the effect of the enzymes papain (0.2%) and microbial transglutaminase (MTG) (1%) on the texture properties of beef and chicken burgers to...
The present study investigated the effect of the enzymes papain (0.2%) and microbial transglutaminase (MTG) (1%) on the texture properties of beef and chicken burgers to develop a meat product with significant increase in softness due to the physiological limitations of the elderly. The products were characterized for pH, objective color, water activity, texture profile analysis (TPA), shear force, compression test, electrophoretic profile, cooking loss, and diameter reduction. A pronounced increase in softness was observed for both raw materials containing papain. An increase in shear force was observed for the beef burger containing only MTG, while the chicken burger showed a reduction of this parameter. The compression tests showed papain alone or combined with MTG decreased the hardness of the burgers. The results showed that the combination of the enzymes papain and MTG can be an effective strategy to develop beef and chicken burgers much softer, contributing to the future studies focused on the physiological needs of the elderly.
Topics: Animals; Cattle; Chickens; Cooking; Food Handling; Hardness; Meat Products; Papain; Shear Strength; Transglutaminases
PubMed: 33429336
DOI: 10.1016/j.meatsci.2020.108421 -
Protein Science : a Publication of the... May 2020Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus that is involved in severe diarrhea disease in piglets, causing considerable agricultural and...
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus that is involved in severe diarrhea disease in piglets, causing considerable agricultural and economic loss in China. The emergence of this new coronavirus increases the importance of understanding SADS-CoV as well as antivirals. Coronaviral proteases, including main proteases and papain-like proteases (PLP), are attractive antiviral targets because of their essential roles in polyprotein processing and thus viral maturation. Here, we describe the biochemical and structural identification of recombinant SADS papain-like protease 2 (PLP2) domain of nsp3. The SADS-CoV PLP2 was shown to cleave nsp1 proteins and also peptides mimicking the nsp2|nsp3 cleavage site and also had deubiquitinating and deISGynating activity by in vitro assays. The crystal structure adopts an architecture resembling that of PLPs from other coronaviruses. We characterize both conserved and unique structural features likely directing the interaction of PLP2 with the substrates, including the tentative mapping of active site and other essential residues. These results provide a foundation for understanding the molecular basis of coronaviral PLPs' catalytic mechanism and for the screening and design of therapeutics to combat infection by SADS coronavirus.
Topics: Alphacoronavirus; Animals; Coronavirus; Coronavirus Papain-Like Proteases; Crystallography, X-Ray; Diarrhea; Models, Molecular; Papain; Sus scrofa; Swine; Swine Diseases; Viral Nonstructural Proteins
PubMed: 32216114
DOI: 10.1002/pro.3857 -
Frontiers in Cellular and Infection... 2019Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no...
Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in India. HEV-protease is a pivotal enzyme responsible for ORF1 polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. HEV-protease region encoding 432-592 amino acids of Genotype-1 was amplified, expressed in Sf21 cells and purified in its native form. The recombinant enzyme was biochemically characterized using SDS-PAGE, Western blotting and Immunofluorescence. The enzyme activity and the inhibition studies were conducted using Zymography, FTC-casein based protease assay and ORF1 polyprotein digestion. To conduct ORF1 digestion assay, the polyprotein, natural substrate of HEV-protease, was expressed in and purified. Cleavage of 186 kDa ORF1 polyprotein by the recombinant HEV-protease lead to appearance of non-structural proteins viz. Methyltransferase, Protease, Helicase and RNA dependent RNA polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. FTC-casein substrate was used for kinetic studies to determine Km and Vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. A 95% inhibition was observed with E-64 which was validated through analysis. The correlation coefficient between inhibition and docking score of Inhibitors was found to have a significant value of = 0.75. The predicted 3D model showed two domain architecture structures similar to Papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. Hence, we propose that HEV-protease has characteristics of "Papain-like cysteine protease," as determined through structural homology, active site residues and class-specific inhibition. However, conclusive nature of the enzyme remains to be established.
Topics: Amino Acid Sequence; Animals; Baculoviridae; Catalytic Domain; Cysteine Proteases; DNA Helicases; Epitopes; Escherichia coli; Hepatitis E virus; Kinetics; Methyltransferases; Molecular Docking Simulation; Open Reading Frames; Papain; Peptide Hydrolases; Protease Inhibitors; Protein Conformation; RNA-Dependent RNA Polymerase; Recombinant Proteins; Sf9 Cells; Virus Replication
PubMed: 32039053
DOI: 10.3389/fcimb.2019.00478