Search Results (157)

Protein turnover is essential for cellular metabolism, organelle biogenesis, stress adaptation, and ultimately the viability of cells and tissues. Papain-like cysteine proteases (PLCPs) are one of the vital components in protein degradation. PLCPs have been reported to act in senescence-associated proteolysis, but their roles in vegetative growth remain unclear. We identified PtCP1, an AALP-like PLCP in Populus tomentosa, localized to the vacuole and acid-triggered activated. CRISPR/Cas9-generated loss-of-function mutant (d7) showed dwarfism and non-stomatal photosynthetic limitations. On the other hand, the gain-of-function line (EM, deleted ERFNIN domain) exhibited accelerated growth and enhanced photosynthetic parameters. We showed d7 had the accumulation of Rubisco, which was the most important protein in photosynthetic carbon fixation. Transcriptomics revealed dysregulated carbon metabolism in d7. This data supported PtCP1-mediated proteolysis regulated photosynthetic carbon assimilation via altered Rubisco turnover, and then it increased the biomass accumulation during vegetative growth in woody plants. Full article

Papain-like cysteine proteases (PLCPs) are vital enzymes involved in plant development, acting as key regulators of processes such as seed germination, nutrient mobilization, senescence, and programmed cell death. In the present study, we analyzed active PLCPs in various barley organs, including roots, leaves, stems, and seeds at different stages of plant development. Protein extracts obtained from barley samples (4-day-old seedlings; plants at 2, 4, 7, and 11 weeks after sowing; developing seeds from 11-week-old plants; and mature dry seeds) were subjected to anion-exchange chromatography. Fractions containing active PLCPs were pooled, biotinylated using the DCG-04 probe, affinity-purified using streptavidin-agarose, and subsequently analyzed via SDS-PAGE. Bands corresponding to biotinylated PLCPs (detected using streptavidin-peroxidase and a chemiluminescent substrate) were excised from the gel and analyzed by tandem mass spectrometry, enabling the identification of up to 23 distinct PLCPs belonging to nine known PLCP subfamilies. Among the identified PLCPs, HvPap-6 from the L-like D subfamily proved to be the most abundant across all barley samples. In seedlings, B-like and L-like D proteases constituted the largest proportion of all PLCP classes, and their levels continued to increase as the plants developed. Although the relative abundance of L-like B and L-like C proteases was high in seedlings, their levels declined in the roots and leaves of developing plants, as three PLCPs from the L-like B subfamily were identified only during the seedling stage. These results suggest that L-like B and L-like C proteases play an important role in seed germination and seedling development. Organ-specific expression was also observed for certain PLCPs: HvPap-26 from the L-Like C subfamily was identified only in the shoots and roots of seedlings; four PLCPs of the L-like E subfamily were detected solely in the roots, whereas two other proteases from this subfamily were identified exclusively in the leaves and shoots under our experimental conditions. Thus, our results suggest that certain active PLCPs are organ-specific, and that the relative importance of identified PLCPs varies within these organs during plant development. Full article

Papain-like cysteine proteases (PLCPs) are central immune hubs frequently targeted by pathogen effectors. Sugarcane smut, caused by Sporisorium scitamineum, threatens global sugarcane yield, yet effector manipulation of host PLCPs remains unclear. Genome-wide analysis of Saccharum spontaneum AP85-441 identified 61 PLCP-encoding genes, which were classified into nine conserved subfamilies. Among these, ScRD21A, a member of the RD21 subfamily, was prioritized for functional characterization. Two Pit2 homologs, SsPit2A and SsPit2B, were identified from S. scitamineum. Yeast two-hybrid, BiFC and pull-down assays demonstrated that both effectors interact with ScRD21A, and that this interaction depends on a conserved LXRR motif within their PID14-like region. In total protein extracts from Nicotiana benthamiana, co-expression of SsPit2A or SsPit2B reduced ScRD21A-associated cysteine protease activity. Transient expression of ScRD21A enhanced flg22-induced ROS production, attenuated Pst DC3000-induced hypersensitive response-associated necrosis, and increased resistance to Botrytis cinerea. Together, these results support a conserved PLCP-targeting strategy in smut fungi and identify the ScRD21A–SsPit2A/B module as a tractable framework for studying effector–protease interactions relevant to sugarcane smut. Full article

The SARS-CoV-2 papain-like protease (PL^pro) is an essential viral enzyme that promotes viral polyprotein processing while simultaneously suppressing the host innate immune response, which makes it a primary target for developing antiviral drugs. The present study employs a comprehensive approach integrating untargeted metabolomic profiling, in silico molecular docking and dynamics simulations, Molecular Mechanics Generalized Born Surface Area (MM-GBSA) energetic assessments, and biochemical enzyme assays. This integrated method aims to discover natural PL^pro inhibitors from two ethnomedicinal plants, Lippia javanica and Acorus calamus, which have long been utilized in African traditional medicine to treat respiratory diseases. Comprehensive metabolite profiling using untargeted Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS) and Global Natural Products Social (GNPS) molecular networking revealed flavonoid glucuronides and phenylpropanoid derivatives as the major constituents in both plant species. In situ histochemical staining further offered spatial validation of phenolic- and lignin-associated tissues, supporting the phenolic-dominated molecular families detected by GNPS molecular networking. In silico evaluation of six selected compounds demonstrated spontaneous and thermodynamically favorable binding to PL^pro, with ΔG_bind values ranging from −5.63 to −6.43 kcal/mol. Catechin-7-glucoside emerged as the lead compound, establishing multiple hydrogen bond networks with Asp164, Gln269, Tyr264, and Asn267, supplemented by hydrophobic engagement with Pro247 and Pro248, and π-π stacking with the blocking loop 2 (BL2 loop). Molecular dynamics simulations confirmed the stability of the protein–ligand complexes. Biochemical enzyme assays confirmed concentration-dependent inhibition of PL^pro proteolytic and deubiquitinating activity by both crude plant extracts and isolated bioactive compounds. However, S-adenosyl-methionine showed comparatively high PL^pro proteolytic activity (IC₅₀ 5.872 µM) compared to catechin-7-glucoside, with an IC₅₀ of 7.493 µM, exhibiting efficacy similar to the reference inhibitor GRL0617. Both the extracts of L. javanica and A. calamus have shown significant inhibitory activity while maintaining cell viability in Human embryonic kidney 293T cell (HEK293T) culture models, indicating a favorable safety profile of the tested concentrations. Based on these results, catechin-based polyphenols and phenylpropanoid derivatives appear as promising lead compounds for the development of PL^pro inhibitors. To progress toward therapeutic use, further work is necessary in pharmacokinetics, structural optimization, and antiviral validation in cell models. Full article

Papain-like protease (PLpro), a crucial functional domain of the SARS-CoV-2 non-structural protein 3 (nsp3), plays a dual role in both hydrolyzing viral polyprotein precursors and modulating host immune responses. These critical functions position PLpro as a key target in the ongoing development of antiviral therapies for SARS-CoV-2. This review analyzes more than 100 PLpro-ligand co-crystal structures and summarizes the major binding modes between these ligands and PLpro. Most of these ligands bind to sites analogous to those targeted by the classical non-covalent inhibitor GRL0617, primarily involving the P3 and P4 subsites and the BL2 loop. Based on these structural insights, optimized inhibitors have expanded targeting beyond the canonical binding site to auxiliary regions such as the BL2 groove and the Val70 site, and in some cases toward the catalytic Cys111 buried within a narrow pocket. Certain ligands identified through various screening approaches bind to non-canonical or allosteric regions, such as the S1 and S2 sites or the zinc-finger domain, engaging PLpro through distinct interaction modes and thereby offering additional opportunities for PLpro inhibitor design. The review also discusses potential strategies for future PLpro inhibitor development informed by recent structural advances. Taken together, these structural and functional insights support ongoing efforts in the structure-guided design and optimization of PLpro inhibitors. Full article

Despite growing interest in South African medicinal plants, advanced metabolomic workflows that integrate positive (ESI+) and negative (ESI−) ionization modes in UPLC-MS/MS remain sparsely applied to South African flora, and especially to Acorus calamus and Lippia javanica species. Herein, application of a dual-polarity (positive (ESI+) and negative (ESI−) ionization modes) using an untargeted UPLC–MS/MS workflow, integrated with HEK293T cytotoxicity screening, to map their metabolomes, and rank potential signature metabolites for targeted antiviral follow-up. SwissADME supported in silico drug-likeness. Neither plant extract was cytotoxic across the concentration range, with absorbance-based cell viability of 73.82% for L. javanica and 77.23% for A. calamus at 250 µg/mL, and fluorescence-based cell viability ≥59.87% and ≥55.89%, respectively. Dual-polarity expanded coverage with ESI− yielded 312 features, compared with 225 with ESI+, consistent with the predominance of acidic phenolics in plant species. Unsupervised and supervised models segregated the plant species (PCA PC1/PC2 variance: ESI+ 89.4%/3.0%; ESI− 93.5%/1.8%; R²X(cum) = 0.799). Differential analysis identified 118 significant features in ESI+ with 80 up-regulated, 38 down-regulated, and 139 in ESI− with 96 up-regulated, 43 down-regulated. The ESI− showed the wider dynamic range. Chemotypes enriched among significant metabolites include flavonols of 3-O-methylkaempferol, apigenin, and conjugates of Pollenin A, iridoid glycosides of oleoside, forsythoside B, and jasmonate-pathway oxylipins of 7-epi-12-hydroxyjasmonic acid and its glucoside. These also include caryoptosidic acid and catechin-7-glucoside, which are ionized in both modes, pinning the increase in biomarker robustness. In conclusion, a dual-mode UPLC–MS/MS approach, integrated with cytotoxicity exploration, delivers a complementary metabolome coverage and a safety awareness for shortlisting of potential signature metabolites from L. javanica and A. calamus. Moreover, in vitro inhibition of SARS-CoV-2 papain-like protease (PL^pro) by these plants links chemical signatures to antiviral relevance. Shortlisted significant metabolites that demonstrated favorable drug-likeness include flavonol scaffolds of 3-O-methylkaempferol, Pollenin A, and jasmonate-pathway derivatives of 7-epi-12-hydroxyjasmonic acid. Moreover, the dual ionization mode may eliminate ionization bias, broaden metabolome coverage, and yield a mechanism-ready shortlist of metabolites from South African medicinal plants for downstream antiviral investigation. Full article

SARS-CoV-2 virus contains two highly conserved domains, the papain-like protease (PLpro) and main protease (Mpro), which play important roles in virus replication, immune suppression, and the induction of inflammation in host tissue. In this study, we applied small-molecule chip screening, enzymatic assays, SARS-CoV-2 spike pseudotyped virus detection and molecular docking to find potential Mpro or PLpro inhibitors. Two small molecules, oxytocin and risedronate sodium, stood out in drug repurposing. Oxytocin and risedronate sodium were shown to influence the activities of Mpro and PLpro, thereby preventing the virus from replication, which may alleviate SARS-CoV-2 infection. Thus, oxytocin, risedronate sodium, and cephalosporins may expand the drug library for treating coronavirus infection. Full article

Human adenovirus infections are typically self-limiting but can become life-threatening in pediatric populations and immunocompromised individuals. Despite this clinical importance, efforts to develop antiviral drugs against adenoviruses remain limited. A promising strategy is to target the adenovirus protease (AVP), an enzyme essential for viral maturation and infectivity. Yet, research on AVP has lagged far behind that on other viral proteases. In this work, we aimed to reposition AVP as a viable target for antiviral therapy. We first discuss why AVP research has fallen behind and emphasize the need to redirect attention toward this protease. Building on advances in SARS-CoV-2 drug discovery, we evaluated the potential of repurposing inhibitors of the main protease (M^pro) and papain-like protease (PL^pro) as modulators of AVP. Additionally, we examined the untapped potential of phytochemicals as novel scaffolds. These analyses were supported by original molecular docking studies. Our results revealed that previously reported SARS-CoV-2 inhibitors, such as the M^pro inhibitor ensitrelvir and the PL^pro inhibitor (compound) 19, engage the catalytic site of AVP and may serve as starting scaffolds for inhibitor design. Screening of phytochemicals further identified promising candidates, including apigenin, camptothecin, kaempferol, and piperine. Together, these findings highlight AVP’s druggability and suggest that both repurposed antivirals and natural products provide complementary avenues for inhibitor development. Finally, we provide some recommendations to facilitate efforts in the discovery of novel AVP inhibitors. Full article

Human immunodeficiency virus (HIV-1) and SARS-CoV-2 continue to co-burden global health, motivating discovery of broad-spectrum small molecules. Conessine, a steroidal alkaloid, has reported membrane-active and antimicrobial properties but remains underexplored as a dual antiviral chemotype. To interrogate conessine’s multi-target antiviral potential against key enzymatic and entry determinants of HIV-1 and SARS-CoV-2 and to benchmark performance versus approved comparators. Eight targets were modeled: HIV-1 reverse transcriptase (RT, 3V81), protease (PR, 1HVR), integrase (IN, 3LPT), gp120–gp41 trimer (4NCO); and SARS-CoV-2 main protease (M^pro, 6LU7), papain-like protease (PL^pro, 6W9C), RNA-dependent RNA polymerase (RdRp, 7BV2), spike RBD (6M0J). Ligands (conessine; positive controls: dolutegravir for HIV-1, nirmatrelvir for SARS-CoV-2) were prepared with standard protonation, minimized, and docked using AutoDock Vina v 1.2.0exhaustiveness 4; 20 poses). Binding modes were profiled in 2D/3D. Protocol robustness was verified by re-docking co-crystallized ligands (RMSD ≤ 2.0 Å). Atomistic MD (explicit TIP3P, OPLS4, 300 K/1 atm, NPT; 50–100 ns) assessed pose stability (RMSD/RMSF), pocket compaction (Rg, volume), and interaction persistence; MM/GBSA provided qualitative energy decomposition. ADMET was predicted in silico. Conessine showed coherent, hydrophobically anchored binding across both viral panels. Best docking scores (kcal·mol⁻¹) were: HIV-1—PR −6.910, RT −6.672, IN −5.733; SARS-CoV-2—spike RBD −7.025, M^pro −5.745, RdRp −5.737, PL^pro −5.024. Interaction maps were dominated by alkyl/π-alkyl packing to catalytic corridors (e.g., PR Ile50/Val82, RT Tyr181/Val106; M^pro His41/Met49; RBD L455/F486/Y489) with occasional carbon-/water-mediated H-bonds guiding orientation. MD sustained low ligand RMSD (typically ≤1.6–2.2 Å) and damped RMSF at catalytic loops, indicating pocket rigidification; MM/GBSA trends (≈ −30 to −40 kcal·mol⁻¹, dispersion-driven) supported persistent nonpolar stabilization. Benchmarks behaved as expected: dolutegravir bound strongly to IN (−6.070) and PR (−7.319) with stable MD; nirmatrelvir was specific for M^pro and displayed weaker, discontinuous engagement at PL^pro/RdRp/RBD under identical settings. ADMET suggested conessine has excellent permeability/BBB access (high logP), but liabilities include poor aqueous solubility, predicted hERG risk, and CYP2D6 substrate dependence.Conessine operates as a hydrophobic, multi-target wedge with the most favorable computed engagement at HIV-1 PR/RT and the SARS-CoV-2 spike RBD, while maintaining stable poses at M^pro and RdRp. The scaffold merits medicinal-chemistry optimization to improve solubility and de-risk cardiotoxicity/CYP interactions, followed by biochemical and cell-based validation against prioritized targets. Full article

Background/Objectives: Naegleria fowleri is a free-living protozoan that causes primary amoebic meningoencephalitis, a rapidly progressing central nervous system infection with high mortality rates and limited treatment options. Targeting virulence-associated proteins is essential for effective drug development. Fowlerpain-1 (FWP1), a papain-like cysteine protease (CP) implicated in extracellular matrix degradation and host–cell cytotoxicity, has been investigated as a therapeutic target. This study aimed to evaluate the FWP1 pocket geometry and stefin binding using an integrated in silico structural biology approach. Methods: A computational pipeline was used, including AlphaFold2-Multimer modeling of FWP1–stefin complexes, 20-ns molecular dynamics simulations under NPT conditions for conformational sampling, and molecular mechanics Poisson–Boltzmann surface area free energy calculations. Three natural CP inhibitors (stefins) were investigated. Structural stability was assessed using root mean square deviations, and binding profiles were characterized using protein–protein interaction analysis. Results: Stable FWP1–stefin interaction interfaces were predicted, with human stefin A showing favorable binding free energy. Two conserved motifs (PG and QVVAG) were identified as critical mediators of active-site recognition. Druggability analysis revealed a concave pocket with both hydrophobic and polar characteristics, consistent with a high-affinity ligand-binding site. Conclusions: This computational study supports a structural hypothesis for selective FWP1 inhibition and identifies stefins as promising scaffolds for developing structure-guided protease-targeted therapeutics against N. fowleri. Full article

Leaf senescence is a developmental process that allows nutrients to be remobilized and transported to sink organs. Previously, papain-like cysteine proteases (PLCPs) have been found to be highly expressed during leaf senescence in different plant species. In this study, we analyzed active PLCPs in barley (Hordeum vulgare L.) leaves during the terminal stage of natural senescence. Anion exchange chromatography of protein extracts from barley leaves, harvested six weeks after anthesis, followed by activity assays using the substrates Z-FR-AMC and Z-RR-AMC, revealed a single prominent peak corresponding to active PLCPs. This hydrolytic activity was completely inhibited by E-64, a potent and irreversible inhibitor of cysteine proteases. Fractions enriched for PLCP activity were affinity-labeled with DCG-04 and subjected to SDS-PAGE fractionation, separating two major bands at 43 and 38 kDa. These bands were analyzed using tandem mass spectrometry, allowing the identification of eleven PLCPs. Identified enzymes belong to eight PLCP subfamilies, including CTB/cathepsin B-like (HvPap-19 and -20), RD19/cathepsin F-like (HvPap-1), ALP/cathepsin H-like (HvPap-12 or aleurain), SAG12/cathepsin L-like A (HvPap-17), CEP/cathepsin L-like B (HvPap-14), RD21/cathepsin L-like D (HvPap-6 and -7), cathepsin L-like E (HvPap-13 and -16), and XBCP3 (HvPap-8). Among the identified PLCPs, HvPap-6 was the most abundant. Peptides corresponding to HvPap-6 were identified in both the 43 kDa and 38 kDa bands in approximately the same quantity based on total spectral count. Thus, our results indicate that two active HvPap-6 isoforms can be isolated from barley leaves at late senescence. Full article

Novel therapies to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of respiratory coronavirus disease 2019 (COVID-19), would be of great clinical value to combat the current and future pandemics. Two viral proteases, papain-like protease (PL^pro) and the main protease 3-chymotrypsin-like protease (3CL^pro), are vital in processing the SARS-CoV-2 polyproteins (pp1a and pp1ab) and in releasing 16 nonstructural proteins, making them attractive antiviral drug targets. In this study, we investigated the degradation of the SARS-CoV-2 main protease 3CL^pro by matrix metalloproteinase-14 (MMP14). MMP14 is known to recognize over 10 distinct substrate cleavage sequences. Through sequence analysis, we identified 17 and 10 putative MMP14 cleavage motifs within the SARS-CoV-2 3CL^pro and PL^pro proteases, respectively. Despite the presence of potential sites in both proteins, our in vitro proteolysis assays demonstrated that MMP14 selectively binds to and degrades 3CL^pro, but not PL^pro. This selective proteolysis by MMP14 results in the complete loss of 3CL^pro enzymatic activity. In addition, SARS-CoV-2 pseudovirus replication was inhibited in 293 T cells when either full-length MMP14 or its catalytic domain (cat-MMP14) were overexpressed, presumably due to 3CL^pro degradation by MMP14. Finally, to prevent MMP14 from degrading off-target proteins, we propose a new recombinant pro-PL-MMP14 construct that can be activated only by another SARS-CoV-2 protease, PL^pro. These findings could open the potential of an alternative therapeutic strategy against SARS-CoV-2 infection. Full article

Feline coronaviruses (FCoVs) are divided into two groups: feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). FIPV is responsible for the severe disease known as feline infectious peritonitis, while FECV typically causes mild symptoms, such as diarrhea, and often does not lead to any disease at all. Currently, it is not possible to distinguish between FIPV and FECV at the molecular level. Therefore, there is an urgent need to understand the molecular features of FIPV. Here, we generated a recombinant virus by replacing the ORF1ab region and the coding sequence for the spike (S) protein of an FECV with the corresponding sequences from FIPVs. The recombinant virus (recFECV-S_DF-2-1ab_FIPV) exhibited similar growth kinetics to its parental strain. Our analysis revealed that the replacement of the ORF1ab in the FECV caused significant alterations in protein expression within the host cells. Furthermore, the presence of the ORF1ab from the FIPV strain resulted in enhanced suppression of the innate immune response compared to the parental strain, as determined through proteomic and transcriptomic studies. Additionally, we demonstrated that the papain-like protease 2 (PL2^pro) of the non-structural protein 3 (NSP3) from both FIPV and FECV functions in immune suppression, and the protease activity is required for this function. Full article

The apoplast is often the first point of contact between plant cells and invading pathogens, serving as an important site for defense signaling. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein from Phytolacca americana (pokeweed), is localized to the apoplast and is hypothesized to accompany a pathogen to the cytosol, where it would inactivate host ribosomes to prevent pathogen spread. However, it is not known whether PAP interacts with other proteins in the apoplast. In this study, we identified Phytolacca americana cysteine protease 1 (PaCP1), an extracellular cysteine protease, as a novel PAP interactor. Sequence and structural analyses classified PaCP1 as a member of the C1A subfamily of papain-like cysteine proteases. Immunoprecipitation, mass spectrometry, and yeast two-hybrid analysis showed that PAP specifically binds the mature, active form of PaCP1. Curiously, PaCP1 cleaves PAP at its N- and C-termini, generating peptides that enhance MAPK phosphorylation in pokeweed leaves, indicating their potential role in stress signaling. PaCP1 processing of PAP to generate bioactive peptides diversifies the function of a ribosome-inactivating protein beyond its canonical inhibition of translation. Our findings present a novel extracellular role for PAP and advance our understanding of how protein interactions in the apoplast contribute to plant immune responses. Full article

The global impact of the COVID-19 crisis has underscored the need for novel therapeutic candidates capable of efficiently targeting essential viral proteins. Existing therapeutic strategies continue to encounter limitations such as reduced efficacy against emerging variants, safety concerns, and suboptimal pharmacodynamics, which emphasize the potential of natural-origin compounds as supportive agents with immunomodulatory, anti-inflammatory, and antioxidant benefits. The present study significantly advances prior molecular docking research through comprehensive virtual screening of structurally related analogs derived from antiviral phytochemicals. These compounds were evaluated specifically against the SARS-CoV-2 main protease (3CLpro) and papain-like protease (PLpro). Utilizing chemical similarity algorithms via the ChEMBL database, over 600 candidate molecules were retrieved and subjected to automated docking, interaction pattern analysis, and comprehensive ADMET profiling. Several analogs showed enhanced binding scores relative to their parent scaffolds, with CHEMBL1720210 (a shogaol-derived analog) demonstrating strong interaction with PLpro (−9.34 kcal/mol), and CHEMBL1495225 (a 6-gingerol derivative) showing high affinity for 3CLpro (−8.04 kcal/mol). Molecular interaction analysis revealed that CHEMBL1720210 forms hydrogen bonds with key PLpro residues including GLY163, LEU162, GLN269, TYR265, and TYR273, complemented by hydrophobic interactions with TYR268 and PRO248. CHEMBL1495225 establishes multiple hydrogen bonds with the 3CLpro residues ASP197, ARG131, TYR239, LEU272, and GLY195, along with hydrophobic contacts with LEU287. Gene expression predictions via DIGEP-Pred indicated that the top-ranked compounds could influence biological pathways linked to inflammation and oxidative stress, processes implicated in COVID-19’s pathology. Notably, CHEMBL4069090 emerged as a lead compound with favorable drug-likeness and predicted binding to PLpro. Overall, the applied in silico framework facilitated the rational prioritization of bioactive analogs with promising pharmacological profiles, supporting their advancement toward experimental validation and therapeutic exploration against SARS-CoV-2. Full article