Cysteine Protease Research Articles

Malaria parasite cysteine and aspartic proteases as key drug targets for antimalarial therapy.

Cysteine and aspartic proteases are enzyme families that play crucial roles in the life cycle of Plasmodium, the parasite responsible for malaria. These proteases are involved in vital biological processes, such as hemoglobin degradation within the host's red blood cells, protein turnover, and regulation of parasite development. Inhibiting these proteases with small molecule drugs can block the parasite's growth and survival. Chemically, these enzymes have specific active sites where inhibitors can bind, preventing the breakdown of key proteins, making them attractive targets for the design of novel antimalarial compounds. Understanding the structure and catalytic mechanisms of these proteases is critical for developing selective and potent inhibitors. The degradation of hemoglobin occurs in the parasite's digestive vacuole, and disruption of this process by targeting these proteases can inhibit parasite development, leading to the death of the parasite. Hence, these proteases are critical for maintaining the parasite's metabolic functions, and inhibiting them can disrupt the parasite's life cycle. Malaria remains a major global health problem, particularly in tropical and subtropical regions, where resistance to existing antimalarial drugs, such as chloroquine and artemisinin-based therapies, is an escalating issue. The emergence of drug-resistant Plasmodium strains highlights the urgent need for new therapeutic strategies. Targeting cysteine and aspartic proteases offers a novel approach to antimalarial drug development, as these enzymes are crucial for parasite survival and have not been widely exploited in current therapies. By inhibiting these proteases, researchers aim to develop new antimalarial treatments that could overcome resistance mechanisms and provide more effective options for malaria control and eradication. The application of computational methods such as molecular docking, dynamics simulations, and quantum mechanical calculations, combined with powerful molecular modeling tools, provides a comprehensive framework for discovering and optimizing inhibitors targeting Plasmodium cysteine and aspartic proteases. These methods facilitate the rational design of novel antimalarial drugs, offering a pathway to overcome drug resistance and improve therapeutic outcomes.

A Puccinia striiformis f. sp. tritici Effector with DPBB Domain Suppresses Wheat Defense

Wheat (Triticum aestivum L.) is a primary crop globally. Among the numerous pathogens affecting wheat production, Puccinia striiformis f. sp. tritici (Pst) is a significant biotic stress agent and poses a major threat to world food security by causing stripe rust or yellow rust disease. Understanding the molecular basis of plant–pathogen interactions is crucial for developing new means of disease management. It is well established that the effector proteins play a pivotal role in pathogenesis. Therefore, studying effector proteins has become an important area of research in plant biology. Our previous work identified differentially expressed candidate secretory effector proteins of stripe rust based on transcriptome sequencing data from susceptible wheat (Avocet S) and resistant wheat (Avocet YR10) infected with Pst. Among the secreted effector proteins, PSTG_14090 contained an ancient double-psi beta-barrel (DPBB) fold, which is conserved in the rare lipoprotein A (RlpA) superfamily. This study investigated the role of PSTG_14090 in plant immune responses, which encodes a protein, here referred to as Pst-DPBB, having 131 amino acids with a predicted signal peptide (SP) of 19 amino acids at the N-terminal end, and the DNA sequence of this effector is highly conserved among different stripe rust races. qRT-PCR analysis indicated that expression levels are upregulated during the early stages of infection. Subcellular localization studies in Nicotiana benthamiana leaves and wheat protoplasts revealed that it is distributed in the cytoplasm, nucleus, and apoplast. We demonstrated that Pst-DPBB negatively regulates the immune response by functioning in various compartments of the plant cells. Based on Co-IP and structural predictions and putative interaction analyses by AlphaFold 3, we propose the probable biological function(s). Pst-DPBB behaves as a papain inhibitor of wheat cysteine protease; Pst-DPBB has high structural homology to kiwellin, which is known to interact with chorismate mutase, suggesting that Pst-DPBB inhibits the native function of the host chorismate mutase involved in salicylic acid synthesis. The DPBB fold is also known to interact with DNA and RNA, which may suggest its possible role in regulating the host gene expression.

Advances in Cysteine Protease B Inhibitors for Leishmaniasis Treatment.

The expression and release of cysteine proteases by Leishmania spp. and their virulence factors significantly influence the modulation of host immune responses and metabolism, rendering cysteine proteases intriguing targets for drug development. This review article explores the substantial role of cysteine protease B (CPB) in medicinal chemistry from 2001 to 2024, particularly concerning combatting Leishmania parasites. We delve into contemporary advancements and potential prospects associated with targeting cysteine proteases for therapeutic interventions against leishmaniasis, emphasizing drug discovery in this context. Computational analysis using the pkCSM tool assessed the physicochemical properties of compounds, providing valuable insights into their molecular characteristics and drug-like potential, enriching our understanding of the pharmacological profiles, and aiding rational inhibitor design. Our investigation highlights that while nonpeptidic compounds constitute the majority (69.2%, 36 compounds) of the dataset, peptidomimetic- based derivatives (30.8%, 16 compounds) also hold promise in medicinal chemistry. Evaluating the most promising compounds based on dissociation constant (Ki) and half maximal inhibitory concentration (IC50) values revealed notable potency, with 41.7% and 80.0% of nonpeptidic compounds exhibiting values < 1 μM, respectively. On the other hand, all peptidic compounds evaluated for Ki (43.8%) and IC50 (31.3%) obtained values < 1 μM, respectively. Further analysis identified specific compounds within both categories (nonpeptidic: 1, 2, and 4; peptidic: 48-52) as particularly promising, warranting deeper investigation into their structure-activity relationships. These findings underscore the diverse landscape of inhibitors in medicinal chemistry and highlight the potential of both nonpeptidic and peptide-based compounds as valuable assets in therapeutic development against leishmaniasis.

Nanomolar Activity of Coumarin-3-thiosemicarbazones Targeting Trypanosoma cruzi Cruzain and T. brucei Cathepsin-L-like Protease

Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei) urgently demand innovative drug development due to their impact on public health worldwide. Their cysteine proteases, Cruzain (CRZ) and T. brucei Cathepsin L-like protease (TbrCATL) are established drug targets for these parasites. In this study, our coumarin-3-thiosemicarbazones demonstrated nanomolar IC50 values (22-698 nM) toward these proteases. Against T. cruzi amastigotes and T. brucei trypomastigotes, LASF-01 displayed a promising result. Herein, MCG-02, the most potent TbrCATL inhibitor, underwent comprehensive analyses, including cytotoxicity assessments and in vitro tests. Molecular dynamics (MD) simulations and a multiscale Quantum Mechanics/Quantum Mechanics (QM/QM) approach were used to generate insights into their binding modes. These suggested that MCG-02 could be a reversible, competitive covalent inhibitor. Further, confirmatory assays were experimentally performed changing different parameters to prove its efficacy. Additionally, the predicted pharmacokinetic profile showed that there is no violation of the Lipinski rule of five. Notably, coumarin-3-thiosemicarbazone hybrids emerged as promising candidates for designing highly active inhibitors against CRZ and TbrCATL. Overall, the integration of in silico and experimental approaches enhanced our understanding regarding the binding modes of MCG-02, which were experimentally corroborated, providing valuable insights for future drug development.

Structural insights into the role of the prosegment binding loop in a papain-superfamily cysteine protease from Treponema denticola.

Periodontal diseases afflict 20-50% of the global population and carry serious health and economic burdens. Chronic periodontitis is characterized by inflammation of the periodontal pocket caused by dysbiosis. This dysbiosis is coupled with an increase in the population of Treponema denticola, a spirochete bacterium with high mobility and invasivity mediated by a number of virulence factors. One such virulence factor is TDE0362, a multidomain protein with a carboxy-terminal papain-superfamily cysteine protease (C0362). Most papain-superfamily cysteine proteases are produced as proenzymes with a prodomain that interacts with the prosegment binding loop (PBL), requiring proteolytic processing for full activation. Previous studies have indicated that C0362 is not produced as a proenzyme, suggesting an alternative regulatory mechanism. We previously determined the crystal structure of C0362 captured in an inactive conformation with an oxidized catalytic cysteine and a disordered PBL. In this follow-up study, we evaluated the active-site architecture and the PBL in two mutant (Y559A and C412S) structures and an inhibitor-bound (E64) structure to provide insight into the role that the PBL plays in the generation of active enzyme. Our results implicate Tyr559 as playing a critical role in the transition of the enzyme to an active state. We subsequently utilized the structural information to generate models of C0362 bound to human complement factors C3 and C4. Collectively, our results provide insight into the regulatory mechanism and putative substrate-binding interfaces of C0362, highlighting avenues of further research towards inhibition of this essential virulence factor.

The Pruritus Mediator Recombinant Mucunain Induced Apoptosis Through Caspase 8 Signaling Pathway in the Skin Epithelial Cells

Background Mucunain, a cysteine protease extracted from the spicules of cowhage, can induce intense itching as a pruritus mediator independent of histamine. It is well known that erythema, edema and vesicles on skin may occur immediately following contact with Mucunain. A recent study has demonstrated that Mucunain may trigger pruritus and inflammation on the left or right groin of each Maltese–Beagle atopic dog. The aim of this study was to investigate the cytotoxic effect of Mucunain on a cocultured skin epithelial cell model in vitro. Methods Functional recombination Mucunain (rtMucunain) were obtained following induction, purification, and renaturation. Human keratinocytes and dermal fibroblasts were cocultured to construct a cocultured cell model skin epithelium cell model. The growth inhibition was measured using an MTS test. Microscopy was used to observe morphological alterations in cell architecture. The DCFH-DA assay was used to evaluate reactive oxygen species production, the cells death type was identified using a Flow cytometry assay, and the pro-inflammatory cytokines IL-6 and −8 were detected using an ELISA assay. The ability to form an apoptosis network was investigated by a Western blot assay to assess apoptosis-related protein expression. Results Following purification and renaturation, rtMucunain was obtained with a yield of up to 50 mg/L. In the cocultured skin epithelial cells treated with rtMucunain, cell growth was inhibited in a dose- and time-dependent manner, with doses of 2.5 µM or higher resulting in significant cytotoxicity. Morphological analysis revealed increased cell dispersion and abnormal morphology, particularly following prolonged exposure. Levels of reactive oxygen species were elevated, indicating oxidative stress. Flow cytometry and Western-blot data demonstrated that the cells underwent apoptosis, with extrinsic apoptosis pathways (NF-κB and Caspase-8) being activated by rtMucunain. Furthermore, treatment with rtMucunain markedly enhanced levels of IL-6 and IL-8, suggesting a robust pro-inflammatory response. Conclusion rtMucunain showed a strong inflammation and apoptosis effect in a cocultured skin epithelium cell model. This effect may be due to the activated Caspase 8 signaling pathway, on which NF-κB might play an important role. It was the first time to clarify the cytotoxic effect of rtMucunain on the skin epithelial cell model, and the comprehensive understanding the damaged mechanisms of rtMucunain on normal skin cells can provide a data basis for the skin irritated experiment of rtMucunain and its analogues.

Triticain alpha represents the major active papain-like cysteine protease in naturally senescing and ozone-treated leaves of wheat

Current background tropospheric ozone (O3) concentrations have significant adverse effects on wheat. O3 generally induces oxidative damages and premature leaf senescence leading to important yield losses. As leaf protein degradation and recycling is involved in both maintaining cell longevity during abiotic stresses and performing efficient nitrogen remobilization during senescence, we aimed to identify proteases involved in acidic endoproteolytic activities during natural and O3-induced leaf senescence in wheat. Field-grown plants of two winter wheat cultivars were exposed to ambient and semi-controlled chronic O3 concentrations, from pre-anthesis to grain harvest. Yield parameters were impacted by the most elevated O3 exposure for both cultivars. At the cellular level, our analysis revealed that both natural leaf senescence and O3 treatments induced a stimulation of acidic (pH 5.5) endoproteolytic activities, mostly due to papain-like cysteine proteases (PLCPs). Identification of active PLCPs using activity-based protein profiling (ABPP) revealed that Triticain α was the major active PLCP in senescing flag leaves and the only PLCP whose abundance increased with O3 stress, a result of positive transcriptional regulation. Our study provides novel insight into the implication of PLCP-mediated proteolysis in O3 sensitivity in a major crop.

In Silico Study of Two Linear Furanocoumarins for Dual Inhibition of Human Rhinovirus and SARS‐CoV‐2

AbstractIn the discovery of antiviral drugs, targeting the 3‐chymotrypsin‐like cysteine protease (3CLpro) of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is advantageous for developing a broad‐spectrum antiviral agent for the treatment of coronaviruses and other respiratory viruses, such as rhinoviruses. The promising biological activities of linear furanocoumarins reported in the literature, in addition to their quantum electronic properties, led to the in silico study of these compounds. Analysis of chemical reactivity descriptors of 3‐methoxypsoralen (1) and 3,5‐dimethoxypsoralen (2) using DFT and ab initio Hartree–Fock (HF) methods indicates that the compounds are more water‐soluble, which is a favorable characteristic for clinical applications. Vibrational spectroscopy data are also presented. Molecular docking results revealed that 1 and 2 strongly bind to 3CLpro with relative affinities of −5.5 and −5.6 kcal mol−1, respectively. They were also docked against the human rhinovirus HRV3C main protease, with relative affinities of −8.4 and −7.3 kcal mol−1, respectively.

Physiological impact of secondary nanoplastics on aquatic inhabitants in special reference to immunotoxicity.

Nanoplastic (NP) pollution poses serious health hazards to aquatic ecosystems, impacting various physiological systems of aquatic organisms. This review examines the complex interplay between NPs and different physiological systems. In the digestive system, NPs downregulate the hsp70-like gene in Mytilus galloprovincialis, leading to decreased metabolic processes and impaired digestion. Neural system exposure to NPs induces abnormal expression of genes like neurogenin1, GFAP, FBJ murine osteosarcoma viral oncogene, GAP-43, synapsin IIa, apoptosis regulator a, Bcl2 and Caspase a, and apoptosis-related cysteine peptidase. These genes play a crucial role in neurodevelopment, synaptic function and apoptosis regulation, potentially impacting neurobiology and cancer biology. NPs also affect reproduction, including gametogenesis, spawning, fertilization, embryogenesis and larval survivability. In the respiratory system, treatment with these causes inflammation in the lungs and gills, resulting in respiratory dysfunction. Moreover, this review investigates the complex interaction between NPs and the immune systems of both invertebrates (e.g., molluscs, arthropods, echinoderms) and vertebrates (e.g., zebrafish). NPs-induced alterations in immune cell function heightened the susceptibility to pathogens and disrupted immune signalling pathways. Subcellular inflammatory responses have been characterized by the secretion of inflammation-promoting and chemotactic cytokines such as irg1l, interleukin 1, interferon, interleukin 6, C-C motif chemokine ligand 20a and tumour necrosis factor. The assessment of the combined effects of NPs and other xenobiotics highlighted their possible synergistic impacts on aquatic fauna and the environment. This comprehensive review emphasizes the urgent need for further research to understand the cumulative effects of NPs on organism health and fitness across multiple physiological systems.

Pan-cancer and experimental analyses reveal the immunotherapeutic significance of CST2 and its association with stomach adenocarcinoma proliferation and metastasis

PurposeCystatin 2 (CST2) is a cysteine protease inhibitor, and recent research suggests its potential involvement in cancer development. However, its role in the occurrence, progression, and prognosis of pan-cancer has not been systematically investigated.Materials and methodsThis study comprehensively analyzes the differential expression of CST2 in pan-cancer. The expression distribution patterns of CST2 were examined using single-cell datasets. Furthermore, we conducted a comprehensive evaluation of the correlation between CST2 expression and various factors. These factors include prognosis, immune cell infiltration, immune-related genes, mutations, methylation, tumor mutation burden (TMB), and microsatellite instability (MSI). In addition, we analyzed the sensitivity of drugs dependent on CST2 expression. We utilized gene set enrichment analysis (GSEA) analysis to explore the biological functions of CST2 across different cancer types. Finally, in gastric cancer cell lines, we will investigate the impact of CST2 knockout on expression levels, clonal proliferation, cell apoptosis, and cell migration.ResultsCST2 exhibits abnormal overexpression in multiple tumors. Single-cell analysis reveals high expression of CST2 in fibroblasts. CST2 is closely associated with prognosis, immune cell infiltration, immune-related genes, mutations, methylation, TMB, and MSI. Enrichment analysis demonstrated a significant correlation between CST2 and immune-related pathways. In stomach adenocarcinoma (STAD), CST2-related risk models are associated with prognosis and demonstrate strong predictive capabilities, while also being closely linked to the immune microenvironment. Drug sensitivity analysis indicates the correlation between CST2 and 21 chemotherapy drugs. Finally, experimental validation revealed significantly elevated expression of CST2 in STAD, indicating its role as a driver gene in regulating malignant cell proliferation and migration.ConclusionCST2 serves as a potential tumor immune biomarker, playing a critical facilitating role in the proliferation and migration processes of STAD.

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating the interferon-inducible ubiquitin-like modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) that incorporate unnatural amino acids into the C-terminal tail of ISG15, enabling the selective detection of USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) such as USP5 and USP14. Combined with a ubiquitin-based DUB ABP, the USP18 ABP is employed in a chemoproteomics screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

Fungal evasion of Drosophila immunity involves blocking the cathepsin-mediated cleavage maturation of the danger-sensing protease

Entomopathogenic fungi play a critical role in regulating insect populations, and representative species from the Metarhizium and Beauveria genera have been developed as eco-friendly biocontrol agents for managing agricultural insect pests. Relative to the advances in understanding antifungal immune responses in Drosophila, knowledge of how fungi evade insect immune defenses remains limited. In this study, we report the identification and characterization of a virulence-required effector Fkp1 in Metarhizium robertsii. Library screening and protein pull-down analysis unveiled that Fkp1 targets the cathepsin protease CtsK1 to inhibit its cleavage maturation of the danger-sensing serine protease Persephone (Psh), thereby facilitating fungal evasion of the Drosophila immune defenses. The Fkp1-like gene is also required in Beauveria bassiana for insect infection. Transgenic expression of Fkp1 in Drosophila suppressed hemolymph cysteine protease activity and down-regulated the expression of antifungal genes. Fkp1 can also mask the Psh cleavage site without interfering with its ability to bait fungal subtilisin proteases. Given the evident compensatory relationship, our data indicate that the protease cascade is more crucial than the molecular pattern pathway in defending flies against fungal infections. This work reveals that Metarhizium fungi have evolved distinct effectors to block the dual recognition pathways of flies for immune evasion and sheds lights on the effector mechanisms mediating microbe-animal interactions.

Species Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with in Vivo Anti-alphaviral Activity.

RA-0002034 (1) is a potent covalent inhibitor targeting the alphavirus nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S-transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing CHIKV viral loads in multiple tissues. Initial estimations of human hepatic clearance and half-life extrapolated from animal data indicate that b.i.d. dosing of 1 will be possible to maintain concentrations sufficient for antiviral activity in humans. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a promising anti-alphaviral therapeutic.

Dual Inhibitors of SARS-CoV-2 3CL Protease and Human Cathepsin L Containing Glutamine Isosteres Are Anti-CoV-2 Agents.

SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P2 position of a peptidomimetic inhibitor. At the P1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from Ki = 0.6-18 nM (cathepsin L) and Ki = 2.6-124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC50 = 0.47-15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys145, and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

Protein Phosphatase 2A B'α and B'β promote pollen wall construction partially through BZR1-activated CEP1 in Arabidopsis.

A well-constructed pollen wall is essential for pollen fertility, which relies on the contribution of tapetum. Our results demonstrate an essential role of the tapetum-expressed protein phosphatase 2A (PP2A) B'α and B'β in pollen wall formation. The b'aβ double mutant pollen grains harbored sticky remnants and tectum breakages, resulting in failed release. B'α and B'β function partially through dephosphorylating and activating BZR1. The bzr1 bes1 double and higher-order mutants of this family displayed similar defects in pollen wall, while bzr1-1D, having an active mBZR1 exhibited fertile pollen grains in a B'α and B'β dependent manner. Correspondingly, the level of phospho-BZR1 is increased and dephospho-BZR1 is decreased in b'aβ and bzr1-1D/b'aβ at anther stages 8-9 as compared with Col-0 and bzr1-1D, respectively. A cysteine protease gene CEP1 was identified as a BZR1 target, whose transcriptional activation necessitates BRREs in the promoter region and BZR1 DNA binding domain. The mRNA level of CEP1 at stages 8-9 was extremely low in bzr1 and bzr1 bes1, while higher in Col-0 and bzr1-1D depending on B'α and B'β. Furthermore, cep1 mutants displayed similar defects in pollen wall. In brief, this study uncovered a PP2A-BZR1-CEP1 regulatory module, providing a new insight into pollen maturation mechanism.

Various Options for Covalent Immobilization of Cysteine Proteases-Ficin, Papain, Bromelain.

This study explores various methods for the covalent immobilization of cysteine proteases (ficin, papain, and bromelain). Covalent immobilization involves the formation of covalent bonds between the enzyme and a carrier or between enzyme molecules themselves without a carrier using a crosslinking agent. This process enhances the stability of the enzyme and allows for the creation of preparations with specific and controlled properties. The objective of this study is to evaluate the impact of covalent immobilization under different conditions on the proteolytic activity of the enzymes. The most favorable results were achieved by immobilizing ficin and bromelain through covalent bonding to medium and high molecular weight chitosans, using 5 and 3.33% glutaraldehyde solutions, respectively. For papain, 5 and 6.67% glutaraldehyde solutions proved to be more effective as crosslinking agents. These findings indicate that covalent immobilization can enhance the performance of these enzymes as biocatalysts, with potential applications in various biotechnological fields.

Activation and Reactivity of the Deubiquitinylase OTU Cezanne-2 from MD Simulations and QM/MM Calculations.

Cezanne-2 (Cez2) is a deubiquitinylating (DUB) enzyme involved in the regulation of ubiquitin-driven cellular signaling and selectively targets Lys11-linked polyubiquitin chains. As a representative member of the ovarian tumor (OTU) subfamily DUBs, it performs cysteine proteolytic isopeptide bond cleavage; however, its exact catalytic mechanism is not yet resolved. In this work, we used different computational approaches to get molecular insights into the Cezanne-2 catalytic mechanism. Extensive molecular dynamics (MD) simulations were performed for 12 μs to model free Cez2 and the diubiquitin (diUb) substrate-bound protein-protein complex in two different charge states of Cez2, each corresponding to a distinct reactive state in its catalytic cycle. The simulations were analyzed in terms of the relevant structural parameters for productive enzymatic catalysis. Reactive diUb-Cez2 complex configurations were identified, which lead to isopeptide bond cleavage and stabilization of the tetrahedral oxyanion intermediate. The reliability of these complexes was further assessed by quantum mechanics/molecular mechanics (QM/MM) optimizations. The results show that Cez2 follows a modified cysteine protease mechanism involving a catalytic Cys210/His367 dyad, with the oxyanion hole to be a part of the "C-loop," and polarization of His367 by the formation of a strictly conserved water bridge with Glu173. The third residue has a dual role in catalysis as it mediates substrate binding and polarization of the catalytic dyad. A similar mechanism was identified for Cezanne-1, the paralogue of Cez2. In general, our simulations provide valuable molecular information that may help in the rational design of selective inhibitors of Cez2 and closely related enzymes.

Insights into the catalytic mechanism of archaeal peptidoglycan endoisopeptidases from methanogenic phages.

Archaeal peptidoglycan, a crucial component of the cell walls of Methanobacteria and Methanopyri, enhances the tightness of methanogenic cells and their resistance to known lytic enzymes and antibiotics. Although archaeal peptidoglycan endoisopeptidases (Pei) can reportedly degrade archaeal peptidoglycan, their biochemistry is still largely unknown. In this study, we investigated the activity and catalytic properties of the endoisopeptidases PeiW and PeiP using synthesized isopeptides identical to natural substrates. Enzymatic assays demonstrated their distinct substrate specificity and cleavage efficiency. The crystal structure of Pei revealed a catalytic mechanism resembling that of cysteine peptidases that use the 'CHD' triad to cleave isopeptide bonds. We also identified several key residues in the substrate binding site that confer recognition specificity, including Y174, V252 and C265. Based on the residues present in the active site and their influence on activity, we propose a classification of the archaeal peptidoglycan endoisopeptide family into four categories to facilitate the identification of new archaeal peptidases in the future. These insights into the structure and function of Pei suggest new strategies for use in methanogen biotechnology.

Targeting pleuro-alveolar junctions reverses lung fibrosis in mice

Lung fibrosis development utilizes alveolar macrophages, with mechanisms that are incompletely understood. Here, we fate map connective tissue during mouse lung fibrosis and observe disassembly and transfer of connective tissue macromolecules from pleuro-alveolar junctions (PAJs) into deep lung tissue, to activate fibroblasts and fibrosis. Disassembly and transfer of PAJ macromolecules into deep lung tissue occurs by alveolar macrophages, activating cysteine-type proteolysis on pleural mesothelium. The PAJ niche and the disassembly cascade is active in patient lung biopsies, persists in chronic fibrosis models, and wanes down in acute fibrosis models. Pleural-specific viral therapeutic carrying the cysteine protease inhibitor Cystatin A shuts down PAJ disassembly, reverses fibrosis and regenerates chronic fibrotic lungs. Targeting PAJ disassembly by targeting the pleura may provide a unique therapeutic avenue to treat lung fibrotic diseases.

Identification, molecular characterization and expression patterns of Cathepsin L in Yesso scallop (Patinopecten yessoensis) shell-infested by Polydora.

Patinopecten yessoensis (Yesso scallop), one of the most important aquaculture molluscs in China, has recently suffered severe Polydora disease, causing economic losses. Cathepsin L (CatL), a cysteine protease, has important functions in immune responses in vertebrates and invertebrates. However, little is known regarding the structure and function of CatL in scallops. In this study, a CatL gene named PyCatL was first identified in the genome of P. yessoensis. Gene structure analysis of PyCatL revealed it had 8 exons and 7 introns and a full length of 7916 bp. The gene sequence was analysed, and typically conserved functional domains (signal peptide, inhibitor I29 domain, and peptidase C1 domain) and motifs (ERWNIN, GNYD and GCXGG) of CatL were all predicted in PyCatL, confirming the sequence as belonging to a CatL gene. Phylogenetic analysis showed the evolutionary status of CatL was consistent with the species taxonomy. PyCatL was expressed ubiquitously in all the tested tissues in this study, suggesting its involvement in a wide range of physiological processes. After Polydora infestation, PyCatL exhibited significant upregulation in various mantle regions at both mRNA and protein levels, contrasting with a notable decrease in gene expression in hemocytes. Additionally, the enzyme activity of PyCatL showed a significant increase in the mantle of diseased P. yessoensis. The results suggested a role for mantle tissue in response to Polydora infestation by upregulating expression of PyCatL. The study offers novel insights into the function of CatL in innate immunity in scallops.