Dengue viruses(DENV)have spread throughout the world and pose a huge threat to human life.The most widespread serotype is type 2 DENV(DENV 2),which has no specific treatment.NS2B-NS3 protease plays a pivotal role in D...Dengue viruses(DENV)have spread throughout the world and pose a huge threat to human life.The most widespread serotype is type 2 DENV(DENV 2),which has no specific treatment.NS2B-NS3 protease plays a pivotal role in DENV replication because of its function in cleavage of the viral polyprotein;thus,it is considered a promising target for antiviral discovery.In this study,we developed a high-throughput screening system based on the NS2B-NS3 protease to identify candidates from an FDA-approved drug library.Eltrombopag was screened out of 3273 drugs,and demonstrated inhibition on DENV 2 at the micromolar level in vitro,significantly reducing viral loads in the targeted organs of challenged mice following intraperitoneal injection.Further mechanistic analysis showed that eltrombopag allosterically binds to the DENV 2 NS2B-NS3 protease in a reversible,noncompetitive manner,therefore inhibiting DENV 2 at the post-infection stage.In addition,eltrombopag inhibited the NS2B-NS3 proteases of DENV 4 and Zika virus,suggesting its potential as a broadspectrum antiviral agent.This study repurposed eltrombopag as a promising antiviral agent against DENV,providing an alternative for antiviral development against flaviviruses.展开更多
Objective:Small cell lung cancer(SCLC)is commonly recognized as the most fatal lung cancer type.Despite substantial advances in immune checkpoint blockade therapies for treating solid cancers,their benefits are limite...Objective:Small cell lung cancer(SCLC)is commonly recognized as the most fatal lung cancer type.Despite substantial advances in immune checkpoint blockade therapies for treating solid cancers,their benefits are limited to a minority of patients with SCLC.In the present study,novel indicators for predicting the outcomes and molecular targets for SCLC treatment were elucidated.Methods:We conducted bioinformatics analysis to identify the key genes associated with tumor-infiltrating lymphocytes in SCLC.The functional role of the key gene identified in SCLC was determined both in vitro and in vivo.Results:A significant correlation was observed between patient survival and CD56dim natural killer(NK)cell proportion.Furthermore,we noted that the hub gene ubiquitin-specific protease 1(USP1)is closely correlated with both CD56dim NK cells and overall survival in SCLC.Bioinformatics analysis revealed that USP1 is upregulated in SCLC.In addition,gene set enrichment analysis revealed that USP1 overexpression hinders NK cell-mediated immune responses.By co-cultivating NK-92 cells with SCLC cells,we demonstrated that NK cell cytotoxicity against SCLC could be improved either via USP1 knock-down or pharmacological inhibition.Furthermore,using a nude-mice xenograft tumor model,we noted that USP1 inhibition effectively suppressed tumor proliferation and increased the expression of NK cell-associated markers.Conclusions:Our study findings highlight the importance of NK cells in regulating SCLC.USP1 overexpression can inhibit NK cell-mediated immunity;therefore,USP1 may serve not only as a prognostic biomarker but also as a potential molecular target of SCLC therapy.展开更多
Proteases,with their extensive sources and remarkable characteristics such as high catalytic efficiency,substrate specificity,and species diversity,have long attracted widespread attention and are widely applied in va...Proteases,with their extensive sources and remarkable characteristics such as high catalytic efficiency,substrate specificity,and species diversity,have long attracted widespread attention and are widely applied in various fields including food processing,detergent production,pharmaceutical,and environmental protection;these enzymes can hydrolyze proteins into peptides and amino acids,thereby participating in crucial physiological activities like digestion and immune regulation,and playing an indispensable role in maintaining the health and daily life of organisms.Moreover,through artificial synthesis of the required proteases,it is possible to achieve efficient large-scale expression and production,which significantly reduces industrial costs,making them more economically viable in practical applications.This paper provides a comprehensive review of proteases,covering their classification,sources,structure-activity relationships,and industrial applications,and constructs a closed-loop analytical framework based on“basic characteristics,production technology,and practical application”to systematically organize and analyze the relevant information;in particular,it quantitatively compares the advantages and defects of expression systems using Escherichia coli,yeast,and Bacillus subtilis,which not only deepens the understanding of these systems but also provides valuable theoretical support for the rational selection of expression vectors in different scenarios,and introduces their development prospects in food,medicine,environmental protection,and related fields.The insights provided herein offer specific directions for future applied research on artificially engineered proteases.展开更多
The 3CL protease, a highly conserved enzyme in the coronavirus, plays a crucial role in the viral life cycle by facilitating viral replication through precise cleavage of polyproteins. Beyond its proteolytic function,...The 3CL protease, a highly conserved enzyme in the coronavirus, plays a crucial role in the viral life cycle by facilitating viral replication through precise cleavage of polyproteins. Beyond its proteolytic function, the 3CL protease also engages in intricate interactions with host cell proteins involved in critical cellular processes such as transcription, translation, and nuclear-cytoplasmic transport, effectively hijacking cellular machinery to promote viral replication. Additionally, it disrupts innate immune signaling pathways, suppresses interferon activity and cleaves antiviral proteins. Furthermore, it modulates host cell death pathways including pyroptosis and apoptosis, interferes with autophagy and inhibits stress granule formation to maintain viral infection and exacerbate viral pathogenesis. This review highlights the molecular mechanisms by which the 3CL protease orchestrates virus-host interactions, emphasizing its central role in coronavirus pathogenesis and highlighting potential therapeutic targets for future interventions.展开更多
Streptococcus suis serotype 2(SS2)is an emerging zoonotic pathogen that causes meningitis in humans and pigs.This pathogen generates substantial economic losses in the swine industry while posing a significant threat ...Streptococcus suis serotype 2(SS2)is an emerging zoonotic pathogen that causes meningitis in humans and pigs.This pathogen generates substantial economic losses in the swine industry while posing a significant threat to public health security.The mechanisms through which SS2 penetrates the brain and induces meningitis remain incompletely understood.This study examines the role and mechanism of SS2 collagenase-like protease(Clp)in facilitating bacterial passage across the blood-brain barrier(BBB).The research demonstrates that SS2 Clp enhanced virulence and tissue colonization while promoting BBB degradation in mice.The Δclp mutant exhibited reduced ability to traverse human brain microvascular endothelial(hCMEC/D3)cell monolayers compared to wild-type SS2,while the addition of recombinant protein rClp increased permeability.Furthermore,rClp significantly enhanced SS2 adhesion to hCMEC/D3,suppressed the expression of intercellular tight junction proteins ZO-1,Occludin,and Claudin-5 independent of its enzyme activity,and triggered hCMEC/D3 apoptosis through cell receptor ligand apoptosis and mitochondrial apoptosis pathways,partially dependent on its enzyme activity,leading to BBB disruption and enhanced permeability.Additionally,Clp enhanced the infiltration of macrophages(F4/80+),monocytes(F4/80-Ly6C+),and neutrophils(Ly6G+)into the brain following SS2 infection.These findings establish that SS2 Clp is essential for bacterial passage across the BBB,offering a theoretical foundation for improved prevention and treatment strategies for SS2-induced meningitis.展开更多
Soybean meal(SBM)prepared by soybean crushing is the most popular protein source in the poultry and livestock industries(Cai et al.,2015)due to its economic manufacture,high protein content,and good nutritional value....Soybean meal(SBM)prepared by soybean crushing is the most popular protein source in the poultry and livestock industries(Cai et al.,2015)due to its economic manufacture,high protein content,and good nutritional value.Despite these benefits,SBM contains various antigen proteins such as glycinin andβ-conglycinin,which account for approximately 70%of the total proteins of the SBM and reduce digestibility and damage intestinal function(Peng et al.,2018).展开更多
Remodeling plant intracellular nucleotide-binding leucine-rich repeat immune receptors(NLRs)to engineer synthetic disease-resistance genes has emerged as a promising approach to achieving broad-spectrum disease resist...Remodeling plant intracellular nucleotide-binding leucine-rich repeat immune receptors(NLRs)to engineer synthetic disease-resistance genes has emerged as a promising approach to achieving broad-spectrum disease resistance.But strategies for expanding NLR recognition spectra[[1],[2],[3],[4],[5]]are often limited by the rapid evolution of pathogens and pests.In our recent study,we developed an innovative strategy to engineer broad-spectrum,durable and complete disease resistance in plants by remodeling autoactive NLRs into protease-activated switches[6].展开更多
Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious gastrointestinal disease characterized by vomiting, diarrhea, and dehydration, with mortality rates approaching...Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious gastrointestinal disease characterized by vomiting, diarrhea, and dehydration, with mortality rates approaching 100% among suckling piglets. The PEDV 3C-like protease (3CLpro) is essential for viral replication and regarded as a critical target for antiviral inhibitor development. In this study, we aimed to identify small-molecule inhibitors of PEDV by targeting 3CLpro. Virtual screening of 1.6 million compounds from the ChemDiv library identified four potential candidates. Molecular dynamics simulations, specifically analyzing RMSD, RMSF, and Rg, demonstrated increased structural stability of the compound-protease complexes compared to the monomeric enzyme. All compounds had low cytotoxicity in Vero cells (CC_(50) > 200 μM). Fluorescence resonance energy transfer-based assays demonstrated dose-dependent inhibitory activity of the compounds against 3CLpro. Among the candidates, compound F366-0161 exhibited the weakest inhibition, with an IC_(50) value of 151.5 μM. Two analogues, 3238-0395 (IC_(50) of 121.4 μM) and L878-0493 (IC_(50) of 123.6 μM), exhibited moderately enhanced activity. Y041-1672 was identified as the most effective inhibitor, with an IC_(50) of 86.48 μM. In viral replication inhibition assays, Y041-1672 reduced PEDV replication, with an EC_(50) of 17.97 μM and a selectivity index (SI) of 15.5 (CC_(50) /EC_(50) ). These results were validated by RT-qPCR, plaque assays, immunofluorescence, and Western blot analyses. In vitro validation confirmed Y041-1672 as the optimal antiviral candidate, and time-of-addition experiments indicated that inhibition primarily occurred during viral replication. This study identifies scaffold molecules for PEDV antiviral drug development, providing strategic insights for PED treatment.展开更多
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of novel coronavirus disease 2019,can cause acute respiratory symptoms and even death globally.However,the immune escape mechanism and vi...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of novel coronavirus disease 2019,can cause acute respiratory symptoms and even death globally.However,the immune escape mechanism and viral pathogenesis remain poorly understood.Here,we report that the SARS-CoV-23C-like(3CL)protease specifically cleaves gasdermin D(GSDMD)at Q29 and Q193,producing two N-terminal fragments,GSDMD1-29 and GSDMD1-193.We also found that SARS-CoV-2 infection induced the cleavage of GSDMD.Then,we demonstrated that the ability to cleave GSDMD was dependent on the protease activity of the 3CL protease.Interestingly,unlike the GSDMD1-275 fragment cleaved by caspase-1,GSDMD1-29 and GSDMD1-193 did not trigger pyroptosis or inhibit SARS-CoV-2 replication.Additionally,various RNA viral proteases display different preferences for cleaving GSDMD at Q29 and Q193.Our findings reveal a mechanism by which SARS-CoV-2 and other RNA viruses inhibit pyroptosis,highlighting the critical role of the 3CL protease in immune evasion and viral replication.展开更多
Human enterovirus A71(EV-A71)is a major causative agent of hand,foot and mouth disease(HFMD),which poses a significant public health threat,particularly among young children.Mitochondrial antiviral signaling protein(M...Human enterovirus A71(EV-A71)is a major causative agent of hand,foot and mouth disease(HFMD),which poses a significant public health threat,particularly among young children.Mitochondrial antiviral signaling protein(MAVS)and interferon regulatory factor 3(IRF3)are vital proteins for the induction of type I interferons(IFN-I)and downstream interferon-stimulated genes(ISGs)during EVA71 infection.While posttranslational modifications are known to critically influence viral infection processes,the mechanisms by which EV-A71 exploits host deubiquitinases(DUBs)for immune evasion remain poorly understood.In this study,we demonstrated that EV-A71 infection upregulated ubiquitinspecific protease 5(USP5)expression.Knockdown of USP5 not only inhibited EV-A71 replication but also observably increased the production of IFN-I and ISGs.Furthermore,USP5 also regulated the replication of EV-D68 and CVA16 and the production of IFN-I and ISGs.Mechanistically,USP5 physically interacted with MAVS and IRF3 and reduced the K63-linked polyubiquitination of MAVS and IRF3.Conversely,USP5 knockdown increased the K63-linked polyubiquitination of MAVS and IRF3,thereby accelerating the phosphorylation of IRF3 and increasing IFN-I production during EV-A71 infection.Furthermore,pharmacological inhibition of USP5 with the small-molecule inhibitor PR-619 significantly potentiated the antiviral effects of IFN against EV-A71.Collectively,our findings reveal a previously unrecognized role of USP5 in facilitating EV-A71 immune evasion by dampening MAVSand IRF3-mediated antiviral signaling.These insights provide a novel therapeutic avenue for combating EV-A71 infection through targeted modulation of the USP5-IRF3 axis.展开更多
Proteases are essential for homeostasis,and their primary function is proteolytic in extracellular and intracellular compartments.The deregulation of expression,abundance,and activity of proteases has been related to ...Proteases are essential for homeostasis,and their primary function is proteolytic in extracellular and intracellular compartments.The deregulation of expression,abundance,and activity of proteases has been related to several pathologies,including cancer.This deregulation contributes to their pro-tumorigenic activity since they participate in the degradation of extracellular matrix components and adhesion molecules,and the activation of growth factors.However,some proteases,such as ADAM metallopeptidase with thrombospondin type 1 motif 8 and kallikrein-related peptidases 5 and 10,have emerged as tumor suppressors due to their antitumoral actions in specific cancer contexts.In this article,we discuss the antitumoral effects of ADAM metallopeptidase with thrombospondin type 1 motif 8,kallikrein-related peptidases 5 and 10 that have been described to date,suggesting their potential use as novel biomarkers and therapeutic targets in cancer.展开更多
Snake venoms,especially those from the two subfamilies,Crotalinae and Viperinae,contained a lot of serine proteases. They were responsible for the hemorrhage,shock,or disorder of blood coagulation after envenomation. ...Snake venoms,especially those from the two subfamilies,Crotalinae and Viperinae,contained a lot of serine proteases. They were responsible for the hemorrhage,shock,or disorder of blood coagulation after envenomation. They acted,by activating,inactivating,or other converting effects,on almost all the components of hemostatic and fibrinolytic systems. Their sequences were homologous to trypsin-kallikrein serine proteases. Variation of primary sequences out of active center results in the difference of substrate specificities and the further difference of biological and pharmacological activities. Because of their common and unique properties compared to their physiological corresponding factors,snake venom proteases are proved to be an excellent model for the study of protease substrate discriminating mechanism. Furthermore,they have found an important position both in basic research and application of hemostasis and thrombosis in clinic.展开更多
[Objective] The study aimed to investigate the activity and gene expression of caspase-like proteases in tobacco leaves growing under different light qualities. [Method] By covering tobacco plants with white, red, yel...[Objective] The study aimed to investigate the activity and gene expression of caspase-like proteases in tobacco leaves growing under different light qualities. [Method] By covering tobacco plants with white, red, yellow, blue and purple films to obtain different light quality, the changes of chlorophyll content, activity and gene expression of caspase-like proteases in the tobacco leaves were studied. [Results] Compared with treatments of white, red and yellow film, blue and purple films delayed the decrease of chlorophyll content and senescence of tobacco leaves at the late growth stage, and relatively lowered the activity and gene expression of caspase-like proteases during growth, development and senescence periods. [Conclusion] Different light qualities exhibited various effects on the growth, development and senescence of tobacco leaves, possibly by affecting the activity and gene expression of caspase-like proteases to some extent.展开更多
[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed v...[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.展开更多
[Objective] The aim of the study was to provide the basis for researching the pathogenicity mechanism of Rhizoctonia solani.[Method] The extracellular protease was purified after ammonium sulfate precipitation through...[Objective] The aim of the study was to provide the basis for researching the pathogenicity mechanism of Rhizoctonia solani.[Method] The extracellular protease was purified after ammonium sulfate precipitation through DEAE-Sephrase Fast Flow,Phenyl-Sepharose Fast Flow and Sephadex G-75 ch rom atography. [Result] The extracellular protease with molecular weight of 49.5 ku was obtained from fermentation liquid of R. solani. The optimal temperature and pH value for its activity were 6.4 and 30 ℃ respectively. Zn^2＋,Fe^3＋,Cu^2＋had inhibition on enzyme activity,while Mg^2＋,Mn^2＋had no effect on enzyme activity,and Ca^2＋ could activate enzymatic activity in low concentration.[Conclusion] R. solani could secrete extracellular protease,but the relationship between the extracellular protease and the pathogenicity of R. solani required further study.展开更多
[Objective] The aim of this study was to study effects of metal ions on the protease activities in digestive tissues and gland of red-white ornamental carp（Cyprinus carpio L）.[Method] Effects of four kinds of metal ...[Objective] The aim of this study was to study effects of metal ions on the protease activities in digestive tissues and gland of red-white ornamental carp（Cyprinus carpio L）.[Method] Effects of four kinds of metal ions （K＋,Na＋,Mg2＋ and Ca2＋） on protease activities in hepatopancreas,foregut,midgut,hindgut of red-white ornamental carp were studied by enzyme analysis method.[Result] Effects of four kinds of metal ions on protease activities of red-white ornamental carp were different in the range of experimental concentration from 25 mmol/L to 150 mmol/L.K＋ could promote protease activities in hepatopancreas and hindgut at different levels.Especially,K＋ had the promoting effect at low-concentration level,but the inhibitory effect at high-concentration level in midgut and the inhibitory effect in foregut.Na＋ had the promoting effect on protease activities in hepatopancreas,foregut and hindgut at different levels,but the inhibitory effect in midgut.Mg2＋ and Ca2＋ had the inhibitory effect on protease activities in intestinal and hepatopancreas at different levels.[Conclusion] This study provides basic data and theoretical foundation for researches on the digestive physiology of red-white ornamental carp or the development and optimization of compound feed.展开更多
[Objective] This study aimed to investigating properties of alkaline protease produced by strain Ⅰ 13.[Method] Crude enzyme of alkaline protease was obtained from alkaline protease produced by strain I 13,while effec...[Objective] This study aimed to investigating properties of alkaline protease produced by strain Ⅰ 13.[Method] Crude enzyme of alkaline protease was obtained from alkaline protease produced by strain I 13,while effects of temperature and pH value on enzyme activity were also investigated in this study.[Result] The optimal temperature of alkaline protease produced by strain Ⅰ 13 was 40 ℃,while enzyme activity maintains a higher level from 30 to 60 ℃ and over 40% of the largest enzyme activity still maintained within the range from 20 to 30 ℃.The optimal pH value was 10.5,and over 90% of the largest enzyme activity still maintained within the range from 8.0 to 11.0,which had broader pH value spectrum.[Conclusion] This alkaline protease has huge potential to be developed into washing-powder additive.展开更多
Celiac disease(CD)is a globally prevalent autoimmune disorder,affecting 1.4%of the population with 7.5%annual incidence increase.In genetically predisposed individuals,gliadin antigen can trigger an immune response in...Celiac disease(CD)is a globally prevalent autoimmune disorder,affecting 1.4%of the population with 7.5%annual incidence increase.In genetically predisposed individuals,gliadin antigen can trigger an immune response in the small intestine,causing villous atrophy that leads to severe symptoms.Current treatment of CD relies on strict gluten-free diets.Neprosin from Nepenthes species is one of the most promising enzymes for the detoxification of gliadin antigen.However,the potential of neprosin in gluten-safe food production has yet to be explored.In this study,neprosin from Nepenthes rafflesiana(NrNpr1)was recombinantly expressed with chicken lysozyme signal peptide in the baker's yeast Saccharomyces cerevisiae expression host.The culture media of the bioengineered S.cerevisiae can degrade pure gliadin substrate and gliadin in wheat flour.The live yeast culture was demonstrated to degrade gliadin in wheat flour dough.The purified recombinant NrNpr1(rNrNpr1)enzyme was successfully characterized to have an optimal pH of 2.5,optimal temperature of 50℃,thermal tolerance of 0-65℃,and can digest all immunogenic epitopes inα-gliadin 33-mer when tested in vitro.ELISA test proved that rNrNpr1 can detoxify 750 mg/L gliadin to a gluten-free level(<20 mg/L)after 6 h of incubation.This study characterized neprosin from N.rafflesiana and report the bioengineering of S.cerevisiae with recombinant neprosin.We demonstrated that rNrNpr1 has promising potential applications in revolutionary gluten-safe food production,which needs further optimization and formulation in bakery.展开更多
Objectives Dysregulated osteoclast function contributes to skeletal diseases.However,the specific ubiquitination regulators of the osteoclastogenesis repressor MafB,particularly at the post-translational level,remain ...Objectives Dysregulated osteoclast function contributes to skeletal diseases.However,the specific ubiquitination regulators of the osteoclastogenesis repressor MafB,particularly at the post-translational level,remain undefined.This study aims to identify ubiquitin-specific proteases(USPs)that deubiquitinate MafB and enhance its stability.Methods We constructed a MafB-conjugated luciferase and overexpressed 40 individual USPs,measuring changes in luciferase activity.The identified USP was overexpressed in human CD14^(+) peripheral blood mononuclear cells(PBMCs)to evaluate its effect.Osteoclast differentiation was assessed through osteoclast marker Integrin alpha-V(CD51)staining and Western blot analysis.Co-immunoprecipitation(co-IP)was performed to assess the interplay.The influence on MafB ubiquitination and degradation was evaluated via immunoprecipitation and Western blot.Finally,MafB was knocked down in the USP-overexpressing PBMCs to analyze its effect on osteoclast differentiation.Results Overexpression of ubiquitin-specific protease 29(USP29)significantly increased MafB expression by approximately 75%(p<0.0001).Elevated USP29 levels strongly inhibited osteoclastic differentiation in CD14^(+) PBMCs(p<0.0001).USP29 was found to interact with MafB,markedly reducing its ubiquitination and subsequent degradation in PBMCs(p<0.001).Knocking down MafB in USP29-overexpressing PBMCs alleviated the inhibitory effect of USP29 on osteoclastogenesis.Conclusion USP29 acts as a potent stabilizer of MafB,inhibiting osteoclastogenesis in human CD14^(+) PBMCs,at least in part,by enhancing MafB stability.These findings expand our understanding of USP29’s role and the post-translational regulation of MafB.Furthermore,USP29 serves as a vital factor that controls osteoclast differentiation,and its regulatory function is at least partially mediated by deubiquitinating and stabilizing MafB.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.82130101)the Youth Innovation Promotion Association of CAS(Grant No.2021333).
文摘Dengue viruses(DENV)have spread throughout the world and pose a huge threat to human life.The most widespread serotype is type 2 DENV(DENV 2),which has no specific treatment.NS2B-NS3 protease plays a pivotal role in DENV replication because of its function in cleavage of the viral polyprotein;thus,it is considered a promising target for antiviral discovery.In this study,we developed a high-throughput screening system based on the NS2B-NS3 protease to identify candidates from an FDA-approved drug library.Eltrombopag was screened out of 3273 drugs,and demonstrated inhibition on DENV 2 at the micromolar level in vitro,significantly reducing viral loads in the targeted organs of challenged mice following intraperitoneal injection.Further mechanistic analysis showed that eltrombopag allosterically binds to the DENV 2 NS2B-NS3 protease in a reversible,noncompetitive manner,therefore inhibiting DENV 2 at the post-infection stage.In addition,eltrombopag inhibited the NS2B-NS3 proteases of DENV 4 and Zika virus,suggesting its potential as a broadspectrum antiviral agent.This study repurposed eltrombopag as a promising antiviral agent against DENV,providing an alternative for antiviral development against flaviviruses.
基金supported by grants from the Dongguan Science and Technology of Social Development Program(No.20231800940192)the Talent Development Foundation of the First Dongguan Affiliated Hospital of Guangdong Medical University(No.PU2023002).
文摘Objective:Small cell lung cancer(SCLC)is commonly recognized as the most fatal lung cancer type.Despite substantial advances in immune checkpoint blockade therapies for treating solid cancers,their benefits are limited to a minority of patients with SCLC.In the present study,novel indicators for predicting the outcomes and molecular targets for SCLC treatment were elucidated.Methods:We conducted bioinformatics analysis to identify the key genes associated with tumor-infiltrating lymphocytes in SCLC.The functional role of the key gene identified in SCLC was determined both in vitro and in vivo.Results:A significant correlation was observed between patient survival and CD56dim natural killer(NK)cell proportion.Furthermore,we noted that the hub gene ubiquitin-specific protease 1(USP1)is closely correlated with both CD56dim NK cells and overall survival in SCLC.Bioinformatics analysis revealed that USP1 is upregulated in SCLC.In addition,gene set enrichment analysis revealed that USP1 overexpression hinders NK cell-mediated immune responses.By co-cultivating NK-92 cells with SCLC cells,we demonstrated that NK cell cytotoxicity against SCLC could be improved either via USP1 knock-down or pharmacological inhibition.Furthermore,using a nude-mice xenograft tumor model,we noted that USP1 inhibition effectively suppressed tumor proliferation and increased the expression of NK cell-associated markers.Conclusions:Our study findings highlight the importance of NK cells in regulating SCLC.USP1 overexpression can inhibit NK cell-mediated immunity;therefore,USP1 may serve not only as a prognostic biomarker but also as a potential molecular target of SCLC therapy.
文摘Proteases,with their extensive sources and remarkable characteristics such as high catalytic efficiency,substrate specificity,and species diversity,have long attracted widespread attention and are widely applied in various fields including food processing,detergent production,pharmaceutical,and environmental protection;these enzymes can hydrolyze proteins into peptides and amino acids,thereby participating in crucial physiological activities like digestion and immune regulation,and playing an indispensable role in maintaining the health and daily life of organisms.Moreover,through artificial synthesis of the required proteases,it is possible to achieve efficient large-scale expression and production,which significantly reduces industrial costs,making them more economically viable in practical applications.This paper provides a comprehensive review of proteases,covering their classification,sources,structure-activity relationships,and industrial applications,and constructs a closed-loop analytical framework based on“basic characteristics,production technology,and practical application”to systematically organize and analyze the relevant information;in particular,it quantitatively compares the advantages and defects of expression systems using Escherichia coli,yeast,and Bacillus subtilis,which not only deepens the understanding of these systems but also provides valuable theoretical support for the rational selection of expression vectors in different scenarios,and introduces their development prospects in food,medicine,environmental protection,and related fields.The insights provided herein offer specific directions for future applied research on artificially engineered proteases.
基金supported by the National Natural Science Foundation of China(grant no.82370015).
文摘The 3CL protease, a highly conserved enzyme in the coronavirus, plays a crucial role in the viral life cycle by facilitating viral replication through precise cleavage of polyproteins. Beyond its proteolytic function, the 3CL protease also engages in intricate interactions with host cell proteins involved in critical cellular processes such as transcription, translation, and nuclear-cytoplasmic transport, effectively hijacking cellular machinery to promote viral replication. Additionally, it disrupts innate immune signaling pathways, suppresses interferon activity and cleaves antiviral proteins. Furthermore, it modulates host cell death pathways including pyroptosis and apoptosis, interferes with autophagy and inhibits stress granule formation to maintain viral infection and exacerbate viral pathogenesis. This review highlights the molecular mechanisms by which the 3CL protease orchestrates virus-host interactions, emphasizing its central role in coronavirus pathogenesis and highlighting potential therapeutic targets for future interventions.
基金supported by the National Key Research and Development Program of China(2021FYD1800405)the National Natural Science Foundation of China(32072823).
文摘Streptococcus suis serotype 2(SS2)is an emerging zoonotic pathogen that causes meningitis in humans and pigs.This pathogen generates substantial economic losses in the swine industry while posing a significant threat to public health security.The mechanisms through which SS2 penetrates the brain and induces meningitis remain incompletely understood.This study examines the role and mechanism of SS2 collagenase-like protease(Clp)in facilitating bacterial passage across the blood-brain barrier(BBB).The research demonstrates that SS2 Clp enhanced virulence and tissue colonization while promoting BBB degradation in mice.The Δclp mutant exhibited reduced ability to traverse human brain microvascular endothelial(hCMEC/D3)cell monolayers compared to wild-type SS2,while the addition of recombinant protein rClp increased permeability.Furthermore,rClp significantly enhanced SS2 adhesion to hCMEC/D3,suppressed the expression of intercellular tight junction proteins ZO-1,Occludin,and Claudin-5 independent of its enzyme activity,and triggered hCMEC/D3 apoptosis through cell receptor ligand apoptosis and mitochondrial apoptosis pathways,partially dependent on its enzyme activity,leading to BBB disruption and enhanced permeability.Additionally,Clp enhanced the infiltration of macrophages(F4/80+),monocytes(F4/80-Ly6C+),and neutrophils(Ly6G+)into the brain following SS2 infection.These findings establish that SS2 Clp is essential for bacterial passage across the BBB,offering a theoretical foundation for improved prevention and treatment strategies for SS2-induced meningitis.
基金supported by the Ten-thousand Talents Plan of Zhejiang Province(No.2022R52021)the Key Research and Development Program of Zhejiang Province(No.2021C04016)the Pioneer and Leading Goose R&D Program of Zhejiang(No.2022C04020),China.
文摘Soybean meal(SBM)prepared by soybean crushing is the most popular protein source in the poultry and livestock industries(Cai et al.,2015)due to its economic manufacture,high protein content,and good nutritional value.Despite these benefits,SBM contains various antigen proteins such as glycinin andβ-conglycinin,which account for approximately 70%of the total proteins of the SBM and reduce digestibility and damage intestinal function(Peng et al.,2018).
基金supported by the Biological Breeding-National Science and Technology Major Project(2024ZD04077).
文摘Remodeling plant intracellular nucleotide-binding leucine-rich repeat immune receptors(NLRs)to engineer synthetic disease-resistance genes has emerged as a promising approach to achieving broad-spectrum disease resistance.But strategies for expanding NLR recognition spectra[[1],[2],[3],[4],[5]]are often limited by the rapid evolution of pathogens and pests.In our recent study,we developed an innovative strategy to engineer broad-spectrum,durable and complete disease resistance in plants by remodeling autoactive NLRs into protease-activated switches[6].
基金supported by the National Key Research and Development Program of China(2021YFD1800303)the National Natural Science Foundation of China(32473044).
文摘Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious gastrointestinal disease characterized by vomiting, diarrhea, and dehydration, with mortality rates approaching 100% among suckling piglets. The PEDV 3C-like protease (3CLpro) is essential for viral replication and regarded as a critical target for antiviral inhibitor development. In this study, we aimed to identify small-molecule inhibitors of PEDV by targeting 3CLpro. Virtual screening of 1.6 million compounds from the ChemDiv library identified four potential candidates. Molecular dynamics simulations, specifically analyzing RMSD, RMSF, and Rg, demonstrated increased structural stability of the compound-protease complexes compared to the monomeric enzyme. All compounds had low cytotoxicity in Vero cells (CC_(50) > 200 μM). Fluorescence resonance energy transfer-based assays demonstrated dose-dependent inhibitory activity of the compounds against 3CLpro. Among the candidates, compound F366-0161 exhibited the weakest inhibition, with an IC_(50) value of 151.5 μM. Two analogues, 3238-0395 (IC_(50) of 121.4 μM) and L878-0493 (IC_(50) of 123.6 μM), exhibited moderately enhanced activity. Y041-1672 was identified as the most effective inhibitor, with an IC_(50) of 86.48 μM. In viral replication inhibition assays, Y041-1672 reduced PEDV replication, with an EC_(50) of 17.97 μM and a selectivity index (SI) of 15.5 (CC_(50) /EC_(50) ). These results were validated by RT-qPCR, plaque assays, immunofluorescence, and Western blot analyses. In vitro validation confirmed Y041-1672 as the optimal antiviral candidate, and time-of-addition experiments indicated that inhibition primarily occurred during viral replication. This study identifies scaffold molecules for PEDV antiviral drug development, providing strategic insights for PED treatment.
基金supported by the National Natural Science Foundation of China(82370015).
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of novel coronavirus disease 2019,can cause acute respiratory symptoms and even death globally.However,the immune escape mechanism and viral pathogenesis remain poorly understood.Here,we report that the SARS-CoV-23C-like(3CL)protease specifically cleaves gasdermin D(GSDMD)at Q29 and Q193,producing two N-terminal fragments,GSDMD1-29 and GSDMD1-193.We also found that SARS-CoV-2 infection induced the cleavage of GSDMD.Then,we demonstrated that the ability to cleave GSDMD was dependent on the protease activity of the 3CL protease.Interestingly,unlike the GSDMD1-275 fragment cleaved by caspase-1,GSDMD1-29 and GSDMD1-193 did not trigger pyroptosis or inhibit SARS-CoV-2 replication.Additionally,various RNA viral proteases display different preferences for cleaving GSDMD at Q29 and Q193.Our findings reveal a mechanism by which SARS-CoV-2 and other RNA viruses inhibit pyroptosis,highlighting the critical role of the 3CL protease in immune evasion and viral replication.
基金supported by the National Natural Science Foundation of China(32300133 to SZ.and 32100106 to YR)the China Postdoctoral Science Foundation(2023M730965 to SZ.)+3 种基金the Science and Technology Department of Henan Province(232102311103 to SZ.)the Chinese Academy of Sciences(CAS)Youth Innovation Promotion Association(2023351 to YR)the Hubei Province Natural Science Funds(2023AFA008 and 2023AFB582 to YR)the Open project of the State Key Laboratory of Antiviral Drugs,Henan University(FX3020A030002).
文摘Human enterovirus A71(EV-A71)is a major causative agent of hand,foot and mouth disease(HFMD),which poses a significant public health threat,particularly among young children.Mitochondrial antiviral signaling protein(MAVS)and interferon regulatory factor 3(IRF3)are vital proteins for the induction of type I interferons(IFN-I)and downstream interferon-stimulated genes(ISGs)during EVA71 infection.While posttranslational modifications are known to critically influence viral infection processes,the mechanisms by which EV-A71 exploits host deubiquitinases(DUBs)for immune evasion remain poorly understood.In this study,we demonstrated that EV-A71 infection upregulated ubiquitinspecific protease 5(USP5)expression.Knockdown of USP5 not only inhibited EV-A71 replication but also observably increased the production of IFN-I and ISGs.Furthermore,USP5 also regulated the replication of EV-D68 and CVA16 and the production of IFN-I and ISGs.Mechanistically,USP5 physically interacted with MAVS and IRF3 and reduced the K63-linked polyubiquitination of MAVS and IRF3.Conversely,USP5 knockdown increased the K63-linked polyubiquitination of MAVS and IRF3,thereby accelerating the phosphorylation of IRF3 and increasing IFN-I production during EV-A71 infection.Furthermore,pharmacological inhibition of USP5 with the small-molecule inhibitor PR-619 significantly potentiated the antiviral effects of IFN against EV-A71.Collectively,our findings reveal a previously unrecognized role of USP5 in facilitating EV-A71 immune evasion by dampening MAVSand IRF3-mediated antiviral signaling.These insights provide a novel therapeutic avenue for combating EV-A71 infection through targeted modulation of the USP5-IRF3 axis.
基金Supported by Colegio de Ciencia y Tecnologia de la Universidad Autónoma de la Ciudad de México,No.UACM-CCyT-2025-CON-11.
文摘Proteases are essential for homeostasis,and their primary function is proteolytic in extracellular and intracellular compartments.The deregulation of expression,abundance,and activity of proteases has been related to several pathologies,including cancer.This deregulation contributes to their pro-tumorigenic activity since they participate in the degradation of extracellular matrix components and adhesion molecules,and the activation of growth factors.However,some proteases,such as ADAM metallopeptidase with thrombospondin type 1 motif 8 and kallikrein-related peptidases 5 and 10,have emerged as tumor suppressors due to their antitumoral actions in specific cancer contexts.In this article,we discuss the antitumoral effects of ADAM metallopeptidase with thrombospondin type 1 motif 8,kallikrein-related peptidases 5 and 10 that have been described to date,suggesting their potential use as novel biomarkers and therapeutic targets in cancer.
文摘Snake venoms,especially those from the two subfamilies,Crotalinae and Viperinae,contained a lot of serine proteases. They were responsible for the hemorrhage,shock,or disorder of blood coagulation after envenomation. They acted,by activating,inactivating,or other converting effects,on almost all the components of hemostatic and fibrinolytic systems. Their sequences were homologous to trypsin-kallikrein serine proteases. Variation of primary sequences out of active center results in the difference of substrate specificities and the further difference of biological and pharmacological activities. Because of their common and unique properties compared to their physiological corresponding factors,snake venom proteases are proved to be an excellent model for the study of protease substrate discriminating mechanism. Furthermore,they have found an important position both in basic research and application of hemostasis and thrombosis in clinic.
基金Supported by National Natural Science Foundation of China(30460016)Science and Technology Plan from Yunnan Branch Office of China National Tobacco Corporation(2011YN03,2010YN03,07A01)~~
文摘[Objective] The study aimed to investigate the activity and gene expression of caspase-like proteases in tobacco leaves growing under different light qualities. [Method] By covering tobacco plants with white, red, yellow, blue and purple films to obtain different light quality, the changes of chlorophyll content, activity and gene expression of caspase-like proteases in the tobacco leaves were studied. [Results] Compared with treatments of white, red and yellow film, blue and purple films delayed the decrease of chlorophyll content and senescence of tobacco leaves at the late growth stage, and relatively lowered the activity and gene expression of caspase-like proteases during growth, development and senescence periods. [Conclusion] Different light qualities exhibited various effects on the growth, development and senescence of tobacco leaves, possibly by affecting the activity and gene expression of caspase-like proteases to some extent.
文摘[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.
基金Supported by High Academic Youth Backbone Support Projects in Heilongjiang Province(1152G022)Scientific Research Foundation for Doctor in Heilongjiang August First Land Reclamation University~~
文摘[Objective] The aim of the study was to provide the basis for researching the pathogenicity mechanism of Rhizoctonia solani.[Method] The extracellular protease was purified after ammonium sulfate precipitation through DEAE-Sephrase Fast Flow,Phenyl-Sepharose Fast Flow and Sephadex G-75 ch rom atography. [Result] The extracellular protease with molecular weight of 49.5 ku was obtained from fermentation liquid of R. solani. The optimal temperature and pH value for its activity were 6.4 and 30 ℃ respectively. Zn^2＋,Fe^3＋,Cu^2＋had inhibition on enzyme activity,while Mg^2＋,Mn^2＋had no effect on enzyme activity,and Ca^2＋ could activate enzymatic activity in low concentration.[Conclusion] R. solani could secrete extracellular protease,but the relationship between the extracellular protease and the pathogenicity of R. solani required further study.
文摘[Objective] The aim of this study was to study effects of metal ions on the protease activities in digestive tissues and gland of red-white ornamental carp（Cyprinus carpio L）.[Method] Effects of four kinds of metal ions （K＋,Na＋,Mg2＋ and Ca2＋） on protease activities in hepatopancreas,foregut,midgut,hindgut of red-white ornamental carp were studied by enzyme analysis method.[Result] Effects of four kinds of metal ions on protease activities of red-white ornamental carp were different in the range of experimental concentration from 25 mmol/L to 150 mmol/L.K＋ could promote protease activities in hepatopancreas and hindgut at different levels.Especially,K＋ had the promoting effect at low-concentration level,but the inhibitory effect at high-concentration level in midgut and the inhibitory effect in foregut.Na＋ had the promoting effect on protease activities in hepatopancreas,foregut and hindgut at different levels,but the inhibitory effect in midgut.Mg2＋ and Ca2＋ had the inhibitory effect on protease activities in intestinal and hepatopancreas at different levels.[Conclusion] This study provides basic data and theoretical foundation for researches on the digestive physiology of red-white ornamental carp or the development and optimization of compound feed.
基金Supported by National Natural Science Foundation of China(30670067)~~
文摘[Objective] This study aimed to investigating properties of alkaline protease produced by strain Ⅰ 13.[Method] Crude enzyme of alkaline protease was obtained from alkaline protease produced by strain I 13,while effects of temperature and pH value on enzyme activity were also investigated in this study.[Result] The optimal temperature of alkaline protease produced by strain Ⅰ 13 was 40 ℃,while enzyme activity maintains a higher level from 30 to 60 ℃ and over 40% of the largest enzyme activity still maintained within the range from 20 to 30 ℃.The optimal pH value was 10.5,and over 90% of the largest enzyme activity still maintained within the range from 8.0 to 11.0,which had broader pH value spectrum.[Conclusion] This alkaline protease has huge potential to be developed into washing-powder additive.
基金supported by the Malaysian Ministry of Higher Education Fundamental Research Grant Scheme(FRGS/1/2024/STG01/UKM/02/5)Universiti Kebangsaan Malaysia Research University Grant(GUP-2024-092)awarded to Hoe-Han Gohsupported by Universiti Kebangsaan Malaysia postdoctoral research scheme。
文摘Celiac disease(CD)is a globally prevalent autoimmune disorder,affecting 1.4%of the population with 7.5%annual incidence increase.In genetically predisposed individuals,gliadin antigen can trigger an immune response in the small intestine,causing villous atrophy that leads to severe symptoms.Current treatment of CD relies on strict gluten-free diets.Neprosin from Nepenthes species is one of the most promising enzymes for the detoxification of gliadin antigen.However,the potential of neprosin in gluten-safe food production has yet to be explored.In this study,neprosin from Nepenthes rafflesiana(NrNpr1)was recombinantly expressed with chicken lysozyme signal peptide in the baker's yeast Saccharomyces cerevisiae expression host.The culture media of the bioengineered S.cerevisiae can degrade pure gliadin substrate and gliadin in wheat flour.The live yeast culture was demonstrated to degrade gliadin in wheat flour dough.The purified recombinant NrNpr1(rNrNpr1)enzyme was successfully characterized to have an optimal pH of 2.5,optimal temperature of 50℃,thermal tolerance of 0-65℃,and can digest all immunogenic epitopes inα-gliadin 33-mer when tested in vitro.ELISA test proved that rNrNpr1 can detoxify 750 mg/L gliadin to a gluten-free level(<20 mg/L)after 6 h of incubation.This study characterized neprosin from N.rafflesiana and report the bioengineering of S.cerevisiae with recombinant neprosin.We demonstrated that rNrNpr1 has promising potential applications in revolutionary gluten-safe food production,which needs further optimization and formulation in bakery.
文摘Objectives Dysregulated osteoclast function contributes to skeletal diseases.However,the specific ubiquitination regulators of the osteoclastogenesis repressor MafB,particularly at the post-translational level,remain undefined.This study aims to identify ubiquitin-specific proteases(USPs)that deubiquitinate MafB and enhance its stability.Methods We constructed a MafB-conjugated luciferase and overexpressed 40 individual USPs,measuring changes in luciferase activity.The identified USP was overexpressed in human CD14^(+) peripheral blood mononuclear cells(PBMCs)to evaluate its effect.Osteoclast differentiation was assessed through osteoclast marker Integrin alpha-V(CD51)staining and Western blot analysis.Co-immunoprecipitation(co-IP)was performed to assess the interplay.The influence on MafB ubiquitination and degradation was evaluated via immunoprecipitation and Western blot.Finally,MafB was knocked down in the USP-overexpressing PBMCs to analyze its effect on osteoclast differentiation.Results Overexpression of ubiquitin-specific protease 29(USP29)significantly increased MafB expression by approximately 75%(p<0.0001).Elevated USP29 levels strongly inhibited osteoclastic differentiation in CD14^(+) PBMCs(p<0.0001).USP29 was found to interact with MafB,markedly reducing its ubiquitination and subsequent degradation in PBMCs(p<0.001).Knocking down MafB in USP29-overexpressing PBMCs alleviated the inhibitory effect of USP29 on osteoclastogenesis.Conclusion USP29 acts as a potent stabilizer of MafB,inhibiting osteoclastogenesis in human CD14^(+) PBMCs,at least in part,by enhancing MafB stability.These findings expand our understanding of USP29’s role and the post-translational regulation of MafB.Furthermore,USP29 serves as a vital factor that controls osteoclast differentiation,and its regulatory function is at least partially mediated by deubiquitinating and stabilizing MafB.
