A dicistrovirus increases pupal mortality in Spodoptera frugiperda by suppressing protease activity and inhibiting larval diet consumption

Understanding interactions between viruses and their hosts is conducive to enabling better application of viruses as biocontrol agents.Certain viruses carried by parasitic wasps enhance the parasitic efficiency of wasp-larvae by protecting them against the immune system of their Lepidopteran host.However,the relationship between prey pests and viruses found in predatory natural enemies remains unclear.Herein,we report the interaction between Arma chinensis virus-1(AcV-1),originally isolated from a predatory natural enemy,Arma chinensis(Hemiptera:Pentatomidae),and one of its prey species,Spodoptera frugiperda(Lepidoptera:Noctuidae).The results showed that the AcV-1 virus appeared harmful to the novel host S.frugiperda by inhibiting larval diet consumption and increasing pupal mortality.Meanwhile,sequencing data indicated that the virus altered the gene expression profiles of S.frugiperda.KEGG analysis showed that the proteasome and phagosome pathways related to protein degradation and immune response were significantly enriched.Although the expression levels of digestive enzyme genes did not change significantly,the total protease activity of AcV-1 virus-positive individuals was significantly decreased,suggesting that the virus inhibited diet consumption of S.frugiperda via the down-regulation of digestive enzyme activities.These results indicate that a virus initially isolated in a predatory natural enemy can decrease the fitness of its prey species.The virus was found to impact the host proteasome and phagosome pathways related to protein degradation and immunity,providing a potential mechanism to enhance controlling efficiency.

Extraction of Natural Nanostructured Hydroxyapatite from Pacific Cod(Gadus macrocephalus)Bone with a Thermostable Collagenolytic Protease and Its ex vivo Intestinal Bioavailability

Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃and pH 7.0,and a desir-able extraction efficiency was achieved by using the crude collagenolytic protease.Dynamic light scattering,transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical(about 110nm)particles mainly com-prised of calcium and phosphorus at an approximate ratio of 5:3.As evaluated with the mouse ex vivo intestinal segments,the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl_(2).By treating with inhibitors(NaN3,ami-loride)and low temperature(4℃),clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA.Over-all,the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.

Effect of Metal Ions on Protease Activities in the Intestines and Hepatopancreas of Red-white Ornamental Carp (Cyprinus carpio L)

[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.

Effects of temperature, pH and NaCl on protease activity in digestive tract of young turbot, Scophthalmus maximus

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish’s internal organs. The optimal activity in the fish’s stomach was at pH of 2.2, while that in the intes- tinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40℃ in stomach, intestine and hepato- pancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an op- posite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.

Comparison in effect of different metal ions, pH and reducing agent on the protease activity in human hyper mature and mature cataract

This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at -20 ℃. The total protein extract was isolated from 5 samples in each case （mature and hyper mature cataract） and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 ℃. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. β mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates.

Protease activated receptor 2 and epidermal growth factor receptor are involved in the regulation of human sperm motility

Aim： To investigate mechanisms of tryptase-induced reduction of sperm motility and explore whether epidermal growth factor receptor （EGF-R） and protease activated receptor 2 （PAR-2）- associated pathways are involved. Methods： Fresh semen was collected from healthy donors （n = 15）. Semen parameters and quality were assessed in accordance with the World Health Organization （WHO） criteria. Swim-up sperm were fixed and subjected to immunocytochemistry and immunoelectronmicroscopy with specific antibodies directed against PAR-2 and EGF-R. Protein extractions from swim-up spermatozoa were analyzed by Western blotting with antibodies for both receptors. Motility of spermatozoa was evaluated by computer-assisted semen analysis. Results： Immunocytochemistry found PAR-2 and EGF-R in approximately 30% of examined human ejaculated spermatozoa. Both receptors were localized in the plasma membrane. Like tryptase, the PAR-2 synthetic agonist SLIGKV reduced sperm motility, and this effect was inhibited by application of two specific EGF-R pathway blockers （AG1478 and PD168393）. Conclusion： The observed reduction of sperm motility by tryptase through the PAR-2 receptor involves EGF-R pathways.

Hippocampal expression of apoptotic protease activating factor-1 following diffuse axonal injury under mild hypothermia

The influence of mild hypothermia on neural cell apoptosis remains poorly understood. Therefore, the present study established rat models of diffuse axonal injury （DAI） at 33℃. Morris water maze results demonstrated significantly better learning and memory functions in DAI rats with hypothermia compared with DAI rats with normothermia. Expression of apoptotic protease activating factor-1 in the hippocampal CA1 region was significantly lower in the DAI hypothermia group compared with the DAI normothermia group. Expression of apoptotic protease activating factor-1 positively correlated with latency, but negatively correlated with platform location times and time of swimming in the quadrant area. Results suggested that post-traumatic mild hypothermia in a rat model of DAI could provide cerebral protection by attenuating expression of apoptotic protease activating factor-1.

Role of Protease Activated Receptor-2 Expression in Renal Interstitial Fibrosis Model in Mice

Summary： The role of protease activated receptor-2 （PAR-2） in the renal tubulointerstitial lesion induced by unilateral ureteral obstruction （UUO） was explored. Mice were sacrificed on the day 1, 3, 5, 7, 10, 14 and 21 after UUO. The expression of PAR-2 mRNA and protein and a-smooth muscle actin （α-SMA） protein in tubuloin,terstitium was detected by RT-PCR and immunohistochemistry at each time point, respedtively. The results showed that the PAR-2 expression in renal tubulointerstitium was increased progressively starting from 24 h to the day 14 post-ligation, and it was significantly associated with the relative volume of interstitium and the positive area of α-SMA. PAR-2 was mainly expressed in renal tubule epithelial cells, especially in proximal tubular cells. It also located in renal capillary ansa, interstitial infiltrate cells and fibroblasts. It was concluded that PAR-2 was active in interstitial and tubular cells in the early phase of fibrotic process and played an important role in mediating the tubulointerstitial lesion after UUO.

Feed nutritional value of brewers’ spent grain residue resulting from protease aided protein removal

Background:This study was conducted to evaluate the feed nutritional value of brewers’spent grain(BSG)residue resulting from protease aided protein removal.The nutritional value was measured as nutrient content,gas production,nutrient digestibility and fermentation characteristics in batch culture.Results:Protein extraction process decreased content of crude protein but concentrated the neutral detergent fibre(NDF)and ferulic acid in BSG residue.The changes in the chemical composition of BSG residue varied with enzyme and enzyme dosage.Digestibility of dry matter(DMD)and NDF of residue differed among proteases.Increasing alcalase dosage linearly decreased DMD,whereas,the DMD linearly increased as everlase or flavourzyme dosage increased.Compared with BSG,the DMD,gas production and fermentation acid concentration of BSG residues were lower,whereas NDF digestibility was higher.Conclusions:The substantially increased NDF content and improved in vitro NDF digestibility due to protease hydrolysis suggest that BSG residue can be potentially exploited as a viable fibre source for ruminant feeding.

The Flavivirus Protease As a Target for Drug Discovery

Many flaviviruses are significant human pathogens causing considerable disease burdens,including encephalitis and hemorrhagic fever,in the regions in which they are endemic.A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication,such as the viral protease.During viral replication,the flavivirus genome is translated as a single polyprotein precursor,which must be cleaved into individual proteins by a complex of the viral protease,NS3,and its cofactor,NS2B.Because this cleavage is an obligate step of the viral life-cycle,the flavivirus protease is an attractive target for antiviral drug development.In this review,we will survey recent drug development studies targeting the NS3 active site,as well as studies targeting an NS2B/NS3interaction site determined from flavivirus protease crystal structures.

Hordatines as a Potential Inhibitor of COVID-19 Main Protease and RNA Polymerase: An In-Silico Approach

Total 40 natural compounds were selected to perform the molecular docking studies to screen and identify the potent antiviral agents specifically for Severe Acute Respiratory Syndrome Coronavirus 2 that causes coronavirus disease 2019(COVID-19).The key targets of COVID-19,protease(PDB ID:7BQY)and RNA polymerase(PDB ID:7bV2)were used to dock our target compounds by Molecular Operating Environment(MOE)version 2014.09.We used 3 different conformations of protease target(6M0K,6Y2F and 7BQY)and two different score functions to strengthen the probability of inhibitors discovery.After an extensive screening analysis,20 compounds exhibit good binding affinities to one or both COVID-19 targets.7 out of 20 compounds were predicted to overcome the activity of both targets.The top 7 hits are,flacourticin(3),sagerinic acid(16),hordatine A(23),hordatine B(24),N-feruloyl tyramine dimer(25),bisavenanthramides B-5(29)and vulnibactins(40).According to our results,all these top hits was found to have a better binding scores than remdesivir,the native ligand in RNA polymerase target(PDB ID:7bV2).Hordatines are phenolic compounds present in barley,were found to exhibit the highest binding affinity to both protease and polymerase through forming strong hydrogen bonds with the catalytic residues,as well as significant interactions with other receptor-binding residues.These results probably provided an excellent lead candidate for the development of therapeutic drugs against COVID-19.Eventually,animal experiment and accurate clinical trials are needed to confirm the preventive potentials of these compounds.

Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation

Objective： To construct the point mutation plasmids expressing HCV NS3/4A with different secondary structures at the N-terminus, and to analyze their serine protease activities. Methods： The point mutation plasmid constructs were generated by using the QuickChange site-directed mutagenesis kit with the backbone of M-H05-5 （AI-1）, and were named as subgroup A1-2, A2-1, A2-2, BI-1, B1-2, B2-1, and B2-2 respectively. The transient expression of the constructs was investigated by immunofluorescence assay and Western blot analysis. The difference in in cis and in trans NS3 serine protease activity between each subgroup was determined by Western blot analysis. Luciferase reporter assay was used to observe the inhibitory effects of the constructs on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation. Results： The point mutation plasmid constructs were verified for the correct sequence by DNA sequencing. The immunofluorescence assay revealed 4 subcellular localization patterns of NS3, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blot analysis indicated that the incomplete cleavage of NS3/4A appeared in subgroups A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker when compared with the other subgroups. By using NS5A/SBAC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared the other subgroups. Differences in inhibitory effects of HCV NS3 on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion： The results suggest that subgroup A2-1 and B2-1 has weaker serine protease activities and weaker inhibitory activities on host cell functions than the other subgroups, which might be explained by the different secondary structure of the 120-aa sequence at N-terminus of NS3.

Intracellular protease activation in apoptosis and cell- mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clini- cal efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

Covariation of mutation pairs expressed in HIV-1 protease and reverse transcriptase genes subjected to varying treatments

A previous study, focused on the correlation of muta-tion pairs of synonymous (S) and asynonymous (A) mutations, distinguished only between the treated and untreated data of protease and reverse tran-scriptase (RT) of HIV-1 subtype B. It is well known that single mutation patterns in HIV-1 are treat-ment-specific. It logically follows that covariation between mutations will also be treatment specific. Thus, our motivation is to give a more in depth study of the covariation between mutation pairs, analyzing not only treated and untreated, but what specific treatments were used, and how they affected the co-variation between the mutations differently. We in-tended to further deepen this study by analyzing the covariation of mutations in protease and RT in dif-ferent subtypes of HIV-1. We found that virus sam-ples subjected to antiretroviral Protease- and RT- inhibitors do show different patterns of mutation covariation in B-subtype protease and RT of HIV-1, while maintaining the same overall trend. covariation will tend to be higher and more distinct from and covariation after treatment. The same trend continues in protease and RT re-gardless of subtype. We also found the highly cova-ried codon positions, position pairs, and position- covariation clusters in protease, affected by different treatments. Most of them are well known major drug-resistance sites for these treatments.

Correlation of carotid plaque vulnerability with lipid metabolism, inflammatory response and protease activity in patients with coronary artery disease

Objective: To study the correlation of carotid plaque vulnerability with lipid metabolism, inflammatory response and protease activity in patients with coronary artery disease. Methods: Patients who were diagnosed with coronary heart disease combined with carotid atherosclerosis in People's Hospital of Dongxihu District Wuhan City between April 2015 and October 2017 were selected and divided into vulnerable group and stable group according to ultrasonic judgment of carotid plaque vulnerability;the healthy volunteers who underwent physical examination during the same period were selected as the control group. The serum was collected to determine the contents of lipid metabolism, inflammatory response and protease activity indexes, and the peripheral blood was collected to determine the expression of inflammatory response indexes. Results: LDL-C, Lp(a), CXCL5, E-selectin, CatK and Meprin- levels in serum as well as ERK1/2, NF-κB and TNF-α expression in peripheral blood of stable group and vulnerable group were significantly higher than those of control group whereas ATGL, Omentin-1, Vaspin, PAI-1, TIMP1 and TIMP2 levels were significantly lower than those of control group;LDL-C, Lp(a), CXCL5, E-selectin, CatK and Meprin-levels in serum as well as ERK1/2, NF-κB and TNF-α expression in peripheral blood of vulnerable group were significantly higher than those of stable group whereas ATGL, Omentin-1, Vaspin, PAI-1, TIMP1 and TIMP2 levels were significantly lower than those of stable group. Conclusion: The changes of carotid plaque vulnerability in patients with coronary artery disease are closely related to the changes in lipid metabolism, inflammatory response and protease activity in the course of disease.

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics

A titrant for the SARS-CoV-2 main protease(M^(pro))was developed that enables,for the first time,the exact determination of the concentration of the enzymatically active M^(pro) by active-site titration.The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme–titrant complex.Four fluorogenic substrates of M^(pro),including a prototypical,internally quenched Dabcyl-EDANS peptide,were compared in terms of solubility under typical assay conditions.By exploiting the new titrant,key kinetic parameters for the M^(pro)-catalyzed cleavage of these substrates were determined.

Ubiquitin-specific protease 21 promotes tumorigenicity and stemness of colorectal cancer by deubiquitinating and stabilizing ZEB1

BACKGROUND Colorectal cancer(CRC)is one very usual tumor together with higher death rate.Ubiquitin-specific protease 21(USP21)has been confirmed to take part into the regulation of CRC progression through serving as a facilitator.Interestingly,the promotive function of USP21 has also discovered in the progression of CRC.ZEB1 has illustrated to be modulated by USP7,USP22 and USP51 in cancers.However,the regulatory functions of USP21 on ZEB1 in CRC progression need more invest-igations.AIM To investigate the relationship between USP21 and ZEB1 in CRC progression.METHODS The mRNA and protein expressions were assessed through RT-qPCR,western blot and IHC assay.The interaction between USP21 and ZEB1 was evaluated through Co-IP and GST pull down assays.The cell proliferation was detected through colony formation assay.The cell migration and invasion abilities were determined through Transwell assay.The stemness was tested through sphere formation assay.The tumor growth was evaluated through in vivo mice assay.RESULTS In this work,USP21 and ZEB1 exhibited higher expression in CRC,and resulted into poor prognosis.Moreover,the interaction between USP21 and ZEB1 was further investigated.It was demonstrated that USP21 contributed to the stability of ZEB1 through modulating ubiquitination level.In addition,USP21 streng-thened cell proliferation,migration and stemness through regulating ZEB1.At last,through in vivo assays,it was illustrated that USP21/ZEB1 axis aggravated tumor growth.CONCLUSION For the first time,these above findings manifested that USP21 promoted tumorigenicity and stemness of CRC by deubiquitinating and stabilizing ZEB1.This discovery suggested that USP21/ZEB1 axis may provide novel sights for the treatment of CRC.

Elucidation of potential relationship between endogenous proteases and key flavor substances in dry-cured pork coppa

Dry-cured meat products are considerably popular around the world due to unique flavor.Proteolysis is one of the enzymatic reactions from which flavor substances are derived,which is affected by endogenous proteases.The purpose aimed to reveal the potential relationship between endogenous proteases and key flavor substances in dry-cured pork coppa in this paper.The dynamic changes of endogenous proteases activity,free amino acids,and volatiles during dry-cured pork coppa processing were characterized.The results showed that 5 kinds of free amino acids,Glu,Lys,Val,Ala,and Leu,were identified as significant contributors to taste.Meanwhile,key volatiles,such as hexanal,nonanal,octanal,benzaldehyde,3-methyl butanoic acid,2-methyl propanoic acid,and ethyl octanoate,greatly contributed to the flavor characteristics of dry-cured pork coppa.Further partial correlation analysis was performed to better elucidate the relationship among parameters.The results revealed that close relationship between endogenous proteases and key substances.RAP not only significantly affected the accumulation of key active-amino acids,but also affected the accumulation of ethyl octanoate,2,3-pentanedione,and 2,3-octanedione by regulating the accumulation of octanoic acid and Leu.In addition,cathepsin B and D,DPP II,DPP IV and RAP notably affected accumulation of hexanal.

Roles of host proteases in the entry of SARS-CoV-2

The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.

Transmembrane serine protease 4 expression in the prognosis of radical resection for biliary tract cancer

BACKGROUND Recent advancements in biliary tract cancer(BTC)treatment have expanded beyond surgery to include adjuvant therapy,yet the prognosis remains poor.Identifying prognostic biomarkers could enhance the assessment of patients who have undergone radical resection for BTC.AIM To determine transmembrane serine protease 4(TMPRSS4)utility as a prognostic biomarker of radical resection for BTC.METHODS Medical records of patients who underwent radical resection for BTC,excluding intrahepatic cholangiocarcinoma,were retrospectively reviewed.The associations between TMPRSS4 expression and clinicopathological factors,overall survival,and recurrence-free survival were analyzed.RESULTS Among the 85 patients undergoing radical resection for BTC,46(54%)were TMPRSS4-positive.The TMPRSS4-positive group exhibited significantly higher preoperative carbohydrate antigen 19-9(CA19-9)values and greater lymphatic invasion than the TMPRSS4-negative group(P=0.019 and 0.039,respectively).Postoperative overall survival and recurrence-free survival were significantly worse in the TMPRSS4-positive group(median survival time:25.3 months vs not reached,P<0.001;median survival time:28.7 months vs not reached,P=0.043,respectively).Multivariate overall survival analysis indicated TMPRSS4 positivity,pT3/T4,and resection status R1 were independently associated with poor prognosis(P=0.032,0.035 and 0.030,respectively).TMPRSS4 positivity correlated with preoperative CA19-9 values≥37 U/mL and pathological tumor size≥30 mm(P=0.016 and 0.038,respectively).CONCLUSION TMPRSS4 is a potential prognostic biomarker of radical resection for BTC.