Evaluation of cytotoxic and anti-tumor activity of partially purified serine protease isolate from the Indian earthworm Pheretima posthuma

Objective:To isolate,partially purify and evaluate the cytotoxic and antitumor activity of a serine protease from the chosen Indian earthworm Pheretima posthuma.Methods:Whole animal extract was prepared and purified its protein constituents by size and charge based chromatographic separation techniques using Sephadex G-50 and DEAE-Cellulose resin respectively.Average molecular weight of the protein isolate was determined and analyzed for its cytotoxic property against Vero cells in different dilutions(1:20 and 1:40)and anti-tumor activity by MTT assay(a colorimetric assay)using breast cancer cell line MCF-7,with tamoxifen as standard.Results:One of the protein constituents after purification was characterized as serine protease by Caseinolytic plate diffusion assay.Average molecular weight of this purified isolate was determined,by SDS-PAGE analysis with standard protein ladder,as of 15 kDa.The performed tests suggested that the 15kDa fraction has potent cytotoxic activity and satisfactory antitumor activity as well in vitro.Conclusions:Exact molecular mechanism of the cytotoxic and antitumor activities is yet to be explored and currently we are working on ultra-purification and biophysical characterization of this fraction.Further investigation into the mechanism(s)of cytotoxic and antitumor activities at molecular level would be useful in treatment of various classes of cancer and viral infections in future.

Cloning,Expression and Activity Analysis of a Bacterial Serine Protease

In the study, a serine protease gene from Pantoea ananatis was cloned and expressed in prokaryotic cells. The activity of recombinant serine protease was analyzed. The results showed that the recombinant serine protease gene was 1 062 bp, encoding 352 amino acids ; the optimal reaction temperature for recombinant serine protease was 50 ℃, and the optimal pH was 5. 0. The serine protease could be developed into a new tool enzyme in biological engineering and food processing.

Cloning, Expression and Activity Analysis of a Novel Fibrinolytic Serine Protease from Arenicola cristata

The full-length c DNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of c DNA ends technique and sequenced. The size of the c DNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity(< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

Characteristics of Mycobacterium tuberculosis serine protease Rv1043c in enzymology and pathogenicity in mice

The serine proteases of Mycobacteria tuberculosis(Mtb)are important contributors to the process of bacterial invasion and its pathogenesis.In the present study,we systematically characterized the role of the Rv1043c protein in Mycobacterium infection by purifying the Rv1043c protein in Escherichia coli and constructing a Mycobacterium smegmatis(Msg)strain overexpressing Rv1043c(Msg_Rv1043c).We found that Rv1043c had serine protease activity and localized to the surface of Mtb.We determined that the optimal pH and temperature for the Rv1043c serine protease were 9.0 and 45°C,respectively.Moreover,the serine protease activity of Rv1043c was enhanced by divalent metal ions of Ca^(2+)and Mg^(2+).Site-directed mutagenesis studies demonstrated that the serine 279 residue in Rv1043c plays a catalytic role.Additionally,mouse model studies confirmed that Rv1043c significantly enhanced the survival of Msg in vivo,induced pulmonary injury and lung cell apoptosis,and promoted the release of pro-inflammatory cytokines interleukin-1βand interleukin-6 in mice.This study presents novel insights into the relationship between mycobacterial serine protease and the pathogenesis of the disease.

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.

Treatment of Scrapie Pathogen 263K With Tetracycline Partially Abolishes Protease-resistant Activity in vitro and Reduces Infectivity in vivo

Objective To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K. Methods Scrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrP^5c in brain tissues of the infected animals was detected by PrP specific Western blot assays. Results Protease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75±0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5±1.73 and 59.5±0.58 days （P〈0.05）. Conclusion Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.

Transmembrane serine protease 2 and angiotensin-converting enzyme 2 anti-inflammatory receptors for COVID-19/inflammatory bowel diseases treatment

long-term,and relapsing inflammatory disorders.IBD may spontaneously grow in the colon,and in severe cases may result in tumor lesions such as invasive carcinoma in inflamed regions of the intestine.Recent epidemiological reports indicate that old age and underlying diseases such as IBD contribute to severity and mortality in patients with coronavirus disease 2019(COVID-19).Currently,the ongoing COVID-19 pandemic caused serious morbidity and mortality worldwide.It has also been shown that the transmembrane serine protease 2 is an essential factor for viral activation and viral engulfment.Generally,viral entry causes a'cytokine storm'that induces excessive generation of proinflammatory cytokines/chemokines including interleukin(IL)-6,IL-2,IL-7,tumor necrosis factor-α,and interferon-γ.Future research could concentrate on developing inflammatory immunological responses that are efficient to encounter COVID-19.Current analysis elucidates the role of inflammation and immune responses during IBD infection with COVID-19 and provides a list of possible targets for IBD-regulated therapies in particular.Data from clinical,in vitro,and in vivo studies were collected in English from PubMed,Google Scholar,Scopus,and the Cochrane library until May 2021.

Exploring QSARs for Inhibitory Activity of Cyclic Urea and Nonpeptide-Cyclic Cyanoguanidine Derivatives HIV-1 Protease Inhibitors by Artificial Neural Network

Quantitative structure–activity relationship study using artificial neural network (ANN) methodology were conducted to predict the inhibition constants of 127 symmetrical and unsymmetrical cyclic urea and cyclic cyanoguanidine derivatives containing different substituent groups such as: benzyl, isopropyl, 4-hydroxybenzyl, ketone, oxime, pyrazole, imidazole, triazole and having anti-HIV-1 protease activities. The results obtained by artificial neural network give advanced regression models with good prediction ability. The two optimal artificial neural network models obtained have coefficients of determination of 0.746 and 0.756. The lowest prediction’s root mean square error obtained is 0.607. Artificial neural networks provide improved models for heterogeneous data sets without splitting them into families. Both the external and cross-validation methods are used to validate the performances of the resulting models. Randomization test is employed to check the suitability of the models.

Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population:Role of serine protease inhibitor Kazal 1type and alcohol

AIM: To test the hypothesis that calcium sensing receptor (CASR) polymorphisms are associated with chronic pancreatitis (CP), and to determine whether serine protease inhibitor Kazal 1type (SPINK1) N34S oralcohol are necessary co-factors in its etiology. METHODS: Initially, 115 subjects with pancreatitis and 66 controls were evaluated, of whom 57 patients and 21 controls were predetermined to carry the high-risk SPINK1 N34S polymorphism. We sequenced CASR gene exons 2, 3, 4, 5 and 7, areas containing the majority of reported polymorphisms and novel mutations. Based on the initial results, we added 223 patients and 239 controls to analyze three common nonsynonymous single nucleotide polymorphisms (SNPs) in exon 7 (A986S, R990G, and Q1011E). RESULTS: The CASR exon 7 R990G polymorphism was signifi cantly associated with CP (OR, 2.01; 95% CI, 1.12-3.59; P = 0.015). The association between CASR R990G and CP was stronger in subjects who reported moderate or heavy alcohol consumption (OR, 3.12; 95% CI, 1.14-9.13; P = 0.018). There was no association between the various CASR genotypes and SPINK1 N34S in pancreatitis. None of the novel CASR polymorphisms reported from Germany and India was detected. CONCLUSION: Our United States-based study confirmed an association of CASR and CP and for the first time demonstrated that CASR R990G is a signifi cant risk factor for CP. We also conclude that the risk of CP with CASR R990G is increased in subjects with moderate to heavy alcohol consumption.

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.

Crystal Structures of the Serine Protease Domain of Murine Plasma Kallikrein

Plasma kallikrein（PK）, a serine protease in the trypsin clan（SA）, plays critical roles in many physiological and pathological pathways. Regulating the abnormal activity of PK has been successfully used in the clinical therapy of hereditary angioedema. In this study, the serine protease domain of murine plasma kallikrein（m PK） was expressed in the pichia pastoris system. The recombinant protein was a glycosylated active enzyme after purification by the cation exchange and size-exclusion chromatography, and was crystallized at the precipitant condition of 25% PEG 3350, 0.1 M Tris-HCl pH 8.5 and 0.1 M Na Cl. The crystal structure of m PK was determined at 2.6 . This is the first published crystal structure of m PK, showing some distinctive features at S2＇ and S3＇ pockets when compared to its human analogue（human plasma kallikrein, h PK）. In addition, m PK show unique structural features in the non-conservative 67-72 and 76-81 loops when compared to other serine proteases. These results provide insights for the design of potent and selective PK inhibitors.

Effects of Dietary Soybean Stachyose and Phytic Acid on Gene Expressions of Serine Proteases in Japanese Flounder(Paralichthys olivaceus)

Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively.Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY (P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL (P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes'expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

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°C. 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 °C. 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.

Preparation and analysis of immunocompetence of recombinant fusion protein of the immunodominant region in chlamydial protease-like activity factor from Chlamydophila pneumoniae and its application in serodiagnosis

To clone the gene coding the immunodominant region in the chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae,to analyze immunocompetence of the expressed protein, and to evaluate its value in serodiagnosis,the CPAF immunodominant region gene was amplified,ligated into a pGEX6p-2 vector,and then the expressed recombinant protein was purified with glutathione S- transferase(GST)agarose gel FF after renaturation,then identified by SDS-PAGE and Western blot.A new indirect ELISA was developed with the purified protein as coating antigen.The immunogenicity of the recombinant protein was evaluated by immunization to New Zealand rabbits,and its immunoreactivity was analyzed by reacting with anti-C,pneumoniae antibody.300 clinical sera samples were respectively de- tected by microimmunofluorescence(MIF)as reference method and the indirect ELISA,and the differ- ence between the two methods was analyzed.Cross-reactivity against Chlamydia trachomatis was investi- gated with the indirect ELISA to detect anti-C,trachomatis positive antisera.The results indicated that a 51.3 kDa recombinant protein was obtained.Western blot assay proved that the recombinant protein could merely specifically react with human anti-C.pneumoniae antisera.The titers of the specific IgG an- tibodies in the immunized New Zealand rabbits were above 1:16 000.Anti-C.pneumoniae IgG positive and negative reference sere were detected with the indirect ELISA,and the concordance rate of negative and positive results were both 100%(40/40).The sensitivity and specificity of the indirect ELISA in comparison with MIF were 93.8%(45/48)and 100%(252/252)separately by detecting 300 clinical sera samples,and the concordance rate between the two methods was 99.0%.No cross reaction against C.trachomatis was found with the indirect ELISA to detect anti-C,trachomatis positive antisera.In con- clusion,the prepared recombinant protein of the CPAF immunodominant region shows excellent immuno- competence and can be used to develop a new indirect ELISA as a method to detect anti-C.pneumoniae antibody for diagnosis of C.pneumoniae infection.

Nerve growth factor activity of several immunal related serine proteases

Rabbit was immuned by the previously purified protein with high nerve growth factor (NGF) bioactivity (NGF_like protease) from \%Agkistrodon halys Pallas\% and the antisera were collected. The polyclonal antibodies were tentatively purified and then used as ligands of an affinity column. The \%A.h.Pallas\% crude venom was fractionated by this affinity column and then by Mono Q on fast protein liquid chromatography (FPLC). As a result, fraction Ⅱ and fraction Ⅲ were purified respectively, whose N_terminal amino acid sequences show high homology with the serine proteases in snake venoms, as well as the previous NGF_like protease. However, they possessed different levels of NGF bioactivity. The NGF activity of the previous NGF_like protease is equivalent to that of NGF, while the activity of fraction Ⅱ seems relatively low in contrast to fraction Ⅲ which had no NGF activity.

<i>In Vitro</i>Anti-HIV Activity of Partially Purified Coumarin(s) Isolated from Fungal Endophyte, <i>Alternaria</i>Species of <i>Calophyllum inophyllum</i>

5 totally different endophytic fungal species were isolated from bark and leaf parts of Calophyllum inophyllum. Leaf part yielded Trichoderma harzianum and Alternaria species, whereas bark showed the presence of Fusarium species, Aspergillus species and unidentified fungi. Two solvents (hexane and methanol) were used for endophytic fungal extraction and the Alternaria species had shown the presence of coumarin whereas Trichoderma harzianum in methanol extract and Fusarium species in hexane extract had shown the coumarin(s) in all the four methods tested. The total coumarin yield was more in microwave assistance method, the methanol Alternaria species (3.941 ± 0.082) stood first, followed by hexane extract of Alternaria species (3.254 ± 0.082), Fusarium species (2.532 ± 0.082) and Trichoderma harzianum (2.294 ± 0.082), the plant extract showed 4.149 + 0.053. The methanol extract of Alternaria species inhibited the activity of HIV-Reverse Transcriptase (RT) (82.81 ± 1.0), integrase (98%) and protease (78) in maximum level followed by hexane extract of Alternaria species (71.12 ± 0.9, 89, 68), Fusarium species (63.92 ± 1.8, 67, 66) and Trichoderma harzianum (56.69 ± 0.9, 71, 63). The endophytic fungi Alternaria species inhibited all the three viral enzymes at maximum level and it was more than standard drug. However, in order to know possible anti-HIV, it is necessary to isolate active coumarin from the Alternaria species and the mechanism of action will be studied in future studies.

Design, Synthesis and Antifungal Activity of 6-Fluoro-3,3a,4,5-tetrahydro-2H-pyrazolo[4,3-c]-quinoline-2-carboxamide Derivatives

A series of 6-fluoro-3,3a,4,5-tetrahydro-2H-pyrazolo[4,3-c]quinoline-2-carboxamide derivatives was designed based on the bioisosterism and combination principle in drug design. The target compounds were synthesized from substituted aniline through Michael addition, cyclization, Mannich reaction and condensation with 4-substituted semicarbazides, and the structures were confirmed by mass spectrometry（MS） and 1H NMR. The antifungal assay was carried out in vitro by two-fold dilution. The result shows that all the compounds are of antifungal activities against the tested fungi at different levels.

Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis(Meyen)via the Toll pathway

Female adults of the migratory locust,Locusta migratoria manilensis(Meyen),can sense seasonal photoperiod changes,which induces embryonic diapause as a key strategy to overwinter.Serine protease inhibitor genes(SPNs)were thought to play key roles during diapause,while few SPNs were functionally characterized.LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs;however,its biological function remained to be explored.So,we conducted RNAi knockdown of LmSPN2,resulting in a significant decrease of the egg diapause rate by 29.7%.Using yeast two-hybrid assays,co-immunoprecipitation,and pull-down methods,we found an interaction between LmSPN2 and LmSPN3,which was proved to be mediated by a glutamate(E331)binding site of LmSPN2.RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%,indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation.Double knockdown of two SPN genes resulted in a 26.4%reduction in diapause rate,indicating that LmSPN2 was the dominant regulatory signal.Moreover,we found four Toll pathway genes(easter,spätzle,pelle,and dorsal)upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3.Therefore,we speculate that two SPNs regulate diapause through the Toll pathway.Our results indicated that LmSPN2 positively regulates locust egg entry into diapause,while LmSPN3 is a negative regulator of embryonic commitment to diapause.Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway.This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

Potato protease inhibitors,a functional food material with antioxidant and anticancer potential

Potato protease inhibitors(PPIs),as the main component of potato protein isolate,have good safety,nutrition and great market potential.The antioxidant and anticancer properties of PPIs were evaluated with cellbased biological assays.The results showed that when the concentration of PPIs was 5 mg/mL,the peroxyl radical scavenging value was(2119±204)mg VCE/100 g,and the cellular antioxidant activity values were(45.83±3.5)(no PBS wash)and(33.25±4.4)μmol QE/100 g(PBS wash).Cells pretreated with PPIs could significantly prevent the oxidative damage induced by H_(2)O_(2),inhibit the morphological changes of cells and maintain the integrity.Furthermore,PPIs had selective anti-proliferative effects on GIST882 cells(IC50=(10.53±3.87)mg/mL)and demonstrated potent inhibition of the growth,migration and invasion of cancer cells.These findings provide a scientific basis for PPIs as promising candidates for functional foods to aid in the prevention of oxidative damage and cancer.