MyMedR

Displaying publications 61 - 80 of 126 in total

Abstract:

Sort:

Fulltext Rapid ecotoxicological tests using bioassay systems - a review

Halmi, M.I.E.

Journal of Biochemistry, Microbiology and Biotechnology, 2016;4(1):29-37.
MyJurnal

The rise in pollution cases globally is expected to increase in line with industrialization.
Monitoring activities for pollutants have been hampered by the astronomical costs of
instrumental-based approach. This has resulted in the intense research on low cost
biomonitoring systems using enzymes, organisms including microorganisms. Only positive
samples are sent for instrumental analysis; dramatically cutting the cost of instrumental
analysis. This review attempts to outline and give due recognition to several selected bioassay
systems that have been tested for their applicability using polluted water samples as a routine
first line-of-defense. This includes small aquatic organisms-based assays, enzymes especially
proteases and bacterial-based systems using respiratory dye or luminescence systems as a
method for toxicant detection.

Matched MeSH terms: Endopeptidases
Fulltext Combining docking and molecular dynamic of protease from Bacillus lehensis G1

Noorul Aini Sulaiman, Nur Zazarina Ramly, Shuhaila Mat-Sharani, Nor Muhammad Mahadi

Journal of Engineering and Science Research, 2018;2(1):1-5.
MyJurnal

Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use.

Matched MeSH terms: Endopeptidases
Fulltext Inhibition of hyphae formation and SIR2 expression in Candida albicans treated with fresh Allium sativum (garlic) extract

Low CF, Chong PP, Yong PV, Lim CS, Ahmad Z, Othman F

J Appl Microbiol, 2008 Dec;105(6):2169-77.
PMID: 19120662 DOI: 10.1111/j.1365-2672.2008.03912.x

The aims of the present study were to determine whether Allium sativum (garlic) extract has any effect on the morphology transformation of Candida albicans, and to investigate whether it could alter the gene expression level of SIR2, a morphogenetic control gene and SAP4, a gene encoding secreted aspartyl proteinase.

Matched MeSH terms: Aspartic Acid Endopeptidases/genetics; Aspartic Acid Endopeptidases/metabolism
Molecular investigation of a gene encoding organic solvent-tolerant alkaline protease from Pseudomonas aeruginosa strain K

Rahman RN, Geok LP, Wong CF, Basri M, Salleh AB

J Basic Microbiol, 2010 Apr;50(2):143-9.
PMID: 20082370 DOI: 10.1002/jobm.200900133

A gene encoding an organic solvent-stable protease was amplified from Pseudomonas aeruginosa strain K by polymerase chain reaction using consensus primers based on multiple sequence alignment of alkaline and metalloprotease genes from Pseudomonas species. The gene, which consisted of 1440 bp nucleotides and deduced 479 amino acid residues, was successfully expressed in pGEX-4T-1 expression system in the presence of 1.0 mM IPTG, after an incubation of 6 h at 37 degrees C. Under these conditions, the recombinant strain K protease was, subsequently, released into the periplasm of E. coli BL21 (DE3) with an optimum proteolytic activity detected at 1.0112 U/ml. To date, this is the first reported expression of alkaline protease (aprA) with such remarkable property in Escherichia coli.

Matched MeSH terms: Endopeptidases/genetics*; Endopeptidases/isolation & purification; Endopeptidases/metabolism*
Complete genome sequence of Bacillus Licheniformis NWMCC0046, a candidate for the laundry industry

Zhou W, Zeng S, Yu J, Xiang J, Zhang F, Takriff MS, et al.

J Basic Microbiol, 2023 Feb;63(2):223-234.
PMID: 36538731 DOI: 10.1002/jobm.202200528

In this study, selected properties of protease and the complete genome sequence of Bacillus licheniformis NWMCC0046 were investigated, to discover laundry applications and other potential probiotic properties of this strain. Partial characterization of B. licheniformis NWMCC0046 showed that its protease has good activity both in alkaline environments and at low temperatures. Also, the protease is compatible with commercial detergents and can be used as a detergent additive for effective stain removal at low temperatures. The complete genome sequence of B. licheniformis NWMCC0046 is comprised of a 4,321,565 bp linear chromosome with a G + C content of 46.78% and no plasmids. It had 4504 protein-encoding genes, 81 transfer RNA (tRNA) genes, and 24 ribosomal RNA (rRNA) genes. Genomic analysis revealed genes involved in exocellular enzyme production and probiotic properties. In addition, genomic sequence analysis revealed specific genes encoding carbohydrate metabolism pathways, resistance, and cold adaptation capacity. Overall, protease properties show its potential as a detergent additive enzyme. The complete genome sequence information of B. licheniformis NWMCC0046 was obtained, and functional prediction revealed its numerous probiotic properties.

Matched MeSH terms: Endopeptidases/genetics
Possible role of inter-domain salt bridges in oligopeptidase B from Trypanosoma brucei: critical role of Glu172 of non-catalytic β-propeller domain in catalytic activity and Glu490 of catalytic domain in stability of OPB

Fukumoto J, Ismail NI, Kubo M, Kinoshita K, Inoue M, Yuasa K, et al.

J. Biochem., 2013 Nov;154(5):465-73.
PMID: 23946505 DOI: 10.1093/jb/mvt077

Oligopeptidase B (OPB) is a member of the prolyl oligopeptidase (POP) family of serine proteases. OPB in trypanosomes is an important virulence factor and potential pharmaceutical target. Characteristic structural features of POP family members include lack of a propeptide and presence of a β-propeller domain (PD), although the role of the β-PD has yet to be fully understood. In this work, residues Glu(172), Glu(490), Glu(524) and Arg(689) in Trypanosoma brucei OPB (Tb OPB), which are predicted to form inter-domain salt bridges, were substituted for Gln and Ala, respectively. These mutants were evaluated in terms of catalytic properties and stability. A negative effect on kcat/Km was obtained following mutation of Glu(172) or Arg(689). In contrast, the E490Q mutant exhibited markedly decreased thermal stability, although this mutation had less effect on catalytic properties compared to the E172Q and R689A mutants. Trypsin digestion showed that the boundary regions between the β-PD and catalytic domains (CDs) of the E490Q mutant are unfolded with heat treatment. These results indicated that Glu(490) in the CD plays a role in stabilization of Tb OPB, whereas Glu(172) in the β-PD is critical for the catalytic activity of Tb OPB.

Matched MeSH terms: Serine Endopeptidases/metabolism*; Serine Endopeptidases/chemistry*
Fulltext Protegrin-1 inhibits dengue NS2B-NS3 serine protease and viral replication in MK2 cells

Rothan HA, Abdulrahman AY, Sasikumer PG, Othman S, Rahman NA, Yusof R

J Biomed Biotechnol, 2012;2012:251482.
PMID: 23093838 DOI: 10.1155/2012/251482

Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein in E. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC(50) of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P < 0.001) at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly (P < 0.01) higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.

Matched MeSH terms: Serine Endopeptidases/metabolism*
Lactobacillus Spp.-Enhanced Memory is Strain-Dependent and Associated, in Part, with Amyloidogenic and anti-Oxidant/Oxidative Stress Interplay in Amyloid Beta Precursor Protein Transgenic Mice

Ahmad Alwi NA, Lim SM, Mani V, Ramasamy K

J Diet Suppl, 2023;20(5):717-734.
PMID: 35876040 DOI: 10.1080/19390211.2022.2103608

This study explored mechanisms underpinning enhanced memory in amyloid precursor protein (APP) transgenic mice (male; 10-12 months; n = 6/group) supplemented with Lactobacillus plantarum LAB12 (LAB12)/Lactobacillus casei Shirota (LcS). Morris Water Maze test was performed before brains were harvested for gene expression and biochemical studies. LAB-supplemented mice exhibited reduced escape latency and distance but significant increased time spent in platform zone. This was associated with downregulated beta-site APP cleaving enzyme-1 (BACE1) mRNA and significant reduced nitric oxide in brains. LAB12 also significantly increased glutathione. The LAB-enhanced memory is strain-dependent and could be mediated, in part, through amyloidogenic pathway and anti-oxidant/oxidative stress interplay.

Matched MeSH terms: Aspartic Acid Endopeptidases/genetics; Aspartic Acid Endopeptidases/metabolism
Phaleria macrocarpa (Scheff.) Boerl fruit aqueous extract enhances LDL receptor and PCSK9 expression in vivo and in vitro

Chong SC, Dollah MA, Chong PP, Maha A

J Ethnopharmacol, 2011 Sep 1;137(1):817-27.
PMID: 21763412 DOI: 10.1016/j.jep.2011.06.041

Phaleria macrocarpa (Scheff.) Boerl (Pm) has been shown to reduce cholesterol level in vitro and in vivo experiment.

Matched MeSH terms: Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism*
Protease activated receptor 2 and matriptase expression in the joints of cats with and without osteoarthritis

Ariffin SMZ, Bennett D, Ferrell WR, Lockhart JC, Dunning L, Clements DN, et al.

J Feline Med Surg, 2021 08;23(8):794-803.
PMID: 33284033 DOI: 10.1177/1098612X20977796

OBJECTIVES: The aim of this study was to determine the presence of protease-activated receptor 2 (PAR2) and matriptase proteins and quantify PAR2 and matriptase mRNA expression in the articular cartilage and synovial membrane of cats with and without osteoarthritis (OA).
METHODS: A total of 28 articular cartilage samples from adult cats (14 OA and 14 normal), 10 synovial membranes from adult cats (five OA and five normal) and three cartilage samples from 9-week-old fetal cats were used. The presence of PAR2 and matriptase in the cartilage and synovial membrane of the adult samples was detected by immunohistochemical (IHC) staining, while real-time PCR was used for mRNA expression analyses in all samples.
RESULTS: PAR2 was detected in all OA and normal articular cartilage and synovial membrane samples but confined to only a few superficial chondrocytes in the normal samples. Matriptase was only detected in OA articular cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression were, however, detected in all cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression levels in OA articular cartilage were five (P <0.001) and 3.3 (P <0.001) times higher than that of the healthy group, respectively. There was no significant difference (P = 0.05) in the OA synovial membrane PAR2 and matriptase mRNA expression compared with the normal samples.
CONCLUSIONS AND RELEVANCE: Detection of PAR2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding, and supports the hypothesis that serine proteases are involved in the pathogenesis of feline OA. The consistent presence of PAR2 and matriptase protein in the cytoplasm of OA chondrocytes suggests a possible involvement of proteases in cartilage degradation. Further investigations into the PAR2 and matriptase pathobiology could enhance our understanding of the proteolytic cascades in feline OA, which might lead to the development of novel therapeutic strategies.

Matched MeSH terms: Serine Endopeptidases
Epicatechin and scopoletin rich Morinda citrifolia (Noni) leaf extract supplementation, mitigated Osteoarthritis via anti-inflammatory, anti-oxidative, and anti-protease pathways

Wan Osman WN, Che Ahmad Tantowi NA, Lau SF, Mohamed S

J Food Biochem, 2019 03;43(3):e12755.
PMID: 31353568 DOI: 10.1111/jfbc.12755

The scopoletin (coumarin) and epicatechin (flavonoid) rich Morinda citrifolia L. (MC) Noni leaves are non-toxic (unlike the fruits) and consumed as vegetables. The anti-osteoarthritis effects of the MC leaf extract against joint cartilage degradation and inflammation were investigated through cartilage explant cultures and pre-clinical animal study. Osteoarthritis were induced by intra-articular monosodium iodoacetate injection into the right knee. The extract, scopoletin and epicatechin, suppressed glycosaminoglycan and nitric oxide release from the cartilage explant in the presence of Interleukin-1β. After 28 days, the extract treatment reduced the in vivo serum levels and joint tissues mRNA expressions for joint cartilage degradation, aggrecanase, and collagenase biomarkers. The extract increased the bone formation marker PINP levels, besides improving the articular cartilage structure and chondrocytes cellularity. The extract improved bone formation/repair, subchondral bone structure, strength and integrity, as well as cartilage synthesis by suppressing inflammation, nitric oxide production, joint catabolism by proteases, and oxidative stress. PRACTICAL APPLICATIONS: The scopoletin (coumarin) and epicatechin (flavonoid) rich Morinda citrifolia (Noni) leaves may be used as vegetables, functional food ingredient, or dietary supplements to suppress osteoarthritis progression against joint cartilage degradation and inflammation. The extract, scopoletin, or epicatechin, suppressed glycosaminoglycan, and nitric oxide release from the cartilage. The Morinda citrifolia leaf extract suppressed inflammation, nitric oxide production, tissues catabolism by proteases and oxidative stress to help reduce joint cartilage degradation, besides improving the articular cartilage structure, chondrocytes health, subchondral bone structure, bone formation/repair, and cartilage synthesis.

Matched MeSH terms: Endopeptidases/genetics; Endopeptidases/immunology
Generation, Fractionation, and Characterization of Iron-Chelating Protein Hydrolysate from Palm Kernel Cake Proteins

Zarei M, Ghanbari R, Tajabadi N, Abdul-Hamid A, Bakar FA, Saari N

J Food Sci, 2016 Feb;81(2):C341-7.
PMID: 26720491 DOI: 10.1111/1750-3841.13200

Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively.

Matched MeSH terms: Endopeptidases/metabolism
Fulltext ACE inhibitory activity of pangasius catfish (Pangasius sutchi) skin and bone gelatin hydrolysate

Mahmoodani F, Ghassem M, Babji AS, Yusop SM, Khosrokhavar R

J Food Sci Technol, 2014 Sep;51(9):1847-56.
PMID: 25190839 DOI: 10.1007/s13197-012-0742-8

Skin and bone gelatins of pangasius catfish (Pangasius sutchi) were hydrolyzed with alcalase to isolate Angiotensin Converting Enzyme (ACE) inhibitory peptides. Samples with the highest degree of hydrolysis (DH) were separated into different fractions with molecular weight cut-off (MWCO) sizes of 10, 3 and 1 kDa, respectively and assayed for ACE inhibitory activity. Skin and bone gelatins had highest DH of 64.87 and 68.48 % after 2 and 1 h incubation, respectively. Results from this study indicated that by decreasing the molecular weight of fractions, ACE inhibitory activity was increased. Therefore, F3 permeates (MWCO

Matched MeSH terms: Endopeptidases
Fulltext Kinetic characterization of Channa striatus muscle sarcoplasmic and myofibrillar protein hydrolysates

Ghassem M, Fern SS, Said M, Ali ZM, Ibrahim S, Babji AS

J Food Sci Technol, 2014 Mar;51(3):467-75.
PMID: 24587521 DOI: 10.1007/s13197-011-0526-6

This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h with an increase in DH. The major amino acids were glutamic acid (16.6%) in thermolysin- myofibrillar hydrolysate followed by aspartic acid (11.1%) in sarcoplasmic protein fraction with no enzyme treatment and lysine (10%) in thermolysin-myofibrillar hydrolysate. The apparent Michaelis constant of proteinase K was lower than thermolysin for both sarcoplasmic and myofibrillar proteins. However, rate of turnover and enzyme efficiency suggested that sarcoplasmic and myofibrillar proteins are suitable substrates for proteinase K and thermolysin hydrolytic reaction, respectively.

Matched MeSH terms: Endopeptidases
Fulltext The encapsulation of flavourzyme in nanoliposome by heating method

Jahadi M, Khosravi-Darani K, Ehsani MR, Mozafari MR, Saboury AA, Pourhosseini PS

J Food Sci Technol, 2015 Apr;52(4):2063-72.
PMID: 25829586 DOI: 10.1007/s13197-013-1243-0

The main objective of this study was to use heating method (HM) to prepare liposome without employing any chemical solvent or detergent. Plackett-Burman design (PBD) was applied for the screening of significant process variables including the lecithin proportion, the cholesterol/lecithin ratio, the pH of solution for liposome preparation, the enzyme/lecithin ratio, the stirring time, the process temperature, the speed of stirrer, the ratio of stirrer to the tank diameter, the application of homogenization, the method of adding enzyme and centrifugation conditions on the encapsulation efficiency (EE %) of liposome and the activity of liposomal Flavourzyme (LAPU(-1)) (P

Matched MeSH terms: Endopeptidases
A new organic solvent tolerant protease from Bacillus pumilus 115b

Rahman RN, Mahamad S, Salleh AB, Basri M

J Ind Microbiol Biotechnol, 2007 Jul;34(7):509-17.
PMID: 17492323

Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.

Matched MeSH terms: Endopeptidases/genetics; Endopeptidases/metabolism*; Endopeptidases/chemistry
Binding specificity of polypeptide substrates in NS2B/NS3pro serine protease of dengue virus type 2: A molecular dynamics Study

Yotmanee P, Rungrotmongkol T, Wichapong K, Choi SB, Wahab HA, Kungwan N, et al.

J Mol Graph Model, 2015 Jul;60:24-33.
PMID: 26086900 DOI: 10.1016/j.jmgm.2015.05.008

The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values.

Matched MeSH terms: Serine Endopeptidases/metabolism*; Serine Endopeptidases/chemistry
Allosteric pocket of the dengue virus (serotype 2) NS2B/NS3 protease: In silico ligand screening and molecular dynamics studies of inhibition

Mukhametov A, Newhouse EI, Aziz NA, Saito JA, Alam M

J Mol Graph Model, 2014 Jul;52:103-13.
PMID: 25023665 DOI: 10.1016/j.jmgm.2014.06.008

The allosteric pocket of the Dengue virus (DENV2) NS2B/NS3 protease, which is proximal to its catalytic triad, represents a promising drug target (Othman et al., 2008). We have explored this binding site through large-scale virtual screening and molecular dynamics simulations followed by calculations of binding free energy. We propose two mechanisms for enzyme inhibition. A ligand may either destabilize electronic density or create steric effects relating to the catalytic triad residues NS3-HIS51, NS3-ASP75, and NS3-SER135. A ligand may also disrupt movement of the C-terminal of NS2B required for inter-conversion between the "open" and "closed" conformations. We found that chalcone and adenosine derivatives had the top potential for drug discovery hits, acting through both inhibitory mechanisms. Studying the molecular mechanisms of these compounds might be helpful in further investigations of the allosteric pocket and its potential for drug discovery.

Matched MeSH terms: Serine Endopeptidases/metabolism*
Reelin (RELN) DNA methylation in the peripheral blood of schizophrenia

Nabil Fikri RM, Norlelawati AT, Nour El-Huda AR, Hanisah MN, Kartini A, Norsidah K, et al.

J Psychiatr Res, 2017 05;88:28-37.
PMID: 28086126 DOI: 10.1016/j.jpsychires.2016.12.020

The epigenetic changes of RELN that are involved in the development of dopaminergic neurons may fit the developmental theory of schizophrenia. However, evidence regarding the association of RELN DNA methylation with schizophrenia is far from sufficient, as studies have only been conducted on a few limited brain samples. As DNA methylation in the peripheral blood may mirror the changes taking place in the brain, the use of peripheral blood for a DNA methylation study in schizophrenia is feasible due to the scarcity of brain samples. Therefore, the aim of our study was to examine the relationship of DNA methylation levels of RELN promoters with schizophrenia using genomic DNA derived from the peripheral blood of patients with the disorder. The case control studies consisted of 110 schizophrenia participants and 122 healthy controls who had been recruited from the same district. After bisufhite conversion, the methylation levels of the DNA samples were calculated based on their differences of the Cq values assayed using the highly sensitive real-time MethyLight TaqMan® procedure. A significantly higher level of methylation of the RELN promoter was found in patients with schizophrenia compared to controls (p = 0.005) and also in males compared with females (p = 0.004). Subsequently, the RELN expression of the methylated group was 25 fold less than that of the non-methylated group. Based upon the assumption of parallel methylation changes in the brain and peripheral blood, we concluded that RELN DNA methylation might contribute to the pathogenesis of schizophrenia. However, the definite effects of methylation on RELN function during development and also in adult life still require further elaboration.

Matched MeSH terms: Serine Endopeptidases/blood*; Serine Endopeptidases/genetics*
Degradation of MicroRNA miR-466d-3p by Japanese Encephalitis Virus NS3 Facilitates Viral Replication and Interleukin-1β Expression

Jiang H, Bai L, Ji L, Bai Z, Su J, Qin T, et al.

J Virol, 2020 07 16;94(15).
PMID: 32461319 DOI: 10.1128/JVI.00294-20

Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1β (IL-1β) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation.IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1β expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.

Matched MeSH terms: Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism

Filters

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links