Displaying publications 1 - 20 of 123 in total

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  1. Design and synthesis of mono and bicyclic tetrapeptides thioester as potent inhibitor of histone deacetylases
    Hoque MA, Islam MS, Islam MN, Kato T, Nishino N, Ito A, et al.
    Amino Acids, 2014 Oct;46(10):2435-44.
    PMID: 25048030 DOI: 10.1007/s00726-014-1800-5
    Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that have an effect on gene regulation. The naturally occurring cyclic depsipeptide FK228 containing disulfide and Largazole possessing thioester functionalities act as pro-drugs and share the same HDAC inhibition mechanism in cell. Inspired from these facts, we have reported bicyclic tetrapeptide disulfide HDAC inhibitors resembling FK228 with potent activity and enhanced selectivity. In the present study, we report the design and synthesis of several mono and bicyclic tetrapeptide thioester HDAC inhibitors that share the inhibition mechanism similar to Largazole. Most of the compounds showed HDAC1 and HDAC4 inhibition and p21 promoting activity in nanomolar ranges. Among these the monocyclic peptides 1, 2 and bicyclic peptide, 4 are notable demanding more advanced research to be promising anticancer drug candidates.
    Matched MeSH terms: Recombinant Proteins/metabolism
  2. Styryl-lactone goniothalamin inhibits TNF-α-induced NF-κB activation
    Orlikova B, Schumacher M, Juncker T, Yan CC, Inayat-Hussain SH, Hajjouli S, et al.
    Food Chem Toxicol, 2013 Sep;59:572-8.
    PMID: 23845509 DOI: 10.1016/j.fct.2013.06.051
    (R)-(+)-Goniothalamin (GTN), a styryl-lactone isolated from the medicinal plant Goniothalamus macrophyllus, exhibits pharmacological activities including cytotoxic and anti-inflammatory effects. In this study, GTN modulated TNF-α induced NF-κB activation. GTN concentrations up to 20 μM showed low cytotoxic effects in K562 chronic myelogenous leukemia and in Jurkat T cells. Importantly, at these concentrations, no cytotoxicity was observed in healthy peripheral blood mononuclear cells. Our results confirmed that GTN inhibited tumor necrosis factor-α (TNF-α)-induced NF-κB activation in Jurkat and K562 leukemia cells at concentrations as low as 5 μM as shown by reporter gene assays and western blots. Moreover, GTN down-regulated translocation of the p50/p65 heterodimer to the nucleus, prevented binding of NF-κB to its DNA response element and reduced TNF-α-activated interleukin-8 (IL-8) expression. In conclusion, GTN inhibits TNF-α-induced NF-κB activation at non-apoptogenic concentrations in different leukemia cell models without presenting toxicity towards healthy blood cells underlining the anti-leukemic potential of this natural compound.
    Matched MeSH terms: Recombinant Proteins/metabolism
  3. Paeonol protects against endoplasmic reticulum stress-induced endothelial dysfunction via AMPK/PPARδ signaling pathway
    Choy KW, Mustafa MR, Lau YS, Liu J, Murugan D, Lau CW, et al.
    Biochem Pharmacol, 2016 09 15;116:51-62.
    PMID: 27449753 DOI: 10.1016/j.bcp.2016.07.013
    Endoplasmic reticulum (ER) stress in endothelial cells often leads to endothelial dysfunction which underlies the pathogenesis of cardiovascular diseases. Paeonol, a major phenolic component extracted from Moutan Cortex, possesses various medicinal benefits which have been used extensively in traditional Chinese medicine. The present study investigated the protective mechanism of paeonol against tunicamycin-induced ER stress in isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). Vascular reactivity in aorta was measured using a wire myograph. The effects of paeonol on protein expression of ER stress markers, reactive oxygen species (ROS) production, nitric oxide (NO) bioavailability and peroxisome proliferator-activated receptor δ (PPARδ) activity in the vascular wall were assessed by Western blot, dihydroethidium fluorescence (DHE) or lucigenin enhanced-chemiluminescence, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA) and dual luciferase reporter assay, respectively. Ex vivo treatment with paeonol (0.1μM) for 16h reversed the impaired endothelium-dependent relaxations in C57BJ/6J and PPARδ wild type (WT) mouse aortas following incubation with tunicamycin (0.5μg/mL). Elevated ER stress markers, oxidative stress and reduction of NO bioavailability induced by tunicamycin in HUVECs, C57BJ/6J and PPARδ WT mouse aortas were reversed by paeonol treatment. These beneficial effects of paeonol were diminished in PPARδ knockout (KO) mouse aortas. Paeonol increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and PPARδ expression and activity while restoring the decreased phosphorylation of eNOS. The present study delineates that paeonol protects against tunicamycin-induced vascular endothelial dysfunction by inhibition of ER stress and oxidative stress, thus elevating NO bioavailability via the AMPK/PPARδ signaling pathway.
    Matched MeSH terms: Recombinant Proteins/metabolism
  4. Butyrylcholinesterase: A Multifaceted Pharmacological Target and Tool
    Ha ZY, Mathew S, Yeong KY
    Curr Protein Pept Sci, 2020;21(1):99-109.
    PMID: 31702488 DOI: 10.2174/1389203720666191107094949
    Butyrylcholinesterase is a serine hydrolase that catalyzes the hydrolysis of esters in the body. Unlike its sister enzyme acetylcholinesterase, butyrylcholinesterase has a broad substrate scope and lower acetylcholine catalytic efficiency. The difference in tissue distribution and inhibitor sensitivity also points to its involvement external to cholinergic neurotransmission. Initial studies on butyrylcholinesterase showed that the inhibition of the enzyme led to the increment of brain acetylcholine levels. Further gene knockout studies suggested its involvement in the regulation of amyloid-beta, a brain pathogenic protein. Thus, it is an interesting target for neurological disorders such as Alzheimer's disease. The substrate scope of butyrylcholinesterase was recently found to include cocaine, as well as ghrelin, the "hunger hormone". These findings led to the development of recombinant butyrylcholinesterase mutants and viral gene therapy to combat cocaine addiction, along with in-depth studies on the significance of butyrylcholinesterase in obesity. It is observed that the pharmacological impact of butyrylcholinesterase increased in tandem with each reported finding. Not only is the enzyme now considered an important pharmacological target, it is also becoming an important tool to study the biological pathways in various diseases. Here, we review and summarize the biochemical properties of butyrylcholinesterase and its roles, as a cholinergic neurotransmitter, in various diseases, particularly neurodegenerative disorders.
    Matched MeSH terms: Recombinant Proteins/metabolism
  5. The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata)
    Kuah MK, Jaya-Ram A, Shu-Chien AC
    Biochim. Biophys. Acta, 2015 Mar;1851(3):248-60.
    PMID: 25542509 DOI: 10.1016/j.bbalip.2014.12.012
    The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.
    Matched MeSH terms: Recombinant Proteins/metabolism
  6. Fulltext Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase
    Ranjani V, Janeček S, Chai KP, Shahir S, Abdul Rahman RN, Chan KG, et al.
    Sci Rep, 2014 Jul 28;4:5850.
    PMID: 25069018 DOI: 10.1038/srep05850
    The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.
    Matched MeSH terms: Recombinant Proteins/metabolism
  7. Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens
    Goulter RM, Taran E, Gentle IR, Gobius KS, Dykes GA
    Colloids Surf B Biointerfaces, 2014 Jul 1;119:90-8.
    PMID: 24880987 DOI: 10.1016/j.colsurfb.2014.04.003
    The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.
    Matched MeSH terms: Recombinant Proteins/metabolism
  8. Fulltext Identification and real-time expression analysis of selected Toxoplasma gondii in-vivo induced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients
    Amerizadeh A, Khoo BY, Teh AY, Golkar M, Abdul Karim IZ, Osman S, et al.
    BMC Infect Dis, 2013;13:287.
    PMID: 23800344 DOI: 10.1186/1471-2334-13-287
    Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
    Matched MeSH terms: Recombinant Proteins/metabolism
  9. Fulltext Role of α-helical structure in organic solvent-activated homodimer of elastase strain K
    Rahman RN, Salleh AB, Basri M, Wong CF
    Int J Mol Sci, 2011;12(9):5797-814.
    PMID: 22016627 DOI: 10.3390/ijms12095797
    Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
    Matched MeSH terms: Recombinant Proteins/metabolism
  10. Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein
    Levitskiy SA, Sycheva AM, Kharlampieva DD, Oberto J, Kamashev DE, Serebryakova MV, et al.
    Biochimie, 2011 Jul;93(7):1102-9.
    PMID: 21443922 DOI: 10.1016/j.biochi.2011.03.005
    HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.
    Matched MeSH terms: Recombinant Proteins/metabolism*
  11. Molecular cloning, characterization and gene expression of an antioxidant enzyme catalase (MrCat) from Macrobrachium rosenbergii
    Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S
    Fish Shellfish Immunol, 2012 May;32(5):670-82.
    PMID: 22293093 DOI: 10.1016/j.fsi.2012.01.013
    In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85-100% enzyme activity from 30 °C to 40 °C.
    Matched MeSH terms: Recombinant Proteins/metabolism
  12. In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica
    Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Pook PC, et al.
    J Ethnopharmacol, 2010 Jul 20;130(2):275-83.
    PMID: 20457244 DOI: 10.1016/j.jep.2010.05.002
    ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (CA) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa, and Oceanic countries and traditionally used for wound healing and maintaining normal blood pressure.

    AIM OF THE STUDY: The present study was carried out to examine the potential modulatory effects of three commercially available active components (asiaticoside, asiatic acid and madecassic acid) and four extracts (aqueous, ethanol, dichloromethane and hexane) of CA on three major cDNA-expressed human cytochrome P450 (CYP) isoforms.

    MATERIALS AND METHODS: High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6beta-hydroxylase assays were developed to probe activities of CYP2C9, CYP2D6 and CYP3A4, respectively. Probe substrates were incubated with or without each active component and extract for each isoform, followed by examination of the kinetics parameters, IC(50) and K(i), to characterize modulatory effects.

    RESULTS: CYP2C9 was more susceptible to inhibitory effects by CA extracts compared to CYP2D6 and CYP3A4. Moderate degree of inhibition was observed in ethanol (K(i)=39.1 microg/ml) and dichloromethane (K(i)=26.6 microg/ml) extracts implying potential risk of interaction when CYP2C9 substrates are consumed with CA products. The two extracts however showed negligible inhibition towards CYP2D6 and CYP3A4 (IC(50)'s of 123.3 microg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC(50)'s of 117.9 microg/ml and above). Among the active constituents investigated, asiatic acid and madecassic acid appeared to selectively inhibit CYP2C9 and CYP2D6 more than CYP3A4. Of particular interest is the potent inhibitory effect of asiatic acid on CYP2C9 (K(i)=9.1 microg/ml). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Inhibitions of asiatic acid with the other isoforms and that of madecassic acid with all isoforms were only moderate (K(i)'s ranged from 17.2 to 84.4 microg/ml). On the other hand, the IC(50) values for asiaticoside were high (1070.2 microg/ml or above) for all three isoforms, indicating negligible or low potential of this compound to modulate CYP enzymatic activity.

    CONCLUSION: Centella asiatica extracts and active constituents inhibited CYP2C9, CYP2D6 and CYP3A4 activities with varying potency with CYP2C9 being the most susceptible isoform to inhibition. Significant inhibition was observed for asiatic acid and CA ethanol and dichloromethane extracts, implying involvement of semipolar constituents from CA in the effect. This study suggested that CA could cause drug-herb interactions through CYP2C9 inhibition.

    Matched MeSH terms: Recombinant Proteins/metabolism
  13. Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris
    Lau YL, Fong MY
    Exp Parasitol, 2008 Jul;119(3):373-8.
    PMID: 18457835 DOI: 10.1016/j.exppara.2008.03.016
    The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n=20), acute (IgM positive, IgG positive; n=20) and chronic (IgM negative, IgG positive; n=20) toxoplasmosis patients, and toxoplasmosis negative control patients (n=20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients' serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P<0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.
    Matched MeSH terms: Recombinant Proteins/metabolism
  14. Selection of high affinity ligands to hepatitis B core antigen from a phage-displayed cyclic peptide library
    Ho KL, Yusoff K, Seow HF, Tan WS
    J Med Virol, 2003 Jan;69(1):27-32.
    PMID: 12436474
    M13 phages that display random disulfide constrained heptapeptides on their gpIII proteins were used to select for high affinity ligands to hepatitis B core antigen (HBcAg). Phages bearing the amino acid sequences C-WSFFSNI-C and C-WPFWGPW-C were isolated, and a binding assay in solution showed that these phages bind tightly to full-length and truncated HBcAg with K D rel values less than 25 nM, which is at least 10 orders of magnitude higher than phage carrying the peptide sequence LLGRMK selected from a linear peptide library. Both the phages that display the constrained peptides were inhibited from binding to HBcAg particles by a monoclonal antibody that binds specifically to the immunodominant region of the particles. A synthetic heptapeptide with the amino acid sequence WSFFSNI derived from one of the fusion peptides inhibits the binding of large surface antigen (L-HBsAg) to core particles with an IC50 value of 12 +/- 2 microM. This study has identified a smaller peptide with a greater inhibitory effect on L-HBsAg-HBcAg association.
    Matched MeSH terms: Recombinant Proteins/metabolism
  15. EgRBP42 encoding an hnRNP-like RNA-binding protein from Elaeis guineensis Jacq. is responsive to abiotic stresses
    Yeap WC, Ooi TE, Namasivayam P, Kulaveerasingam H, Ho CL
    Plant Cell Rep, 2012 Oct;31(10):1829-43.
    PMID: 22699852 DOI: 10.1007/s00299-012-1297-x
    RNA-binding proteins (RBPs) have been implicated as regulatory proteins involved in the post-transcriptional processes of gene expression in plants under various stress conditions. In this study, we report the cloning and characterization of a gene, designated as EgRBP42, encoding a member of the plant heterogeneous nuclear ribonucleoprotein (hnRNP)-like RBP family from oil palm (Elaeis guineensis Jacq.). EgRBP42 consists of two N-terminal RNA recognition motifs and a glycine-rich domain at the C-terminus. The upstream region of EgRBP42 has multiple light-responsive, stress-responsive regulatory elements and regulatory elements associated with flower development. Real-time RT-PCR analysis of EgRBP42 showed that EgRBP42 was expressed in oil palm tissues tested, including leaf, shoot apical meristem, root, female inflorescence, male inflorescence and mesocarp with the lowest transcript level in the roots. EgRBP42 protein interacted with transcripts associated with transcription, translation and stress responses using pull-down assay and electrophoretic mobility shift assay. The accumulation of EgRBP42 and its interacting transcripts were induced by abiotic stresses, including salinity, drought, submergence, cold and heat stresses in leaf discs. Collectively, the data suggested that EgRBP42 is a RBP, which responds to various abiotic stresses and could be advantageous for oil palm under stress conditions. Key message EgRBP42 may be involved in the post-transcriptional regulation of stress-related genes important for plant stress response and adaptation.
    Matched MeSH terms: Recombinant Proteins/metabolism
  16. Structural basis for binding uronic acids by family 32 carbohydrate-binding modules
    Teh AH, Sim PF, Hisano T
    Biochem Biophys Res Commun, 2020 12 10;533(3):257-261.
    PMID: 33010888 DOI: 10.1016/j.bbrc.2020.09.064
    The alginate lyase AlyQ from Persicobacter sp. CCB-QB2 is a three-domained enzyme with a carbohydrate-binding module (CBM) from family 32. The CBM32 domain, AlyQB, binds enzymatically cleaved but not intact alginate. Co-crystallisation of AlyQB with the cleaved alginate reveals that it binds to the 4,5-unsaturated mannuronic acid of the non-reducing end. The binding pocket contains a conserved R248 that interacts with the sugar's carboxyl group, as well as an invariant W303 that stacks against the unsaturated pyranose ring. Targeting specifically the non-reducing end is more efficient than the reducing end since the latter consists of a mixture of mannuronic acid and guluronic acid. AlyQB also seems unable to bind these two saturated sugars as they contain OH groups that will clash with the pocket. Docking analysis of YeCBM32, which binds oligogalacturonic acid, shows that the stacking of the pyranose ring is shifted in order to accommodate the sugar's axial C1-OH, and its R69 is accordingly elevated to bind the sugar's carboxyl group. Unlike AlyQB, YeCBM32's binding pocket is able to accommodate both saturated and unsaturated galacturonic acid.
    Matched MeSH terms: Recombinant Proteins/metabolism
  17. Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1
    Atago Y, Shimodaira J, Araki N, Bin Othman N, Zakaria Z, Fukuda M, et al.
    Biosci Biotechnol Biochem, 2016 May;80(5):1012-9.
    PMID: 26828632 DOI: 10.1080/09168451.2015.1127134
    Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.
    Matched MeSH terms: Recombinant Proteins/metabolism
  18. Functional characterisation and product specificity of Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1
    Jaafar NR, Khoiri NM, Ismail NF, Mahmood NAN, Abdul Murad AM, Abu Bakar FD, et al.
    Enzyme Microb Technol, 2020 Oct;140:109625.
    PMID: 32912685 DOI: 10.1016/j.enzmictec.2020.109625
    Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.
    Matched MeSH terms: Recombinant Proteins/metabolism
  19. Fulltext Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions
    Minayoshi Y, Maeda H, Yanagisawa H, Hamasaki K, Mizuta Y, Nishida K, et al.
    Drug Deliv, 2018 Nov;25(1):1067-1077.
    PMID: 29688069 DOI: 10.1080/10717544.2018.1464083
    Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.
    Matched MeSH terms: Recombinant Proteins/metabolism
  20. Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12
    Jaafar NR, Littler D, Beddoe T, Rossjohn J, Illias RM, Mahadi NM, et al.
    Acta Crystallogr F Struct Biol Commun, 2016 11 01;72(Pt 11):831-839.
    PMID: 27827354
    Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.
    Matched MeSH terms: Recombinant Proteins/metabolism
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