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  • Portland Press Ltd.  (9)
  • Biology  (9)
  • 1
    Online Resource
    Online Resource
    Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization
    Portland Press Ltd. ; 2003
    In:  Biochemical Journal Vol. 374, No. 3 ( 2003-09-15), p. 625-632
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 374, No. 3 ( 2003-09-15), p. 625-632
    Abstract: Most microarray slides are manufactured or coated with a layer of poly(l-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/bj20030486
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2003
    detail.hit.zdb_id: 1473095-9
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  • 2
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    Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin
    Portland Press Ltd. ; 2012
    In:  Biochemical Journal Vol. 444, No. 2 ( 2012-06-01), p. 189-197
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 444, No. 2 ( 2012-06-01), p. 189-197
    Abstract: The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA. TbpA functions as a gated channel for the passage of iron into the periplasm. In the present study we have mapped the effect of TbpA from Actinobacillus pleuropneumoniae on pTf (porcine Tf) using H/DX-MS (hydrogen/deuterium exchange coupled to MS) and compare it with a previously determined binding site for TbpB. The proposed TbpA footprint is adjacent to and potentially overlapping the TbpB-binding site, and induces a structural instability in the TbpB site. This suggests that simultaneous binding to pTf by both receptors would be hindered. We demonstrate that a recombinant TbpB lacking a portion of its anchor peptide is unable to form a stable ternary TbpA–pTf–TbpB complex. This truncated TbpB does not bind to a preformed Tf–TbpA complex, and TbpA removes pTf from a preformed Tf–TbpB complex. Thus the results of the present study support a model whereby TbpB ‘hands-off’ pTf to TbpA, which completes the iron removal and transport process.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20112133
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2012
    detail.hit.zdb_id: 1473095-9
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  • 3
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    The role of vicinal tyrosine residues in the function of Haemophilus influenzae ferric-binding protein A
    Portland Press Ltd. ; 2010
    In:  Biochemical Journal Vol. 432, No. 1 ( 2010-11-15), p. 57-67
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 432, No. 1 ( 2010-11-15), p. 57-67
    Abstract: The periplasmic FbpA (ferric-binding protein A) from Haemophilus influenzae plays a critical role in acquiring iron from host transferrin, shuttling iron from the outer-membrane receptor complex to the inner-membrane transport complex responsible for transporting iron into the cytoplasm. In the present study, we report on the properties of a series of site-directed mutants of two adjacent tyrosine residues involved in iron co-ordination, and demonstrate that, in contrast with mutation of equivalent residues in the N-lobe of human transferrin, the mutant FbpAs retain significant iron-binding affinity regardless of the nature of the replacement amino acid. The Y195A and Y196A FbpAs are not only capable of binding iron, but are proficient in mediating periplasm-to-cytoplasm iron transport in a reconstituted FbpABC pathway in a specialized Escherichia coli reporter strain. This indicates that their inability to mediate iron acquisition from transferrin is due to their inability to compete for iron with receptor-bound transferrin. Wild-type iron-loaded FbpA could be crystalized in a closed or open state depending upon the crystallization conditions. The synergistic phosphate anion was not present in the iron-loaded open form, suggesting that initial anchoring of iron was mediated by the adjacent tyrosine residues and that alternate pathways for iron and anion binding and release may be considered. Collectively, these results demonstrate that the presence of a twin-tyrosine motif common to many periplasmic iron-binding proteins is critical for initially capturing the ferric ion released by the outer-membrane receptor complex.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20101043
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2010
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  • 4
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    Online Resource
    Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys10 and His106
    Portland Press Ltd. ; 2009
    In:  Biochemical Journal Vol. 417, No. 1 ( 2009-01-01), p. 55-64
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 417, No. 1 ( 2009-01-01), p. 55-64
    Abstract: GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys10 and His106 as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys10 and His106 to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys10 and His106 leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His106 plays an important role in the catalytic mechanism of the enzyme, whereas Cys10 is involved in binding of the substrate, glutathione. Engineering of the Cys10 site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys10 plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys10 and the substrate, glutathione.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20071702
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2009
    detail.hit.zdb_id: 1473095-9
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  • 5
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    Online Resource
    LRRC8A is responsible for exosome biogenesis and volume regulation in colon cancer cells
    Portland Press Ltd. ; 2023
    In:  Biochemical Journal Vol. 480, No. 9 ( 2023-05-17), p. 701-713
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 480, No. 9 ( 2023-05-17), p. 701-713
    Abstract: Exosomes are vital mediators for intercellular communications in the tumor microenvironment to accelerate colon cancer progression. Leucine-rich repeat-containing 8A (LRRC8A), the core component of the volume-regulated anion channel, is closely associated with acquiring heterogeneity for tumor cells. However, the role of LRRC8A in the exosomes remains largely unknown. Here, we reported that LRRC8A was one of the compositions in the exosomes released from colon cancer HCT116 cells. Down-regulation of LRRC8A proteins inhibited ex vivo cell growth and induced apoptosis. Consistently, chloride channel blockers DCPIB and NPPB inhibited cell growth and induced cell apoptosis in a time or concentration-dependent manner. Interestingly, the total amounts and proportions of different diameter exosomes released in 6 h were not altered by the treatment of DCPIB and NPPB in HCT116 cells. In contrast with the inhibition of LRRC8A, overexpression of LRRC8A proteins in HCT116 cells released significantly more distinct populations of exosomes. Importantly, the switches of ratios for exosomes in a hypotonic challenge were eliminated by DCPIB treatment. Collectively, our results uncovered that LRRC8A proteins were responsible for the exosome generation and sorted into exosomes for monitoring the volume regulation.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BCJ20220614
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2023
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  • 6
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    Online Resource
    High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin
    Portland Press Ltd. ; 2007
    In:  Biochemical Journal Vol. 404, No. 2 ( 2007-06-01), p. 217-225
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 404, No. 2 ( 2007-06-01), p. 217-225
    Abstract: The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q & gt;H9A, E57A & gt;Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L & gt;H9Q & gt;H9A, E57A & gt;Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay & gt;2.0×1018 M−1. These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20070110
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2007
    detail.hit.zdb_id: 1473095-9
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  • 7
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    Trace analysis of methylated and hydroxymethylated cytosines in DNA by isotope-dilution LC–MS/MS: first evidence of DNA methylation in Caenorhabditis elegans
    Portland Press Ltd. ; 2015
    In:  Biochemical Journal Vol. 465, No. 1 ( 2015-01-01), p. 39-47
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 465, No. 1 ( 2015-01-01), p. 39-47
    Abstract: From 1986 to the present, the popular research model organism Caenorhabditis elegans has been thought to completely lack DNA methylation and seems to have lost DNA methylation enzymes from its genomes. In the present study, we report the development of a sensitive and selective assay based on LC–MS/MS to simultaneously measure 5-methyl-2′-deoxycytidine (5-mdC) and 5-hydroxymethyl-2′-deoxycytidine (5-hmdC) in DNA hydrolysates. With the use of isotope internal standards ([2H3] 5-mdC and [2H3]5-hmdC) and online solid-phase extraction, the detection limits of 5-mdC and 5-hmdC were estimated to be 0.01 and 0.02 pg respectively, which correspond to a 0.000006% and 0.00001% methylation and hydroxymethylation level. This method was applied to investigate whether DNA methylation/hydroxymethylation exists in C. elegans. The present study for the first time demonstrates that 5-mdC is present in C. elegans genomic DNA (0.0019–0.0033% of cytosine methylated) using LC–MS/MS, whereas another epigenetic modification, 5-hmdC, is not detectable. Furthermore, we found that C. elegans DNA was hypo- or hyper-methylated in a dose-dependent manner by the DNA methyltransferase (DNMT)-inhibiting drug decitabine (5-aza-2′-deoxycytidine) or cadmium respectively. Our data support the possible existence of an active DNA-methylation mechanism in C. elegans, in which unidentified DNMTs could be involved. The present study highlights the importance of re-evaluating the evolutionary conservation of DNA-methylation machinery in nematodes which were traditionally considered to lack functional DNA methylation.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20140844
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2015
    detail.hit.zdb_id: 1473095-9
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  • 8
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    PPARα and AP-2α regulate bombesin receptor subtype 3 expression in ozone-stressed bronchial epithelial cells
    Portland Press Ltd. ; 2007
    In:  Biochemical Journal Vol. 405, No. 1 ( 2007-07-01), p. 131-137
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 405, No. 1 ( 2007-07-01), p. 131-137
    Abstract: Previously, we found that bombesin receptor subtype 3 (BRS-3) significantly increased in an ozone-stressed airway hyperresponsiveness animal model and resulted in induced wound repair and protection from acute lung injury. In the present study, we determined molecular mechanisms of BRS-3 regulation in human BECs (bronchial epithelial cells) in response to ozone stress. Ten oligonucleotide probes corresponding to various regions of the BRS-3 promoter were used in EMSA (electrophoretic mobilityshift assays). Four were found to have an enhanced mobility shift with extracts from ozone-stressed cells. On the basis of the assay of mutated probes binding with extracts and antibody supershift, they were verified as MTF-1 (metal-regulatory-element-binding transcription factor-1), PPARα (peroxisome-proliferator-activated receptor α), AP-2α (activator protein 2α) and HSF-1 (heat-shock factor 1). Next, ChIP (chromatin immunoprecipitation) assay, site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe these transcription factors associated with the BRS-3 promoter. Only AP-2α and PPARα increased ozone-inducible DNA binding on the BRS-3 promoter and BRS-3 expression. The time courses of AP-2α and PPARα activation, followed by BRS-3 expression, were also examined. It was shown that ozone-inducible BRS-3 expression and AP-2α- and PPARα-binding activity correlated over a 48 h period. The translocation of PPARα was observed by immunofluorescence assay, which showed that PPARα nuclear translocation increased after ozone exposure. Our data suggest that AP-2α and PPARα may be especially involved in this ozone-inducible up-regulation mechanism of BRS-3 expression.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20061754
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2007
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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  • 9
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    The N-terminal ubiquitin-binding region of ubiquitin-specific protease 28 modulates its deubiquitination function: NMR structural and mechanistic insights
    Portland Press Ltd. ; 2015
    In:  Biochemical Journal Vol. 471, No. 2 ( 2015-10-15), p. 155-165
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 471, No. 2 ( 2015-10-15), p. 155-165
    Abstract: The deubiquitinase ubiquitin-specific protease 28 (Usp28) contains a ubiquitin-binding region (UBR) composed of one ubiquitin-associated domain (UBA) and one ubiquitin-interacting motif (UIM) at its N-terminus. It is of interest that an additional small ubiquitin-like modifier (SUMO)-interacting motif (SIM) is located next to its UIM. To date, the functional role of the Usp28 UBR is still not understood. To elucidate the regulatory mechanism of the UBR on the full functional display of Usp28, in the present study, NMR and biochemical approaches were applied. The solution structure of Usp28 UBR was obtained, and the key residues responsible for ubiquitin and SUMO1/2 recognition were identified. In addition, we find that the ubiquitin-binding ability of Usp28 UBR was required for full enzymatic activity of Usp28, whereas binding of SUMO1/2 impaired the catalytic activity of the enzyme by competitively blocking its interactions with ubiquitin substrates. Our findings provide a first insight into understanding how the enzymatic activity of Usp28 is regulated by its non-catalytic UBR and endogenous ligands.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20150088
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2015
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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