Domain structure of growth signalobodies critically affects the outcome of antibody library selection
In this study, we screened naïve scFv library-inserted signalobodies having distinct extracellular and transmembrane (TM) domains. Although the previously constructed signalobody with the extracellular D1/D2 domains of erythropoietin receptor had recovered the clones with high affinity against a target antigen and with low background cell growth, its D1/D2-deficient variant which was tested in this study recovered the clones with low affinity against a target antigen and with considerable background cell growth. In addition, mutagenesis in the TM domain lowered the level of the background cell growth. These results su...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Yoshida, R., Kawahara, M., Nagamune, T. Tags: Regular Papers Source Type: research
An early endosome regulator, Rab5b, is an LRRK2 kinase substrate
Leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for Parkinson’s disease (PD). LRRK2 contains a kinase and a GTPase domain, both of which provide critical intracellular signal-transduction functions. We showed previously that Rab5b, a small GTPase protein that regulates the motility and fusion of early endosomes, interacts with LRRK2 and co-regulates synaptic vesicle endocytosis. Using recombinant proteins, we show here that LRRK2 phosphorylates Rab5b at its Thr6 residue in in vitro kinase assays with mass spectrophotometry analysis. Phosphorylation of Rab5b by LRRK2 on the threonine resid...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Yun, H. J., Kim, H., Ga, I., Oh, H., Ho, D. H., Kim, J., Seo, H., Son, I., Seol, W. Tags: Regular Papers Source Type: research
A highly effective and adjustable dual plasmid system for O-GlcNAcylated recombinant protein production in E. coli
O-GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O-GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O-...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Han, C., Shan, H., Bi, C., Zhang, X., Qi, J., Zhang, B., Gu, Y., Yu, W. Tags: Regular Papers Source Type: research
Structure-based design of a streptavidin mutant specific for an artificial biotin analogue
In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a Kd value of 5.9 x 10–7 M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail ...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Kawato, T., Mizohata, E., Shimizu, Y., Meshizuka, T., Yamamoto, T., Takasu, N., Matsuoka, M., Matsumura, H., Kodama, T., Kanai, M., Doi, H., Inoue, T., Sugiyama, A. Tags: Regular Papers Source Type: research
New roles of DNA and SopB in polymerization of SopA of Escherichia coli F plasmid
This study followed kinetics of polymerization and de-polymerization of SopA by turbidity measurement and found new effects by DNA and SopB. Plasmid DNA, at low concentrations, shortened the lag (nucleation) phase of SopA polymerization and also caused an initial ‘burst’ of turbidity. Results with two non-specific 20-bp DNAs indicated sequence/length dependence of these effects. sopAB operator DNA only showed inhibition of SopA polymerization. Results of turbidity decrease of pre-formed SopA polymer in the presence of ethylenediaminetetraacetic acid showed that SopB also accelerates disassembly of the SopA poly...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Hanai, R., Arai, Y. Tags: Regular Papers Source Type: research
Properties and functions of the storage sites of glycogen phosphorylase
Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits. Each subunit has two distinct maltooligosaccharide binding sites: a storage site and a catalytic site. Our characterization of the properties of these sites suggested that GP activity consists of two activities: (i) binding to the glycogen molecule and (ii) phosphorolysis of the non-reducing-end glucose residues. Activity (i) is mainly due to the activities of the two storage sites, which depended on the ionic strength of the medium and were directly inhibited by cyclodextrins (CDs). Activity (i) is of benefit to GP because a high concentr...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Makino, Y., Fujii, Y., Taniguchi, M. Tags: Regular Papers Source Type: research
Substrate-dependent nitric oxide synthesis by secreted endoplasmic reticulum aminopeptidase 1 in macrophages
In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)- and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. The...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Goto, Y., Ogawa, K., Nakamura, T. J., Hattori, A., Tsujimoto, M. Tags: Regular Papers Source Type: research
MicroRNAs as the fine-tuners of Src oncogenic signalling
The cellular Src (c-Src) tyrosine kinase is upregulated and believed to play a pivotal role in various human cancers. However, the molecular mechanism underlying c-Src-mediated tumour progression remains elusive. Recent studies have revealed that several microRNAs (miRNAs) function as tumour suppressors by regulating the malignant expression of signalling molecules. Aberrant expression of miRNAs is frequently observed in human cancers and should be exploited to seek related molecular targets. In this review, we focus on miRNAs found to be involved in Src signalling in various cancers. We summarize recent findings on Src-re...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Oneyama, C., Okada, M. Tags: JB Reviews Source Type: research
The emerging role of calcium-modulating cyclophilin ligand in posttranslational insertion of tail-anchored proteins into the endoplasmic reticulum membrane
Tail-anchored (TA) proteins, a class of membrane proteins having an N-terminal cytoplasmic region anchored to the membrane by a single C-terminal transmembrane domain, are posttranslationally inserted into the endoplasmic reticulum (ER) membrane. In yeasts, the posttranslational membrane insertion is mediated by the Guided Entry of TA Proteins (GET) complex. Get3, a cytosolic ATPase, targets newly synthesized TA proteins to the ER membrane, where Get2 and Get3 constitute the Get3 receptor driving the membrane insertion. While mammalian cells employ TRC40 and WRB, mammalian homologs of Get3 and Get1, respectively, they lack...
Source: Journal of Biochemistry - May 27, 2015 Category: Biochemistry Authors: Yamamoto, Y., Sakisaka, T. Tags: JB Reviews Source Type: research
Improved solid-phase DNA probe method for tRNA purification: large-scale preparation and alteration of DNA fixation
In this study, we initially devised a large-scale purification system using tetraalkylammonium salts. The yield of tRNA was increased more than 10-fold and the manual steps were decreased as compared with the previous procedure. However, deterioration of column was very rapid owing to shedding of the biotinylated DNA probe. We therefore devised a method of covalent DNA fixation, in which a 5'-aminohexyl (dT)8 oligomer was fixed onto the N-hydroxysuccinimide-activated agarose, and then a DNA oligomer containing the tRNA and repeated A8 sequences was annealed. The probe sequence for tRNA purification was synthesized in colum...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Kazayama, A., Yamagami, R., Yokogawa, T., Hori, H. Tags: Regular Papers Source Type: research
Glucocorticoid augments lipopolysaccharide-induced activation of the I{kappa}B{zeta}-dependent genes encoding the anti-microbial glycoproteins lipocalin 2 and pentraxin 3
Bacterial lipopolysaccharide (LPS), one of the most potent inducers of inflammation, activates the transcription factor NF-B to induce expression of both proinflammatory mediators and anti-microbial glycoproteins such as lipocalin 2 (Lcn2) and pentraxin 3 (PTX3) in macrophages. Glucocorticoids are known to inhibit LPS-induced expression of proinflammatory cytokines via glucocorticoid receptor (GR)-mediated transrepression of NF-B, whereas their effect on induction of anti-microbial effectors has remained to be elucidated. Here we show that the synthetic glucocorticoid dexamethasone (Dex) strongly enhances LPS-induced trans...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Yamazaki, S., Akira, S., Sumimoto, H. Tags: Regular Papers Source Type: research
Redox-dependent DNA distortion in a SoxR protein-promoter complex studied using fluorescent probes
The [2Fe-2S] transcriptional factor SoxR, a member of the MerR family, is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters and functions as a sensor of oxidative stress in Escherichia coli. In the oxidized state, distortion of the target DNA promoter region initiates transcription by RNA polymerase, thereby activating transcription. The inactive reduced state of the protein has remained uncharacterized. Here, we directly observed redox-dependent conformational changes in the promoter DNA by site-specifically replacing selected adenine (A) and cytosine (C) bases in the promoter oligonucleotide with t...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Fujikawa, M., Kobayashi, K., Kozawa, T. Tags: Regular Papers Source Type: research
Identification of two promoters for human D-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2
d-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (–237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformat...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Tran, D. H., Shishido, Y., Chung, S. P., Trinh, H. T. T., Yorita, K., Sakai, T., Fukui, K. Tags: Regular Papers Source Type: research
Effect of nitric oxide on VnfA, a transcriptional activator of VFe-nitrogenase in Azotobacter vinelandii
The transcriptional activator, VnfA, is necessary for the expression of the structural genes encoding vanadium-dependent nitrogenase in Azotobacter vinelandii. We have previously reported that VnfA harbours a Fe-S cluster as a prosthetic group, presumably a 3Fe-4S type, which is vital for the transcriptionally active VnfA. A plausible effector molecule is a reactive oxygen species (ROS), which disassembles the Fe-S cluster switching the active VnfA to become fully inactive. This finding prompted us to investigate the effect of nitric oxide (NO), another physiologically important radical species on the VnfA activity. Unlike...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Miura, Y., Yoshimitsu, K., Takatani, N., Watanabe, Y., Nakajima, H. Tags: Regular Papers Source Type: research
Probe optimization for sequencing by ligation
Sequencing by ligation (SBL) is a straightforward enzymatic method for interrogating DNA sequence, in which the ligation efficiency and specificity of each probe play an essential role. Here, the number of labelled dyes in the probe, probe length and probe constituent were investigated to optimize the ligation efficiency and specificity. First, the performance of double- and single-labelled fluorescent probes in SBL was evaluated. The experimental results showed that double-labelled fluorescent probes could yield a remarkable increase in the fluorescence intensities and avoid higher background compared with single-labelled...
Source: Journal of Biochemistry - April 28, 2015 Category: Biochemistry Authors: Pu, D., Chen, J., Qian, X., Xiao, P. Tags: Regular Papers Source Type: research
