• Molecules and Cells
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• v.21 no.1
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• pp.34-41
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• 2006

The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.

• Mass Spectrometry Letters
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• v.13 no.4
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• pp.115-124
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• 2022

Emerging evidence has shown that RNA-binding proteins (RBPs) dynamically regulate all aspects of RNA in cells and involve in major biological processes of RNA, including splicing, modification, transport, transcription and degradation. RBPs, as powerful and versatile regulatory molecule, are essential to maintain cellular homeostasis. Perturbation of RNA-protein interactions and aberration of RBPs function is associated with diverse diseases, such as cancer, autoimmune disease, and neurological disorders. Therefore, it is crucial to systematically investigate the RNA-binding proteome for understanding interactions of RNA with proteins. Thanks to the development of the mass spectrometry, a variety of proteome-wide methods have been explored to define comprehensively RNA-protein interactions in recent years and thereby contributed to speeding up the study of RNA biology. In this review, we systematically described these methods and summarized the advantages and disadvantages of each method.

• BMB Reports
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• v.40 no.5
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• pp.740-748
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• 2007

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage ${\phi}11$ was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A ~19 kDa protein copurified with intact His-CI (~ 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At ~10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in ${\phi}11$ cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators $O_L$ and $O_R$, respectively. Equilibrium binding studies indicate that His-CI binds to $O_R$ with a little more strongly than $O_L$ and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures ($32-42^{\circ}C$). Both $O_L$ and $O_R$ harbor a nearly identical inverted repeat and studies show that ${\phi}11$ repressor binds to each repeat efficiently. Additional analyses indicate that ${\phi}11$ repressor, like $\lambda$ repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of ${\phi}11$ CI even nearly resembles to that of $\lambda$ phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of ${\phi}11$ repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.

• BMB Reports
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• v.31 no.4
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• pp.370-376
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• 1998

Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

• BMB Reports
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• v.40 no.1
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• pp.44-52
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• 2007

Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubule-binding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.

• Applied Microscopy
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• v.51
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• pp.17.1-17.10
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• 2021

Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69±19 pN for wild-type, 58±19 pN for M2, 53±19 pN for M3, and 49±19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

• BMB Reports
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• v.28 no.3
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• pp.275-280
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• 1995

The respiratory burst oxidase of neutrophils is a multicomponent enzyme, domant in resting cells, that catalyzes the reduction of oxygen to $O_{2}^{-}$ at the expense of NADPH. In the resting neutrophil, some of the components of the oxidase, including proteins p47 and p67, are in the cytosol, while the rest are in the plasma membrane. Recent evidence has suggested that at least some of the cytosolic oxidase components exist as a complex. The cytosolic complex with a molecular weight of ~240 kDa was found to bind to blue-agarose and 2',5'-ADP-agarose, which recognize nucleotide requiring enzymes. In order to identify the nucleotide binding component of the cytosolic complex we purified recombinant p47 and p67 fusion proteins using the pGEX system. Pure recombinant p47 was retained completely on 2',5'-ADP-agarose, whereas pure recombinant p67 did not bind to these affinity beads. On the basis of these results, we infer that p47 may contain the nucleotide binding site.

• Journal of Industrial Technology
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• v.28 no.B
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Molecules and Cells
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• v.39 no.3
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• pp.179-185
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• 2016

Posttranscriptional regulation of RNA metabolism, including RNA processing, intron splicing, editing, RNA export, and decay, is increasingly regarded as an essential step for fine-tuning the regulation of gene expression in eukaryotes. RNA-binding proteins (RBPs) are central regulatory factors controlling posttranscriptional RNA metabolism during plant growth, development, and stress responses. Although functional roles of diverse RBPs in living organisms have been determined during the last decades, our understanding of the functional roles of RBPs in plants is lagging far behind our understanding of those in other organisms, including animals, bacteria, and viruses. However, recent functional analysis of multiple RBP family members involved in plant RNA metabolism and elucidation of the mechanistic roles of RBPs shed light on the cellular roles of diverse RBPs in growth, development, and stress responses of plants. In this review, we will discuss recent studies demonstrating the emerging roles of multiple RBP family members that play essential roles in RNA metabolism during plant growth, development, and stress responses.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.235-240
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• 2010

This study was performed to comprehend the plasma proteins expressed specifically during early pregnancy in pregnant or non-pregnant Hanwoo using proteomic analysis technique. Plasma samples (0, 2, 3, 4, 7, and 11 weeks after AI) were obtained from pregnant (P, n=3) or non-pregnant (NP, n=4) Hanwoo, respectively. To evaluate proteins differentially expressed, 2-dimensional electrophoresis (2DE) was conducted. Normalized protein spots were selected for the significant expression variation deviated over two fold in its expression level between two groups; Molecular functions of the proteins were DNA binding, protein binding, hemoglobin binding, ferrochelatase and transporter activity and arylestera, respectively. According to western blotting, haptoglobin was specifically expressed only in NP group during early pregnancy; however, paraoxonase 1 was highly expressed in pregnant group. Based on these results, pregnancy was maintained successfully by the activation of specific plasma proteins associated with immune system and antioxidant regulation during early pregnancy in Hanwoo.