• BMB Reports
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• v.30 no.5
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• pp.303-307
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• 1997

Grb2, which is composed of a Src homology 2 (SH2) domain and two Src homology 3 (SH3) domains, is known to serve as an adaptor protein in signaling for Ras activation. Thus, a blocker of the Grb2 interactions with other proteins can be a potential candidate for an anticancer drug. In this study, we have developed a high throughput screening method for SH3 domain binding ligands and blockers. Firstly, we made and purified the glutathione S-transferase (GST)-fusion proteins with the Grb2 SH2 and SH3 domains, and the entire Grb2. This method measures the binding of a biotin-labeled oligopeptide, derived from a Grb2/SH3 binding motif in the hSos, to the GST-fusion proteins, which are precoated as glutathione S-transferase fusion protein on a solid phase. When $1\;{\mu}g$ of each fusion protein was used to coat the wells, both N- and C- terminal SH3 the domains as well as the whole of Grb2 were able to interact with the biotin-conjugated ligand peptide, while the SH2 domain and GST alone showed no binding affinity. Although N- and C- terminal SH3 domains showed an increase of binding to the ligand peptide in proportion to the amount of peptide, the GST fusion protein with Grb2 demonstrated much higher binding affinity. GST-Grb2 coating on the solid phase showed a saturation curve; 66 and 84% of the maximal binding was observed at 100 and 300 ng/$100\;{\mu}l$, respectively. This binding assay system was peptide sequence-specific, showing a dose-dependent inhibition with the unlabeled peptide of SH3 binding motif. Several other peptides, such as SH2 domain binding motifs and PTB domain binding motif, were ineffective to inhibit the binding to the biotin-conjugated ligand peptide. These results suggest that our method may be useful to screen for new anticancer drug candidates which can block the signaling pathways mediated by SH3 domain binding.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.607-611
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• 2004

Glycinin, one of the predominant storage proteins in soybeans, was examined as to whether it could be used as a calcium-binding mediator after chemical phosphorylation and enzymatic hydrolysis. Glycinin is composed of six subunits. One of the proglycinin subunits $(A_{la}B_{lb})$ was overexpressed in E. coli to obtain nonphosphorylated proteins with homogeneity. To investigate the enhanced calcium-binding properties of the phosphopeptides, the proglycinin was purified, phosphorylated, and hydrolyzed with trypsin. The proglycinin expressed in E. coli was purified by ammonium sulfate precipitation, ion-exchange chromatography, and cryoprecipitation. Chemical phosphorylation by sodium trimetaphosphate was performed to obtain phosphorylated proglycinin. After the phosphorylation, one-dimensional isoelectric focusing gel electroanalysis confirmed the phosphorylation of the proglycinin. The phosphorylated peptides were then hydrolyzed with trypsin, followed by a binding reaction with calcium chloride. The calcium-bound phosphopeptides were finally separated using immobilized metal $(Ca^{2+})$ chromatography. Consequently, a limited tryptic hydrolysate of the isolated phosphopeptides exhibited an enhanced calcium-binding ability, suggesting the potential of glycinin phosphopeptides as a calcium-binding mediator with greater availability.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.10
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• pp.1484-1489
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• 2006

This study was carried out to investigate the effect of high pressure and the addition of non-meat proteins on the physico-chemical and binding properties of restructured pork. Pressurizations were carried out at up to 200 MPa and non-meat proteins used as a binder were isolated soy protein (ISP), sodium caseinate (SC), whey protein concentrate (WPC) and egg white powder (EWP). The pH values of all treatments were affected by the level of pressure. $L^*$-value of all treatments increased significantly (p<0.05), while both $a^*$-value and $b^*$-value of all treatments showed a significant decrease (p<0.05) with increasing pressure level. Binders could contribute only additive effects on both pH and color of the treatments. It was found that high pressure improved the water binding capacities and binding strength of the treatments. Binders also improved the binding strength of restructured pork. However, SC and WPC had no effect on water binding properties under high pressure. These results indicate that the application of high pressure had more significant effect on restructuring meat than binders.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.66-66
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• 2003

In skeletal muscle cells, depolarization of the transverse tubules (T-tubules) results in Ca$\^$2+/ release from the sarcoplasmic reticulum (SR), leading to elevated cytoplasmic Ca$\^$2+/ and muscle contraction. This process has been known as excitation-contraction coupling (E-C coupling). Several proteins, such as the ryanodine receptor (RyR), triadin, junctin, and calsequestrin (CSQ), have been identified to be involved in the Ca$\^$2＋/ release process. However, the molecular interactions between the SR proteins have not been resolved. In the present study, the mechanisms of interaction between RyRl and triadin have been studied by in vitro protein binding and $\^$45/Ca$\^$2+/ overlay assays. Our data demonstrate that the intraluminal loop II of RyR1 binds to triadin in Ca$\^$2+/-independent manner. Moreover, we could not find any Ca$\^$2+/ binding sites in the loop II region. GST-pull down assay revealed that a KEKE motif of triadin, which was previously identified as a CSQ binding site (Kobayasi et al.,2000 JBC) was also a binding site for RyR1. Our results suggest that the intraluminal loop II of RyR could participate in the RyR-mediated Ca$\^$2+/ release process by offering a direct binding site to luminal triadin.

• Journal of the Korean Magnetic Resonance Society
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• v.25 no.2
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• pp.17-23
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• 2021

High mobility group protein B1 (HMGB1) is a highly conserved, non-histone, chromatin associated nuclear protein encoded by HMGB1 gene. HMGB1 proteins may be general co-factors in Hox-mediated transcriptional activation that facilitate the access of Hox proteins to specific DNA targets. It is unclear that the exact binding interface of Hoxc9DBD and HMGB1. To identify the interface and binding affinity of Hoxc9DBD and HMGB1 A-box, the paramagnetic probe, MTSL was used in NMR titration experiment. It is attached to the N-terminal end of HMGB1 A-box by reaction with thiol groups. The backbone assignment of HMGB1 A-box was achieved with 3D NMR techinques. The ¹⁵N-labeled HMGB1 A-box was titrated with MTSL-labeled Hoxc9DBD respectively. Based on the chemical shift changes we can identify the interacting residues and further map out the binding sites on the protein structure. The NMR titration result showed that the binding interface of HMGB1 A-box is around loop-1 between helix-1 and helix-2. In addition, the additional contacts were found in N- and C-terminus. The N-terminal arm region of Hoxc9DBD is the major binding region and the loop between helix1 and helix2 is the minor binding region.

• Journal of Oral Medicine and Pain
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• v.24 no.3
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• pp.235-244
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• 1999

The present study was performed to investigate the binding between salivary proteins(low-molecular-weight mucin;MG2, amylase, proline-rich proteins;PRPs) and oral pathogens(Streptococcus gordonii, Actinomyces viscosus, Staphylococcus aureus) by using solid-phase assay. In the case of transferring proteins to Immobilon-P, S. gordonii binds to MG2. A. viscosus binds to MG2, amylase, and PRPs, and S. aureus binds to MG2 and amylase. On nitrocellulose membrane, S, gordonii and A. viscosus bind to MG2, amylase, and PRPs. S. aureus binds to MG2 and PRPs. However, rabbit anti-A. viscosus antisera and rabbit anti-S. aureus antisera showed cross reactivity to PRPs adsorbed to only nitrocellulose membrane in negative control experiments, which were done without bacterial overlay. The results were different according to the membrane used as solid-phase, which reflected the assay-sensitive nature of binding experiment. PRPs and amylase are known to be components of tooth enamel pellicle. In addition, there was experimental evidence that PRPs and MG2 may covalently bind to oral mucosal epithelium. Considering above facts, the results of the present study can provide information on the interactions between salivary proteins and oral bacteria on tooth and oral mucosal surfaces.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2019-2029
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• 2016

Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two cysteine and two histidine residues ($Cys_2His_2$) coordinate to the zinc ion for the structural functions to generate a ${\beta}{\beta}{\alpha}$ fold, and this secondary structure supports specific interactions with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In this account, the structural similarity and differences of well-known $Cys_2His_2$-type zinc fingers such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology. These zinc finger proteins have different numbers of domains for their structural roles to maintain biological progress through transcriptional regulations from exogenous stresses. The superimposed structures of these finger domains provide interesting details when these fingers are applied to specific gene binding and editing. The structural information in this study will aid in the selection of unique types of zinc finger applications in vivo and in vitro approaches, because biophysical backgrounds including complex structures and binding affinities aid in the protein design area.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.12
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• pp.5315-5320
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• 2016

Purpose: Tumor cell growth and sensitivity to chemotherapy depend on many factors, among which insulin-like growth factors (IGFs) may play important roles. The aim of the present study was to evaluate the levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in primary tumors and ascites as predictors of response to neoadjuvant chemotherapy in ovarian cancer (OC) patients. Materials and Methods: Tumor tissue samples and ascitic fluid were obtained from 59 patients with advanced OC. The levels of IGF-I, IGF-II, IGFBP-3, IGFBP-4 and PAPP-A were determined using ELISA kits. Taking into account the data on expression of these IGF-related proteins and outcome, logistic regression was performed to identify predictors of response to neoajuvant chemotherapy. Results: Human ovarian tumors expressed IGFs, IGFBP-3, IGFBP-4 and PAPP-A and these proteins were also present in ascites fluid and associated with its volume. IGFs and IGFBPs in ascites and soluble PAPP-A might play a key role in ovarian cancer progression. However, levels of proteins of the IGF system in tumors were not significant predictors of objective clinical response (oCR). Univariate analysis showed that the level of IGF-I in ascites was the only independent predictor for oCR. Conclusion: The level of IGF-I in ascites was shown to be an independent predictor of objective clinical response to chemotherapy for OC patients treated with neoadjuvant chemotherapy and debulking surgery.

• 대한생식의학회:학술대회논문집
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• 2001.11a
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• pp.57-62
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• 2001

One of recent advances of mammalian fertilization is the understanding of the molecular basis of fertilization. Several proteins localized in sperm nucleus or on sperm surface are necessary for the fertilization process. Protamines, sperm nuclear proteins, are required for normal sperm function that leads to fertilization. Fertilin and cyritestin are sperm surface proteins and essential for sperm-egg binding. Fertilin is also required for sperm transport in the female reproductive tracts. Metalloproteses on sperm plasma membrane are found to play a role in sperm-egg fusion. The functional analysis of these proteins provides a new insight into the molecular mechanisms underlying mammalian fertilization and male fertility.

• Molecules and Cells
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• v.20 no.1
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• pp.51-56
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• 2005

Excitation-contraction coupling (ECC) proteins in the human heart were characterized using human atrial tissues from different age groups. The samples were classified into one infant group (Group A: 0.2-7 years old) and three adult groups (Group B: 21-30; Group C: 41-49; Group D: 60-66). Whole homogenates (WH) of atrial tissues were assayed for ligand binding, $^{45}Ca^{2+}$ uptake and content of ECC proteins by Western blotting. Equilibrium [$^3H$]ryanodine binding to characterize the ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR) showed that the maximal [$^3H$]ryanodine binding ($B_{max}$) to RyR was similar in all the age groups, but the dissociation constant ($k_d$) of ryanodine was higher in the infant group than the adult groups. Oxalate-supported $^{45}Ca^{2+}$ uptake into the SR, a function of the SR SERCA2a activity, was lower in the infant group than in the adult groups. Similarly, [$^3H$]PN200-110 binding, an index of dihydropyridine receptor (DHPR) density, was lower in the infant group. Expression of calsequestrin and triadin assessed by Western blotting was similar in the infant and adult groups, but junctin expression was considerably higher in the adult groups. These differences in key ECC proteins could underlie the different $Ca^{2+}$ handling properties and contractility of infant hearts.