[Show abstract][Hide abstract] ABSTRACT: Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the SDS-solubilized membrane proteins from E. coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor type A (ETA) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.
Protein Science 12/2014; 23(12). DOI:10.1002/pro.2575 · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteomic analyses of the excretory/secretory proteins from the RH strain of Toxoplasma gondii have been performed to understand their functions in the host-parasite interaction. A total of 34 proteins were identified from LC/MS/MS analysis and their abundance was estimated by spectral counting methods. Among them, 8 species of micronemal proteins (MICs), 2 species of rhoptry proteins (ROPs), and 6 species of dense granular proteins (GRAs) were confirmed. Besides these, 18 species of protein were newly identified, and their cellular functions were estimated from sequence analysis. The three most abundant of the 34 identified extractor/secretory proteins-GRA1, GRA7 and GRA2-were confirmed to be highly expressed in T. gondii using the spectral count method. This phenomenon is another demonstration of the importance of GRA proteins for the penetration and survival of T. gondii.
Bulletin- Korean Chemical Society 10/2014; 35(10):3071-3076. DOI:10.5012/bkcs.2014.35.10.3071 · 0.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Alzheimer's disease, cytochrome c-dependent apoptosis is a crucial pathway in neuronal cell death. Although beta-amyloid (Aβ) oligomers are known to be the neurotoxins responsible for neuronal cell death, the underlying mechanisms remain largely elusive. Here, we report that the oligomeric form of synthetic Aβ of 42 amino acids elicits death of HT-22 cells. But, when expression of a bcl-2 family protein BAK is suppressed by siRNA, Aβ oligomer-induced cell death was reduced. Furthermore, significant reduction of cytochrome c release was observed with mitochondria isolated from BAK siRNA-treated HT-22 cells. Our in vitro experiments demonstrate that Aβ oligomers bind to BAK on the membrane and induce apoptotic BAK pores and cytochrome c release. Thus, the results suggest that Aβ oligomers function as apoptotic ligands and hijack the intrinsic apoptotic pathway to cause unintended neuronal cell death.
[Show abstract][Hide abstract] ABSTRACT: Rhoptry protein 6 (ROP6) from Toxoplasma gondii is a 480-amino acid protein with no homology to any reported excretory or secretory protein. Especially, unlike the many other rhoptry protein types, ROP6 does not have a kinase domain. The biochemical and biophysical properties of ROP6 are unknown. Here, we investigated its structure using an in silico analysis method and overexpression and purification using an Escherichia coli system. The protein was purified to more than 85% homogeneity using immobilized metal affinity chromatography in denaturing conditions. After purification, ROP6 showed slow migration in SDS-PAGE, including fast proteolysis. This implies that ROP6 has a high percentage of flexible regions or extended loop structures. Secondary structure prediction and prediction of intrinsically disordered regions by using various bioinformatics tools, indicated that approximately 60% of ROP6 is predicted to be intrinsically disordered or random coil regions. These observations indicate that ROP6 is an intrinsically disordered protein.
Protein Expression and Purification 06/2014; 101. DOI:10.1016/j.pep.2014.06.011 · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A derivative of Mycobacterium bovis Bacillus Calmette-Guerin (BCG) has been used for the preparation of tuberculosis vaccines. To establish a Korean tuberculosis vaccine derived from BCG-Pasteur , genome sequencing of a BCG-Korea strain was completed by Joung and coworkers. A comparison analysis of the genome sequences of the BCG-Pasteur and BCG-Korea strains showed marginal increases in the total genome length (~0.05%) and the number of genes (~4%) in the BCG-Korea genome. However, how the genomic changes affect the BCG-Korea protein expression levels remains unknown. Here, we provide evidence of the proteomic alterations in the BCG-Korea strain by using a SWATH-based mass spectrometric approach (Sequential Window Acquisition of all THeoretical mass spectra). Twenty BCG proteins were selected by top-rank identification in the BCG proteome analysis and the proteins were quantified by the SWATH method. Thirteen of 20 proteins showing significant changes were enough to discriminate between the two BCG proteomes. The SWATH method is very straightforward and provides a promising approach owing to its strong reliability and reproducibility during the proteomic analysis.
Bulletin- Korean Chemical Society 03/2014; 35(3). DOI:10.5012/bkcs.2014.35.3.933 · 0.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein kinase CK2 is a ubiquitous kinase that can phosphorylate hundreds of cellular proteins and plays important roles in cell growth and development. Deregulation of CK2 is related to a variety of human cancers, and CK2 is regarded as a suppressor of apoptosis; therefore, it is a target of anticancer therapy. Nucleolar phosphoprotein 140 (Nopp140), which is an intrinsically disordered protein, interacts with CK2 and inhibits the latter's catalytic activity in vitro. Interestingly, the catalytic activity of CK2 is recovered in the presence of d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6). IP6 is widely distributed in animal cells, but the molecular mechanisms that govern its cellular functions in animal cells have not been completely elucidated. In this study, the crystal structure of CK2 in complex with IP6 showed that the lysine-rich cluster of CK2 plays an important role in binding to IP6. The biochemical experiments revealed that a Nopp140 fragment (residues 568-596) and IP6 competitively bind to the catalytic subunit of CK2 (CK2α), and phospho-Ser574 of Nopp140 significantly enhances its interaction with CK2α. Substitutions of K74E, K76E, and K77E in CK2α significantly reduced the interactions of CK2α with both IP6 and the Nopp140-derived peptide. Our study gives an insight into the regulation of CK2. In particular, our work suggests that CK2 activity is inhibited by Nopp140 and reactivated by IP6 by competitive binding at the substrate recognition site of CK2.
Proceedings of the National Academy of Sciences 11/2013; 110. DOI:10.1073/pnas.1304670110 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phase contrast microscopy (PCM) is a widely used analytical method for airborne asbestos, but it is unable to distinguish asbestos from non-asbestos fibers and requires time-consuming and laborious manual counting of fibers. Previously, we developed a high-throughput microscopy (HTM) method that could greatly reduce human intervention and analysis time through automated image acquisition and counting of fibers. In this study, we designed a dual-mode HTM (DM-HTM) device for the combined reflection and fluorescence imaging of asbestos, and automated a series of built-in image processing commands of ImageJ software to test its capabilities. We used DksA, a chrysotile-adhesive protein, for selective detection of chrysotile fibers in the mixed dust-free suspension of crysotile and amosite prepared in the laboratory. We demonstrate that fluorescently-stained chrysotile and total fibers can be identified and enumerated automatically in a high-throughput manner by the DM-HTM system. Combined with more advanced software that can correctly identify overlapping and branching fibers and distinguish between fibers and elongated dust particles, the DM-HTM method should enable fully automated counting of airborne asbestos.
[Show abstract][Hide abstract] ABSTRACT: Uridinediphospho-N-acetylglucosamine enolpyruvyl transferase (MurA, E.C. 188.8.131.52) is an essential bacterial enzyme that catalyzes the first step of the cell wall biosynthetic pathway, which involves the transfer of an enolpyruvyl group from phosphoenolpyruvate to uridinediphospho-Nacetylglucosamine. In this study, novel inhibitors of Haemophilus influenzae MurA (Hi MurA) were identified using high-throughput screening of a chemical library from the Korea Chemical Bank. The identified compounds contain a quinoline moiety and have much lower effective inhibitory concentrations (IC50) than fosfomycin, a wellknown inhibitor of MurA. These inhibitors appear to covalently modify the sulfhydryl group of the active site cysteine (C117), since the C117D mutant Hi MurA was not inhibited by these compounds and excess dithiothreitol abolished their inhibitory activities. The increased mass value of Hi MurA after treatment with the identified inhibitor further confirmed that the active-site cysteine residue of Hi MurA is covalently modified by the inhibitor.
Journal of Microbiology and Biotechnology 03/2013; 23(3):329-34. · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Single-chain variable fragments of antibodies (scFv) specific to 2,4-dinitrotoluene (DNT) were isolated from a phage library displaying synthetic human scFv fragments with 6 diversified complementary determining regions (CDRs). A DNT derivative that contained an extended amine group was synthesized and conjugated to the NHS-group that was linked to magnetic beads. Phages specific to the immobilized DNT derivatives were isolated from the library after 4 rounds of sequential binding and elution processes. The displayed scFv fragments from the isolated phages showed consensus CDR sequences. One DNT-specific scFv was expressed in E. coli and purified using Ni-affinity chromatography. The purified DNT-specific scFv binds specifically to the immobilized DNT-derivative with K D value of 6.0 × 10 −7 M. The scFv and DNT interaction was not disrupted by the addition of 4-nitrotoluene or benzoic acid. These data demonstrate that the screened scFv from the phage displayed library could be used for selective and sensitive detection of explosives such as TNT.
Bulletin of the Korean Chemical Society 02/2013; 34(2):460-464. DOI:10.5012/bkcs.2013.34.2.460 · 0.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Uridinediphospho-N-acetylglucosamine enolpyruvyl transferase (MurA, E.C. 184.108.40.206) is an essential bacterial enzyme that catalyzes the first step of the cell wall biosynthetic pathway, which involves the transfer of an enolpyruvyl group from phosphoenolpyruvate to uridinediphospho-N-acetylglucosamine. In this study, novel inhibitors of Haemophilus influenzae MurA (Hi MurA) were identified using high-throughput screening of a chemical library from the Korea Chemical Bank. The identified compounds contain a quinoline moiety and have much lower effective inhibitory concentrations (IC 50) than fosfomycin, a well-known inhibitor of MurA. These inhibitors appear to covalently modify the sulfhydryl group of the active site cysteine (C117), since the C117D mutant Hi MurA was not inhibited by these compounds and excess dithiothreitol abolished their inhibitory activities. The increased mass value of Hi MurA after treatment with the identified inhibitor further confirmed that the active-site cysteine residue of Hi MurA is covalently modified by the inhibitor. Haemophilus influenzae is a Gram-negative bacterium that causes clinical diseases such as pneumonia and acute bacterial meningitis in infants and young children. The bacterial cell wall is composed mainly of peptidoglycan, and MurA catalyzes the first step in the biosynthesis of peptidoglycan. MurA transfers the enolpyruvyl group of phosphoenolpyruvate (PEP) to the 3'-hydroxyl group of UDP-N-acetylglucosamine (UNAG) to form UDP-N-acetylglucosamine (UDP-GlcNAc)-enolpyruvate . MurA is essential for cell growth, since deletion of the MurA gene in Escherichia coli or Streptococcus pneumoniae is lethal [5, 8]. The indispensability of MurA and its universal presence in bacterial but not in mammalian cells make MurA an attractive target for antibiotic development. The reaction catalyzed by MurA was initiated by the protonation of PEP and the deprotonation of the 3'-hydroxy group of UNAG, followed by nucleophilic addition at the oxocarbenium ion of PEP in a tetrahedral configuration. Inorganic phosphate was released from the tetrahedral adduct to produce enolpyruvyl-UDP-N-acetylglucosamine (EP-UNAG) (Fig. 1) [9, 22]. The crystal structures of MurAs from Escherichia coli. Enterobacter cloacae, and Haemophilus influenzae were highly homologous [21, 23, 25], and the active site was located in the cleft of two domains, and a flexible loop region covered the active site. MurA undergoes a conformational change from an "open" or substrate-free state to a "closed" or substrate-bound state during the catalytic reaction. Upon binding of UNAG to the active site of MurA, the loop region moves toward the active site to form the "closed" state. Studies have shown that substitution of a conserved cysteine residue (Cys117 in Hi MurA) into Ala or Ser completely abolished its catalytic activity ; thus, this residue (Cys117 in Hi MurA) in the loop is critical for the catalytic reaction. MurA acts as a general acid to transfer hydrogen to PEP during catalysis , and/or it is involved in phosphate release . The cysteine residue in the loop region is highly conserved in MurAs from various bacterial species except a few cases, such as in Chlamydia trachomatis or Mycobacterium tuberculosis MurA, in which the cysteine is replaced by an aspartate residue [7, 18]. The cysteine residue is also the target of fosfomycin, an antibiotic produced by Streptomyces . Fosfomycin forms a covalent adduct to the sulfhydryl group of cysteine to irreversibly inhibit MurA [15, 23]. Although fosfomycin can effectively inhibit the catalytic activity of MurA and the growth of some bacteria, fosfomycin resistance frequently occurs through overexpression or substitution of the MurA active-site cysteine [13, 16]. Additionally, modification or reduced uptake of fosfomycin leads to fosfomycin resistance [1, 4].
Journal of Microbiology and Biotechnology 01/2013; 23(3):329-334. DOI:10.4014/jmb.1210.10053 · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human serotonin receptor 3A (5-HT(3A)) is a ligand-gated ion channel regulated by serotonin. A fusion protein (P9-5-HT(3A)) of 5-HT(3A) with the P9 protein, a major envelope protein of bacteriophage phi6, was highly expressed in the membrane fraction of E. coli, and the expressed protein was purified to homogeneity using an affinity chromatography. P9-5-HT(3A) was observed as mixed oligomers in detergents. The purified P9-5-HT(3A) was efficiently reconstituted into proteoliposomes, and the serotonin-dependent ion-channel activity of P9-5-HT(3A) was observed by measuring the increased fluorescence of Fluo-3 attributed to the formation of a complex with the Ca(2+) ions released from the proteoliposomes. Alanine substitution for Trp178 of 5-HT(3A) abolished the serotonin-dependent ion-channel activity, confirming the importance of Trp178 as a ligand-binding site. Furthermore, the ion-channel activity of the reconstituted P9-5-HT(3A) was effectively blocked by treatment with ondansetron, an antagonist of 5-HT(3A). The bacterial expression system of human 5-HT(3A) and the proteoliposomes reconstituted with 5-HT(3A) would provide biophysical and structural analyses of 5-HT(3A).
Protein Expression and Purification 01/2013; DOI:10.1016/j.pep.2013.01.001 · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase that catalyzes the conversion of prostaglandin PGH(2) to PGE(2) and represents a novel target for therapeutic treatment of inflammatory disorders. It is essential to identify mPGES-1 inhibitor with novel scaffold as new hit or lead compound for the purpose of the next-generation anti-inflammatory drugs. Herein we report the discovery of sulfonamido-1,2,3-triazole-4,5-dicarboxylic derivatives as a novel class of mPGES-1 inhibitors identified through fragment-based virtual screening and in vitro assays on the inhibitory activity of the actual compounds. 1-[2-(N-Phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f) inhibits human mPGES-1 (IC(50) of 1.1μM) with high selectivity (ca.1000-fold) over both COX-1 and COX-2 in a cell-free assay. In addition, the activity of compound 6f was again tested at 10μM concentration in presence of 0.1% Triton X-100 and found to be reduced to 1/4 of its original activity without this detergent. Compared to the complete loss of activity of nuisance inhibitor with the detergent, therefore, compound 6f would be regarded as a partial nuisance inhibitor of mPGES-1 with a novel scaffold for the optimal design of more potent mPGES-1 inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Human microsomal prostaglandin E synthase-1 (mPGES-1) is an emerging drug target for inflammatory disorders and cancer suppression. Therefore, it is crucially important to discover mPGES-1 inhibitors with novel structural scaffolds for the development of anti-inflammatory drugs. Here, we report the mPGES-1 inhibitors identified through screening of a chemical library. Initial screening of 1841 compounds out of 200,000 in a master library resulted in 9 primary hits. From the master library, 387 compounds that share the scaffold structure with the 9 primary hit compounds were selected, of which 3 compounds showed strong inhibitory activity against mPGES-1 having IC(50) values of 1-3μM. Notably, a derivative of sulfonylhydrazide, compound 3b, inhibited the LPS-induced PGE(2) production in RAW 264.7 cells. This compound showed novel scaffold structure compared to the known inhibitors of mPGES-1, suggesting that it could be further developed as a potent mPGES-1 inhibitor.
[Show abstract][Hide abstract] ABSTRACT: 본 발명은 mPGES-1에 대하여 활성 저해 효과를 갖는 항염증제에 관한 것으로서, 보다 상세하게는 상기 화학식 1로 표시되는 화합물을 주요 성분으로 함유하는 염증을 일으키는데 관여하는 PGE2를 생성하는 mPGES-1에 대하여 활성 저해 효과를 갖는 항염증제에 관한 것이다. 본 발명의 상기 화학물을 포함하는 항염증제는 프로스타글란딘 E2 (PGE2) 합성 효소인 mPGES-1의 활성을 억제함으로서 프로스타글란딘 E2에 의하여 유발되는 염증을 억제함으로서 류마티즘 등 염증질환 또는 통증의 치료에 사용될 수 있다. 또한, PGE2의 증가는 전립선암이나 난소암 유발과 관련 있으므로, 본 발명의 mPGES-1의 억제화합물은 전립선암이나 난소암 치료제로 사용할 수 있다.
[Show abstract][Hide abstract] ABSTRACT: Nopp140 is a highly phosphorylated protein that resides in the nucleolus of mammalian cell and is involved in the biogenesis of the nucleolus. It interacts with a variety of proteins related to the synthesis and assembly of the ribosome. It also can bind to a ubiquitous protein kinase CK2 that mediates cell growth and prevents apoptosis. We found that Nopp140 is an intrinsically unfolded protein (IUP) lacking stable secondary structures over its entire sequence of 709 residues. We discovered that mitoxantrone, an anticancer agent, was able to enhance the interaction between Nopp140 and CK2 and maintain suppressed activity of CK2. Surface plasma resonance studies on different domains of Nopp140 show that the C-terminal region of Nopp140 is responsible for binding with mitoxantrone. Our results present an interesting example where a small chemical compound binds to an intrinsically unfolded protein (IUP) and enhances protein-protein interactions.
Bulletin of the Korean Chemical Society 06/2012; DOI:10.5012/bkcs.2012.33.6.2005 · 0.84 Impact Factor