• Molecules and Cells
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• 제43권5호
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• pp.469-478
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• 2020

Hepatitis C virus (HCV) propagation is highly dependent on cellular proteins. To identify the host factors involved in HCV propagation, we previously performed protein microarray assays and identified the LIM and SH3 domain protein 1 (LASP-1) as an HCV NS5A-interacting partner. LASP-1 plays an important role in the regulation of cell proliferation, migration, and protein-protein interactions. Alteration of LASP-1 expression has been implicated in hepatocellular carcinoma. However, the functional involvement of LASP-1 in HCV propagation and HCV-induced pathogenesis has not been elucidated. Here, we first verified the protein interaction of NS5A and LASP-1 by both in vitro pulldown and coimmunoprecipitation assays. We further showed that NS5A and LASP-1 were colocalized in the cytoplasm of HCV infected cells. NS5A interacted with LASP-1 through the proline motif in domain I of NS5A and the tryptophan residue in the SH3 domain of LASP-1. Knockdown of LASP1 increased HCV replication in both HCV-infected cells and HCV subgenomic replicon cells. LASP-1 negatively regulated viral propagation and thereby overexpression of LASP-1 decreased HCV replication. Moreover, HCV propagation was decreased by wild-type LASP-1 but not by an NS5A binding-defective mutant of LASP-1. We further demonstrated that LASP-1 was involved in the replication stage of the HCV life cycle. Importantly, LASP-1 expression levels were increased in persistently infected cells with HCV. These data suggest that HCV modulates LASP-1 via NS5A in order to regulate virion levels and maintain a persistent infection.

• 한국축산식품학회지
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• 제40권4호
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• pp.588-600
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• 2020

The effect of age (22, 30, 38, and 46 days) on Beijing duck breast myosin gels was investigated. The results showed that the water holding capacity (WHC) and gel strength were markedly improved at the age of 30 days. Differential scanning calorimetry suggested that the myosin thermal ability increased at the age of 30 and 38 days (p<0.05). A compact myosin gel network with thin cross-linked strands and small regular cavities formed at the age of 30 days, which was resulted from the higher content of hydrophobic interactions and disulfide bonds. Moreover, the surface hydrophobicity of myosin extracted from a 30-day-old duck breast decreased significantly under temperature higher than 80℃ (p<0.05). This study illustrated that myosin extracted from a 30-day-old duck's breast enhanced and stabilized the WHC, thermal stability and molecular forces within the gel system. It concluded that age is an essential influencing factor on the myosin thermal stability and gel quality of Beijing duck due to the transformation of fibrils with different myosin character.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.325-330
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• 2005

We are generally interested in the analysis, detection and prediction of structural motifs in proteins, in order to infer compatibility of amino acid sequence to structure in proteins of known three-dimensional structure available in the Protein Data Bank. In this context, we are analyzing some of the well-characterized structural motifs in proteins. We have analyzed simple structural motifs, such as, ${\beta}$-turns and ${\gamma}$-turns by evaluating the statistically significant type-dependent amino acid positional preferences in enlarged representative protein datasets and revised the amino acid preferences. In doing so, we identified a number of ‘unexpected’ isolated ${\beta}$-turns with a proline amino acid residue at the (i+2) position. We extended our study to the identification of multiple turns, continuous turns and to peptides that correspond to the combinations of individual ${\beta}$ and ${\gamma}$-turns in proteins and examined the hydrogen-bond interactions likely to stabilize these peptides. This led us to develop a database of structural motifs in proteins (DSMP) that would primarily allow us to make queries based on the various fields in the database for some well-characterized structural motifs, such as, helices, ${\beta}$-strands, turns, ${\beta}$-hairpins, ${\beta}$-${\alpha}$-${\beta}$, ${\psi}$-loops, ${\beta}$-sheets, disulphide bridges. We have recently implemented this information for all entries in the current PDB in a relational database called ODSMP using Oracle9i that is easy to update and maintain and added few additional structural motifs. We have also developed another relational database corresponding to amino acid sequences and their associated secondary structure for representative proteins in the PDB called PSSARD. This database allows flexible queries to be made on the compatibility of amino acid sequences in the PDB to ‘user-defined’ super-secondary structure conformation and vice-versa. Currently, we have extended this database to include nearly 23,000 protein crystal structures available in the PDB. Further, we have analyzed the ‘structural plasticity’ associated with the ${\beta}$-propeller structural motif We have developed a method to automatically detect ${\beta}$-propellers from the PDB codes. We evaluated the accuracy and consistency of predicting ${\beta}$ and ${\gamma}$-turns in proteins using the residue-coupled model. I will discuss results of our work and describe databases and software applications that have been developed.

• 대한의생명과학회지
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• 제12권2호
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• pp.73-79
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• 2006

Nebulin is a giant ($600{\sim}900$ kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin filamints in vertebrate skeletal muscles. Recently, There is an evidence that the nebulin also expressed in non muscle tissue, brain and liver. We identified a new isoform of nebulin from adult brain library by PCR screening. It contains two simple-repeats exon 165, 166 and linker-repeats exon $154{\sim}161$ except exon 159. The nebulin modules M160 to M170 (exon 150 to exon 161) has been shown to bind desmin. In mature striated muscle, desmin intermediate filaments surround Z-discs and link individual myofibrils laterally at their Z-discs and to other intracellular structures, including the costameres and the intercalated discs of the sarcolemma, sarcoplasmic reticulum, mitochondria, T-tubules, and nuclei. Therefore, it is an interesting possibility that the differential splice pathways within the linker region of nebulin modify the affinity of nebulin's interaction with desmin. The specific interactions of nebulin and desmin were confirmed in vivo by yeast two hybrid experiments. To verify in the cellular level the interaction between nebulin isoform and desmin, we transfected COS-7 cell with EGFP-tagged nebulin and DsRed-tagged desmin. Based on evidence showing that despite exon 159 was deleted, the new isoform of nebulin was interact with desmin. This suggest that nebulin in brain may interact with another intermediate filament. The conservation of these ligand-binding capacity in brain and skeletal nebulins suggest that nebulins may have conserved roles in brain and skeletal muscle.

• 한국수산과학회지
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• 제28권2호
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• pp.209-218
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• 1995

본 연구에서는 유전자 복제기작을 생화학적, 분자생물학적 방법을 사용하여 bacteriphage T7을 대상으로 연구하였다. Bacteriophage T7의 유전자 복제, 재조합, 수선시 필수 단백질로 작용하는 gene 2.5 단백질의 생체내 기능에 대한 유전학적 연구와 단백질을 분리 정제하여 복제 단백질들과의 상호작용에 대한 연구를 수행하였다. 연구결과 gene 2.5 단백질은 DNA복제시 필수 구성단백질로 작용하며, 복제과정에서 유전자 복제에 관여하는 핵심 단백질들인 DNA polymerase, helicase/primase와 직접 단백질-단백질 상호 협동 작용을 하는 r것을 증명하였다. 특히 gene 2.5 단백질의 C-terminal domain이 절편된 변이체의 경우 복제 단백질들과 상호작용이 결여되었다. 따라서 C-terminal domain이 gene 2.5 단백질의 기능에 필수적으로 관여함을 입증하였다.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2003년도 제2차 연례학술대회 발표논문집
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• pp.26-51
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• 2003

Large scale protein interaction maps provide a new, global perspective with which to analyse protein function. PSIMAP, the Protein Structural Interactome Map, is a database of all the structurally observed interactions between superfamilies of protein domains with known three-dimensional structure in thePDB. PSIMAP incorporates both functional and evolutionary information into a single network. It makes it possible to age protein domains in terms of taxonomic diversity, interaction and function. One consequence of it is to predict the most important protein domain structure in evolution. We present a global analysis of PSIMAP using several distinct network measures relating to centrality, interactivity, fault-tolerance, and taxonomic diversity. We found the following results: ${\bullet}$ Centrality: we show that the center and barycenter of PSIMAP do not coincide, and that the superfamilies forming the barycenter relate to very general functions, while those constituting the center relate to enzymatic activity. ${\bullet}$ Interactivity: we identify the P-loop and immunoglobulin superfamilies as the most highly interactive. We successfully use connectivity and cluster index, which characterise the connectivity of a superfamily's neighbourhood, to discover superfamilies of complex I and II. This is particularly significant as the structure of complex I is not yet solved. ${\bullet}$ Taxonomic diversity: we found that highly interactive superfamilies are in general taxonomically very diverse and are thus amongst the oldest. This led to the prediction of the oldest and most important protein domain in evolution of lift. ${\bullet}$ Fault-tolerance: we found that the network is very robust as for the majority of superfamilies removal from the network will not break up the network. Overall, we can single out the P-loop containing nucleotide triphosphate hydrolases superfamily as it is the most highly connected and has the highest taxonomic diversity. In addition, this superfamily has the highest interaction rank, is the barycenter of the network (it has the shortest average path to every other superfamily in the network), and is an articulation vertex, whose removal will disconnect the network. More generally, we conclude that the graph-theoretic and taxonomic analysis of PSIMAP is an important step towards the understanding of protein function and could be an important tool for tracing the evolution of life at the molecular level.

• BMB Reports
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• 제39권4호
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• pp.339-345
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• 2006

The blood-neural barrier (BNB), including blood-brain barrier (BBB) and blood-retinal barrier (BRB), is an endothelial barrier constructed by an extensive network of endothelial cells, astrocytes and neurons to form functional 'neurovascular units', which has an important role in maintaining a precisely regulated microenvironment for reliable neuronal activity. Although failure of the BNB may be a precipitating event or a consequence, the breakdown of BNB is closely related with the development and progression of CNS diseases. Therefore, BNB is most essential in the regulation of microenvironment of the CNS. The BNB is a selective diffusion barrier characterized by tight junctions between endothelial cells, lack of fenestrations, and specific BNB transporters. The BNB have been shown to be astrocyte dependent, for it is formed by the CNS capillary endothelial cells, surrounded by astrocytic end-foot processes. Given the anatomical associations with endothelial cells, it could be supposed that astrocytes play a role in the development, maintenance, and breakdown of the BNB. Therefore, astrocytes-endothelial cells interaction influences the BNB in both physiological and pathological conditions. If we better understand mutual interactions between astrocytes and endothelial cells, in the near future, we could provide a critical solution to the BNB problems and create new opportunities for future success of treating CNS diseases. Here, we focused astrocyte-endothelial cell interaction in the formation and function of the BNB.

• BMB Reports
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• 제30권5호
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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 Applied Biological Chemistry
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• 제60권2호
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• pp.179-184
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• 2017

톨라신은 1.9 kDa의 펩티드 독소로서 Pseudomonas tolaasii에 의해 생성되며, 재배중 느타리버섯에 갈반병을 일으킨다. 톨라신은 막에 pore를 형성하여 세포 구조를 파괴하고, 버섯 재배의 생산성을 심하게 감소시킨다. 톨라신에 의한 세포독성의 작용 기작은 완전히 밝혀지지 않았지만, 분자다중화에 의해 세포막에 채널구조 형성으로 이루어진다. 그러므로, 톨라신과 작용하는 식품첨가물 중에 톨라신의 다중화결합을 통한 세포막 pore 형성을 저해하는 물질이 있을 것이다. 본 연구에서는, 다양한 물질들이 톨라신의 활성을 저해함을 확인하고, 이들을 톨라신 저해물질(TIF)이라 명명하였다. 대부분의 톨라신 저해물질들은 식품가공과정에 쓰이는 유화제였다. 다양한 종류의 저해물질 중에 지방산과 에스터 결합한 polyglycerol과 지방산과 에스터 결합한 sucrose 화합물이 $10^{-4}-10^{-5}M$ 농도범위에서 톨라신의 세포독성을 효과적으로 저해하였다. 이러한 저해물질들은 균상재배하는 느타리버섯에서 갈반병의 발생을 성공적으로 억제하였다.

• Journal of Microbiology and Biotechnology
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• 제27권6호
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• pp.1138-1149
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• 2017

The use of microalgal biomass is an interesting technology for the removal of heavy metals from aqueous solutions owing to its high metal-binding capacity, but the interactions with bacteria as a strategy for the removal of toxic metals have been poorly studied. The goal of the current research was to investigate the potential of Burkholderia tropica co-immobilized with Chlorella sp. in polyurethane discs for the biosorption of Hg(II) from aqueous solutions and to evaluate the influence of different Hg(II) concentrations (0.041, 1.0, and 10 mg/l) and their exposure to different contact times corresponding to intervals of 1, 2, 4, 8, 16, and 32 h. As expected, microalgal bacterial biomass adhered and grew to form a biofilm on the support. The biosorption data followed pseudo-second-order kinetics, and the adsorption equilibrium was well described by either Langmuir or Freundlich adsorption isotherm, reaching equilibrium from 1 h. In both bacterial and microalgal immobilization systems in the co-immobilization of Chlorella sp. and B. tropica to different concentrations of Hg(II), the kinetics of biosorption of Hg(II) was significantly higher before 60 min of contact time. The highest percentage of biosorption of Hg(II) achieved in the co-immobilization system was 95% at pH 6.4, at 3.6 g of biosorbent, $30{\pm}1^{\circ}C$, and a mercury concentration of 1 mg/l before 60 min of contact time. This study showed that co-immobilization with B. tropica has synergistic effects on biosorption of Hg(II) ions and merits consideration in the design of future strategies for the removal of toxic metals.