• Title, Summary, Keyword: membrane fusion

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The Mechanism of Membrane Fusion During the Infection of HIV

  • Yu Yeon Gyu
    • Proceedings of the Microbiological Society of Korea Conference
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    • pp.97-101
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    • 2001
  • The fusion between viral envelope and target cell membrane is a central step of viral infection, and the fusion proteins located at viral envelope mediate such process. Gp41 of HIV is one of the fusion proteins whose structure and mechanism of membrane fusion had been extensively studied. Functionally important motives of gp41 are the N-terminus fusion peptide, the coiled-coil and the membrane proximal C-peptide regions. The role of these regions during the fusion process had been thoroughly examined. Specially, insertion of the fusion peptide into membrane and conformational change of the coiled-coil and C-peptide regions are assumed to be critical for the fusion mechanism. In addition, the coiled-coil region has been shown to interact with membrane, and the C-peptide region regulates the interaction in a dose dependent manner. Furthermore, fusion defective mutations of the coiled-coil region dramatically changed its binding affinity to membrane. These results suggested that the membrane binding property of the coiled-coil region is important for the fusion activity of gp41, and such property could be modulated by the interaction with the C-peptide region.

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Characterization of the Putative Membrane Fusion Peptides in the Envelope Proteins of Human Hepatitis B Virus

  • Kang, Ha-Tan;Yu, Yeon-Gyu
    • Bulletin of the Korean Chemical Society
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    • v.28 no.10
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    • pp.1756-1762
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    • 2007
  • Envelope proteins of virus contain a segment of hydrophobic amino acids, called as fusion peptide, which triggers membrane fusion by insertion into membrane and perturbation of lipid bilayer structure. Potential fusion peptide sequences have been identified in the middle of L or M proteins or at the N-terminus of S protein in the envelope of human hepatitis B virus (HBV). Two 16-mer peptides representing the N-terminal fusion peptide of the S protein and the internal fusion peptide in L protein were synthesized, and their membrane disrupting activities were characterized. The internal fusion peptide in L protein showed higher activity of liposome leakage and hemolysis of human red blood cells than the N-terminal fusion peptide of S protein. Also, the membrane disrupting activity of the extracellular domain of L protein significantly increased when the internal fusion peptide region was exposed to N-terminus by the treatment of V8 protease. These results indicate that the internal fusion peptide region of L protein could activate membrane fusion when it is exposed by proteolysis.

Analysis of fusogenic activity of autographa californica nuclear polyhedrosis virus (Ac NPV) gp64 envelope glycoprotein

  • Kim, Hee-Jin;Yang, Jai-Myung
    • Journal of Microbiology
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    • v.34 no.1
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    • pp.7-14
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    • 1996
  • Teh baculovirus gp64 glycoprotein is a major component of the envelope of budded virus (BV) and has been shown that it plays an essential role in the infection process, especially virus-cell membrane fusion. We have cloned Autographa californica Nuclear Polyhedrosis Virus (AcNPV) gp64 protein were examined for membrane fusion activity by using a synchtium formation assay under various conditions. The optimal conditions required for inducing membrane fusion are 1) form pH 4.0 to 4.8 2) 15 min exposure of cells to acidic pH 3) at least 1 .mu.g of gp64 cloned plasmid DNA per 3 * 10$^{6}$ cells 4) and an exposure of cells to acidic pH at 72 h post-transfection. In order to investigate the role of hydrophobicity of the gp64 glycoprotein for the membrane fusion, the two leucine residues (amino acid position at 229 and 230) within hydrophobic region I were substituted to alanine by PCR-derived site-directed mutagenisis and the membrane fusion activity of the mutant was anlaysed. The gp64 glycoprotein carrying double alamine substitution mutation showed no significant difference in fusion activity. This result suggested that minor changes in hydrophobicity at the amino acid position 229 and 230 does not affect the acid-induced membrane fusion activity of the gp64 glycoprotein.

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Membrane interaction of the coiled-coil motif of HIV gp41 and its implication in the membrane fusion process

  • Jin, Bong-Suk;Yu, Yeon-Gyu
    • Proceedings of the Korean Biophysical Society Conference
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    • pp.58-58
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    • 2003
  • The envelope glycoprotein of HIV, gp41, mediates the membrane fusion with human cells. The extracellular domain of gp41 has two helical regions. The N-terminus helical region (N-helix) forms trimeric coiled coil, interacts with the C-terminus helical region (C-helix) of gp41 to form a stable helical bundle structure. In this study, we have shown that the N-helix of gp41 has membrane interacting and disrupting abilities. It was localized into the interface of the lipidic phase and head group of the membrane. In contrast, the N-helix region with membrane fusion defective mutations could not bind to membrane. In addition, the N-helix bound on the membrane was released from the membrane by the C-helix, and the complex of the N- and C-helix did not interact with membrane. These results suggested that the membrane binding ability of the N-helix is necessary for the fusion activity of gp41, and such property is possibly controlled by the C-helm.

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Ultrastructural Process of Protoplast Fusion Between Lentinula edodes and Coriolus versicolor

  • Kim, Chae-Kyun;Kim, Byong-Kak
    • Mycobiology
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    • v.29 no.1
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    • pp.15-18
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    • 2001
  • Protoplast fusion is a useful technique for establishing fungal hybrids to overcome the natural barriers. The ultrastructure of protoplast and its fusion process were observed using a scanning electron microscopy(SEM) and a transmission electron microscopy(TEM). The protoplasts were variable in size from $0.5{\sim}15{\mu}m$ in diameter, and the mean diameter was about $3{\sim}5{\mu}m$. It was impossible to discriminate protoplasts of Lentinula edodes from protoplasts of Coriolus versicolor by size and surface structure. Big aggregates of the dehydrated protoplasts were observed, after polyethylene glycol 4000 treatment. Nucleus, mitochondria, lipid granules and various vesicles having granules were scattered in the cytoplasm. The vesicles were heterogeneous in size and vary from one protoplast to another. The fused membrane layer of the two protoplasts was observed. Time protoplast membrane contact and reorganization of membrane components were essential condition for protoplast fusion. Transmission electron micrograph showed fused protoplasts and flattening of the cells in the area of the membrane contact. We hope that our electron microscopic observations provide some insights into the understanding of the fusion process of protoplast in fungi.

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Ultrastructural Studies for Protoplasts and Protoplast Fusion in Streptomyces lavendulae (Streptomyces levendulae의 원형질체와 원형질체 융합에 대한 미세구조)

  • 하영칠;홍순우;유진철;임헌만
    • Korean Journal of Microbiology
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    • v.24 no.3
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    • pp.197-203
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    • 1986
  • Morphology and ultrastructure of protoplast fusion mode in Streptomyces lavendulae were studied by scanning and transmission electron microscopy. The isolated protoplasts were stable in some degree in hypertonic solution except that several protoplasts showed irregular morphology. Fusion events were occurred as follows; contact zone, fusion zone and separation zone were appeared sequentially. After formation of the separation zone, cytoplasm and DNA from both parents were mixed eventually. In the contact zone, two menbranes were still separated by electron transparent space. The contact zone changed to fusion zone by formation of fusion membrane that phospholipid molecules of two membranes were rearranged. Thereafter, nonmembraneous separation zone was formed by disappearance of fusion membrane. These changes were characterized by successive changes in typical membrane structure in fusion areas and by a progressive loss of bispherical shape.

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A possible role of lipopolysaccharides in the prevention of lysosome0symbiosome fusion as studied by microinjection of an anti-LPS monoclonal antibody (리소솜과 공생낭의 융합저해에서의 Lipopolysaccharide의 역할에 관한 연구)

  • Choi, Eui-Yul
    • Korean Journal of Microbiology
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    • v.32 no.4
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    • pp.280-284
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    • 1994
  • Lack of lysosomal fusion with symbiosomes in symbiont-bearing Amoeba proteus may be due either to the presence of a component in the symbiosome membrane or to the absence of a component needed in the fusion process. Using monoclonal antibody as a probe, lipopolysaccharides were identified as symbiosome-membrane components contributed by symbionts and were found to be exposed on the cytoplasmic side of the membrane. In order to test whether lipopolysaccharides may play a role in the prevention of lysosome-symbiosome fusion, the antilipopolysaccharides antibody was microinjected and processed for double immunostaining in conjuction with anti-lysosome antibody as a lysosome-fusion indicator. Microinjection of the anti-LPS antibody caused symbiosomes to fuse with lysosomes, suggesting that X-bacterial lipopolysaccharides could be 'fusion-preventing' factors.

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Effect of Retinoic Acid on Membrane Fusion and Expression of Fibronectin in Chick Embryonic Myoblasts (Retinoic Acid가 배양게배 근원세포의 융합과 Fibronictin의 발현에 미치는 영향)

  • 김혜선;정필중;강만식;정진하;하두봉
    • The Korean Journal of Zoology
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    • v.38 no.4
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    • pp.483-489
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    • 1995
  • Retinoic acid was found to block membrane fusion of chick embryonic myoblasts in culture. This effed was dosedependent and could he reversed upon removal of the agent from the culture medium. Furthermore, the retinoic acid-mediated inhibition of membrane fusion was observed with the fusion competent cells but not with the cells that had already been committed for fusion, indicating that the effect of RA is differentiation stage-specific. However, retinoic acid showed little or no effect on the ability of the cells to form bipolar shape and to align along their axes. Neither the cell proliferation nor accumulation of muscle specific proteins, such as creatine kinase and tropomyosin, was impaired significantly. On the other hand, retinoic acid blocked the differentiation time~ependent loss of fibronectin, whose process is prerequisite for myoblast fusion. These results suggest that retinoic add acts as a specific inhibitor of membrane fusion by preventing the loss of fibronectin from the differentiating myoblasts.

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Enzymatic Properties of a Fusion Protein between Human Cytochrome P450 1A1 and Rat NADPH-P450 Reductase Expressed in Escherichia Coli (대장균에서 발현된 인간 Cytochrome P450 1A1과 Rat NADPH-P450 Reductase와의 Fusion Protein의 효소 특성 연구)

  • 천영진;정태천;이현걸;한상섭;노정구
    • Toxicological Research
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    • v.12 no.2
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    • pp.155-161
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    • 1996
  • The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

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Oligomerization of the substitution mutants of autographa californica nuclear polyhedrosis Virus (AcNPV) gp64 glycoprotein

  • Kim, Ki-Nam;Poo, Ha-Ryoung;Yang, Jai-Myung
    • Journal of Microbiology
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    • v.35 no.1
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    • pp.72-77
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    • 1997
  • The baculovirus gp64 glycoprotein is a major component of the envelope protein of budded virus (BV). It has been shown that the gp64 glycoprotein plays an essential role in the infection process, especialy fusion between virus envelope and cellular endosomic membrane. Recently we reported optimal conditions required for gp64-mediated membrane fusion in pGP64 DNA transfected Spodoptera frugiperda (Sf9) cells (H. J. Kim and J. M. Yang, Jour, Microbiology, 34.7-14). In order to investigate the role of hydrophobicity within the fusion domain of the gp64 glycoprotein for membrane fusion, 13 mutants which have substitution mutation within hydrophobic region I were constructed by PCR-derived site-derected mutagenesis. Each mutated gp64 glycoproteins was transiently expressed by transfecting plasmid DNA into Spodoptera frugiperda (Sf9) cells. Oligomerization of the transisently expressed gp64 glycoproteins was a nalysed by running them on SDS-polyacrylamide gel electrophoresis under non-reducing condition followed by immunoblotting. All of the mutant gp64 glycoproteins expect cysteine-228 were able to form trimers. These results suggest that hydrophobic region I of the gp64 may not be responsible for the oligomerization of the gp64 glycoprotein.

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