• Molecules and Cells
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• 제38권2호
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• pp.180-186
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• 2015

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the ${\alpha}$-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.

• The Korean Journal of Physiology and Pharmacology
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• 제8권3호
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• pp.141-146
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• 2004

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins, composed of two presynaptic membrane proteins [synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin] and a presynaptic vesicular protein [vesicle-associated membrane protein (VAMP)], serve as a core of exocytotic fusion machinery, which can be affected by ischemia. Synaptic protein in core region, striatum and cortex has been shown to alter after focal ischemia, however, little is known in hippocampus. Hippocampus is remote from ischemic core, but it is one of the most vulnerable regions. Using immunohistochemistry, the present study was undertaken to investigate the alteration of expression of SNAP-25, syntaxin, and VAMP in the hippocampus of rats which were subjected to middle cerebral artery occlusion (MCAO) for 2h and allowed to reperfuse. At 2 weeks of reperfusion, the SNAP-25 and syntaxin immunoreactivity was increased in the stratum oriens of the CA1 and the stratum lucidum of the CA3 in the ipsilateral hippocampus. However, VAMP immunoreactivity didn't show significant change. These results demonstrate that the level of the presynatpic plasma membrane proteins (SNAP-25 and syntaxin) in the rat hippocampus is more sensitively affected by focal ischemia than that of the synaptic vesicle protein (VAMP).

• Applied Microscopy
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• 제28권4호
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• pp.577-590
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• 1998

Puromycin aminonucleoside (PAN) nephropathy was induced in a group of Sprague-Dawley rat by a single dose of intraperitoneal Injection to study an ultrastructural change of glomerulus. The experimental rats developed proteinuria three days after PAN injection. Electron microscopic studies of glomeruli showed the loss of epithelial foot processes, formation of cytoplasmic vacuoles, microvillous formation and increased numbers of lysosomes in the cytoplasm of podocytes. It is strongly suggested that proteinuria in PAN nephrosis may be primarily due to a glomerular epithelial lesion, leading to focal disarray of anionic sites or focal defects in the epithelial covering of the basement membrane. The loss of anionic sites in the basement membrane nay be caused by the foot process fusion and the epithelial detachment from the basement membrane.

• BMB Reports
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• 제54권2호
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• pp.118-123
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• 2021

The bacterial effector protein RavZ from a pathogen can impair autophagy in the host by delipidating the mammalian autophagy-related gene 8 (mATG8)-phosphatidylethanolamine (PE) on autophagic membranes. In RavZ, the membrane-targeting (MT) domain is an essential function. However, the molecular mechanism of this domain in regulating the intracellular localization of RavZ in cells is unclear. In this study, we found that the fusion of the green fluorescent protein (GFP) to the MT domain of RavZ (GFP-MT) resulted in localization primarily to the cytosol and nucleus, whereas the GFP-fused duplicated-MT domain (GFP-2xMT) localized to Rab5- or Rab7-positive endosomes. Similarly, GFP fusion to the catalytic domain (CA) of RavZ (GFP-CA) resulted in localization primarily to the cytosol and nucleus, even in autophagy-induced cells. However, by adding the MT domain to GFP-CA (GFP-CA-MT), the cooperation of MT and CA led to localization on the Rab5-positive endosomal membranes in a wortmannin-sensitive manner under nutrient-rich conditions, and to autophagic membranes in autophagy-induced cells. In autophagic membranes, GFP-CA-MT delipidated overexpressed or endogenous mATG8-PE. Furthermore, GFP-CA△α3-MT, an α3 helix deletion within the CA domain, failed to localize to the endosomal or autophagic membranes and could not delipidate overexpressed mATG8-PE. Thus, the CA or MT domain alone is insufficient for stable membrane localization in cells, but the cooperation of MT and CA leads to localization to the endosomal and autophagic membranes. In autophagic membranes, the CA domain can delipidate mATG8-PE without requiring substrate recognition mediated by LC3-interacting region (LIR) motifs.

• 한국자기공명학회논문지
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• 제13권2호
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• pp.96-107
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• 2009

Lactophoricin (LPcin-I) is a cationic amphipathic peptide with 23-mer peptide, and corresponds to the carboxy terminal 113-135 region of Component-3 of proteose-peptone. LPcin-I is a good candidate as a peptide antibiotic, because it has an antibacterial activity, but no hemolytic activity. On the other hand, its shorter analog (LPcin-II), which corresponds to the 119-135 region of PP3, has no antibacterial activity. In order to understand the structure-activity relationship under the membrane environments, we succeed to produce large amounts of LPcin-I and LPcin-II peptides. Peptides were over expressed in the form of fusion protein in Escherichia coli, and purified with several chromatography techniques. In this paper, we introduce the optimizing processes of purification and NMR measurement.

• Journal of Plant Biotechnology
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• 제36권3호
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• pp.268-274
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• 2009

Porphyromonas gingivalis, the gram-negative anaerobic oral bacterium, initiates periodontal disease by binding to saliva-coated oral surface. The cholera toxin B subunit (CTB) genetically linked to FimA1 (1-200 aa) or FimA2 (201-337 aa) of the P. gingivalis fimbrial antigen were introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation method. The integration of CTB-FimA1 or CTB-FimA2 fusion genes were confirmed in the chromosome of transformed leaves by genomic DNA PCR amplification method. Synthesis and assembly of the CTB-FimA fusion proteins into oligomeric structures with pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding activities of CTB-FimA fusion proteins to intestinal epithelial cell membrane receptors were confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA showed that the expression levels of the CTB-FimA1 or CTB-FimA2 fusion proteins were 0.0019, 0.002% of the total soluble protein in transgenic tuber tissues, respectively The synthesis of CTB-FimA monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using edible plants for the production of enterocyte targeted fimbrial antigens that could elicit mucosal immune responses.

• Molecules and Cells
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• 제19권2호
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• pp.191-197
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• 2005

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD) is responsible for highly efficient protein transduction across plasma membranes. In a previous study, we showed that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. In this study, we fused the human SOD gene with a Tat PTD transduction vector at its N- and/or C-terminus. The fusion proteins (Tat-SOD, SOD-Tat, Tat-SOD-Tat) were purified from Escherichia coli and their ability to enter cells in vitro and in vivo compared by Western blotting and immunohistochemistry. The transduction efficiencies and biological activities of the SOD fusion protein with the Tat PTD at either terminus were equivalent and lower than the fusion protein with the Tat PTD at both termini. The availability of a more efficient SOD fusion protein provides a powerful vehicle for therapy in human diseases related to this anti-oxidant enzyme and to reactive oxygen species.

• 한국동물학회지
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• 제36권3호
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• pp.342-346
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• 1993

배양 근원세포의 세포 골격 단백질의 양이 분화과정에 따라 점차 감소하는 것으로 나타났다. 이러한 세포 골격 단백질의 분해는, 세포막에 투과성을 나타내는 calpain의 저해제인 calpeptin의 처리에 의하여 억제될 수 있었다. 또한, calpeptin은 특정 세포 골격 단백질의 분해를 제한적으로 억제하였으나, 전체적인 세포 단백질의 양상에는 별 영향을 주지 않았다. 뿐만 아니라, calpeptin은 농도 의존적으로 근원세포의 융합을 억제하였다. 반면에, calpain의 강력한 저해제이지만 세포막에 투과성을 보이지 않는 E-64는 세포 골격 단백질의 분해와 막 융합에 아무런 효과를 나타내지 못하였다. 이러한 결과는 calpain이 근세포 분화 시기에 따라 세포 골격 단백질의 분해를 촉매하며, 이 분해 과정은 근원세포 융합에 필연적인 것으로 추측된다. 또한, 이 결과는 calpain 저해제들의 선별적 효과가 그들의 세포막에 대한 투과성에 기인함을 시사한다.

• 한국식품과학회지
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• 제20권5호
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• pp.650-658
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• 1988

발아 중인 대두와 두채 자엽부에서의 단백지 유동 현상을 광학현미경(LM)과 주사 전자현미경(TEM)을 이용하여 epidermis, vascular tissue 그리고 storage parenchyma에서 비교 관찰하였다. 발아 초기의 자엽에는 직경이 $0.1-15{\mu}m$에 해당하는 다양한 크기의 단백질 체(protein bodies)로 채워져 있었으며 epidermis와 vascular tissue의 단백질체는 parenchyma 세포보다 훨씬 적은 크기의 $0.1-5{\mu}m$였다. 발아가 진행됨에 따라, 단백질의 분해현상은 epidermis와 vascular tissue에서 먼저 시작되었다. 그 분해 유형은 먼저 침투한 수분에 의해 팽창된 단백질체의 외막에는 손상이 없어 단백질 체 내부와 외부로부터 가수분해가 일어났으며, 단백질이 완전히 분해도기 전에 단백질체 끼리의 융합 또한 관찰 되어졌다. 단백질체 분해의 결과는 세포내에 중심액포를 만든다는 것이며, 두채 성장 말기에는 액포막의 파괴현상도 관찰되었다.

• Molecules and Cells
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• 제41권1호
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• pp.50-54
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• 2018

Atg5 and Atg7 have long been considered as essential molecules for autophagy. However, we found that cells lacking these molecules still form autophagic vacuoles and perform autophagic protein degradation when subjected to certain stressors. During this unconventional autophagy pathway, autophagosomes appeared to be generated in a Rab9-dependent manner by the fusion of vesicles derived from the trans-Golgi and late endosomes. Therefore, mammalian autophagy can occur via at least two different pathways; the Atg5/Atg7-dependent conventional pathway and an Atg5/Atg7-independent alternative pathway.