• Korean Journal of Microbiology
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• v.51 no.4
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• pp.338-346
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• 2015

The present work was undertaken to address the role of protein disulfide isomerase 2 (Pdi2) in the mercury-tolerance of Schizosaccharomyces pombe, using the Pdi2-overexpressing recombinant plasmid pYPDI2 and the corresponding vector plasmid pRS316. When exposed to mercuric chloride, the PDI2 overepxression cells grew significantly better than the vector control cells. They revealed the lower levels of intracellular reactive oxygen species (ROS) and nitric oxide (NO), when incubated with mercuric chloride for 6 h, than the vector control cells. The PDI2 overepxression cells contained the higher levels of total glutathione (GSH) and superoxide dismutase (SOD) activity than the vector control cells, after 6 h of incubation in mercuric chloride. However, the PDI2 overepxression cells contained similar levels of glutathione peroxidase (GPx) activities, compared to those of the vector control cells. Taken together, the S. pombe Pdi2 promotes the tolerance against mercury toxicity through up-regulating total GSH and SOD and subsequently attenuating ROS and NO elevations.

• Archives of Pharmacal Research
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• v.26 no.12
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• pp.1047-1054
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• 2003

Molecular chaperones have a crucial role in the folding of nascent polypeptides in endoplasmic reticulum. Some of them are known to be sensitive to the modification by electrophilic metabolites of organic pro-toxicants. In order to identify chaperone proteins sensitive to alkyators, ER extract was subjected to alkylation by 4-acetamido-4 -maleimidyl-stilbene-2,2 -disulfonate (AMS), and subsequent SDS-PAGE analyses. Protein spots, with molecular mass of 160, 100, 57 and 36 kDa, were found to be sensitive to AMS alkylation, and one abundant chaperon protein was identified to be protein disulfide isomerase (PDI) in comparison with the purified PDI. To see the reactivity of PDI with cysteine alkylators, the reduced form ($PDI_{red}$) of PDI was incubated with various alkylators containing Michael acceptor structure for 30 min at $38^{\circ}C$ at pH 6.3, and the remaining activity was determined by the insulin reduction assay. Iodoacetamide or N-ethylmaleimide at 0.1 mM remarkably inactivated $PDI_{red}$ with N-ethylmaleimide being more potent than iodoacetamide. A partial inactivation of $PDI_{oxid}$ was expressed by iodoacetamide, but not N-ethylmaleimide (NEM) at pH 6.3. Of Michael acceptor compounds tested, 1,4-benzoquinone ($IC_{50}, 15 \mu$ M) was the most potent, followed by 4-hydroxy-2-nonenal and 1,4-naphthoquinone. In contrast, 1,2-naphthoquinone, devoid of a remarkable inactivation action, was effective to cause the oxidative conversion of $PDI_{red}$ to $PDI_{oxid}$. Thus, the action of Michael acceptor compounds differed greatly depending on their structure. Based on these, it is proposed that POI, one of chaperone proteins in ER, could be susceptible to endogenous or xenobiotic Michael acceptor compounds in vivo system.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.85-86
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• 2004

• Proceedings of the KAIS Fall Conference
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• 2012.05a
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• pp.239-242
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• 2012

효모 (Saccharomyces Cervisiae)는 단일 세포의 형태로 존재하는 진핵 세포로써 동물세포와 유사한 기작으로 분비성 단백질을 생성한다. 따라서 박테리아와 달리 효모를 이용하면 당단백질이나 이황결합을 포함하는 분비성 단백질을 경제적으로 대량 합성할 수 있다. 효모의 필수 단백질 중 하나인 단백질 이황이성질화 효소는 소포체에 위치하며 분비성 단백질에 구조적으로 안정한 이황결합을 제공하는 효소이다. 본 연구는 단백질 이황이성질화 효소 (protein disulfide isomerase)가 지니고 있는 두 개의 활성도메인 중 분비성 단백질들의 합성 효율에 직접적으로 관여하는 부위를 찾는 연구이다. 효모 유전체로부터 단백질 이황이성질화 효소의 유전자 (PDI1)을 제거하고 효소의 변이 유전자를 주입한 후 효모의 성장 속도를 측정하였다. 또한 효모의 대표적 분비성 단백질을 각 변이 효소를 지니는 효모에 과발현시켜 합성 및 이황결합 형성 효율을 측정하였다. 단백질 이황이성질화 효소내 두 개의 활성 부위 중 아미노 말단쪽에 위치한 a 도메인에 있는 활성 부위가 분비성 단백질의 활성에 중요한 역할을 한다는 것을 알 수 있었다. 이 결과는 이황결합이나 당을 포함하는 외래 단백질의 고효율 합성을 위한 새로운 효모종 개발에 중요한 정보를 제공할 것으로 기대 된다.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.989-992
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• 2003

Recombinant human tissue plasminogen activator deletion mutein (Reteplase) is a clinically promising thrombolytic drug. Reteplase cDNA was subcloned into a bacteria expression system, and the resultant recombinant was biologically characterized. The Reteplase was expressed in Escherichia coli as an inclusion body, and the downstream processes of the Reteplase inclusion body included denaturation, renaturation, and purification. A protein disulfide isomerase (PDI) was used to assist the refolding of Reteplase, and it was found to increase the refolding rate from less than 2％ to more than 20％. The refolded Reteplase was purified through two chromatography steps, including lysine-coupled agarose affinity chromatography and then CM-sepharose cation-exchange chomatography. The purity of r-PA was analyzed by Western bolt analysis, and N-terminal amino acid and amino acid composition analyses confirmed the end-product. Reteplase showed higher thrombolytic potency in an animal thrombus model.

• Journal of Korean Neurosurgical Society
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• v.59 no.2
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• pp.106-116
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• 2016

Objective : Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. Methods : U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. Results : Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). Conclusion : Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.

• Journal of Life Science
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• v.24 no.7
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• pp.743-749
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• 2014

The aim of this study was to identify the effect of fermented Angelica gigas Nakai (A. gigas) on lipid metabolism in orotic acid-induced fatty liver model rats. Sprague-Dawley male rats were randomly divided into four dietary groups (n=6 per group): a normal (N) group fed a standard diet only, OA control, OA acid plus 5% (w/w) A. gigas (OAG), and OA plus 5% (w/w) fermented A. gigas (OFAG). OA treatment induced enlargement of the liver and accumulation of hepatic triglycerides. The consum ption of fermented A. gigas reduced triglyceride concentrations in the liver and increased the serum lipid concentrations to normal levels. Furthermore, OA treatment significantly decreased serum triglyc eride concentrations without diminishing mRNA expression of microsomal triglyceride transfer protei n (MTP) and protein disulfide isomerase (PDI). Hepatic MTP mRNA expression increased 1.08-fold in response to OA treatment, despite triglyceride accumulation in the liver relative to that of the normal group. OFAG administration was slightly lower as compared to the OA treatment. This result suggests that MTP mRNA expression is not always correlated with hepatic triglyceride accumulation in the OA-induced fatty liver model. However, PDI mRNA expression was significantly increased in the OAG and OFAG groups (1.62-fold and 1.63-fold, respectively) compared with the normal group. The hepatocytes in the OA group contained numerous large fat droplets. These were slightly reduced in the OFAG group.

• Journal of Life Science
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• v.21 no.4
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• pp.613-615
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• 2011

ERp29 is a endoplasmic reticulum (ER) lumenal resident protein that shows sequence similarity to the protein disulfide isomerase family. Its biological function is thought to play a role in the processing of secretory proteins within the ER, possibly by participating in the folding of proteins in the ER. Although some data on ERp29 have been reported, its normal functions are still unclear. To gain insights into the function of ERp29, we identified ARF5 protein as a protein that interacts with ERp29 using yeast two-hybrid screening and GST pull-down assay. Interaction between ERp29 and ARF5 was detected under normal cell conditions but not under ER stress conditions. This result may provide a clue for understanding ERp29 biological functions.

• Journal of Life Science
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• v.20 no.11
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• pp.1577-1581
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• 2010

Granulocyte colony stimulating factor (G-CSF) is a hematopoietic cytokine that stimulates bone marrow cells to proliferate and differentiate into granulocytes. G-CSF is approved and used for therapeutic purposes. The endoplasmic reticulum (ER) signal peptide of hG-CSF was replaced with silkworm-specific signal peptides to express and efficiently secrete recombinant hG-CSF by silkworm cells. Plasmids that contain cDNAs for hG-CSF and hG-CSF fused with silkworm- specific signal peptides of prophenoloxidase activating enzyme (PPAE), protein disulfide isomerase (PDI), and bombyxin (BX) were constructed. The G-CSF protein was expressed in insect cell line BM5 and was detected by western blot analysis. The cells transfected with plasmids containing rhG-CSF genes with silkworm-specific signal sequences released mature rhG-CSF protein more efficiently than the cells transfected with pG-CSF, the plasmid containing human G-CSF gene, including its own signal sequence. The production of hG-CSF reached maximal level at four days post-transfection and remained at a high level until 7 days post-transfection. These data demonstrate that the modification of the human G-CSF mimic to insect proteins synthesized in ER greatly improves the production of the protein.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2001.04a
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• pp.30-30
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• 2001

No Abstract. See Full-text