• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.8
• /
• pp.3517-3522
• /
• 2015

Background: Glucose regulated protein 78 (GRP78) is a type of molecular chaperone. It is a possible candidate protein that contributes to development of drug resistance. We first examined the involvement of GRP78 in chemotherapy-resistance in human ovarian cancer cell. Materials and Methods: The expression of GRP78 mRNA and protein were examined by RT-PCR and western blotting, respectively, in human ovarian cancer cells line (HO-8910). Sensitivity of HO-8910 to paclitaxel was determined with methyl thiazolyl tetrazolium (MTT). Suppression of GRP78 expression was performed using specific small-interfering RNA (siRNA) in HO-8910 cells, and cell apoptosis was assessed by flow cytometry. Statistical analysis was performed using the SPSS 15.0 statistical package. Results: HO-8910 cells, with high basal levels of GRP78, exhibited low sensitivity to paclitaxel. The mRNA and protein levels of GRP78 were dramatically decreased at 24h, 48h and 72h after transfection and the sensitivity to paclitaxel was increased when the GRP78 gene was disturbed by specific siRNA transfection. Conclusions: The results suggested that high GRP78 expression might be one of the molecular mechanisms causing resistance to paclitaxel, and therefore siRNA of GRP78 may be useful in tumor-specific gene therapy for ovarian cancer.

• Molecules and Cells
• /
• v.37 no.12
• /
• pp.851-856
• /
• 2014

ATAD2, a remarkably conserved, yet poorly characterized factor is found upregulated and associated with poor prognosis in a variety of independent cancers in human. Studies conducted on the yeast Saccharomyces cerevisiae ATAD2 homologue, Yta7, are now indicating that the members of this family may primarily be regulators of chromatin dynamics and that their action on gene expression could only be one facet of their general activity. In this review, we present an overview of the literature on Yta7 and discuss the possibility of translating these findings into other organisms to further define the involvement of ATAD2 and other members of its family in regulating chromatin structure and function both in normal and pathological situations.

• Korean Journal of Microbiology
• /
• v.45 no.3
• /
• pp.239-245
• /
• 2009

Translation initiation factor eIF1A performs essential functions in various initiation steps including 43S preinitiation complex formation in eukaryotes, and contains a highly conserved oligonucleotide-binding (OB) fold. In our previous study, we discovered that eIF1A possesses RNA annealing activity and forms a stable complex with double-stranded RNA. In this study, we initiated site-directed mutations in eIF1A to find the active sites for these biochemical activities and to investigate whether they are essential functions for yeast cell growth. A truncated protein, eIF1A($\Delta$T), devoid of both N- and C-terminal domains but containing an intact OB-fold exhibited RNA annealing activity. In contrast, all point mutations in OB-fold domain, except R57D, impaired both RNA annealing and dsRNA binding activities, indicating that the intact OB-fold domain is required for both activities. Viabilities of the mutant yeast cells were not correlated with RNA annealing activity but with the in vivo protein stabilities in the case of R57D and K94D.

• Journal of Microbiology
• /
• v.38 no.3
• /
• pp.145-149
• /
• 2000

GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37$^{\circ}C$. However, GroEL production increased 3.4-fold at 42$^{\circ}C$. GroEL synthesis was not transient but continuous at 42$^{\circ}C$, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly , while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42$^{\circ}C$ were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42$^{\circ}C$.

• Parasites, Hosts and Diseases
• /
• v.55 no.6
• /
• pp.643-652
• /
• 2017

Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P-, and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion, and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed, and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehlis' gland, prostate gland, and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed.

• Molecules and Cells
• /
• v.24 no.2
• /
• pp.210-214
• /
• 2007

The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells.

• KSBB Journal
• /
• v.16 no.1
• /
• pp.1-10
• /
• 2001

Escherichia coli has been used as an expression work horse for foreign genes. This article summarized recent development in genetic engineering techniques for overproduction of medical proteins and industrial enzymes. Special emphasis was placed upon research activities concerning folding and refolding of inclusion bodies at genetic and fermentation levels. Plasmid and mRNA stabilization, development of strong inducible promoters, modification of translational elements and reduction of rpoteolytic degradation were carried out to elevate an expression level of a target protein. Optimization of culture conditions, improvement of denaturation and renaturation steps and coexpression of molecular chaperones or foldase were accomplished to produce active proteins in soluble form. Fusion protein systems with selective separation and surface display technology were also performed in an effort to make the E. coli expression system more effective and versatile.

• Biomedical Science Letters
• /
• v.17 no.1
• /
• pp.1-5
• /
• 2011

Previously, we have identified 14 putative downstream target genes of Hoxc8 homeoprotein in F9 murine embryonic teratocarcinoma cells through proteomics analysis. Among those, we tested a possibility of a DNA-k type molecular chaperone, Grp78, as a direct downstream target of Hoxc8, by cloning a 2.4 kb upstream region of murine Grp78 into a reporter plasmid and by testing if Hoxc8 can regulate its expression. We observed that Hoxc8 proteins could transactivate the reporter gene, which was affected by small interference RNAs (siRNAs) against to Hoxc8, suggesting that Grp78 is a novel downstream target of Hoxc8 in vivo.

• Biomedical Science Letters
• /
• v.12 no.2
• /
• pp.81-89
• /
• 2006

The ischemia-responsive 94 gene (irp94) encoding a 94 kDa endoplasmic reticulum resident protein was investigated its molecular properties associated with unfoled protein responses. First, the expression of irp94 mRNA was tested after the reperfusion of the transient forebrain ischemia induction at the central nervous system in three Mongolian gerbils. Second, irp94 expression in PC12 cells, which are derived from transplantable rat pheochromocytoma cultured in the DMEM media, was tested at transcriptional and translational levels. The half life of irp94 mRNA was also determined In PC12 cells. Last, the changes of irp94 mRNA expression were investigated by the addition of various ER stress inducible chemicals (A23187, BFA, tunicamycin, DTT and $H_2O_2$) and proteasome inhibitors, and heat shock. High level expression of irp94 mRNA was detected after 3 hours reperfusion in the both sites of the cerebral cortex and hippocampus of the gerbil brain. The main regulation of irp94 mRNA expression in PC 12 cells was determined at the transcriptional level. The half life of irp94 mRNA in PC12 cells was approximately 5 hours after the initial translation. The remarkable expression of irp94 mRNA was detected by the treatment of tunicamycin, which blocks glycosylation of newly synthesized polypeptides, and $H_2O_2$, which induces apoptosis. When PC12 cells were treated with the cytosol proteasome inhibitors such as ALLN (N-acetyl-leucyl-norleucinal) and MG 132 (methylguanidine), irp94 mRNA expression was increased. These results indicate that expression of irp94 was induced by ER stress including oxidation condition and glycosylation blocking in proteins. Expression of irp94 was increased when the cells were chased after heat shock, suggesting that irp94 may be involved in recovery rather than protection against ER stresses. In addition, irp94 expression was remarkably increased when cytosol proteasomes were inhibited by ALLN and MG 132, suggesting that irp94 plays an important role for maintaining the ERAD (endoplasmic reticulum associated degradation) function.

• Environmental Analysis Health and Toxicology
• /
• v.29
• /
• pp.2.1-2.7
• /
• 2014

Objectives Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. Methods This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. Results The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Conclusions Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments.