• Nutrition Research and Practice
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• v.17 no.6
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• pp.1128-1142
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• 2023

BACKGROUND/OBJECTIVES: Inonotus obliquus has been used as antidiabetic herb around the world, especially in the Russian and Scandinavian countries. Diabetes is widely believed to be a key factor in Alzheimer's disease (AD), which is widely considered to be type III diabetes. To investigate whether I. obliquus can also ameliorate AD, it would be interesting to identify new clues for AD treatment. We tested the anti-AD effects of raw Inonotus obliquus polysaccharide (IOP) in a mouse model of AD (3×Tg-AD transgenic mice). MATERIALS/METHODS: SPF-grade 3×Tg-AD mice were randomly divided into three groups (Control, Metformin, and raw IOP groups, n = 5 per group). β-Amyloid deposition in the brain was analyzed using immunohistochemistry for AD characterization. Gene and protein expression of pertinent factors of the ubiquitin-proteasome system (UPS) was determined using real-time quantitative polymerase chain reaction and Western blotting. RESULTS: Raw IOP significantly reduced the accumulation of amyloid aggregates and facilitated UPS activity, resulting in a significant reduction in AD-related symptoms in an AD mouse model. The presence of raw IOP significantly enhanced the expression of ubiquitin, E1, and Parkin (E3) at both the mRNA and protein levels in the mouse hippocampus. The mRNA level of ubiquitin carboxyl-terminal hydrolase isozyme L1, a key factor involved in UPS activation, also increased by approximately 50%. CONCLUSIONS: Raw IOP could contribute to AD amelioration via the UPS pathway, which could be considered as a new potential strategy for AD treatment, although we could not exclude other mechanisms involved in counteracting AD processing.

• Biomolecules & Therapeutics
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• v.32 no.1
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• pp.115-122
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• 2024

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90β and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, β-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

• Korean Journal of Biological Psychiatry
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• v.14 no.2
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• pp.115-121
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• 2007

Objetives : Identification of target genes for ethanol in neurons is important for understanding its molecular and cellular mechanism of action and the neuropathological changes seen in alcoholics. The purpose of this study is to identify of altered gene expression after acute treatmet of ethanol in rat gliom cells. Methods : We used high density cDNA microarray chip to measure the expression patterns of multiple genes in cultured rat glioma cells. DNA microarrays allow for the simultaneous measurement of the expression of several hundreds of genes. Results : After comparing hybridized signals between control and ethanol treated groups, we found that treatment with ethanol increased the expression of 15 genes and decreased the expression of 12 genes. Upregulated genes included Orthodenticle(Drosophila) homolog 1, procollagen type II, adenosine A2a receptor, GATA bindning protein 2. Downregulated genes included diacylglycerol kinase beta, PRKC, Protein phosphatase 1, clathrin-associated protein 17, nucleoporin p58, proteasome. Conclusion : The gene changes noted were those related to the regulation of transcription, signal transduction, second messenger systems. modulation of ischemic brain injury, and neurodengeneration. Although some of the genes were previously known to be ethanol responsive, we have for the most part identified novel genes involved in the brain response to ethanol.

• Journal of Life Science
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• v.33 no.3
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• pp.277-286
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• 2023

Sarcopenia is the age-related loss of muscle mass and function. It is a natural part of aging and can lead to decreased mobility and increased frailty. The ubiquitin-proteasome pathway, which is involved in muscle protein degradation, is closely linked to sarcopenia. Germinated Rhynchosia nulubilis hydrolysate (GRH) has been reported to have anti-inflammatory and antioxidant properties, but there have been no reports on its inhibitory effect on muscle reduction. However, no study has yet explored the relationship between GRH and muscle loss inhibition. In this study, we evaluated the effects of GRH on muscle atrophy inhibitory activity in dexamethasone (Dexa)-induced muscle atrophy C2C12 myotubes and mouse models. Moreover, we identified a molecular pathway underlying the effects of GRH on skeletal muscle. May Grunwald-Giemsa staining showed that the length and area of myotubes increased in the groups treated with GRH. In addition, the GRH-treated group significantly reduced the expression of muscle ring finger protein 1 and muscular atrophy F-box (MAFbx) in the Dexa-induced muscular atrophy C2C12 model. GRH also improved muscle strength in C57BL/6 mice with Dexa-induced muscle atrophy, resulting in prolonged running exhaustive time and increased grip strength. We found that muscle strengthening by GRH was correlated with a decreased expression of the MAFbx gene in mouse muscle tissue. In conclusion, GRH can attenuate Dexa-induced muscle atrophy by inhibiting the ubiquitin-proteasome pathway via downregulation of the MAFbx gene expression.

• Tuberculosis and Respiratory Diseases
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• v.48 no.2
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• pp.166-179
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• 2000

Background: The main reason for the failure of anti-cancer chemotherapy is the build up of resistance by cancer cells to apoptosis. The activation of NF-${\kappa}B$ in many cancer cell lines is reported to be underlying mechanism behind the build up of resistance of cancer cells to apoptosis. However, this relationship varied depending on the cells used in the experiments. In this study, the role of NF-${\kappa}B$ activation in the TNF-$\alpha$-induced apoptosis in lung cancer cell line was evaluated. Methods: NCI-H157 cells were used in all experiments. Cells were exposed to a high dose of TNF-$\alpha$(20 ng/ml) for 24 or 48 hours with or without blocking NF-${\kappa}B$ activation. TNF-$\alpha$-induced activation of NF-${\kappa}B$ was inhibited either by overexpression of $I{\kappa}B{\alpha}$-super repressor($I{\kappa}B{\alpha}$-SR) or by pre-treatment with proteasome inhibitor. Cell viability and apoptosis were evaluated with MTT assay and Western blot analysis for PARP fragment, respectively. Results: Cell viability of NCI-H157 cells was not affected by TNF-$\alpha$ treatment alone; however, combined treatment with TNF-$\alpha$ and cycloheximide reduced cell viability significantly, indicating that resistance to TNF-$\alpha$ is mediated by the new proteins synthesized after TNF-$\alpha$ stimulation. To evaluate the role of NF-${\kappa}B$ in the transcription of anti-apoptotic proteins. delete NF-${\kappa}B$ activation was inhibited before TNF-$\alpha$ stimulation. as described above. $AD5I{\kappa}B{\alpha}$-SR-transduction inhibited TNF-$\alpha$-induced nuclear translocation of p65. TNF-$\alpha$-induced cell death and apoptosis increased after inhibition of TNF-$\alpha$-induced activation of NF-${\kappa}$ by methods. Conclusion: These results suggest that TNF-$\alpha$-induced activation of NF-${\kappa}B$ may be closely related to the acquisition of the resistance to TNF-$\alpha$-induced apoptosis in lung cancer cells. Therefore. blocking of NF-${\kappa}B$ pathway can be a useful therapeutic modality in the treatment of lung cancer.

• Journal of Life Science
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• v.28 no.9
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• pp.1007-1015
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• 2018

The E6-associated protein (E6AP) is known to induce the ubiquitination and proteasomal degradation of HCV core protein and thereby directly impair capsid assembly, resulting in a decline in HCV replication. To counteract this anti-viral host defense system, HCV core protein has evolved a strategy to inhibit E6AP expression via DNA methylation. In the present study, we further explored the mechanism by which HCV core protein inhibits E6AP expression. HCV core protein upregulated both the protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b to inhibit E6AP expression via promoter hypermethylation in HepG2 cells but not in Hep3B cells, which do not express p53. Interestingly, p53 overexpression alone in Hep3B cells was sufficient to activate DNMTs in the absence of HCV core protein and thereby inhibit E6AP expression via promoter hypermethylation. In addition, upregulation of p53 was absolutely required for the HCV core protein to inhibit E6AP expression via promoter hypermethylation, as evidenced by both p53 knockdown and ectopic expression experiments. Accordingly, levels of the ubiquitinated forms of HCV core protein were lower in HepG2 cells than in Hep3B cells. Based on these observations, we conclude that HCV core protein evades ubiquitin-dependent proteasomal degradation in a p53-dependent manner.

• Journal of Life Science
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• v.28 no.9
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• pp.1016-1021
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• 2018

Clostridium difficile toxin A causes pseudomembranous colitis. The pathogenesis of toxin A-induced colonic inflammation includes toxin A-dependent epithelial cell apoptosis, resulting in the loss of barrier function provided by epithelial cells against luminal pathogens. Toxin A-dependent epithelial cell apoptosis has been linked to toxin A-induced production of reaction oxygen species and subsequent p38MAPK activation; $p21^{CIP1/WAF1}$ upregulation-dependent cell cycle arrest; cytoskeletal disaggregation; and/or the induction of Fas ligand on epithelial cells. However, the molecular mechanisms underlying toxin A-induced apoptosis remain poorly understood. This study tested whether toxin A could block the Wnt signaling pathway, which is involved in gut epithelial cell proliferation, differentiation and antiapoptotic progression. Toxin A treatment of nontransformed human colonocytes (NCM460) rapidly reduced ${\beta}$-catenin protein, an essential component of the Wnt signaling pathway. Exposure of mouse ileum to toxin A also significantly reduced ${\beta}$-catenin protein levels. MG132 inhibition of proteasome-dependent protein degradation resulted in the recovery of toxin A-mediated reduction of ${\beta}$-catenin, indicating that toxin A may activate intracellular processes, such as $GSK3{\beta}$, to promote degradation of ${\beta}$-catenin. Immunoblot analysis showed that toxin A increased active phosphorylation of $GSK3{\beta}$. Because the Wnt signaling pathway is essential for gut epithelial cell proliferation and anti-apoptotic processes, our results suggest that toxin A-mediated inhibition of the Wnt signaling pathway may be required for maximal toxin A-induced apoptosis of gut epithelial cells.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.60 no.3
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• pp.394-400
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• 2015

The roots of Platycodon grandiflorum species either dried or fresh, are used as an ingredient in salads and traditional cuisine in Korea. To interpret the root proteins, a systematical and targeting analysis were carried out from diploid and tetraploid roots. Two dimensional gels stained with CBB, a total of 39 differential expressed proteins were identified from the diploid root under in vivo condition using image analysis by Progenesis Same Spot software. Out of total differential expressed spots, 39 differential expressed protein spots (${\geq}\;1.5$-fold) were analyzed using LTQ-FTICR mass spectrometry. Except two proteins, the rest of the identified proteins were confirmed as down-regulated such as Isocitrate dehydrogenase, Proteasome subunit alpha type-2-B. However, the most of the identified proteins from the explants were mainly associated with the oxidoreductase activity, nucleic acid binding, transferase activity and catalytic activity. The exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.

• Tuberculosis and Respiratory Diseases
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• v.70 no.2
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• pp.113-124
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• 2011

Background: Macrophages are one of the most important inflammatory cells in innate immunity. PU.1 is a macrophage-specific transcription factor. Ubiquitins are the ultimate regulator of eukaryotic transcription. The ubiquitination process for PU.1 is unknown. This study investigated the lipopolysaccharide (LPS)-induced activation of PU.1 and its relation to ubiquitins in the macrophages. Methods: Raw264.7 cells, the primary cultured alveolar, pulmonary, and bone marrow derived macrophages were used. The Raw264.7 cells were treated with MG-132, $NH_4Cl$, lactacytin and LPS. Nitric oxide and prostaglandin D2 and E2 were measured. Immunoprecipitation and Western blots were used to check ubiquitination of PU.1. Results: The PU.1 ubiquitination increased after LPS ($1{\mu}g$/mL) treatment for 4 hours on Raw264.7 cells. The ubiquitination of PU.1 by LPS was increased by MG-132 or $NH_4Cl$ pretreatment. Two hours of LPS treatment on macrophages, PU.1 activation was not induced nor increased with the inhibition of proteasomes and/or lysosomes. The ubiquitination of PU.1 was increased in LPS-treated Raw264.7 cells at 12- and at 24 hours. LPS-treated cells increased nitric oxide production, which was diminished by MG-132 or $NH_4Cl$. LPS increased the production of $PGE_2$ in the alveolar and peritoneal macrophages of wild type mice; however, $PGE_2$ was blocked or diminished in Rac2 null mice. Pretreatment of lactacystin increased $PGE_2$, however it decreased the $PGD_2$ level in the macrophages derived from the bone marrow of B57/BL6 mice. Conclusion: LPS treatment in the macrophages ubiquitinates PU.1. Ubiquitination of PU.1 may be involved in synthesis of nitric oxide and prostaglandins.

• BMB Reports
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• v.42 no.7
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• pp.444-449
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• 2009

Different neurodegenerative disorders like prion disease, is caused by protein misfolding conformers. Reverse-transfected cytosolic prion protein (PrP) and PrP expressed in the cytosol have been shown to be neurotoxic. To investigate the possible mechanism of neurotoxicity due to accumulation of PrP in cytosol, a PrP mutant lacking the signal and GPI (CytoPrP) was introduced into the SH-SY5Y cell. MTT and trypan blue assays indicated that the viability of cells expressing CytoPrP was remarkably reduced after treatment of MG-132. Obvious apoptosis phenomena were detected in the cells accumulated with CytoPrP, including loss of mitochondrial transmembrane potential, increase of caspase-3 activity, more annexin V/PI-double positive-stained cells and reduced Bcl-2 level. Moreover, DNA fragmentation and TUNEL assays also revealed clear evidences of late apoptosis in the cells accumulated CytoPrP. These data suggest that the accumulation of CytoPrP in cytoplasm may trigger cell apoptosis, in which mitochondrial relative apoptosis pathway seems to play critical role.