• BMB Reports
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• v.55 no.4
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• pp.161-165
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• 2022

The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenance of cellular homeostasis and cell growth. mTOR also functions as an endogenous inhibitor of autophagy. Under nutrient-rich conditions, mTOR complex 1 (mTORC1) phosphorylates the ULK1 complex, preventing its activation and subsequent autophagosome formation, while inhibition of mTORC1 using either rapamycin or nutrient deprivation induces autophagy. Autophagy and proteasomal proteolysis provide amino acids necessary for protein translation. Although the connection between mTORC1 and autophagy is well characterized, the association of mTORC1 inhibition with proteasome biogenesis and activity has not been fully elucidated yet. Proteasomes are long-lived cellular organelles. Their spatiotemporal rather than homeostatic regulation could be another adaptive cellular mechanism to respond to starvation. Here, we reviewed several published reports and the latest research from our group to examine the connection between mTORC1 and proteasome. We have also investigated and described the effect of mTORC1 inhibition on proteasome activity using purified proteasomes. Since mTORC1 inhibitors are currently evaluated as treatments for several human diseases, a better understanding of the link between mTORC1 activity and proteasome function is of utmost importance.

• Bulletin of the Korean Chemical Society
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• v.34 no.5
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• pp.1425-1428
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• 2013

The proteasome, which is highly evolutionarily conserved, is responsible for the degradation of most short-lived proteins in cells. Small-molecule inhibitors targeting the proteasome's degradative activity have been extensively developed as lead compounds for various human diseases. An exemplified molecule is bortezomib, which was approved by FDA in 2003 for the treatment of multiple myeloma. Here, using transiently and stably expressed N-end rule model substrates in mammalian cells, we evaluated and identified that salinosporamide A and salinosporamide B effectively inhibited the proteasomal degradation. Considering that a variety of proteasome substrates are implicated in the pathogenesis of many diseases, they have the potential to be clinically applicable as therapeutic agents.

• Molecules and Cells
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• v.41 no.12
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• pp.1008-1015
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• 2018

$I{\kappa}B$, a cytoplasmic inhibitor of nuclear factor-${\kappa}B$ ($NF-{\kappa}B$), is reportedly degraded via the proteasome. However, we recently found that long-term incubation with proteasome inhibitors (PIs) such as PS-341 or MG132 induces $I{\kappa}B{\alpha}$ degradation via an alternative pathway, lysosome, which results in $NF-{\kappa}B$ activation and confers resistance to PI-induced lung cancer cell death. To enhance the anti-cancer efficacy of PIs, elucidation of the regulatory mechanism of PI-induced $I{\kappa}B{\alpha}$ degradation is necessary. Here, we demonstrated that PI up-regulates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) via both de novo protein synthesis and Kelch-like ECH-associated protein 1 (KEAP1) degradation, which is responsible for $I{\kappa}B{\alpha}$ degradation via macroautophagy activation. PIs increased the protein level of light chain 3B (LC3B, macroautophagy marker), but not lysosome-associated membrane protein 2a (Lamp2a, the receptor for chaperone-mediated autophagy) in NCI-H157 and A549 lung cancer cells. Pretreatment with macroautophagy inhibitor or knock-down of LC3B blocked PI-induced $I{\kappa}B{\alpha}$ degradation. PIs up-regulated Nrf2 by increasing its transcription and mediating degradation of KEAP1 (cytoplasmic inhibitor of Nrf2). Overexpression of dominant-negative Nrf2, which lacks an N-terminal transactivating domain, or knock-down of Nrf2 suppressed PI-induced LC3B protein expression and subsequent $I{\kappa}B{\alpha}$ degradation. Thus, blocking of the Nrf2 pathway enhanced PI-induced cell death. These findings suggest that Nrf2-driven induction of LC3B plays an essential role in PI-induced activation of the $I{\kappa}B$/$NF-{\kappa}B$ pathway, which attenuates the anti-tumor efficacy of PIs.

• Journal of Chest Surgery
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• v.50 no.3
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• pp.144-152
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• 2017

Background: The ubiquitin-proteasome system (UPS) is an important pathway of proteolysis in pathologic hypertrophic cardiomyocytes. We hypothesize that MG132, a proteasome inhibitor, might prevent hypertrophic cardiomyopathy (CMP) by blocking the UPS. Nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$) and androgen receptor (AR) have been reported to be mediators of CMP and heart failure. This study drew upon pathophysiologic studies and the analysis of $NF-{\kappa}B$ and AR to assess the cardioprotective effects of MG132 in a left ventricular hypertrophy (LVH) rat model. Methods: We constructed a transverse aortic constriction (TAC)-induced LVH rat model with 3 groups: sham (TAC-sham, n=10), control (TAC-cont, n=10), and MG132 administration (TAC-MG132, n=10). MG-132 (0.1 mg/kg) was injected for 4 weeks in the TAC-MG132 group. Pathophysiologic evaluations were performed and the expression of AR and $NF-{\kappa}B$ was measured in the left ventricle. Results: Fibrosis was prevalent in the pathologic examination of the TAC-cont model, and it was reduced in the TAC-MG132 group, although not significantly. Less expression of AR, but not $NF-{\kappa}B$, was found in the TAC-MG132 group than in the TAC-cont group (p<0.05). Conclusion: MG-132 was found to suppress AR in the TAC-CMP model by blocking the UPS, which reduced fibrosis. However, $NF-{\kappa}B$ expression levels were not related to UPS function.

• Molecules and Cells
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• v.26 no.3
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• pp.285-290
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• 2008

Estrogen-induced proliferation in estrogen receptor (ER)-positive breast cancer cells is primarily mediated through two distinct intracellular receptors, $ER{\alpha}$ and $ER{\beta}$. Although tumor necrosis factor alpha ($TNF{\alpha}$) and $E2/ER{\alpha}$ are known to exert opposing effects on cell proliferation in MCF-7 cells, the mechanism by which $TNF{\alpha}$ antagonizes $E2/ER{\alpha}$-mediated cell proliferation is not well understood. The present study suggests that reduced cell survival in response to $TNF{\alpha}$ treatment in MCF-7 cells may be associated with the down-regulation of $ER{\alpha}$ protein. The decrease in $ER{\alpha}$ protein level was accompanied by an inhibition of $ER{\alpha}$ gene transcription. Cell viability was decreased synergistically by the combined treatment with $ER{\alpha}$-siRNA and $TNF{\alpha}$. Furthermore, pretreatment of cells with the PI3-kinase (PI3K)/ Akt inhibitor, LY294002, markedly enhanced $TNF{\alpha}$-induced down-regulation of the $ER{\alpha}$ protein, suggesting that the PI3K/Akt pathway might be involved in control of the $ER{\alpha}$ level. Moreover, down-regulation of $ER{\alpha}$ by $TNF{\alpha}$ was not inhibited in cells that were pretreated with the proteasome inhibitors, MG132 and MG152, which suggests that proteasome-dependent proteolysis does not significantly influence $TNF{\alpha}$-induced down-regulation of $ER{\alpha}$ protein. In contrast, the effect of the PI3K/Akt inhibitor on $ER{\alpha}$ was blocked in cells that were treated with LY294002 in the presence of the proteasome inhibitors. Collectively, our findings show that the $TNF{\alpha}$ may partly regulate the growth of MCF-7 breast cancer cells through the down-regulation of $ER{\alpha}$ expression, which is primarily mediated by a PI3K/Akt signaling.

• Biomolecules & Therapeutics
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• v.17 no.1
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• pp.25-31
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• 2009

Clusterin is a heterodimeric sulfated glycoprotein and plays a role in many different types of cancer as a cell survival factor and helps cancerous cells to evade stress-induced apoptosis. To investigate whether the regulation of clusterin expression is involved in the mechanism of anticancer agent, we studied the effect of tamoxifen on clusterin expression in human prostate cancer PC-3 cells. Treatment of PC-3 cells with tamoxifen reduced cellular proliferation. Western blot analyses showed that treatment with tamoxifen suppressed clusterin expression in a concentration-dependent manner. Transfection with clusterin siRNA plasmid showed that clusterin is required for PC-3 cell survival. We found that tamoxifen resulted in a rapid decrease in the phosphorylation of Akt on Ser473 leading to prevent kinase activity. Expression of myristoylated Akt prevented tamoxifen-mediated clusterin downregulation. Interestingly, MG132, a wellknown proteasome inhibitor also recovered clusterin expression suppressed by tamoxifen. These data indicate that clusterin expression may be regulated by activation of Akt and ubiquitin-proteasome pathway plays an important role in tamoxifen-mediated clusterin suppression.

• IMMUNE NETWORK
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• v.22 no.3
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• pp.28.1-28.11
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• 2022

The 26S proteasome irreversibly hydrolyzes polyubiquitylated substrates to maintain protein homeostasis; it also regulates immune responses by generating antigenic peptides. An alternative form of the 26S proteasome is the immunoproteasome, which contains substituted catalytic subunits (β1i/PSMB9, β2i/PSMB10, and β5i/PSMB8) instead of constitutively expressed counterparts (β1/PSMB6, β2/PSMB7, and β5/PSMB5). The immunoproteasome expands the peptide repertoire presented on MHC class I molecules. However, how its activity changes in this context is largely elusive, possibly due to the lack of a standardized methodology to evaluate its specific activity. Here, we describe an assay protocol that measures the immunoproteasome activity of whole-cell lysates using commercially available fluorogenic peptide substrates. Our results showed that the most accurate assessment of immunoproteasome activity could be achieved by combining β5i-targeting substrate Ac-ANW-AMC and immunoproteasome inhibitor ONX-0914. This simple and reliable protocol may contribute to future studies of immunoproteasomes and their pathophysiological roles during viral infection, inflammation, and tumorigenesis.

• Journal of Microbiology and Biotechnology
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• v.23 no.12
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• pp.1785-1790
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• 2013

The synthetic machinery of ATF4 (activating transcription factor 4) is activated in response to various stress conditions involved in nutrient restriction, endoplasmic reticulum homeostasis, and oxidation. Stress-induced inhibition of proteasome activity triggers the unfolded protein response and endoplasmic reticulum stress, where ATF4 is crucial for consequent biological events. In the current study, we showed that the $NAD^+$-dependent deacetylase, SIRT1, suppresses ATF4 synthesis during proteasome inhibition. SIRT1 depletion via transfection of specific siRNA into HeLa cells resulted in a significant increase in ATF4 protein, which was observed specifically in the presence of the proteasome inhibitor MG132. Consistent with SIRT1 depletion data, transient transfection of cells with SIRT1-overexpressing plasmid induced a decrease in the ATF4 protein level in the presence of MG132. Interestingly, however, ATF4 mRNA was not affected by SIRT1, even in the presence of MG132, indicating that SIRT1-induced suppression of ATF4 synthesis occurs under post-transcriptional control. Accordingly, we propose that SIRT1 serves as a negative regulator of ATF4 protein synthesis at the post-transcriptional level, which is observed during stress conditions, such as proteasome inhibition.

• The Korean Journal of Food And Nutrition
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• v.32 no.5
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• pp.565-570
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• 2019

Proteasome inhibitors can improve the efficiency of cancer treatments by inhibiting nuclear factor ${\kappa}B$($NF-{\kappa}B$) activation in cancer cells. Lentils are a type of beans of which consumption of such beans is increasing. The purpose of this study was to investigate the effects of lentils extract (LE) on the proteasomal activities, $NF-{\kappa}B$ activation, and cell cycle in HepG2 human liver cancer cells. LE treatments inhibited proteasomal activities at concentrations of 10, 50, and $100{\mu}g/mL$ respectively, and repressed $NF-{\kappa}B$ activation at concentrations of 1, 10, and $100{\mu}g/mL$ respectively, in HepG2 cells. LE treatments at concentrations of 1, 10, and $100{\mu}g/mL$ respectively, increased sub-G1 cell population in HepG2 cells, which may be the result of apoptosis. The results suggest that LE inhibited $NF-{\kappa}B$ activation partially with its proteasome inhibitory activities, and the increase of sub-G1 cell population was induced partially, by inhibition of $NF-{\kappa}B$ activation in HepG2 cells.

• Molecules and Cells
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• v.24 no.3
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• pp.445-451
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• 2007

Translation initiation factor 4E (eIF4E) is a key regulator of protein synthesis. Abnormal regulation of eIF4E is closely linked to oncogenic transformation. Several regulatory mechanisms affecting eIF4E are discussed, including transcriptional regulation, phosphorylation and binding of an inhibitor protein. However it is not clear how the level of eIF4E protein is regulated under basal conditions. Here we demonstrate that Diap1 (Drosophila Inhibitor of Apoptosis Protein), a cell death inhibitor, binds directly to eIF4E and poly-ubiquitinates it via its E3 ligase activity, promoting its proteasome-dependent degradation. Expression of Diap1 caused a reduction of Cyclin D1 protein level and inhibited the growth stimulation induced by overexpression of eIF4E. Taken together, our results suggest that the level of eIF4E protein is regulated by Diap1, and that IAPs may play a role in cap-dependent translation by regulating the level of eIF4E protein.