• Molecules and Cells
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• 제40권12호
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• pp.897-905
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• 2017

Cellular protein homeostasis is maintained by two major degradation pathways, namely the ubiquitin-proteasome system (UPS) and autophagy. Until recently, the UPS and autophagy were considered to be largely independent systems targeting proteins for degradation in the proteasome and lysosome, respectively. However, the identification of crucial roles of molecular players such as ubiquitin and p62 in both of these pathways as well as the observation that blocking the UPS affects autophagy flux and vice versa has generated interest in studying crosstalk between these pathways. Here, we critically review the current understanding of how the UPS and autophagy execute coordinated protein degradation at the molecular level, and shed light on our recent findings indicating an important role of an autophagy-associated transmembrane protein EI24 as a bridging molecule between the UPS and autophagy that functions by regulating the degradation of several E3 ligases with Really Interesting New Gene (RING)-domains.

• BMB Reports
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• 제45권5호
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• pp.299-304
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• 2012

The ubiquitin-proteasome system is a major proteolytic system for nonlysosomal degradation of cellular proteins. Here, we investigated the response of mouse embryonic stem (ES) cells under proteotoxic stress. Proteasome inhibitors induced expression of heat shock protein 70 (HSP70) in a concentration- and time-dependent manner, and also induced apoptosis of ES cells. Importantly, more apoptotic cells were observed in ES cells compared with other somatic cells. To understand this phenomenon, we further investigated the expression of HSP70 and pluripotent cell markers. HSP70 expression was more significantly increased in somatic cells than in ES cells, and expression levels of pluripotent cell markers such as Oct4 and Nanog were decreased in ES cells. These results suggest that higher sensitivity of ES cells to proteotoxic stress may be related with lower capacity of HSP70 expression and decreased pluripotent cell marker expression, which is essential for the survival of ES cells.

• Biomolecules & Therapeutics
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• 제17권1호
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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.

• Molecules and Cells
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• 제27권3호
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• pp.299-305
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• 2009

Over-expression of phospholipase D (PLD) 1 or PLD2 down-regulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the $CKII{\beta}$ subunit. This PLD-induced $CKII{\beta}$ degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in $CKII{\beta}$ degradation. PLD isozymes interacted with the $CKII{\beta}$ subunit. Immunocytochemical staining revealed that PLD and $CKII{\beta}$ colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to $CKII{\beta}$ inhibited $CKII{\beta}$ autophosphorylation, which is known to be important for $CKII{\beta}$ stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of $CKII{\beta}$ degradation by ubiquitin-proteasome machinery.

• BMB Reports
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• 제53권1호
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• pp.56-63
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• 2020

The ubiquitin-proteasome system (UPS) and autophagy are two major degradative pathways of proteins in eukaryotic cells. As about 30% of newly synthesized proteins are known to be misfolded under normal cell conditions, the precise and timely operation of the UPS and autophagy to remove them as well as their tightly controlled regulation, is so important for proper cell function and survival. In the UPS, target proteins are labeled by small proteins called ubiquitin, which are then transported to the proteasome complex for degradation. Alternatively, many greatly damaged proteins are believed to be delivered to the lysosome for autophagic degradation. Although these autophagy and UPS pathways have not been considered to be directly related, many recent studies proposed their close link and dynamic interconversion. In this review, we'll focus on the several regulatory molecules that function in both UPS and autophagy and their crosstalk. Among the proposed multiple modulators, we will take a closer look at the so-called main connector of UPS-autophagy regulation, p62. Last, the functional role of p62 in the mitophagy and its implication for the pathogenesis of Parkinson's disease, one of the major neurodegenerative diseases, will be briefly reviewed.

• IMMUNE NETWORK
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• 제22권3호
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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.

• 한국식품영양학회지
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• 제32권5호
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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.

• Applied Microscopy
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• 제48권1호
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• pp.1-5
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• 2018

Cryo-transmission electron microscopy (cryo-TEM) allows us to perform structural analysis of a analyses of large protein complexes, which are difficult to analyze using X-ray crystallography or nuclear magnetic resonance. The most common examples of proteins used are ribosomes and proteasomes. In this paper, we briefly describe the advantage of cryo-TEM and the process of two-dimensional classification by considering a human proteasome as an example.

• The Plant Pathology Journal
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• 제24권4호
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• pp.367-374
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• 2008

The genomes of plants contain more than 600 genes encoding a diverse set of proteases and the subunits of proteasomes. These proteases and proteasomes consist of plant proteolytic systems, which are involved in various cellular metabolic processes. Plant proteolytic systems have been shown to have diverse roles in defense responses, such as execution of the attack on the invading organisms, participation in signaling cascades, and perception of the invaders. In order to provide a framework for illustrating the importance of proteolytic systems in plant defense, characteristics of non-proteasome proteases and the 26S proteasome are summarized. The involvement of caspase-like proteases, saspases, apoplastic proteases, and the 26S proteasome in pathogen defense suggests that plant proteolytic systems are essential for defense and further clarity on the roles of plant proteases in defense is challenging but fundamentally important to understand plant-microbe interactions.

• Molecules and Cells
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• 제41권11호
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• pp.933-942
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• 2018

Traditionally, small-molecule or antibody-based therapies against human diseases have been designed to inhibit the enzymatic activity or compete for the ligand binding sites of pathological target proteins. Despite its demonstrated effectiveness, such as in cancer treatment, this approach is often limited by recurring drug resistance. More importantly, not all molecular targets are enzymes or receptors with druggable 'hot spots' that can be directly occupied by active site-directed inhibitors. Recently, a promising new paradigm has been created, in which small-molecule chemicals harness the naturally occurring protein quality control machinery of the ubiquitin-proteasome system to specifically eradicate disease-causing proteins in cells. Such 'chemically induced protein degradation' may provide unprecedented opportunities for targeting proteins that are inherently undruggable, such as structural scaffolds and other non-enzymatic molecules, for therapeutic purposes. This review focuses on surveying recent progress in developing E3-guided proteolysis-targeting chimeras (PROTACs) and small-molecule chemical modulators of deubiquitinating enzymes upstream of or on the proteasome.