• BMB Reports
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• 제48권9호
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• pp.487-488
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• 2015

The ubiquitin-proteasome system and the autophagy lysosome system are the two major protein degradation machineries in eukaryotic cells. These two systems coordinate the removal of unwanted intracellular materials, but the mechanism by which they achieve this synchronization is largely unknown. The ubiquitination of substrates serves as a universal degradation signal for both systems. Our study revealed that the amino-terminal Arg, a canonical N-degron in the ubiquitin-proteasome system, also acts as a degradation signal in autophagy. We showed that many ER residents, such as BiP, contain evolutionally conserved arginylation permissive pro-N-degrons, and that certain inducers like dsDNA or proteasome inhibitors cause their translocation into the cytoplasm where they bind misfolded proteins and undergo amino-terminal arginylation by arginyl transferase 1 (ATE1). The amino-terminal Arg of BiP binds p62, which triggers p62 oligomerization and enhances p62-LC3 interaction, thereby stimulating autophagic delivery and degradation of misfolded proteins, promoting cell survival. This study reveals a novel ubiquitin-independent mechanism for the selective autophagy pathway, and provides an insight into how these two major protein degradation pathways communicate in cells to dispose the unwanted proteins. [BMB Reports 2015; 48(9): 487-488]

• 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.

• Bulletin of the Korean Chemical Society
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• 제30권5호
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• pp.1039-1042
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• 2009

RNA polymerase II C-terminal domain phosphatase 1 containing ubiquitin like domain (UBLCP1) has been identified as a regulatory molecule of RNA polymerase II. UBLCP1 consists of ubiquitin like domain (UBL) and phosphatase domain homologous with UDP and CTD phosphatase. UBLCP1 was cloned into the E.coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using both affinity and gel-filtration chromatography. Domains of UBLCP1 protein were successfully purified as 7 mg/500 mL (UBLCP1, 36.78 KDa), 32 mg/500 mL (UBL, 9 KDa) and 8 mg/500 mL (phosphatase domain, 25 KDa) yielded in LB medium, respectively. Isotope-labeled samples including triple-labeled ($^2H/^{15}N/^{13}C$) UBLCP1 were also prepared for hetero-nuclear NMR experiments. $^{15}N-^{1}H$ 2D-HSQC spectra of UBLCP1 suggest that both UBL and phosphatase domain are properly folded and structurally independent each other. These data will promise us further structural investigation of UBLCP1 by NMR spectroscopy and/or X-ray crystallography.

• 농업과학연구
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• 제41권3호
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• pp.237-243
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• 2014

Human Glucagon like peptide-1 (GLP-1) is an incretin hormone that promotes secretion of insulin. In order to eliminate the formation of the soluble aggregate, Ala19 in GLP-1 was substituted with Thr, resulting in a GLP-1 mutant GLP-1A19T. The gene synthesis of GLP-1A19T and the fusion of 6-lysine tagged ubiquitin gene were accomplished by using the overlap extension polymerase chain reaction. The ubiquitin fused GLP-1A19T (K6UbGLP-1A19T) is expressed as form of inclusion body with little formation of the soluble aggregation in recombinant E. coli. In order to produce K6UbGLP-1A19T in large amounts, fed-batch fermentation was carried out in a pH-stat feeding strategy. Maximum dry cell weight of 87.7 g/L and 20.4% of specific K6UbGLP-1A19T content were obtained. Solid-phase refolding using a cation exchanger was carried out to renature K6UbGLP-1A19T. The refolded K6UbGLP-1A19T aggregated little and was released GLP-1A19T by on-column cleavage with ubiquitin-specific protease-1. The molecular mass of GLP-1A19T showed an accurate agreement with its theoretical molecular mass.

• Molecules and Cells
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• 제37권5호
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• pp.389-398
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• 2014

Siah acts as an E3 ubiquitin ligase that binds proteins destined for degradation. Extensive homology between siah and Drosophila Siah homologue (sina) suggests their important physiological roles during embryonic development. However, detailed functional studies of Siah in vertebrate development have not been carried out. Here we report that Siah2 specifically augments nodal related gene expression in marginal blastomeres at late blastula through early gastrula stages of zebrafish embryos. Siah2 dependent Nodal signaling augmentation is confirmed by cell-based reporter gene assays using 293T cells and 3TP-luciferase reporter plasmid. We also established a molecular hierarchy of Siah as a upstream regulator of FoxH1/Fast1 transcriptional factor in Nodal signaling. Elevated expression of nodal related genes by overexpression of Siah2 was enough to override the inhibitory effects of atv and lft2 on the Nodal signaling. In particular, E3 ubiquitin ligase activity of Siah2 is critical to limit the duration and/or magnitude of Nodal signaling. Additionally, since the embryos injected with Siah morpholinos mimicked the atv overexpression phenotype at least in part, our data support a model in which Siah is involved in mesendoderm patterning via modulating Nodal signaling.

• Animal cells and systems
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• 제16권3호
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• pp.173-182
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• 2012

The p53 protein plays a pivotal role in tumor suppression. The cellular level of p53 is normally kept low by proteasome-mediated degradation, allowing cell cycle progression and cell proliferation. Under stress conditions, such as DNA damage, p53 is stabilized and activated through various post-translational modifications of itself as well as of its regulatory proteins for induction of the downstream genes responsible for cell cycle arrest, DNA repair, and apoptosis. Therefore, the level of p53 should be tightly regulated for normal cell growth and for prevention of the accumulation of mutations in DNA under stress conditions, which otherwise would lead to tumorigenesis. Since the discovery of Mdm2, a critical ubiquitin E3 ligase that destabilizes p53 in mammalian cells, nearly 20 different E3 ligases have been identified and shown to function in the control of stability, nuclear export, translocation to chromatin or nuclear foci, and oligomerization of p53. So far, a large number of excellent reviews have been published on the control of p53 function in various aspects. Therefore, this review will focus only on mammalian ubiquitin E3 ligases that mediate proteasome-dependent degradation of p53.

• BMB Reports
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• 제52권2호
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• pp.119-126
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• 2019

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) initiates the extrinsic apoptotic pathway through formation of the death-inducing signaling complex (DISC), followed by activation of effector caspases. TRAIL receptors are composed of death receptors (DR4 and DR5), decoy receptors (DcR1 and DcR2), and osteoprotegerin. Among them, only DRs activate apoptotic signaling by TRAIL. Since the levels of DR expressions are higher in cancer cells than in normal cells, TRAIL selectively activates apoptotic signaling pathway in cancer cells. However, multiple mechanisms, including down-regulation of DR expression and pro-apoptotic proteins, and up-regulation of anti-apoptotic proteins, make cancer cells TRAIL-resistant. Therefore, many researchers have investigated strategies to overcome TRAIL resistance. In this review, we focus on protein regulation in relation to extrinsic apoptotic signaling pathways via ubiquitination. The ubiquitin proteasome system (UPS) is an important process in control of protein degradation and stabilization, and regulates proliferation and apoptosis in cancer cells. The level of ubiquitination of proteins is determined by the balance of E3 ubiquitin ligases and deubiquitinases (DUBs), which determine protein stability. Regulation of the UPS may be an attractive target for enhancement of TRAIL-induced apoptosis. Our review provides insight to increasing sensitivity to TRAIL-mediated apoptosis through control of post-translational protein expression.

• BMB Reports
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• 제52권3호
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• pp.181-189
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• 2019

Cancer remains a life-threatening disease and accounts for the major mortality rates worldwide. The practice of using biomarkers for early detection, staging, and customized therapy may increase cancer patients' survival. Deubiquitinating enzymes (DUBs) are a family of proteases that remove ubiquitin tags from proteins of interest undergoing proteasomal degradation. DUBs play several functional roles other than deubiquitination. One of the important roles of DUBs is regulation of tumor progression. Several reports have suggested that the DUB family members were highly-elevated in various cancer cells and tissues in different stages of cancer. These findings suggest that the DUBs could be used as drug targets in cancer therapeutics. In this review, we recapitulate the role of the DUB family members, including ubiquitin-specific protease, otubain protease, and important candidates from other family members. Our aim was to better understand the connection between DUB expression profiles and cancers to allow researchers to design inhibitors or gene therapies to improve diagnosis and prognosis of cancers.

• BMB Reports
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• 제54권8호
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• pp.413-418
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• 2021

ErbB3-binding protein 1 (EBP1) is a multifunctional protein associated with neural development. Loss of Ebp1 leads to upregulation of the gene silencing unit suppressor of variegation 3-9 homolog 1 (Suv39H1)/DNA (cytosine 5)-methyltransferase (DNMT1). EBP1 directly binds to the promoter region of DNMT1, repressing DNA methylation, and hence, promoting neural development. In the current study, we showed that EBP1 suppresses histone methyltransferase activity of Suv39H1 by promoting ubiquitin-proteasome system (UPS)-dependent degradation of Suv39H1. In addition, we showed that EBP1 directly interacts with Suv39H1, and this interaction is required for recruiting the E3 ligase MDM2 for Suv39H1 degradation. Thus, our findings suggest that EBP1 regulates UPS-dependent degradation of Suv39H1 to govern proper heterochromatin assembly during neural development.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.189-189
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• 2022

Soil salinity negatively affects plant growth, productivity, and metabolism. Rice is known to have more sensitive phenotypes than other cereal crops, such as wheat, sorghum, and barley. We characterized the molecular function of rice C3HC4 as a really interesting new gene (RING). Oryza sativa RING finger protein H2-3 (OsRFPH2-3) was highly expressed in 100 mM NaCl. To identify the localization of OsRFPH2-3, we fused vectors that include C-terminal GFP protein (35S;;OsRFPH2-3-GFP). OsRFPH2-3 was expressed in the nucleus in rice protoplasts. An in vitro ubiquitin assay demonstrated that OsRFPH2-3 possessed E3-ubiquitin ligase activity. However, the mutated OsRFPH2-3 were not possessed any E3-ubiquitin ligase activity. Under normal conditions, there is no significant phenotypic difference between transgenic plants and WT plants. However, OsRFPH2-3-overexpressing plants exhibited higher fresh weight and length under saline conditions. Also, transgenic plants maintain higher chlorophyll, proline, and soluble sugar contents and lower H₂O₂ and MDA contents than the wild type; these results support transgenic plants with enhanced salinity tolerance phenotypes.