• Molecules and Cells
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• v.27 no.6
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• pp.697-701
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• 2009

We previously identified cancer-upregulated gene 2 (CUG2) as a commonly up-regulated gene in various human cancer tissues, especially in ovary, liver, and lung (Lee et al., 2007a). CUG2 was determined to be a nuclear protein that exhibited high proto-oncogenic activities when overexpressed in NIH3T3 mouse fibroblast cells. To identify other cellular functions of CUG2, we performed yeast two-hybrid screening and identified CENP-T, a component of CENP-A nucleosome complex in the centromere, as an interacting partner of CUG2. Moreover, CENP-A, the principle centromeric determinant, was also found in complex with CENP-T/CUG2. Immunofluorescent staining revealed the co-localization of CUG2 with human centromeric markers. Inhibition of CUG2 expression drastically affected cell viability by inducing aberrant cell division. We propose that CUG2 is a new component of the human centromeric complex that is required for proper chromosome segregation during mitosis.

• BMB Reports
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• v.48 no.8
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• pp.431-437
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• 2015

Notch signaling plays a pivotal role in cell fate determination, cellular development, cellular self-renewal, tumor progression, and has been linked to developmental disorders and carcinogenesis. Notch1 is activated through interactions with the ligands of neighboring cells, and acts as a transcriptional activator in the nucleus. The Notch1 intracellular domain (Notch1-IC) regulates the expression of target genes related to tumor development and progression. The Notch1 protein undergoes modification after translation by posttranslational modification enzymes. Phosphorylation modification is critical for enzymatic activation, complex formation, degradation, and subcellular localization. According to the nuclear cycle, Notch1-IC is degraded by E3 ligase, FBW7 in the nucleus via phosphorylation-dependent degradation. Here, we summarize the Notch signaling pathway, and resolve to understand the role of phosphorylation in the regulation of Notch signaling as well as to understand its relation to cancer. [BMB Reports 2015; 48(8): 431-437]

• Molecules and Cells
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• v.43 no.4
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• pp.313-322
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• 2020

Eukaryotes transport biomolecules between intracellular organelles and between cells and the environment via vesicle trafficking. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE proteins) play pivotal roles in vesicle and membrane trafficking. These proteins are categorized as Qa, Qb, Qc, and R SNAREs and form a complex that induces vesicle fusion for targeting of vesicle cargos. As the core components of the SNARE complex, the SNAP25 Qbc SNAREs perform various functions related to cellular homeostasis. The Arabidopsis thaliana SNAP25 homolog AtSNAP33 interacts with Qa and R SNAREs and plays a key role in cytokinesis and in triggering innate immune responses. However, other Arabidopsis SNAP25 homologs, such as AtSNAP29 and AtSNAP30, are not well studied; this includes their localization, interactions, structures, and functions. Here, we discuss three biological functions of plant SNAP25 orthologs in the context of AtSNAP33 and highlight recent findings on SNAP25 orthologs in various plants. We propose future directions for determining the roles of the less well-characterized AtSNAP29 and AtSNAP30 proteins.

• Molecules and Cells
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• v.19 no.1
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• pp.23-30
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• 2005

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. Using d2EGFP, a short-lived enhanced green fluorescent protein, we investigated whether polyglutamine-expanded ataxin-1 affects the function of the proteasome, a cellular multicatalytic protease that degrades most misfolded proteins and regulatory proteins. In Western blot analysis and immunofluorescence experiments, d2EGFP was less degraded in HEK 293T cells transfected with ataxin-1(82Q) than in cells transfected with lacZ or empty vector controls. To test whether the stability of the d2EGFP protein was due to aggregation of ataxin-1, we constructed a plasmid carrying $ataxin-1-{\Delta}114$, lacking the self-association region (SAR), and examined degradation of the d2EGFP. Both the level of $ataxin-1-{\Delta}114$ aggregates and the amount of d2EGFP were drastically reduced in cells containing $ataxin-1-{\Delta}114$. Furthermore, d2EGFP localization experiments showed that polyglutamine-expanded ataxin-1 inhibited the general function of the proteasome activity. Taken together, these results demonstrate that polyglutamine-expanded ataxin-1 decreases the activity of the proteasome, implying that a disturbance in the ubiquitin-proteasome pathway is directly involved in the development of spinocerebellar ataxia type1.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• BMB Reports
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• v.41 no.12
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• pp.863-867
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• 2008

CD320 has been recently discovered and reported as a follicular dendritic cell (FDC) protein. Although CD320 is known to enhance proliferation of germinal center (GC) B cells, little other information is available. In this study, we investigated its cellular distribution in the GC. Confocal microscopy of human tonsil sections revealed co-localization of CD320 with CD19 and CD38 but not with CD3 indicating that GC B cells expressed CD320 in addition to FDC. In purified GC B cells, CD320 expression was inhibited in the nucleus, membrane and cytoplasm. Reverse transcriptase-polymerase chain reaction confirmed CD320 mRNA expression in B cells. These finding indicate that CD320 is expressed in B cells in addition to FDC, and that its GC activity may be more complicated than previously thought.

• Molecules and Cells
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• v.44 no.5
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• pp.342-355
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• 2021

The microphthalmia-associated transcription factor family (MiT family) proteins are evolutionarily conserved transcription factors that perform many essential biological functions. In mammals, the MiT family consists of MITF (microphthalmia-associated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC). These transcriptional factors belong to the basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor family and bind the E-box DNA motifs in the promoter regions of target genes to enhance transcription. The best studied functions of MiT proteins include lysosome biogenesis and autophagy induction. In addition, they modulate cellular metabolism, mitochondria dynamics, and various stress responses. The control of nuclear localization via phosphorylation and dephosphorylation serves as the primary regulatory mechanism for MiT family proteins, and several kinases and phosphatases have been identified to directly determine the transcriptional activities of MiT proteins. In different immune cell types, each MiT family member is shown to play distinct or redundant roles and we expect that there is far more to learn about their functions and regulatory mechanisms in host defense and inflammatory responses.

• Molecules and Cells
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• v.42 no.7
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• pp.523-529
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• 2019

mRNA quality is controlled by multiple RNA surveillance machineries to reduce errors during gene expression processes in eukaryotic cells. Nonsense-mediated mRNA decay (NMD) is a well-characterized mechanism that degrades error-containing transcripts during translation. The ATP-dependent RNA helicase up-frameshift 1 (UPF1) is a key player in NMD that is mostly prevalent in the cytoplasm. However, recent studies on UPF1-RNA interaction suggest more comprehensive roles of UPF1 on diverse forms of target transcripts. Here we used subcellular fractionation and immunofluorescence to understand such complex functions of UPF1. We demonstrated that UPF1 can be localized to the nucleus and predominantly associated with the chromatin. Moreover, we showed that UPF1 associates more strongly with the chromatin when the transcription elongation and translation inhibitors were used. These findings suggest a novel role of UPF1 in transcription elongation-coupled RNA machinery in the chromatin, as well as in translation-coupled NMD in the cytoplasm. Thus, we propose that cytoplasmic UPF1-centric RNA surveillance mechanism could be extended further up to the chromatin-associated UPF1 and co-transcriptional RNA surveillance. Our findings could provide the mechanistic insights on extensive regulatory roles of UPF1 for many cellular RNAs.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.229-235
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• 2018

Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus.

• Molecules and Cells
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• v.45 no.7
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• pp.435-443
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• 2022

In response to environmental changes, signaling pathways rewire gene expression programs through transcription factors. Epigenetic modification of the transcribed RNA can be another layer of gene expression regulation. N⁶-adenosine methylation (m⁶A) is one of the most common modifications on mRNA. It is a reversible chemical mark catalyzed by the enzymes that deposit and remove methyl groups. m⁶A recruits effector proteins that determine the fate of mRNAs through changes in splicing, cellular localization, stability, and translation efficiency. Emerging evidence shows that key signal transduction pathways including TGFβ (transforming growth factor-β), ERK (extracellular signal-regulated kinase), and mTORC1 (mechanistic target of rapamycin complex 1) regulate downstream gene expression through m⁶A processing. Conversely, m⁶A can modulate the activity of signal transduction networks via m⁶A modification of signaling pathway genes or by acting as a ligand for receptors. In this review, we discuss the current understanding of the crosstalk between m⁶A and signaling pathways and its implication for biological systems.