• Journal of Life Science
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• v.30 no.1
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• pp.10-17
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• 2020

Kinesin-1 is microtubule-dependent plus-end direct molecular motor protein essential for intracellular transport. It is a member of the kinesin superfamily proteins (KIFs) which transport cargo, including organelles, vesicles, neurotransmitter receptors, cell-signaling molecules, and protein complexes through interaction between its light chain subunit and the cargo. Kinesin light chain 1 (KLC1) is a non-motor subunit that associates with the kinesin heavy chain (KHC). Although KLC1 interacts with many different adaptor proteins and scaffolding proteins, its binding proteins have not yet been fully identified. We used the yeast two-hybrid assay to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1, and found an interaction between KLC1 and EH domain-binding protein 1 like 1 (EHBP1L1). EHBP1L1 bound to the region containing all six TPR repeats of KLC1 and did not interact with KIF5B (a motor protein of kinesin 1) or KIF3A (a motor protein of kinesin 2) in the yeast two-hybrid assay. The carboxyl-terminus of the coiled-coil domain of EHBP1L1 is essential for interaction with KLC1. However, another EHBP1L1 isoform, EHBP1, did not interact with KLC1 in the yeast two-hybrid assay. KLC1 interacted with GST-EHBP1L1 and its coiled-coil domain but not with GST only. When co-expressed in HEK-293T cells, EHBP1L1 co-localized with KLC1 and co-immunoprecipitated with KLC1 and KIF5B but not KIF3A. These results suggest that kinesin 1 motor protein may transport EHBP1L1-associated cargo in cells.

• Journal of Life Science
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• v.26 no.3
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• pp.282-288
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• 2016

Protein-protein interactions regulate the subcellular localization and function of receptors, enzymes, and cytoskeletal proteins. Proteins containing the postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain have potential to act as scaffolding proteins and play a pivotal role in various processes, such as synaptic plasticity, neural guidance, and development, as well as in the pathophysiology of many diseases. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, has a scaffolding function in the clustering of surface receptors, organization of signaling complexes, and coordination of cytoskeletal dynamics. However, the cellular function of MUPP1 has not been fully elucidated. In the present study, a yeast two-hybrid system was used to identify proteins that interacted with the N-terminal PDZ domain of MUPP1. The results revealed an interaction between MUPP1 and Wdpcp (formerly known as Fritz). Wdpcp was identified as a planar cell polarity (PCP) effector, which is known to have a role in collective cell migration and cilia formation. Wdpcp bound to the PDZ1 domain but not to other PDZ domains of MUPP1. The C-terminal end of Wdpcp was essential for the interaction with MUPP1 in the yeast two-hybrid assay. This interaction was further confirmed in a glutathione S-transferase (GST) pull-down assay. When coexpressed in HEK-293T cells, Wdpcp was coimmunoprecipitated with MUPP1. In addition, MUPP1 colocalized with Wdpcp at the same subcellular region in cells. Collectively, these results suggest that the MUPP1-Wdpcp interaction could modulate actin cytoskeleton dynamics and polarized cell migration.

• Korean Journal of Medicinal Crop Science
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• v.15 no.3
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• pp.170-176
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• 2007

This study was performed to anticancer activities and immune modulatary activities according to the parts of the S. japonica Sieb. et Zucc. The cytotoxicity on human kidney cell (HEK 293) was showed below 27.4% in adding the methanol extracts. The anticancer activity were increased in over 60% by barks extracts in AGS and MCF-7 cells. The immune cell growth using human immune B and T cells was improved by the barks extracts of S. japonica Sieb. et Zucc. in adding 1.0mg/ml concentration. The secretion of the IL-6 and TNF-${\alpha}$ from human immune B and T cells was showed secretion for the amount of cytokines by bark extracts of S. japonica Sieb. et Zucc. NK cell growth was increased against control all of the extracts of S. japonica Sieb. et Zucc. Densitometric analysis of Bcl-2 revealed that possible to decrease potentialities of taking cancer in adding of extracts from S. japonica Sieb. et Zucc. From the results, the roots and barks extracts of S. japonica Sieb. et Zucc. were showed useful biological activities.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.17 no.3
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• pp.127-135
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• 2016

Two-pore domain potassium (K2P) channels are the targets of physiological stimuli, such as intracellular pH, bioactive lipids, and neurotransmitters, and they set the resting membrane potential. Some types of K2P channels play a critical role in both apoptosis and tumoriogenesis. Among the K2P channels, no antagonists of the TREK2 channel have been reported. The aim of the present study was to determine if the TREK2 channel is blocked and whether cell proliferation is influenced by flavonoids in the TREK2 overexpressing HEK293 cells (HEKT2). The electrophysiological current was recorded using single channel patch clamp techniques and cell proliferation was measured using a XTT assay. The electrophysiological results showed that the TREK2 channel activity was reduced to $91.5{\pm}13.1%$ (n=5) and $82.2{\pm}13.7%$ (n=5) by flavonoids, such as epigallocatechin-3-gallate (EGCG) and quercetin in HEKT2 cells, respectively. In contrast, the EGCG analogue, epicatechin (EC), had no significant inhibitory effects on the TREK2 single channel activity. In addition, cell proliferation was reduced to $69.4{\pm}14.0%$ (n=4) by ECGG in the HEKT2 cells. From these results, EGCG and quercetin represent the first known TREK2 channel inhibitors and only EGCG reduced HEKT2 cell proliferation. This suggests that the flavonoids may work primarily by inhibiting the TREK2 channel, leading to a change in the resting membrane potential, and triggering the initiation of a change in intracellular signaling for cell proliferation. TREK2 channel may, at least in part, contribute to cell proliferation.

• Korean Journal of Food Science and Technology
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• v.41 no.5
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• pp.552-559
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• 2009

In this study, the anticancer activity of the water extract at $100^{\circ}C$ was compared to that of the callus extracts via a ultrasonification extraction process. All the extracts were utilized to evaluate cytotoxicity, antioxidant and immune activities. The callus extracted via ultrasonification extraction showed relatively low cytotoxicity on normal human cell lines, HEK293 and HEL299, showing 13.17% and 21.78%, respectively. The callus extract has 59.82% which was similar to 61.70% for water extracts. It was also found that callus extract yielded higher nitric oxide secretion form macrophage than other extracts. The growths of both human stomach adenocarcinoma (AGS) cell and human lung carcinoma (A549) were inhibited up to 70% by adding 1.0 mg/mL of the callus extracts with ultrasonification extraction. This inhibition ratio (70%) was almost close to that of water extract. Human hepatoma carcinoma (HEP3B) cell growth was most significantly inhibited up to 75% by adding 1.0 mg/mL of callus extracts, and its selectivity was highest compared to other extracts. It indicates that the callus extracts could selectively inhibit growth of digestive system-related cancer cells. It can be also concluded from the results of this study that the callus extracts associated with ultrasonification extraction process have the potential for anticancer activity.

• Journal of Life Science
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• v.25 no.5
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• pp.594-600
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• 2015

Protein-protein interactions have a critical role in the regulation of many cellular functions. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain is one of domains that mediate protein-protein interactions. PDZ domains typically bind to the specific motif at the carboxyl (C)-terminal end of partner proteins. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, serves a scaffolding function for structure proteins and signaling proteins, but the cellular function of MUPP1 has not been fully elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and muskelin. Muskelin was recently identified as a GABA_A receptor (GABA_AR) α1 subunit binding protein and known to have a role in receptor endocytosis and degradation. Muskelin bound to the 3^rd PDZ domain, but not to other PDZ domains of MUPP1. The C-terminal end of muskelin was essential for the interaction with MUPP1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, muskelin but not the C-terminal deleted muskelin was co-immunoprecipitated with MUPP1. In addition, MUPP1 co-localized with muskelin at the same subcellular region in cells. These findings collectively suggest that MUPP1 or its interacting proteins could modulate GABA_AR trafficking and turnover through the interaction with muskelin.

• Journal of Life Science
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• v.16 no.4
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• pp.571-578
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• 2006

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

• Journal of Life Science
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• v.26 no.2
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• pp.147-154
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• 2016

Osteosarcoma (OS) is the most common and malignant bone tumors. Although many types of resection surgery and experimental agents were developed, median survival and clinical prognosis are poorly investigated. Recently, several researches have reported that Eucheuma cottonii has potent as protective effects of coal dust-induced lung damage via inhibition of malondialdehyde (MDA) and oxidative stress in bronchoalveolar lavage fluids (BALF). However, anti-cancer effects and specific molecular mechanism of extract from Eucheuma cottonii (EE) has not been clearly studied yet. This study evaluated that anti-cancer potential of EE in human osteosarcoma Saos-2 cells. EE indicated cytotoxicity on Saos-2 cells in a dose-dependent manner. Morphological degradation and nucleic condensation were also observed under the EE treatment. However, it did not significantly affect on non-cancerous kidney HEK-293 cells under the same concentration which is shown cytotoxicity on Saos-2 cells. The phosphorylation of Fas-Associated Death Domain (FADD) and expression of cleaved caspase-8, -7 and -3 were upregulated in a dose-dependent manner. In immunofluorescence staining, expression level of Fas and cleaved PARP were upregulated by EE treatment. Furthermore, treatment of EE induces upregulation of sub G1 phase by flow cytometry analysis. The results demonstrated that EE has a therapeutic potential against osteosarcoma via FADD mediated caspase cascade apoptosis signal pathway.

• Journal of Life Science
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• v.31 no.3
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• pp.257-265
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• 2021

Heterotrimeric kinesin-2 is a molecular motor protein of the kinesin superfamily (KIF) that moves along a microtubule plus-end directed motor protein. It consists of three different motor subunits (KIF3A, KIF3B, and KIF3C) and a kinesin-associated protein 3 (KAP3) that form a heterotrimeric complex. Heterotrimeric kinesin-2 interacts with many different binding proteins through the cargo-binding domain of the KIF3s. The activity of heterotrimeric kinesin-2 is regulated to ensure that the cargo is directed to the right place at the right time. How this regulation occurs, however, remains in question. To identify the regulatory proteins for heterotrimeric kinesin-2, we performed yeast two-hybrid screening and found a specific interaction with creatine kinase B (CKB), which is the brain isoform of cytosolic creatine kinase enzyme. CKB bound to the cargo-binding domain of KIF3A but did not interact with the KIF3B, KIF5B, or KAP3 in the yeast two-hybrid assay. The carboxyl (C)-terminal region of CKB is essential for the interaction with KIF3A. Another protein kinase, CaMKIIa, interacted with KIF3A, but GSK3a did not interact with KIF3A in the yeast two-hybrid assay. KIF3A interacted with GST-CKB-C but not with GSK-CKB-N or GST alone. When co-expressed in HEK-293T cells, CKB co-localized with KIF3A and co-immunoprecipitated with KIF3A and KIF3B but not KIF5B. These results suggest that the CKB-KIF3A interaction may regulate the cargo transport of heterotrimeric kinesin-2 under energy-compromised conditions in cells.

• Journal of Life Science
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• v.33 no.1
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• pp.1-7
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• 2023

Intracellular cargo transport is mediated by molecular motor proteins, such as kinesin and cytoplasmic dynein. Kinesins make up a large subfamily of molecular motors. Kinesin-1 is a plus-end-directed molecular motor protein that moves various cargoes, such as organelles, protein complexes, and mRNAs, along a microtubule track. It consists of the kinesin superfamily protein (KIF) 5A, 5B, and 5C (also called kinesin heavy chains) and kinesin light chains (KLCs). Kinesin-1 interacts with many different binding proteins through its carboxyl (C)-terminal region of KIF5s and KLCs, but their binding proteins have not yet been fully identified. In this study, a yeast two-hybrid assay was used to identify the proteins that interact with the KIF5A specific C-terminal region. The assay revealed an interaction between KIF5A and glutamate-rich 4 (ERICH4). ERICH4 bound to the KIF5A specific the C-terminal region but did not interact with the C-terminal region of KIF5B or KIF3A (a motor protein of kinesin-2). In addition, KIF5A did not interact with another isoform, ERICH1. Glutathione S-transferase (GST) pull-downs showed that KIF5A interacts with GST-ERICH4 and GST-ERICH4-amino (N)-terminal but not with GST-ERICH4-C or GST alone. When co-expressed in HEK-293T cells, ERICH4 co-localized with KIF5A and co-immunoprecipitated with KIF5A and KLC but not KIF3B. Together, our findings suggest that ERICH4 is capable of binding to KIF5A and that it may serve as an adaptor protein that links kinesin-1 with cargo.