• Korean Journal of Medicinal Crop Science
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• v.17 no.3
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• pp.210-216
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• 2009

This study was performed to compare the effects of immuno-modulating activities of Rhodiola sachalinensis A. Bor. fractionized by consecutive solvent separation. The Cytotoxicity of all fractionized extracts on human kidney cell (HEK293) was lower than crude extracts. Generally, the butanol and chloroform extracts showed less cytotoxicity on about 10.07% and 9.67% than the crude extracts. For human immune B and T cell growth, chloroform fraction showed the highest cell growth compared to the control. The secretion of cytokines (IL-6, $TNF-\alpha$) on human B and T cells were increased by adding chloroform extracts. Also, NK cell growth was significantly improved up to nearly 30% by adding the supernatant of B cell medium grown with the chloroform fraction. It was also found that chloroform fraction could yield higher nitric oxide production from macrophage than untreated control cells. Differentiation of HL-60 cells was increased up to 131.9% after treatment with chloroform fraction extracts, compared to the control. These results indicate that the chloroform fraction of R. sachalinensis have high immune activation activity than others fractions and the crude extracts, implying that this chloroform fractions could be used a new functional material.

• 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.

• 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.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.32 no.3
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• pp.189-195
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• 2022

Kinesin-2 comprises two subfamilies of the heterotrimeric or homodimeric motors found in mammalian cells. Heterotrimeric kinesin-2 consists of kinesin superfamily proteins (KIFs) 3A and 3B and kinesin-associated protein 3 (KAP3), which is a molecular motor protein that moves along microtubules. It plays diverse roles in cargo transport, including anterograde trafficking in cilia, and interacts with many different cargoes and proteins, but their binding proteins have not yet been fully identified. In this study, the yeast two-hybrid assay was used to identify the proteins that interact with the cargo-binding domain (CBD) of KIF3A, and an interaction between KIF3A and brain expressed X-linked 2 (Bex2) was found. Bex2 bound to the CBD-containing C-terminal tail region of KIF3A but did not interact with the same region of KIF3B or KIF5A (a motor protein of kinesin-1). KIF3A interacted with another isoform, Bex1, but did not interact with Bex3. In addition, glutathione S-transferase (GST) pull-downs showed that KIF3A specifically interacts with GST-Bex1 and GST-Bex2 but not with GST alone. When co-expressed in HEK-293T cells, Bex2 co-localized with KIF3A and co-immunoprecipitated with KIF3A and KIF3B but not KIF5B. In combination, these results suggest that Bex2 is capable of binding to heterotrimeric kinesin-2 and may serve as an adaptor protein that links heterotrimeric kinesin-2 with cargo.

• BMB Reports
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• v.55 no.6
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• pp.275-280
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• 2022

The treatment of atopic dermatitis (AD) is challenging due to its complex etiology. From epidermal disruption to chronic inflammation, various cells and inflammatory pathways contribute to the progression of AD. As with immunosuppressants, general inhibition of inflammatory pathways can be effective, but this approach is not suitable for long-term treatment due to its side effects. This study aimed to identify a plant extract (PE) with anti-inflammatory effects on multiple cell types involved in AD development and provide relevant mechanistic evidence. Degranulation was measured in RBL-2H3 cells to screen 30 PEs native to South Korea. To investigate the anti-inflammatory effects of Parasenecio auriculatus var. matsumurana Nakai extract (PAE) in AD, production of cytokines and nitric oxide, activation status of FcεRI and TLR4 signaling, cell-cell junction, and cell viability were evaluated using qRT-PCR, western blotting, confocal microscopy, Griess system, and an MTT assay in RBL-2H3, HEK293, RAW264.7, and HaCaT cells. For in vivo experiments, a DNCBinduced AD mouse model was constructed, and hematoxylin and eosin, periodic acid-Schiff, toluidine blue, and F4/80-staining were performed. The chemical constituents of PAE were analyzed by HPLC-MS. By measuring the anti-degranulation effects of 30 PEs in RBL-2H3 cells, we found that Paeonia lactiflora Pall., PA, and Rehmannia glutinosa (Gaertn.) Libosch. ex Steud. show an inhibitory activity of more than 50%. Of these, PAE most dramatically and consistently suppressed cytokine expression, including IL-4, IL-9, IL-13, and TNF-α. PAE potently inhibited FcεRI signaling, which mechanistically supports its basophil-stabilizing effects, and PAE downregulated cytokines and NO production in macrophages via perturbation of toll-like receptor signaling. Moreover, PAE suppressed cytokine production in keratinocytes and upregulated the expression of tight junction molecules ZO-1 and occludin. In a DNCB-induced AD mouse model, the topical application of PAE significantly improved atopic index scores, immune cell infiltration, cytokine expression, abnormal activation of signaling molecules in FcεRI and TLR signaling, and damaged skin structure compared with dexamethasone. The anti-inflammatory effect of PAE was mainly due to integerrimine. Our findings suggest that PAE could potently inhibit multi-inflammatory cells involved in AD development, synergistically block the propagation of inflammatory responses, and thus alleviate AD symptoms.

• Korean Journal of Food Science and Technology
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• v.41 no.3
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• pp.313-319
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• 2009

The marine microalga Spirulina maxima was extracted using water or ethanol at 100 or $80^{\circ}C$ and by ultrasonification in water at $60^{\circ}C$. The ultrasonification technique generated the highest yield (19.8%). To be therapeutically useful, the extraction should yield a product with low cytotoxicity and high immunity against skin infections. The cytotoxicity of all extracts (1.0 mg/mL) was below 25%. Moreover, the cytotoxicity of the extract generated by ultrasonification was 5%. Extracts prepared in the described manners could inhibit hyaluronidase activity by up to 40% compared to the control. Increased growth of human B, T and NK cells and an increase in cytokine secretion were observed, confirming the interrelationship between both human immune and skin immune activity. The extract prepared by ultrasonification increased the growth of human B, T and NK cells up to $10.3{\times}10^4$ cells/mL, $11.3{\times}10^4$ cells/mL and $19.1{\times}10^4$ cells/mL, respectively. The extract prepared by ultrasonification also greatly increased the secretion of both IL-6 and $TNF-{\alpha}$. Moreover, it was estimated that protein, Na and leucine occupy a high ratio. Accordingly, this study has confirmed that extracts prepared as described have the potential to effectively increase skin immunity.

• Korean Journal of Medicinal Crop Science
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• v.17 no.5
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• pp.311-320
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• 2009

The low quality fresh ginseng was fermented by Phelinus linteus or Hericium erinaceum mycelium. This fermented ginseng was extracted by water at $100^{\circ}C$ or water with ultrasonification at $60^{\circ}C$. Total phenolic compounds was improved by ultrasonification extraction process, compare to conventional water extraction. All extracts enhanced the growth of human B and T cells, showing 2.68 times and 3.43 times higher, respectively, than the control. The secretion of TNF-$\alpha$ and IL-6 from human immune cells was enhanced as $3.53{\times}10^{-4}\;pg/cell$, $3.40{\times}10^{-4}\;pg/cell$ by adding H. erinaceum mycelium fermented ginseng. H. erinaceum mycelium fermented ginseng yielded higher nitric oxide production from macrophage than Lipopolysaccharides (LPS). The cytotoxicity on human normal kidney cell (HEK293) was as low as 20.5% in adding the maximum concentration of $1.0\;mg/m{\ell}$ of fermented ginseng. Generally, the extracts from ultrasonification extraction process showed 10% lower toxicity than that by conventional process. H. erinaceum mycelium fermented ginseng had the highest anticancer activity on human lung cancer and stomach cancer cells as 69.33% and 75.32%, respectively at $1.0\;mg/m{\ell}$. It can be concluded that, in general, H. erinaceum mycelium fermented ginseng has relatively better immune and anticancer activities than P. linteus fermented ginseng. Expecially, the extracts treated with ultrasonification had higher activities than that from conventional extraction process.

• 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.