• Journal of Integrative Natural Science
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• v.2 no.2
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• pp.55-58
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• 2009

A ligand-receptor docking program is an indispensible tool in modern pharmaceutical design. An accurate prediction of small molecular docking pose to a receptor is essential in drug design as well as molecular recognition. An effective docking program requires the ability to locate a correct binding pose in a surprisingly complex conformational space. However, there is an inherent difficulty to predict correct binding pose. The odds are more demanding than finding a needle in a haystack. This mainly comes from the flexibility of both ligand and receptor. Because the searching space to consider is so vast, receptor rigidity has been often applied in docking programs. Even nowadays the receptor may not be considered to be fully flexible although there have been some progress in search algorithm. Improving the efficiency of searching algorithm is still in great demand to explore other applications areas with inherently flexible ligand and/or receptor. In addition to classical search algorithms such as molecular dynamics, Monte Carlo, genetic algorithm and simulated annealing, rather recent algorithms such as tabu search, stochastic tunneling, particle swarm optimizations were also found to be effective. A good search algorithm would require a good balance between exploration and exploitation. It would be a good strategy to combine algorithms already developed. This composite algorithms can be more effective than an individual search algorithms.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.315-321
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• 2018

Molecular and functional characterization of proteins and their levels is of great interest in understanding the mechanism of diverse cellular processes. In this study, we report on the convenient Escherichia coli-based protein expression system that allows recombinant of soluble proteins expression and cytosolic domain of membrane-localised kinases, followed by the detection of autophosphorylation activity in protein kinases. This approach is applied to regulatory proteins of Arabidopsis thaliana, including 14-3-3, calmodulin, calcium-dependent protein kinase, TERMINAL FLOWER 1(TFL1), FLOWERING LOCUS T (FT), receptor-like cytoplasmic kinase and cytoplasmic domain of leucine-rich repeat-receptor like kinase proteins. Our Western blot analysis which uses phospho-specific antibodies showed that five putative LRR-RLKs and two putative RLCKs have autophosphorylation activity in vitro on threonine and/or tyrosine residue(s), suggesting their potential role in signal transduction pathways. Our findings were also discussed in the broader context of recombinant expression and biochemical analysis of soluble and membrane-localised receptor kinases in microbial systems.

• Biomolecules & Therapeutics
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• v.23 no.3
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• pp.218-224
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• 2015

Endocannabinoids can affect multiple cellular targets, such as cannabinoid (CB) receptors, transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and peroxisome proliferator-activated receptor ${\gamma}$($PPAR{\gamma}$). The stimuli to induce adipocyte differentiation in hBM-MSCs increase the gene transcription of the $CB_1$ receptor, TRPV1 and $PPAR{\gamma}$. In this study, the effects of three endocannabinoids, N-arachidonoyl ethanolamine (AEA), N-arachidonoyl dopamine (NADA) and 2-arachidonoyl glycerol (2-AG), on adipogenesis in hBM-MSCs were evaluated. The adipocyte differentiation was promoted by AEA whereas inhibited by NADA. No change was observed by the treatment of non-cytotoxic concentrations of 2-AG. The difference between AEA and NADA in the regulation of adipogenesis is associated with their effects on $PPAR{\gamma}$ transactivation. AEA can directly activate $PPAR{\gamma}$. The effect of AEA on $PPAR{\gamma}$ in hBM-MSCs may prevail over that on the $CB_1$ receptor mediated signal transduction, giving rise to the AEA-induced promotion of adipogenesis. In contrast, NADA had no effect on the $PPAR{\gamma}$ activity in the $PPAR{\gamma}$ transactivation assay. The inhibitory effect of NADA on adipogenesis in hBM-MSCs was reversed not by capsazepine, a TRPV1 antagonist, but by rimonabant, a $CB_1$ antagonist/inverse agonist. Rimonabant by itself promoted adipogenesis in hBM-MSCs, which may be interpreted as the result of the inverse agonism of the $CB_1$ receptor. This result suggests that the constantly active $CB_1$ receptor may contribute to suppress the adipocyte differentiation of hBM-MSCs. Therefore, the selective $CB_1$ agonists that are unable to affect cellular $PPAR{\gamma}$ activity inhibit adipogenesis in hBM-MSCs.

• Molecules and Cells
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• v.43 no.11
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• pp.935-944
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• 2020

Aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in maintaining cellular homeostasis in response to environmental stress. Under conditions of hypoxia or xenobiotic exposure, ARNT regulates the subset of genes involved in adaptive responses, by forming heterodimers with hypoxia-inducible transcription factors (HIF1α and HIF2α) or aryl hydrocarbon receptor (AhR). Here, we have shown that ARNT interacts with DDB1 and CUL4-associated factor 15 (DCAF15), and the aryl sulfonamides, indisulam and E7820, induce its proteasomal degradation through Cullin-RING finger ligase 4 containing DCAF15 (CRL4^DCAF15) E3 ligase. Moreover, the two known neo-substrates of aryl sulfonamide, RNA-binding motif protein 39 (RBM39) and RNA-binding motif protein 23 (RBM23), are not required for ARNT degradation. In line with this finding, aryl sulfonamides inhibited the transcriptional activities of HIFs and AhR associated with ARNT. Our results collectively support novel regulatory roles of aryl sulfonamides in both hypoxic and xenobiotic responses.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.2
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• pp.105-109
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• 2015

NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.

• Molecules and Cells
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• v.40 no.10
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• pp.787-795
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• 2017

Avoiding ingestion of excessively salty food is essential for cation homeostasis that underlies various physiological processes in organisms. The molecular and cellular basis of the aversive salt taste, however, remains elusive. Through a behavioral reverse genetic screening, we discover that feeding suppression by $Na^+$-rich food requires Ionotropic Receptor 76b (Ir76b) in Drosophila labellar gustatory receptor neurons (GRNs). Concentrated sodium solutions with various anions caused feeding suppression dependent on Ir76b. Feeding aversion to caffeine and high concentrations of divalent cations and sorbitol was unimpaired in Ir76b-deficient animals, indicating sensory specificity of Ir76b-dependent $Na^+$ detection and the irrelevance of hyperosmolarity-driven mechanosensation to Ir76b-mediated feeding aversion. Ir76b-dependent $Na^+$-sensing GRNs in both L- and s-bristles are required for repulsion as opposed to the previous report where the L-bristle GRNs direct only low-$Na^+$ attraction. Our work extends the physiological implications of Ir76b from low-$Na^+$ attraction to high-$Na^+$ aversion, prompting further investigation of the physiological mechanisms that modulate two competing components of $Na^+$-evoked gustation coded in heterogeneous Ir76b-positive GRNs.

• Archives of Pharmacal Research
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• v.20 no.6
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• pp.572-578
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• 1997

To gain further insight into how antiestrogens modulate cell function, the effects of antiestrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of trans-tamoxifen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Trans-tamoxifen $(1{\mu}M)$ markedly inhibited the estrogen stimulated proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor $(1.15{\pm}0.03 pmole/mg protein)$ over that of control. In T47D cells that contained low levels of estrogen receptor $(0.23{\pm}0.05 pmole/mg protein)$, trans-tamoxifen $(1{\mu}M)$ showed minimal inhibition of estrogen stimulated cell proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by trans-tamoxifen treatment. These results showed their sensitivity to growth inhibition by antiestrogen conrrelated well with their estrogen receptor content. Also we examined the effect of antiestrogen on cellular progestrone receptor level as well as plasminogen activator activity in MCF-7 cells. Trans-tamoxifen $(1{\mu}M)$ showed maximal inhibition of estrogen stimulated progestrone receptor level as well as plasminogen activator activity in MCF-7 cells that were stimulated by estrogen. It is not clear whether these inhibitions of progestrone receptor and plasminogen activator activity by estrogen are related to the antiestrogen inhibition of cell proliferation of MCF-7 cells. From the results of this study, it is clearly demonstrated that trans-tamoxifen is an antiestrogen in MCF-7 human breast cancer cells. Our data suggest that the biological effectiveness of trans-tamoxifen appear to result from its affinity of interaction with the estrogen receptor.

• BMB Reports
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• v.41 no.2
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• pp.139-145
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• 2008

We cloned and pharmacologically characterized the guinea pig cysteinyl leukotriene (CysLT) 2 receptor (gpCysLT2). gpCysLT2 consists of 317 amino acids with 75.3%, 75.2%, 73.3% identity to those of humans, mice and rats, respectively. The gpCysLT2 gene is highly expressed in the lung, moderately in eosinophils, skin, spleen, stomach, colon, and modestly in the small intestine. CysLTs accelerated the proliferation of gpCysLT2-expressing HEK293. Leukotriene C4 (LTC4) and Leukotriene D4 (LTD4) enhanced the cell proliferation higher than Bay-u9773, a CysLT2 selective partial agonist and a nonselective antagonist for CysLT receptors. Bay-u9773 did not antagonize the cell proliferation by LTC4 and LTD4. Despite the equipotency of the mitogenic effect among these chemicals, calcium mobilization (CM) levels were variable (LTC4 > LTD4 >> Bay-u9773), and Bay-u9773 antagonized the CM by LTC4. Moreover, the Gi/o inhibitor pertussis toxin perfectly inhibited agonist-induced cell proliferation. These results reveal that cell proliferation via CysLT2 signaling was mediated by Gi/o signaling but independent of calcium mobilization.

• Biomolecules & Therapeutics
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• v.27 no.5
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• pp.474-483
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• 2019

Vascular endothelial growth factor (VEGF) plays a pivotal role in pathologic ocular neovascularization and vascular leakage via activation of VEGF receptor 2 (VEGFR2). This study was undertaken to evaluate the therapeutic mechanisms and effects of the tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a VEGFR2 inhibitor, in the development of vascular permeability and choroidal neovascularization (CNV). In cultured human retinal microvascular endothelial cells (HRMECs), treatment with RLYE blocked VEGF-A-induced phosphorylation of VEGFR2, Akt, ERK, and endothelial nitric oxide synthase (eNOS), leading to suppression of VEGF-A-mediated hyper-production of NO. Treatment with RLYE also inhibited VEGF-A-stimulated angiogenic processes (migration, proliferation, and tube formation) and the hyperpermeability of HRMECs, in addition to attenuating VEGF-A-induced angiogenesis and vascular permeability in mice. The anti-vascular permeability activity of RLYE was correlated with enhanced stability and positioning of the junction proteins VE-cadherin, ${\beta}$-catenin, claudin-5, and ZO-1, critical components of the cortical actin ring structure and retinal endothelial barrier, at the boundary between HRMECs stimulated with VEGF-A. Furthermore, intravitreally injected RLYE bound to retinal microvascular endothelium and inhibited laser-induced CNV in mice. These findings suggest that RLYE has potential as a therapeutic drug for the treatment of CNV by preventing VEGFR2-mediated vascular leakage and angiogenesis.

• Molecules and Cells
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• v.37 no.3
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• pp.257-263
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• 2014

A mammalian cell renovates itself by autophagy, a process through which cellular components are recycled to produce energy and maintain homeostasis. Recently, the abundance of gap junction proteins was shown to be regulated by autophagy during starvation conditions, suggesting that transmembrane proteins are also regulated by autophagy. Transient receptor potential vanilloid type 1 (TRPV1), an ion channel localized to the plasma membrane and endoplasmic reticulum (ER), is a sensory transducer that is activated by a wide variety of exogenous and endogenous physical and chemical stimuli. Intriguingly, the abundance of cellular TRPV1 can change dynamically under pathological conditions. However, the mechanisms by which the protein levels of TRPV1 are regulated have not yet been explored. Therefore, we investigated the mechanisms of TRPV1 recycling using HeLa cells constitutively expressing TRPV1. Endogenous TRPV1 was degraded in starvation conditions; this degradation was blocked by chloroquine (CLQ), 3MA, or downregulation of Atg7. Interestingly, a glucocorticoid (cortisol) was capable of inducing autophagy in HeLa cells. Cortisol increased cellular conversion of LC3-I to LC-3II, leading autophagy and resulting in TRPV1 degradation, which was similarly inhibited by treatment with CLQ, 3MA, or downregulation of Atg7. Furthermore, cortisol treatment induced the colocalization of GFP-LC3 with endogenous TRPV1. Cumulatively, these observations provide evidence that degradation of TRPV1 is mediated by autophagy, and that this pathway can be enhanced by cortisol.