• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.247-254
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• 2015

In this presentation, I describe the expression and purification of the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a commercially available double cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the soluble form. I describe here the expression and characterization of a biologically active heterodimer composed of the liver X receptor β-ligand binding domain and retinoid X receptor α-ligand binding domain. Although many of these proteins were previously seen to be produced in E. coli as insoluble aggregates or "inclusion bodies", I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests that co-expression of the liver X receptor β-ligand binding domain with its binding partner improves the solubility of the complex and probably assists in their correct folding, thereby functioning as a type of molecular chaperone.

• Journal of Korean Society of Water and Wastewater
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• v.20 no.6
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• pp.893-904
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• 2006

To improve sensitivity of biosensor as yeast two-hybrid detection system for estrogenic activity of suspected chemicals, we tested effects of several combinations of the bait and fish components in the two-hybrid system on Saccharomyces cerevisiae inducted a chromosome-integrated lacZ reporter gene that was under the control of CYC1 promoter and the upstream Gal4p-binding element $UAS_{GAL}$. The bait components that were fused with the Gal4p DNA binding domain are full-length human estrogen receptor ${\alpha}$ and its ligand-binding domain. The fish components that were fused with the Gal4p transcriptional activation domain were nuclear receptor-binding domains of co-activators SRC1 and TIF2. We found that the combination of the full-length human estrogen receptor ${\alpha}$ with the nuclear receptor-binding domain of co-activator SRC1 was most effective for the estrogen-dependent induction of reporter activity among the two-hybrid systems so far reported. The relative strength of transcriptional activation by representative natural and xenobiotic chemicals was well correlated with their estrogenic potency that had been reported with other assay systems.

• BMB Reports
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• v.40 no.5
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• pp.783-790
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• 2007

Receptor binding plays an important role in determining host specificity of the Bacillus thuringiensis Cry $\delta$-endotoxins. Mutations in domains II and III have suggested the participation of certain residues in receptor recognition and insect specificity. In the present study, we expressed the cloned domain II-III fragment of Cry4Ba and examined its binding characteristics to mosquito-larval midgut proteins. The 43-kDa Cry4Ba-domain II-III protein over-expressed in Escherichia coli as inclusion bodies was only soluble when carbonate buffer, pH 10.0 was supplemented with 4M urea. After renaturation via stepwise dialysis and subsequent purification, the refolded domain II-III protein, which specifically reacts with anti Cry4Ba-domain III monoclonal antibody, predominantly exists as a $\beta$-sheet structure determined by circular dichroism spectroscopy. In vitro binding analysis to both histological midgut tissue sections and brush border membrane proteins prepared from susceptible Aedes aegypti mosquito-larvae revealed that the isolated Cry4Ba-domain II-III protein showed binding functionality comparable to the 65-kDa full-length active toxin. Altogether, the data present the 43-kDa Cry4Ba fragment comprising domains II and III that was produced in isolation was able to retain its receptor-binding characteristics to the target larval midgut proteins.

• Environmental Mutagens and Carcinogens
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• v.22 no.3
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• pp.175-182
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• 2002

Endocrine disruptors (EDs) are the chemicals that affect endocrine systems through activation or inhibition of steroid hormone response. It is necessary to have a good system to evaluate rapidly and accurately endocrine-disrupting activities of suspected chemicals and their degradation products. The key targets of EDs are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We constructed a co-expression system of Gal4p DNA binding domain (DBD)- ligand binding domain of human estrogen receptor $\alpha$ or $\beta$, and Gal4p transactivation domain (TAD)-co-activator AIB-1, SRC-1 or TIF-2 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of CYC1 promoter and Gal4p binding site (GAL4 upstream activating sequence, GAL4$_{UAS}$). Expression of this reporter gene was dependent on the presence of estrogen or EDs in the culture medium. We found that the two-hybrid system with combination of the hER$\beta$ LBD and co-activator SRC-1 was most effective in the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by those chemicals correlated with their estrogenic activities measured by other assay systems, indicating that this assay system is efficient and reliable for measuring estrogenic activity. The data in this research demonstrated that the yeast detection system using steroid hormone receptor and co-activator is a useful tool for identifying chemicals that interact with steroid receptors.s.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.6
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• pp.913-919
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• 2015

In vertebrates, the vitamin D receptor (VDR), a member of the nuclear receptor superfamily, binds the biologically active ligand $1{\alpha},25-(OH)_2$-vitamin $D_3$ (1,25 $D_3$). Nearly all vertebrates, including Agnatha, possess a VDR with high ligand selectivity for 1,25 $D_3$ and related metabolites. Although a putative ancestral VDR gene is present in the genome of the chordate invertebrate Ciona intestinalis, the functional characteristics of marine invertebrate VDR are still obscure. To elucidate the ascidian Halocynthia roretzi VDR (HrVDR), we cloned full-length HrVDR cDNA and investigated the transcriptional activity of HrVDR in HEK293 cells. HrVDR consists of 1,680 nucleotides (559 amino acids [aa]), including a short N-terminal region (A/B domain; 26 aa), DNA-binding domain (C domain; 72 aa), hinge region (D domain; 272 aa), and C-terminal ligand-binding domain (E domain; 161 aa). The amino acid sequence identity of HrVDR was greatest to that of C. intestinalis VDR (56%). In the luciferase reporter assays, the transcriptional activity of HrVDR was not significantly increased by 1,25 $D_3$, whereas the farnesoid X receptor agonist GW4064 increased the transactivation of HrVDR. These results suggest the presence of a novel ligand for and a distinct ligand-binding domain in ascidian VDR.

• Molecules and Cells
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• v.44 no.1
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• pp.1-12
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• 2021

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.

• Microbiology and Biotechnology Letters
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• v.39 no.2
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• pp.140-145
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• 2011

Porcine epidemic diarrhea virus (PEDV) infection causes acute enteritis with lethal watery diarrhea resulting in a high mortality rate in piglets. As with the other members of group 1 coronaviruses, PEDV also utilizes the host aminopeptidase N (APN) as the major cellular receptor for entry into target cells. The coronavirus spike (S) protein is known to interact with the cellular surface for viral attachment and the S1 domain of all characterized coronaviruses contains a receptor-binding domain (RBD) that mediates a specific high-affinity interaction with their respective cellular receptors. Although the RBDs of several coronaviruses have been mapped, the location of the PEDV RBD has to date not been defined. As a first step toward the identification of the region of the S protein of the PEDV that is critical for recognition with the cellular receptor, we generated a series of S1-truncated variants and examined their abilities to bind to the porcine APN (pAPN) receptor. Our data indicate that the N-terminus of the S1 domain is required for pAPN association. The results from the present study may assist in our understanding of the molecular interactions between the PEDV S protein and the pAPN receptor.

• KSBB Journal
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• v.7 no.3
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• pp.201-208
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• 1992

Cell surface binding of epidermal growth factor(EGF) to EGF receptors was studied for a series of site-directed receptor mutants transfected into B82 mouse fibroblasts. Scatchard plots for truncation mutant receptors significantly lost nonlinearity for truncations below residue 1022. Transient plots of dissociation kinetics exhibited biphasic behavior for all receptor types, but the fraction of receptor in slow-dissociating form was reduced by an order of magnitude for the truncation mutants below residue 1022. Comparison of dissociation kinetics between control cells and cells treated with Triton X-100 revealed no significant variation for the slow-dissociating receptor form, but a noticeable variation was observed for the fast-dissociating receptor form when EGF receptors were truncated below residue 991. These results suggest that high affinity of EGF binding at cell surface depend on the EGF receptor cytoplasmic region.

• Genomics & Informatics
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• v.21 no.3
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• pp.30.1-30.13
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• 2023

Ephs belong to the largest family of receptor tyrosine kinase and are highly conserved both sequentially and structurally. The structural organization of Eph is similar to other receptor tyrosine kinases; constituting the extracellular ligand binding domain, a fibronectin domain followed by intracellular juxtamembrane kinase, and SAM domain. Eph binds to respective ephrin ligand, through the ligand binding domain and forms a tetrameric complex to activate the kinase domain. Eph-ephrin regulates many downstream pathways that lead to physiological events such as cell migration, proliferation, and growth. Therefore, considering the importance of Eph-ephrin class of protein in tumorigenesis, 7,620 clinically reported missense mutations belonging to the class of variables of unknown significance were retrieved from cBioPortal and evaluated for pathogenicity. Thirty-two mutations predicted to be pathogenic using SIFT, Polyphen-2, PROVEAN, SNPs&GO, PMut, iSTABLE, and PremPS in-silico tools were found located either in critical functional regions or encompassing interactions at the binding interface of Eph-ephrin. However, seven were reported in nonsmall cell lung cancer (NSCLC). Considering the relevance of receptor tyrosine kinases and Eph in NSCLC, these seven mutations were assessed for change in the folding pattern using molecular dynamic simulation. Structural alterations, stability, flexibility, compactness, and solvent-exposed area was observed in EphA3 Trp790Cys, EphA7 Leu749Phe, EphB1 Gly685Cys, EphB4 Val748Ala, and Ephrin A2 Trp112Cys. Hence, it can be concluded that the evaluated mutations have potential to alter the folding pattern and thus can be further validated by in-vitro, structural and in-vivo studies for clinical management.

• Molecules and Cells
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• v.27 no.6
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• pp.629-634
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• 2009

G protein-coupled receptors (GPCRs) are part of multi-protein networks called 'receptosomes'. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.