ABSTRACT
To characterize the potential endocrine-disrupting chemicals (EDCs) in the environment that interact with the crustacean ecdysone receptor (EcR), we established a method involving in silico modeling/molecular docking and in vitro reporter gene assay. Cherry shrimp (Neocaridina davidi) EcR (NdEcR) and retinoid X receptor (NdRxR) were identified and cloned for use in this method. A theoretical 3D model of NdEcR ligand-binding domain (LBD) was built in silico based on sequence homology with the established X-ray structure of insect EcR. The interaction of the NdEcR LBD with ecdysteroids, diacylhydrazine (DAH) pesticides, and other potential EDCs was evaluated using molecular docking programs. The results revealed that the ligand-binding pocket in the NdEcR LBD was flexible and adaptive for accommodating ligands of different shapes. The agonistic and antagonistic activities of the candidate compounds were further assessed by in vitro reporter gene assay using human cell lines transiently transfected with NdEcR and NdRxR expression plasmids and a reporter plasmid containing synthesized ecdysone response element. The assay was validated by the dose-dependent responses of EcR-mediated gene transcription after treating the transfected cell lines with ecdysteroids, 20-hydroxyecdysone, and ponasterone A. Examination of the candidate compounds using the reporter gene assay revealed restricted functional specificity to ecdysteroids and DAHs. Three of the tested DAH pesticides originally targeting the insect EcR were found to be weak agonists and strong antagonists of NdEcR. These results suggest that DAHs are potential EDCs for crustaceans that disrupt their ecdysteroid signals by functioning as EcR agonists or antagonists.
Subject(s)
Crustacea/drug effects , Ecdysteroids/pharmacology , Pesticides/toxicity , Receptors, Steroid/metabolism , Animals , Binding Sites , Cell Line , Computer Simulation , Crustacea/metabolism , Decapoda/genetics , Ecdysone/metabolism , Ecdysone/pharmacology , Ecdysteroids/toxicity , Ecdysterone/analogs & derivatives , Ecdysterone/pharmacology , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Molecular Docking Simulation , Pesticides/chemistry , Pesticides/metabolism , Phylogeny , Receptors, Steroid/agonists , Receptors, Steroid/antagonists & inhibitors , Receptors, Steroid/genetics , Retinoid X Receptors/chemistry , Retinoid X Receptors/genetics , Retinoid X Receptors/metabolismABSTRACT
The liquid crystalline chromosomes of dinoflagellates are the alternative to the nucleosome-based organization of chromosomes in the eukaryotes. These nucleosome-less chromosomes have to devise novel ways to maintain active parts of the genome. The dinoflagellate histone-like protein HCc3 has significant sequence identity with the bacterial DNA-binding protein HU. HCc3 also has a secondary structure resembling HU in silico. We have examined HCc3 in its recombinant form. Experiments on DNA-cellulose revealed its DNA-binding activity is on the C-terminal domain. The N-terminal domain is responsible for intermolecular oligomerization as demonstrated by cross-linking studies. However, HCc3 could not complement Escherichia coli HU-deficient mutants, suggesting functional differences. In ligation assays, HCc3-induced DNA concatenation but not ring closure as the DNA-bending HU does. The basic HCc3 was an efficient DNA condensing agent, but it did not behave like an ordinary polycationic compound. HCc3 also induced specific structures with DNA in a concentration-dependent manner, as demonstrated by atomic force microscopy (AFM). At moderate concentration of HCc3, DNA bridging and bundling were observed; at high concentrations, the complexes were even more condensed. These results are consistent with a biophysical role for HCc3 in maintaining extended DNA loops at the periphery of liquid crystalline chromosomes.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/ultrastructure , Dinoflagellida/genetics , Protozoan Proteins/chemistry , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Computational Biology , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Microscopy, Atomic Force , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sequence AlignmentABSTRACT
Complementary DNA regions coding for two different mature goldfish growth hormones (gfGH-I and gfGH-II) with four and five cysteine residues were cloned into the bacterial expression vector, pRSETA. The recombinant gfGH-I (five cysteines) and -II (four cysteines) were produced in Escherichia coli as the fusion proteins carrying N-terminal 6XHis tag, which facilitates purification by using metal chelating affinity chromatography under denaturing condition with urea. The recombinant hormones were further refolded by gradually removing the urea. Native gfGH was also purified from goldfish pituitary glands and served as a positive control in the present study. The native and recombinant hormones were tested in goldfish hepatic radioreceptor assay and in vitro Spi 2.1 promoter activation assay. Our results showed that the two recombinant gfGHs are biologically active, and they have similar biological activities despite their having different cysteine contents.