Ziwei Chang

Influence of divalent metal ions on E2-induced ER pathway in goldfish (Carassius auratus) hepatocytes

Metal ions existing in the environment could influence the estrogen pathway in aquatic animal, but the detailed mechanism is still delusive. We here showed that in male Carassius auratus hepatocytes, copper (Cu) or cadmium (Cd), did not directly induce vitellogenin (VTG) expression. Interestingly, co-exposure with Cd²⁺ (or Cu²⁺) and 17-β-estradiol (E2) greatly increased the VTG level, comparing with single treatment of E2. Meanwhile, Cd²⁺ or Cu²⁺ (but not E2) triggers HSP70 expression. But, mixture of Cd²⁺ or Cu²⁺ with E2 did not obviously raise HSP70 level. E2 also had no obvious effect on reactive oxygen species. Co-treatment of Cd²⁺ and E2 showed no obvious increase compared to single treatment with Cd²⁺. We further assume that Cd²⁺-involved oxidative stress generates misfolded proteins, resulting in the competition of HSP70 proteins from a heterocomplex (with estrogen receptor). Thus, dissociation of the heterocomplex actives the receptor-ligand binding activity and promotes the E2-induced VTG expression.

Potential role of HSP90 in mediating the interactions between estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) signaling pathways

The estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) are ligand-activated transcription factors involved in estrogen or xenobiotic exposure, whereas the 90-kDa heat shock protein (HSP90), which is a ubiquitously expressed molecular chaperone, is involved in the signal transduction process. Although the interactions between these pathways have been under investigation, the mechanisms are unclear and the potential role of HSP90 in these interactions has not been reported. The results of goldfish primary hepatocytes showed that exposure to PCB77 and 17β-estradiol (E2) alone induced significant protein expression of cytochrome P450 1A (CYP1A) and vitellogenin (VTG), respectively. On the other hand, the combined exposure to PCB77 and E2 led to the reduction of CYP1A and VTG compared to the single treatments. Although the AhRs and ERs were naturally induced during the co-treatment, the total amount of HSP90 binding to the receptors was not changed. Furthermore, while the HSP90 chaperon activity was blocked by the specific inhibitor (geldanamycin), reciprocal inhibition between AhR and ER pathways was not observed. These findings indicate a potential role of HSP90 where competition between AhR and ER for binding to HSP90 can occur and cause reciprocal inhibition.

Molecular characterization of the aryl hydrocarbon receptor (AhR) pathway in goldfish (Carassius auratus) exposure to TCDD: the mRNA and protein levels.

In bony fish or other aquatic vertebrates, the aryl hydrocarbon receptor (AhR) signaling pathway is initiated by exposure to polycyclic (or/and halogenated) aromatic hydrocarbons (PAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), which subsequently induces the up-regulated expression of a series of related genes (such as cytochrome P4501A (CYP1A)). However, a lack of applicable protein reagents hinders our further understanding of the AhR signaling pathway, which focuses only on gene-based investigations. The goldfish (Carassius auratus) is an ideal model for a study of environmental pollution in whole-Asian fresh water. Here, three sensitive and specific polyclonal antisera against goldfish AhR1, AhR2, and CYP1A proteins were developed. These antisera not only bound the in-vitro synthesized target proteins, but recognized the real proteins expressed in goldfish tissues, with minimal cross-reactivity to non-specific proteins. Together with the analysis of semi-quantitative RT-PCR and polyclonal-antibody-based sandwich ELISA, we confirmed that goldfish AhRs differed in the expression (mRNA and protein levels) patterns among test tissues. Importantly, the relative abundance of each AhR mRNA levels from the different tissues showed no obvious consistency with their protein levels. After exposure to TCDD, goldfish AhR2 showed a more sensitivity than AhR1, and stimulated CYP1A expression directly, similar with the other reported fish models. Overall, development of these antibodies in this study will allow valuable and versatile investigations to further understand the AhR signaling pathway, and different expression (mRNA and protein) patterns represent the first step in determining the regulatory mechanisms underlying the TCDD-exposed aquatic environment.

Functional expression of Carassius auratus cytochrome P4501A in a novel Shewanella oneidensis expression system and application for the degradation of benzo[a]pyrene

Cytochrome P4501A (CYP1A) in fish has attracted an increasing interest because of its important roles in the metabolic activation of certain xenobiotics such as aromatic hydrocarbons. CYPs are reported to be expressed in yeast, insect cells and Escherichia coli, but are critical for high-level expression. Besides, this study found that the purification of recombinant goldfish CYP1A would result in a loss of enzyme activity. Because large quantities of functional CYP1A are required, it is necessary to find a suitable host with high-level expression. In the present study, a novel expression system using Shewanella oneidensis was established successfully for the production of goldfish CYP1A. A signal peptide in the expression vector leads to the high-level expression in the periplasmic space of Shewanella. The recombinant CYP1A in Shewanella reached up to 1μmol per liter of culture, and showed the typical P450 hemoprotein spectra. An ethoxyresorufin O-deethylase assay revealed the amount of functional proteins in Shewanella to be almost ten times more than those in other expression systems. Furthermore, the CYP1A-mediated degradation of benzo[a]pyrene was also observed by (1)H NMR spectroscopy. These results indicate the possible application of periplasmic fractions to obtain sufficient quantities of functional CYP1A for further studies.

Functional HSF1 Requires Aromatic-Participant Interactions in Protecting Mouse Embryonic Fibroblasts against Apoptosis Via G2 Cell Cycle Arrest

The present study highlighted the aromatic-participant interactions in in vivo trimerization of HSF1 and got an insight into the process of HSF1 protecting against apoptosis. In mouse embryonic fibroblasts (MEFs), mutations of mouse HSF1 (W37A, Y60A and F104A) resulted in a loss of trimerization activity, impaired binding of the heat shock element (HSE) and lack of heat shock protein 70 (HSP70) expression after a heat shock. Under UV irradiation, wild-type mouse HSF1 protected the MEFs from UV-induced apoptosis, but none of the mutants offered protection. We found that normal expression of HSF1 was essential to the cell arrest in G2 phase, assisting with the cell cycle checkpoint. The cells that lack normal HSF1 failed to arrest in the G2 phase, resulting in the process of cell apoptosis. We conclude that the treatment with UV or heat shock stresses appears to induce the approach of HSF1 monomers directly via aromatic-participant interactions, followed by the formation of a HSF1 trimer. HSF1 protects the MEFs from the stresses through the expression of HSPs and a G2 cell cycle arrest.

Erratum to Expression Procedure Optimization of Carassius aurantus CYP1A in Shewanella Using Plasmid Construction Strategy

In the 2018 issue of Biotechnology and Bioprocess Engineering (BBE), an error occurred in the research article: Yunqi Ma, Ming Lu, Ziwei Chang, Chu Lee, Hae-Kyun Yoo, and Jang-Su Park (2018) Expression Procedure Optimization of Carassius aurantus CYP1A in Shewanella Using Plasmid Construction Strategy. Biotechnol. Bioprocess Eng. 23: 86-92. — In the AcknowlegmentOriginal wording: This study was funded by a grant from the National Fisheries Research and Development Institute, Korea (R2016003).This should be replaced by: This study was funded by a grant from Aquaculture technology development for the species inhabiting Korean coasts of the East Sea (R2018012).Received: 14 March 2018