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Characterization of Xenopus laevis iodothyronine deiodinase (DIO3) enzyme and chemical inhibition comparison with human DIO3
Citation:
Mayasich, S., J. Denny, J. Korte, P. Kosian, J. Olker, P. Degoey, J. O'Flanagan, S. Degitz, AND M. Hornung. Characterization of Xenopus laevis iodothyronine deiodinase (DIO3) enzyme and chemical inhibition comparison with human DIO3. North American Society for Comparative Endocrinology, Gainsville, FL, May 24 - 28, 2019.
Impact/Purpose:
This work helps to address EPA’s need to determine whether chemicals can adversely affect thyroid hormones, which are necessary for amphibian development and metamorphosis. An in vitro assay based on production of amphibian deiodinase enzymes in cultured cell lines has been developed to screen chemicals for their ability to inhibit these enzymes that regulate active thyroid hormone levels in the organism. The results from this effort will provide a basis for making better informed decisions about the predictive potential of screening assays between mammalian and amphibian species.
Description:
Thyroid hormones are necessary for normal sequential development and metamorphosis of amphibian tissues and organs. These processes are regulated in part by activity of deiodinase (DIO) enzymes in peripheral tissues where deiodinases catalyze the removal of an iodine from thyroid hormones to either activate or inactivate the hormone. As part of an effort to evaluate chemicals for thyroid hormone disrupting potential, we previously conducted chemical screening using recombinant human DIO enzymes. To determine whether screening results from human proteins can be a surrogate for inhibition in other species, empirical cross-species comparisons are needed. Initial efforts to address this by expressing the Xenopus laevis DIO Type 3 in a human cell line (HEK293) produced low activity. Because deiodinases are selenoproteins containing the rare amino acid selenocysteine, they require not only the protein-coding region of the gene but also the selenocysteine insertion sequence (SECIS) within the 3′-untranslated region (UTR) to produce a functional protein. Replacing the X. laevis 3’-UTR with the 3’-UTR from the human DIO3 gene in the construct transfected into HEK293 cells resulted in doubling of X. laevis enzyme specific activity. Although the highest X. laevis DIO3 activity achieved was 1/20th that of human DIO3, the enzymes’ affinities (measured Km) for the thyroid hormone substrate were similar. Amino acid identity is highly conserved between these species within the enzymes’ catalytic sites, which can often be predictive of similar sensitivity to chemicals. Indeed, chemicals we tested for inhibition of DIO3 in concentration-response mode most often resulted in highly similar or identical curves between species. However, curves for some chemicals were significantly different. These results suggest that: (1) amino acid identity conservation within protein active sites generally informs cross-species predictions of chemical sensitivity, but more empirical data are needed to identify other contributing structure-activity factors, and (2) to accomplish high throughput chemical screening, species-specific cellular compatibility issues must be resolved to increase non-mammalian protein expression in mammalian cells.