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Site-directed mutagenesis of human type 3 iodothyronine deiodinase to evaluate SeqAPASS predictions of cross-species susceptibility to chemical inhibition
Citation:
Mayasich, S., M. Goldsmith, K. Mattingly, AND C. LaLone. Site-directed mutagenesis of human type 3 iodothyronine deiodinase to evaluate SeqAPASS predictions of cross-species susceptibility to chemical inhibition. SETAC, Pittsburgh, PA, November 13 - 17, 2022. https://doi.org/10.23645/epacomptox.21437319
Impact/Purpose:
As part of EPA’s efforts to reduce live-animal testing, new approach methods (NAMs) are being developed including in vitro assays and computational methods. The study being presented is an example of how these methods can complement each other. The SeqAPASS (Sequence Alignment to Predict Across Species Susceptibility) tool is useful in cross-species comparisons of critical amino acids involved in protein function at ligand-protein interaction sites. Molecular modeling supported the in vitro finding of potential competitive inhibitors binding at the putative catalytic site identified from the literature, and other chemicals interacting at the proposed cofactor site. Differences in response to chemicals among the variants representing cross-species variations in critical amino acids were partially explained by the SeqAPASS prediction. The study demonstrates the value in further development of computational methods to advance effective and efficient cross-species comparisons for ultimate use in ecological risk assessments and Adverse Outcome Pathway (AOP) development.
Description:
The potential for U.S. EPA’s SeqAPASS (Sequence Alignment to Predict Across-Species Susceptibility) tool to predict susceptibility of type 3 iodothyronine deiodinase (DIO3) enzyme to chemical inhibition was investigated. The SeqAPASS tool identifies whether known critical amino acids involved in catalytic function are exact, partial, or not matches across species compared to a template species. Predictions of susceptibility are based on side chain properties including molecular weight and side chain classification. From this evaluation, amino acid differences identified between vertebrates guided the selection of amino acids for site-directed mutagenesis (SDM) studies. Site-directed mutagenesis of the wildtype (WT) human DIO3 gene sequence was used to create six variant proteins expressed in cell culture which were tested in vitro for chemical inhibition. We found significant differences in in vitro IC50 results, but generally within an order of magnitude, among variants for a set of chemicals selected as potential competitive inhibitors based on a previous screening of ToxCast chemicals. Three variants predicted by SeqAPASS as partial matches showed significant differences from WT IC50 for 5 to 6 of the 9 chemicals screened, one amino acid change that was not a match in SeqAPASS resulted in differences from WT for 5 of the 9 chemicals, whereas another differed significantly for 8 chemicals. Another amino acid change that was not a match in SeqAPASS differed from WT for only one chemical. Hill slopes for the concentration-response curves did not differ significantly among variants for most chemicals, but steeper slopes for some chemicals indicated potential binding to more than one site. Virtual docking of the chemicals to structural models representing the WT and amino acid substitutions in other species support the hypothesis that certain chemicals are competitive inhibitors at the catalytic site and others may interact at a proposed cofactor site (interaction site of the reducing cofactor, dithiothreitol [DTT]). Differences in inhibition were only partially explained by side-chain properties of the substituted amino acid. Chemical characteristics and the location and putative function of the substituted amino acid in the hDIO3 enzyme are important considerations in understanding the potential for chemical susceptibility across species. This abstract neither constitutes nor necessarily reflects US EPA policy.
URLs/Downloads:
DOI: Site-directed mutagenesis of human type 3 iodothyronine deiodinase to evaluate SeqAPASS predictions of cross-species susceptibility to chemical inhibition
SETAC2022 HDIO3 SDM MAYASICH_STICS_FINAL.PDF (PDF, NA pp, 24126.666 KB, about PDF)