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Human DIO3 cross-species-based site-directed mutagenesis modeling & docking
Citation:
LaLone, C. AND S. Mayasich. Human DIO3 cross-species-based site-directed mutagenesis modeling & docking. National Institutes of Health and University of Montana Collaborators Webinar, Duluth, MN, March 12, 2021. https://doi.org/10.23645/epacomptox.14166647
Impact/Purpose:
This presentation will describe to collaborators our efforts to utilize site directed mutagenesis, a technique in molecular biology, to evaluate changes in individual amino acids that make the human deiodinase 3 enzyme look like other vertebrate species. This is an effort to understand how well predictive approaches for predicting chemical susceptibility across species are performing when compared to laboratory studies that capture changes in proteins observed in a variety of species and evaluates whether or not those changes alter bioactivity.
Description:
A goal of this study is to use the molecular technique called site-directed mutagenesis to address the important question of whether differences in the critical amino acids forming the binding pocket, or neighboring amino acids, may result in differences in chemical binding to the human deiodinase 3 (DIO3) enzyme. In order to test the predictive capability of the molecular modeling, site-directed mutagenesis will be used to design different plasmids, each with a single mutated amino acid in the human DIO3 sequence, and to express each protein in human cell culture. These mutations are based on a search to compare amino acids at the critical positions across species using the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. The plasmids will each be transfected into HEK-293 cells and the expressed enzyme proteins harvested. An in vitro deiodinase enzyme assay that was developed for high-throughput screening of hundreds of ToxCast chemicals will be used to measure activity of enzymes produced from transfection of each plasmid construct. Differences in activity among the mutants and wildtype enzymes may be found, which may or may not be attributable to the change in amino acid sequence. Causes of relative differences in activity may also be related to transfection efficiency, unfolded protein response, or other causes that are not the goal of this study to determine, and are not expected to affect chemical binding affinity. We will focus on differences in inhibitory potency of known DIO3 inhibitors among the hDIO3 mutants and the hDIO3 wildtype protein to potentially infer sensitivity across species with the specific critical amino acid substitution. Results from this in vitro screening of a selected set of chemical inhibitors and known non-inhibitors will be compared to docking scores from in silico binding of these chemicals to models based on the mutated sequences. Additionally, models will be constructed for species with amino acid variations in critical positions as well as in neighboring positions within the active site, including Xenopus laevis.
