Catalysis leads to posttranslational inactivation of the type 1 deiodinase and alters its conformation

• Published in: Journal of endocrinology Vol. 214; no. 1; pp. 87 - 94
• Main Authors: Zhu, Bo, Shrivastava, Ashutosh, Luongo, Cristina, Chen, Ting, Harney, John W, Marsili, Alessandro, Tran, Thuy-Van, Bhadouria, Anulika, Mopala, Radhika, Steen, Amanda I, Larsen, P Reed, Zavacki, Ann Marie
• Format: Journal Article
• Language: English
• Published: Bristol BioScientifica 01.07.2012
• Subjects: Biocatalysis - drug effects; Biological and medical sciences; Dose-Response Relationship, Drug; Enzyme Activation - drug effects; Fluorescence Resonance Energy Transfer; Fundamental and applied biological sciences. Psychology; HEK293 Cells; Hep G2 Cells; Humans; Iodide Peroxidase - chemistry; Iodide Peroxidase - genetics; Iodide Peroxidase - metabolism; Luciferases - genetics; Luciferases - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mutation; Propylthiouracil - pharmacology; Protein Conformation; Protein Multimerization; Protein Processing, Post-Translational - drug effects; Regular papers; Reverse Transcriptase Polymerase Chain Reaction; Sonication; Substrate Specificity; Triiodothyronine - metabolism; Triiodothyronine - pharmacology; Vertebrates: endocrinology
• ISSN: 0022-0795; 1479-6805
• DOI: 10.1530/JOE-11-0459

Previously, it was shown that the type 1 deiodinase (D1) is subject to substrate-dependent inactivation that is blocked by pretreatment with the inhibitor of D1 catalysis, propylthiouracil (PTU). Using HepG2 cells with endogenous D1 activity, we found that while considerable D1-mediated catalysis of reverse tri-iodothyronine (rT3) is observed in intact cells, there was a significant loss of D1 activity in sonicates assayed from the same cells in parallel. This rT3-mediated loss of D1 activity occurs despite no change in D1 mRNA levels and is blocked by PTU treatment, suggesting a requirement for catalysis. Endogenous D1 activity in sonicates was inactivated in a dose-dependent manner in HepG2 cells, with a ∼50% decrease after 10 nM rT3 treatment. Inactivation of D1 was rapid, occurring after only half an hour of rT3 treatment. D1 expressed in HEK293 cells was inactivated by rT3 in a similar manner. 75Se labeling of the D1 selenoprotein indicated that after 4 h rT3-mediated inactivation of D1 occurs without a corresponding decrease in D1 protein levels, though rT3 treatment causes a loss of D1 protein after 8–24 h. Bioluminescence resonance energy transfer studies indicate that rT3 exposure increases energy transfer between the D1 homodimer subunits, and this was lost when the active site of D1 was mutated to alanine, suggesting that a post-catalytic structural change in the D1 homodimer could cause enzyme inactivation. Thus, both D1 and type 2 deiodinase are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms.