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

Abstract

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 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 D1mRNA 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 ½ 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 hours 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 hours. Bioluminescence Resonance Energy Transfer (BRET) 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 (D2) are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms.

New insights into the structure and mechanism of iodothyronine deiodinases

“…Similar observations were made with Dio1, which has been shown to exist in a substrate-induced inactive form (Zhu et al 2012). Substrate-induced inactivation increased the fluorescence resonance energy transfer (FRET) signal in Dio1 fusion proteins indicating a conformational change in the inactivated dimer.…”

“…Substrate-induced inactivation increased the fluorescence resonance energy transfer (FRET) signal in Dio1 fusion proteins indicating a conformational change in the inactivated dimer. Both inactivation and increased FRET signal depended on a functional active site (Zhu et al 2012), suggesting that substrate turnover, rather than binding, is required for inactivation and is compatible with a selenenylsulfide contributing to this mechanism.…”

Current concepts and challenges to unravel the role of iodothyronine deiodinases in human neoplasias

“…However, DIO2 can also be reactivated through deubiquitination by ubiquitin-specific peptidase 33 (USP33) (Curcio-Morelli et al 2003b). DIO1 activity is also regulated by rT3 in a post-translational level through a mechanism that possibly involves post-catalytic structural changes in the DIO1 homodimer inactivating the enzyme (Zhu et al 2012). The mechanism of substrate-induced inactivation of DIO2 and DIO1 suggests that this regulation might be applicable to all three deiodinases (Schweizer & Steegborn 2015).…”

Thyroid Hormone Metabolism and Transport

“…106 Catalysis-induced inactivation of D1 has also been described, whereas the 3'-untranslated region of the human D1 mRNA has been proposed as a target for the micro-RNA MiR-224. 107,108 Finally, synthesis of the D2 protein appears to be impaired in the setting of endoplasmic reticulum stress. 109 Consi derable progress has been made in defining the molecular mechanisms involved in the transcriptional control of the deiodinases, and this topic has been reviewed by Gereben and colleagues.…”