Ubiquitination-Induced Conformational Change within the Deiodinase Dimer Is a Switch Regulating Enzyme Activity

Abstract

Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination. Our model system is the thyroid hormone-activating type 2 deiodinase (D2), an endoplasmic reticulum-resident type 1 integral membrane enzyme. D2 exists as a homodimer maintained by interacting surfaces at its transmembrane and globular cytosolic domains. The D2 dimer associates with the Hedgehog-inducible ubiquitin ligase WSB-1, the ubiquitin conjugase UBC-7, and VDU-1, a D2-specific deubiquitinase. Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2's globular interacting surfaces that are critical for dimerization and catalytic activity. This state of transient inactivity and change in dimer conformation persists until deubiquitination. The continuous association of D2 with this regulatory protein complex supports rapid cycles of deiodination, conjugation to ubiquitin, and enzyme reactivation by deubiquitination, allowing tight control of thyroid hormone action.Thyroid hormone receptors are ligand-dependent transcription factors that can either activate or repress transcription. Deiodination is a critical process that can convert the minimally active T4 molecule to the ligand for thyroid hormone receptors, T3. While there are three known deiodinases, the type 2 iodothyronine deiodinase (D2) is the key enzyme necessary for intracellular T3 production, a process shown to be critical in development (8,16,24), neuroendocrine gonadal control (33), and energy homeostasis (27,31). Given the fact that D2 catalyzes intracellular T3 production, it is not surprising that changes in D2 expression can modulate the thyroid hormone signaling pathway in a tissue-specific fashion, without affecting serum T3 concentrations (5).The D2 pathway, and hence tissue-specific thyroid hormone signaling, is regulated by ubiquitination, a critical posttranslational regulator of protein stability (11,25). D2 ubiquitination is accelerated in proportion to T4 concentration, thus creating a feedback loop controlling T3 production. D2 is inactivated upon conjugation to ubiquitin and, like other ubiquitinated proteins, targeted to the proteasome system (18, 29). The physiological relevance of this pathway is illustrated in the tibial growth plate of developing chickens, where ubiquitination of D2 and thus thyroid hormone signaling are under the control of the Hedgehog signaling cascade. In response to Hedgehog signaling, the D2-specific ubiquitin ligase WD repeat and SOCS box-containing 1 (WSB-1) is induced in perichondrial cells, thus accelerating D2 ubiquitination (16). The resulting decrease in D2 activity and local T3 concentration is thought to contribute to the Hedgehog induction of parathyroid hormone-related peptide (PTHrP) (16). The link between D2 activity and PTH...

Coupling between Nutrient Availability and Thyroid Hormone Activation

“…cAMP, FOXO3, and NF-B) (14,15), translational (i.e. ER stress) (16), and post-translational mechanisms, such as ubiquitination and proteasomal degradation (17). In addition, D2 activity in rat BAT is up-regulated by growth factors such as IGF-1 and the multifunctional protein insulin (18,19), which promotes cellular glucose uptake and growth by acting through various nutrient-sensing pathways, such as the PI3K-mTOR signaling pathway.…”

The molecular basis of the non-thyroidal illness syndrome

“…D2 is the main enzyme involved in the production of tissue T 3 and is therefore heavily involved in local TH metabolism. D2 is negatively regulated by TH, both pre-and posttranscriptionally, as T 3 down regulates Dio2 mRNA expression (Burmeister et al 1997), while T 4 as well as rT 3 (both substrates for D2) affect D2 activity via increasing D2 ubiquitination and subsequent proteasomal degradation (Sagar et al 2007).…”

“…This mechanism was first described by Gereben et al (2000) and was shown to be induced by the major substrate of D2, T 4 . Ubiquitination of D2 by the ubiquitin conjugating enzymes UBC6 and UBC7 is mediated via WSB-1 (a D2 specific E3 ubiguitin ligase adaptor subunit) which changes the conformation of the D2 dimer and thus its catalytic activity (Sagar et al 2007). D2 can be reactivated by de-ubiquitination by the deubiquitinating enzyme USP-33 (Curcio-Morelli et al 2003).…”

New insights into the structure and mechanism of iodothyronine deiodinases

“…The inactivation process involves ubiquitination of the active enzyme, which leads to an inactive Dio2 conformation (Sagar et al 2007), and is followed by proteasomal degradation (Steinsapir et al 1998(Steinsapir et al , 2000 or reactivation through deubiquitination (Sagar et al 2007). Several proteins are involved in ubiquitinylation of Dio2 (Dentice et al 2005) and a still unresolved question is how the ubiquitin-ligase is able to distinguish a Dio2 from a Dio2 that has already seen the substrate.…”