Minimal requirements for ubiquitination-mediated regulation of thyroid hormone activation

Activation of thyroxine by outer ring deiodination is the crucial first step of thyroid hormone action. Substrate-induced ubiquitination of type 2 deiodinase (D2) is the most rapid and sensitive mechanism known to regulate thyroid hormone activation. While the molecular machinery responsible for D2 ubiquitination has been extensively studied, the combination of molecular features sufficient and required to allow D2 ubiquitination have not previously been determined. To address this question, we constructed chimeric deiodinases by introducing different combinations of D2-specific elements into type 1 deiodinase (D1), another member of the deiodinase enzyme family, which, however, does not undergo ubiquitination in its native form. Studies on the chimeric proteins expressed transiently in HEK-293T cells revealed that combined insertion of the D2-specific instability loop and the K237/K244 D2 ubiquitin carrier lysines into the corresponding positions of D1 could not ubiquitinate D1 unless the chimera was directed to the endoplasmic reticulum (ER). Fluorescence resonance energy transfer measurements demonstrated that the C-terminal globular domain of the ER-directed chimera was able to interact with the E3 ligase subunit WSB1. However, this interaction did not occur between the chimera and the TEB4 (MARCH6) E3 ligase, although a native D2 could readily interact with the N-terminus of TEB4. In conclusion, insertion of the instability loop and ubiquitin carrier lysines in combination with direction to the ER are sufficient and required to govern WSB1-mediated ubiquitination of an activating deiodinase enzyme.

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Investigation of Receptor Heteromers Using NanoBRET Ligand Binding

International Journal of Molecular Sciences ◽

10.3390/ijms22031082 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1082

Author(s):

Elizabeth K. M. Johnstone ◽

Heng B. See ◽

Rekhati S. Abhayawardana ◽

Angela Song ◽

K. Johan Rosengren ◽

...

Keyword(s):

Angiotensin Ii ◽

Ligand Binding ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Receptor Heteromers ◽

Intracellular Proteins ◽

Β2 Adrenergic Receptor ◽

First Time

Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the β2 adrenergic receptor (AT1-β2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).

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The Thyroid Hormone-Inactivating Deiodinase Functions as a Homodimer

Molecular Endocrinology ◽

10.1210/me.2007-0490 ◽

2008 ◽

Vol 22 (6) ◽

pp. 1382-1393 ◽

Cited By ~ 32

Author(s):

G. D. Vivek Sagar ◽

Balázs Gereben ◽

Isabelle Callebaut ◽

Jean-Paul Mornon ◽

Anikó Zeöld ◽

...

Keyword(s):

Energy Transfer ◽

Thyroid Hormone ◽

Transmembrane Domain ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Globular Domain ◽

Intact Cells ◽

Euthyroid Sick Syndrome ◽

Hormone Inactivation

Abstract The type 3 deiodinase (D3) inactivates thyroid hormone action by catalyzing tissue-specific inner ring deiodination, predominantly during embryonic development. D3 has gained much attention as a player in the euthyroid sick syndrome, given its robust reactivation during injury and/or illness. Whereas much of the structure biology of the deiodinases is derived from studies with D2, a dimeric endoplasmic reticulum obligatory activating deiodinase, little is known about the holostructure of the plasma membrane resident D3, the deiodinase capable of thyroid hormone inactivation. Here we used fluorescence resonance energy transfer in live cells to demonstrate that D3 exists as homodimer. While D3 homodimerized in its native state, minor heterodimerization was also observed between D3:D1 and D3:D2 in intact cells, the significance of which remains elusive. Incubation with 0.5–1.2 m urea resulted in loss of D3 homodimerization as assessed by bioluminescence resonance energy transfer and a proportional loss of enzyme activity, to a maximum of approximately 50%. Protein modeling using a D2-based scaffold identified potential dimerization surfaces in the transmembrane and globular domains. Truncation of the transmembrane domain (ΔD3) abrogated dimerization and deiodinase activity except when coexpressed with full-length catalytically inactive deiodinase, thus assembled as ΔD3:D3 dimer; thus the D3 globular domain also exhibits dimerization surfaces. In conclusion, the inactivating deiodinase D3 exists as homo- or heterodimer in living intact cells, a feature that is critical for their catalytic activities.

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Inhibition of Type 1 Iodothyronine Deiodinase by Bisphenol A

Hormone and Metabolic Research ◽

10.1055/a-0919-3879 ◽

2019 ◽

Vol 51 (10) ◽

pp. 671-677 ◽

Cited By ~ 7

Author(s):

Maurício Martins da Silva ◽

Carlos Frederico Lima Gonçalves ◽

Leandro Miranda-Alves ◽

Rodrigo Soares Fortunato ◽

Denise P. Carvalho ◽

...

Keyword(s):

Adipose Tissue ◽

Thyroid Hormone ◽

Bisphenol A ◽

Brown Adipose Tissue ◽

Tissue Type ◽

Deiodinase Activity ◽

Brown Adipose

AbstractPlastics are ubiquitously present in our daily life and some components of plastics are endocrine-disrupting chemicals, such as bisphenol A and phthalates. Herein, we aimed to evaluate the effect of plastic endocrine disruptors on type 1 and type 2 deiodinase activities, enzymes responsible for the conversion of the pro-hormone T4 into the biologically active thyroid hormone T3, both in vitro and in vivo. Initially, we incubated rat liver type 1 deiodinase and brown adipose tissue type 2 deiodinase samples with 0.5 mM of the plasticizers, and the deiodinase activity was measured. Among them, only BPA was capable to inhibit both type 1 and type 2 deiodinases. Then, adult male Wistar rats were treated orally with bisphenol A (40 mg/kg b.w.) for 15 days and hepatic type 1 deiodinase and brown adipose tissue type 2 deiodinase activities and serum thyroid hormone concentrations were measured. In vivo bisphenol A treatment significantly reduced hepatic type 1 deiodinase activity but did not affect brown adipose tissue type 2 deiodinase activity. Serum T4 levels were higher in bisphenol A group, while T3 remained unchanged. T3/T4 ratio was decreased in rats treated with bisphenol A, reinforcing the idea that peripheral metabolism of thyroid hormone was affected by bisphenol A exposure. Therefore, our results suggest that bisphenol A can affect the metabolism of thyroid hormone thus disrupting thyroid signaling.

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Relation of thyroid hormone abnormalities with subclinical inflammatory activity in patients with type 1 and type 2 diabetes mellitus

Endocrine ◽

10.1007/s12020-015-0651-5 ◽

2015 ◽

Vol 51 (1) ◽

pp. 63-71 ◽

Cited By ~ 12

Author(s):

Arnaldo Moura Neto ◽

Maria Candida Ribeiro Parisi ◽

Sarah Monte Alegre ◽

Elizabeth Joao Pavin ◽

Marcos Antonio Tambascia ◽

...

Keyword(s):

Diabetes Mellitus ◽

Type 2 Diabetes ◽

Type 2 Diabetes Mellitus ◽

Thyroid Hormone ◽

Inflammatory Activity

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Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

Journal of Virology ◽

10.1128/jvi.02545-08 ◽

2009 ◽

Vol 83 (14) ◽

pp. 7322-7336 ◽

Cited By ~ 49

Author(s):

Ian B. Hogue ◽

Adam Hoppe ◽

Akira Ono

Keyword(s):

Human Immunodeficiency Virus ◽

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Membrane Binding ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 structural polyprotein Pr55Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.

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