Thyronamines Are Isozyme-Specific Substrates of Deiodinases

Abstract

3-Iodothyronamine (3-T 1 AM) and thyronamine (T AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T(3)) actions. Their proposed biosynthesis from thyroid hormones would require decarboxylation and more or less extensive deiodination. Deiodinases (Dio1, Dio2, and Dio3) catalyze the removal of iodine from their substrates. Because a role of deiodinases in thyronamine biosynthesis requires their ability to accept thyronamines as substrates, we investigated whether thyronamines are converted by deiodinases. Thyronamines were incubated with isozyme-specific deiodinase preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3',5'-triiodothyronamine (rT3AM), 3',5'-diiodothyronamine (3',5'-T2AM), and 3,3'-diiodothyronamine (3,3'-T2AM) as well as tyrosyl ring deiodinations of 3,5,3'-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) were observed with Dio1. These reactions were completely inhibited by the Dio1-specific inhibitor 6n-propyl-2-thiouracil (PTU). Dio2 containing preparations also deiodinated rT(3)AM and 3',5'-T2AM at the phenolic rings but in a PTU-insensitive fashion. All thyronamines with tyrosyl ring iodine atoms were 5(3)-deiodinated by Dio3-containing preparations. In functional competition assays, the newly identified thyronamine substrates inhibited an established iodothyronine deiodination reaction. By contrast, thyronamines that had been excluded as deiodinase substrates in LC-MS/MS experiments failed to show any effect in the competition assays, thus verifying the former results. These data support a role for deiodinases in thyronamine biosynthesis and contribute to confining the biosynthetic pathways for 3-T 1 AM and T 0 AM.

Type 1 iodothyronine deiodinase in human physiology and disease

“…Recently, attention has been turned to a potential role of D1 in the biosynthesis of thyronamines (3-iodothyronamine, 3-T 1 AM, and thyronamine, T 0 AM), a newly identified class of endogenous compounds that seem to be isozyme-specific substrates of deiodinases (Scanlan et al 2004, Piehl et al 2008, Scanlan 2009). These molecules are chemical derivatives of thyronines, the principal chemical form of T 4 that appears to antagonize the typical actions of thyroid hormones.…”

“…These molecules are chemical derivatives of thyronines, the principal chemical form of T 4 that appears to antagonize the typical actions of thyroid hormones. It has been shown that the iodothyronamines have V max /K m values comparable to the corresponding iodothyronine, indicating that they would be as readily deiodinated as iodothyronines by the appropriate deiodinase isoenzyme (Piehl et al 2008). Nevertheless, unlike T 4 , T 4 AM is not a substrate for D1 or D2 enzymes.…”

Essential Molecular Determinants for Thyroid Hormone Transport and First Structural Implications for Monocarboxylate Transporter 8

“…The uptake of T 3 , T 3 AM, and TRIAC by wild-type MCT8 and the mutants R445A and D498A was directly measured by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) (17,18). The cells were incubated for 3 min in DMEM-F12 (1:1) with a final concentration of 50 M. Then the medium was removed, and the cells were washed once with ice-cold PBS and harvested.…”

“…Data processing was performed using Bio Analyst version 1.5. software (Applied Biosystems). Liquid-liquid extraction of all compounds for LC-MS/MS analysis was performed as described previously (17). For LC-MS/MS analysis, 4 pmol of deuterated 3-T 1 AM-d 4 was used as internal standard.…”

“…However, this type of experiment represents an indirect approach. We took advantage of our LC-MS/MS assays for iodothyronines and TAM (17,18) and directly measured uptake of unlabeled ligands into MCT8-transfected MDCK1 cells. In a first step, we validated the LC-MS/MS method in direct comparison with the established [ 125 I]T 3 uptake assay (Fig.…”