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.

Figure 1

Structure and nomenclature of thyronamines.

Figure 2

Representative LC-MS/MS chromatogram of thyronamine standard solutions. Thirty picomoles of T₀AM, 3-T₁AM, 3′-T₁AM, 3,5-T₂AM, and 3,3′-T₂AM were injected each. Due to the lower intensity of T₃AM, rT₃AM and T₄AM in this assay, 150 pmol of these compounds had to be injected to obtain peaks that were high enough to present them together with T₀AM and mono- and diiodothyronamines in one chromatogram. T₁AM-d4 (internal standard) and 3′,5′-T₂AM were not injected because their retention times (13.0 and 15.6 min, respectively) were almost identical to that of 3-T₁AM and 3,3′-T₂AM, respectively (A). Identification of thyronamines and compound specific retention times (rt) (B). cps, Counts per second.

Figure 3

LC-MS/MS-based deiodinase assays. In each reaction presented here, 500 nm substrate was used. Each chromatogram represents three separate experiments performed in triplicate. rT₃ (A) as well as rT₃AM and 3′,5′-T₂AM (B and C) were deiodinated at the phenolic rings by HepG2 lysates in a PTU-sensitive fashion. rT₃AM and 3′,5′-T₂AM were deiodinated at the phenolic rings in a PTU-insensitive way by Dio2-containing MSTO-211H lysates (D and E). The positive control substrate T₃ was deiodinated at the tyrosyl ring by Dio3 from ECC-1 lysates yielding 3′,5′-T₂ (F). ECC-1 lysates also deiodinated T₄AM, rT₃AM, T₃AM (G–I), 3,3′-T₂AM, 3,5-T₂AM (data not shown), and 3-T₁AM (J) at the tyrosyl rings. cps, Counts per second; iS, Internal standard (3-T₁AM-d4).

Figure 4

Thyronamine substrates of Dio1 inhibit the phenolic ring deiodination of rT₃ by Dio1 in ¹²⁵I⁻ release assays. In Eadie-Hofstee plots (left panels), incubations with dimethylsulfoxide are designated (♦), whereas the various thyronamine concentrations are presented as follows: ⋄, 0.1 μm; ▴, 0.5 μm; ▵, 1.0 μm; •, 2.0 μm; ○, 3.0 μm □, 5.0 μm; ×, 7.0 μm; ▪, 10.0 μm. The effect of thyronamines on the K_m and V_max of the phenolic ring deiodination of rT₃ by Dio1 was analyzed using Friedman test followed by Dunn’s post test, and P < 0.05 was considered significant. In Dixon plots (right panels), the various rT₃ concentrations are presented as follows: ♦, 0.2 μm; ⋄, 0.5 μm; ▴, 1.0 μm; ▵, 2.3 μm; •, 3.3 μm; ○, 3.8 μm; □, 4.4 μm. 3′,5′-T₂AM acted as a mixed inhibitor (A), whereas rT₃AM caused noncompetitive inhibition (B). The mean ± sd values reported for apparent K_m, apparent V_max, and K_i values are from three separate experiments performed in triplicate.

Figure 5

Summary of the newly identified thyronamine deiodination reactions.