Since the realization that the cellular homologs of a gene found in the retrovirus that contributes to erythroblastosis in birds (v-erbA), i.e. the proto-oncogene c-erbA encodes the nuclear receptors for thyroid hormones (THs), most of the interest for THs focalized on their ability to control gene transcription. It was found, indeed, that, by regulating gene expression in many tissues, these hormones could mediate critical events both in development and in adult organisms. Among their effects, much attention was given to their ability to increase energy expenditure, and they were early proposed as anti-obesity drugs. However, their clinical use has been strongly challenged by the concomitant onset of toxic effects, especially on the heart. Notably, it has been clearly demonstrated that, besides their direct action on transcription (genomic effects), THs also have non-genomic effects, mediated by cell membrane and/or mitochondrial binding sites, and sometimes triggered by their endogenous catabolites. Among these latter molecules, 3,5-diiodo-L-thyronine (3,5-T2) has been attracting increasing interest because some of its metabolic effects are similar to those induced by T3, but it seems to be safer. The main target of 3,5-T2 appears to be the mitochondria, and it has been hypothesized that, by acting mainly on mitochondrial function and oxidative stress, 3,5-T2 might prevent and revert tissue damages and hepatic steatosis induced by a hyper-lipid diet, while concomitantly reducing the circulating levels of low density lipoproteins (LDL) and triglycerides. Besides a summary concerning general metabolism of THs, as well as their genomic and non-genomic effects, herein we will discuss resistance to THs and the possible mechanisms of action of 3,5-T2, also in relation to its possible clinical use as a drug.

https://www.mdpi.com/1422-0067/21/11/4140/pdf

Genomic and Non-Genomic Mechanisms of Action of Thyroid Hormones and Their Catabolite 3,5-Diiodo-L-Thyronine in Mammals.

On the Potential of Machine Learning to Examine the Relationship Between Sequence, Structure, Dynamics and Function of Intrinsically Disordered Proteins

Background: Many physiological effects of thyroid hormone (TH) are mediated by its canonical action via nuclear receptors (TH receptor α and β [TRα and TRβ]) to regulate transcription of target genes. Heterozygous dominant negative mutations in human TRα mediate resistance to thyroid hormone alpha (RTHα), characterized by features of hypothyroidism (e.g., skeletal dysplasia, neurodevelopmental retardation, constipation) in specific tissues, but near-normal circulating TH concentrations. Hitherto, 41 RTHα cases have been recorded worldwide. Methods: RTHα cases (n = 10) attending a single center underwent cutaneous assessment, recording skin lesions. Lesions excised from different RTHα patients were analyzed histologically and profiled for cellular markers of proliferation and oncogenic potential. Proliferative characteristics of dermal fibroblasts and inducible pluripotent stem cell (iPSC)-derived keratinocytes from patients and control subjects were analyzed. Results: Multiple skin tags and nevi were recorded in all cases, mainly in the head and neck area with a predilection for flexures. The affected patients had highly deleterious mutations (p.E403X, p.E403K, p.F397fs406X, p.A382PfsX7) involving TRα1 alone or mild/moderate loss-of-function mutations (p.A263V, p.L274P) common to TRα1 and TRα2 isoforms. In four patients, although lesions excised for cosmetic reasons were benign intradermal melanocytic nevi histologically, they significantly overexpressed markers of cell proliferation (K17, cyclin D1) and type 3 deiodinase. In addition, oncogenic markers typical of basal cell carcinoma (Gli-1, Gli-2, Ptch-1, n = 2 cases) and melanoma (c-kit, MAGE, CDK4, n = 1) were markedly upregulated in skin lesions. Cell cycle progression and proliferation of TRα mutation-containing dermal fibroblasts and iPSC-derived keratinocytes from patients were markedly increased. Conclusions: Our observations highlight frequent occurrence of skin tags and benign melanocytic nevi in RTHα, with cutaneous cells from patients being in a hyperproliferative state. Such excess of skin lesions, including nevi expressing oncogenic markers, indicates that dermatologic surveillance of RTHα patients, monitoring lesions for features that are suspicious for neoplastic change, is warranted.

Germ Line Mutations in the Thyroid Hormone Receptor Alpha Gene Predispose to Cutaneous Tags and Melanocytic Nevi

Resistance to thyroid hormone alpha occurs due to pathogenic, heterozygous variants in THRA. The entity was first described in 2012 and to date only a small number of patients with varying severity have been reported. In this review, we summarize and interpret the heterogeneous clinical and laboratory features of all published cases, including ours. Many symptoms and findings are similar to those seen in primary hypothyroidism. However, thyroid-stimulating hormone levels are normal. Free triiodothyronine (T3) levels are in the upper half of normal range or frankly high and free thyroxine (T4) levels are low or in the lower half of normal range. Alterations in free T3 and free T4 may not be remarkable, particularly in adults, possibly contributing to underdiagnosis. In such patients, low reverse T3 levels, normo- or macrocytic anemia or, particularly in children, mildly elevated creatine kinase levels would warrant THRA sequencing. Treatment with L-thyroxine results in improvement of some clinical findings.

The Clinical Spectrum of Resistance to Thyroid Hormone Alpha in Children and Adults

The nuclear thyroid hormone receptors (THRs) are key mediators of thyroid hormone function on the cellular level via modulation of gene expression. Two different genes encode THRs (THRA and THRB), and are pleiotropically involved in development, metabolism, and growth. The THRA1 and THRA2 isoforms, which result from alternative splicing of THRA, differ in their C-terminal ligand-binding domain (LBD). Most published disease-associated THRA variants are located in the LBD of THRA1 and impede triiodothyronine (T3) binding. This keeps the nuclear receptor in an inactive state and inhibits target gene expression. Here, we investigated a new dominant THRA variant (chr17:g.38,241,010A > G, GRCh37.13 | c.518A > G, NM_199334 | p.(E173G), NP_955366), which is located between the DNA- and ligand-binding domains and affects both splicing isoforms. Patients presented partially with hypothyroid (intellectual disability, motor developmental delay, brain atrophy, and constipation) and partially with hyperthyroid symptoms (tachycardia and behavioral abnormalities) to varying degrees. Functional characterization of THRA1p.(E173G) by reporter gene assays revealed increased transcriptional activity in contrast to THRA1(WT), unexpectedly revealing the first gain-of-function mutation found in THRA1. The THRA2 isoform does not bind T3 and antagonizes THRA1 action. Introduction of p.(E173G) into THRA2 increased its inhibitory effect on THRA1, which helps to explain the hypothyroid symptoms seen in our patients. We used protein structure models to investigate possible underlying pathomechanisms of this variant with a gain-of-antagonistic function and suggest that the p.(E173G) variant may have an influence on the dimerization domain of the nuclear receptor.

https://www.mdpi.com/1422-0067/22/10/5338/pdf

A New Mechanism in THRA Resistance: The First Disease-Associated Variant Leading to an Increased Inhibitory Function of THRA2.

Resistance to thyroid hormone alpha (RTHα) is a rare and under-recognized genetic disease caused by mutations of THRA, the gene encoding thyroid hormone receptor α1 (TRα1). We report here two novel THRA missense mutations (M259T, T273A) in patients with RTHα. We combined biochemical and cellular assays with in silico modeling to assess the capacity of mutant TRα1 to bind triiodothyronine (T3), to heterodimerize with RXR, to interact with transcriptional coregulators, and to transduce a T3 transcriptional response. M259T, and to a lower extent T273A, reduces the affinity of TRα1 for T3. Their negative influence is only reverted by large excess of T3. The severity of the two novel RTHα cases originates from a reduction in the binding affinity of TRα1 mutants to T3 and thus correlates with the incapacity of corepressors to dissociate from TRα1 mutants in the presence of T3.

Two Novel Cases of Resistance to Thyroid Hormone Due to THRA Mutation.

https://hal.archives-ouvertes.fr/hal-03047222/document

Structural Insights into the Interaction of the Intrinsically Disordered Co-activator TIF2 with Retinoic Acid Receptor Heterodimer (RXR/RAR)

Retinoid X receptors (RXRα, β, and γ) are essential members of the nuclear receptor (NR) superfamily of ligand-dependent transcriptional regulators that bind DNA response elements and control the expression of large gene networks. As obligate heterodimerization partners of many NRs, RXRs are involved in a variety of pathophysiological processes. However, despite this central role in NR signaling, there is still no consensus regarding the precise biological functions of RXRs and the putative role of the endogenous ligands (rexinoids) previously proposed for these receptors. Based on available crystal structures, we introduced a series of amino acid substitutions into the ligand-binding pocket of all three RXR subtypes in order to alter their binding properties. Subsequent characterization using a battery of cell-based and in vitro assays led to the identification of a double mutation abolishing the binding of any ligand while keeping the other receptor functions intact, and a triple mutation that selectively impairs interaction with natural rexinoids but not with some synthetic ligands. We also report crystal structures that help understand the specific ligand-binding capabilities of both variants. These RXR variants, either fully disabled for ligand binding or retaining the property of being activated by synthetic compounds, represent unique tools that could be used in future studies to probe the presence of active endogenous rexinoids in tissues/organs and to investigate their role in vivo. Last, we provide data suggesting a possible involvement of fatty acids in the weak interaction of RXRs with corepressors.

Design and in vitro characterization of RXR variants as tools to investigate the biological role of endogenous rexinoids.

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved α-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a cooperative binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.

https://biorxiv.org/content/10.1101/2020.10.28.359091v1.full.pdf

Structural insights into the cooperative interaction of the intrinsically disordered co-activator TIF2 with retinoic acid receptor heterodimer (RXR/RAR)

Laura Guee

Papers

Two Novel Cases of Resistance to Thyroid Hormone Due to THRA Mutation.

Structural Insights into the Interaction of the Intrinsically Disordered Co-activator TIF2 with Retinoic Acid Receptor Heterodimer (RXR/RAR)

Design and in vitro characterization of RXR variants as tools to investigate the biological role of endogenous rexinoids.

Structural insights into the cooperative interaction of the intrinsically disordered co-activator TIF2 with retinoic acid receptor heterodimer (RXR/RAR)