# Iodothyronine deiodinase

Iodothyronine deiodinases (EC 1.21.99.4 and EC 1.21.99.3) are a subfamily of deiodinase enzymes important in the activation and deactivation of thyroid hormones. Thyroxine (T$_4$), the precursor of 3,5,3'-triiodothyronine (T$_3$) is transformed into T$_3$ by deiodinase activity. T$_3$, through binding a nuclear thyroid hormone receptor, influences the expression of genes in practically every vertebrate cell. Iodothyronine deiodinases are unusual in that these enzymes contain selenium, in the form of an otherwise rare amino acid selenocysteine.
These enzymes are not to be confused with the iodotyrosine deiodinases that are also deiodinases, but not members of the iodothyronine family. The iodotyrosine deiodinases (unlike the iodothyronine deiodinases) do not use selenocysteine or selenium. The iodotyrosine enzymes work on iodinated single tyrosine residue molecules to scavenge iodine, and do not use as substrates the double-tyrosine residue molecules of the various iodothyronines.

## Activation and inactivation

In tissues, deiodinases can either activate or inactivate thyroid hormones:
* Activation occurs by conversion of thyroxine (T$_4$) to the active hormone triiodothyronine (T$_3$) through the removal of an iodine atom on the outer ring.
* Inactivation of thyroid hormones occurs by removal of an iodine atom on the inner ring, which converts thyroxine to the inactive reverse triiodothyronine (rT$_3$), or which converts the active triiodothyronine to diiodothyronine (T$_2$).
The major part of thyroxine deiodination occurs within the cells.
Deiodinase 2 activity can be regulated by ubiquitination:
* The covalent attachment of ubiquitin inactivates D2 by disrupting dimerization and targets it to degradation in the proteosome.
* Deubiquitination removing ubiquitin from D2 restores its activity and prevents proteosomal degradation.
* The Hedgehog cascade acts to increase D2 ubiquitination through WSB1 activity, decreasing D2 activity.
D-propranolol inhibits thyroxine deiodinase, thereby blocking the conversion of T$_4$ to T$_3$, providing some though minimal therapeutic effect.

## Reactions

| Reactions catalyzed by specific deiodinase isoforms | Iodothyronine deiodinase activity and regulation |

## Structure

The three deiodinase enzymes share certain structural features in common although their sequence identity is lower than 50%. Each enzyme weighs between 29 and 33kDa. Deiodinases are dimeric integral membrane proteins with single transmembrane segments and large globular heads (see below). They share a TRX fold that contains the active site including the rare selenocysteine amino acid and two histidine residues. Selenocysteine is coded by a UGA codon, which generally signifies termination of a peptide through a stop codon. In point mutation experiments with Deiodinase 1 changing UGA to the stop codon TAA resulted in a complete loss of function, while changing UGA to cysteine (TGT) caused the enzyme to operate at around 10% normal efficiency. In order for UGA to be read as a selenocysteine amino acid instead of a stop codon, it is necessary that a downstream stem loop sequence, the selenocysteine insertion sequence (SECIS), be present to bind with SECIS binding protein-2 (SBP-2), which binds with elongation factor EFsec. The translation of selenocysteine is not efficient, even though it is important to the functioning of the enzyme. Deiodinase 2 is localized to the ER membrane while Deiodinase 1 and 3 are found in the plasma membrane.
The related catalytic domains of Deiodinases 1-3 feature a thioredoxine-related peroxiredoxin fold. The enzymes catalyze a reductive elimination of iodine, thereby oxidizing themselves similar to Prx, followed by a reductive recycling of the enzyme.

## Types

In most vertebrates, there are three types of enzymes that can deiodinate thyroid hormones:
| Type | Location | Function |
| type I (DI) | is commonly found in the liver and kidney | DI can deiodinate both rings |
| type II deiodinase (DII) | is found in the heart, skeletal muscle, CNS, fat, thyroid, and pituitary | DII can only deiodinate the outer ring of the prohormone thyroxine and is the major activating enzyme (the already inactive reverse triiodothyronine is also degraded further by DII) |
| type III deiodinase (DIII) | found in the fetal tissue and the placenta; also present throughout the brain, except in the pituitary | DIII can only deiodinate the inner ring of thyroxine or triiodothyronine and is the major inactivating enzyme |

## Function

Deiodinase 1 both activates T$_4$ to produce T$_3$ and inactivates T$_4$. Besides its increased function in producing extrathyroid T$_3$ in patients with hyperthyroidism, its function is less well understood than D2 or D3 Deiodinase 2, located in the ER membrane, converts T$_4$ into T$_3$ and is a major source of the cytoplasmic T$_3$ pool. Deiodinase 3 prevents T$_4$ activation and inactivates T$_3$. D2 and D3 are important in homeostatic regulation in maintaining T$_3$ levels at the plasma and cellular levels. In hyperthyroidism D2 is down regulated and D3 is upregulated to clear extra T$_3$, while in hypothyroidism D2 is upregulated and D3 is downregulated to increase cytoplasmic T$_3$ levels.
Serum T$_3$ levels remain fairly constant in healthy individuals, but D2 and D3 can regulate tissue specific intracellular levels of T$_3$ to maintain homeostasis since T$_3$ and T$_4$ levels may vary by organ. Deiodinases also provide spatial and temporal developmental control of thyroid hormone levels. D3 levels are highest early in development and decrease over time, while D2 levels are high at moments of significant metamorphic change in tissues. Thus D2 enables production of sufficient T$_3$ at necessary time points while D3 may shield tissue from overexposure to T$_3$.
Also, iodothyronine deiodinases (type 2 y 3; DIO2 and DIO3, respectively) respond to seasonal changes in photoperiod-driven melatonin secretion and govern peri-hypothalamic catabolism of the prohormone thyroxine (T4). In long summer days, the production of hypothalamic T3 increase due to DIO-2-mediated conversion of T4 to the biologically active hormone. This process allows to active anabolic neuroendocrine pathways that maintain reproductive competence and increase body weight. However, during the adaptation to reproductively inhibitory photoperiods, the levels of T3 decrease due to peri-hypothalamic DIO3 expression that catabolizes T4 and T3 into receptor inactive amines.
Deiodinase 2 also plays a significant role in thermogenesis in brown adipose tissue (BAT). In response to sympathetic stimulation, dropping temperature, or overfeeding BAT, D2 increases oxidation of fatty acids and uncouples oxidative phosphorylation via uncoupling protein, causing mitochondrial heat production. D2 increases during cold stress in BAT and increases intracellular T$_3$ levels. In D2 deficient models, shivering is a behavioral adaptation to the cold. However, heat production is much less efficient than uncoupling lipid oxidation.

## Disease relevance

In cardiomyopathy the heart reverts to a fetal gene programming due to the overload of the heart. Like during fetal development, thyroid hormone levels are low in the overloaded heart tissue in a local hypothyroid state, with low levels of deiodinase 1 and deiodinase 2. Although deiodinase 3 levels in a normal heart are generally low, in cardiomyopathy deiodinase 3 activity is increased to decrease energy turnover and oxygen consumption.
Hypothyroidism is a disease diagnosed by decreased levels of serum thyroxine (T$_4$). Presentation in adults leads to decreased metabolism, increased weight gain, and neuropsychiatric complications. During development, hypothyroidism is considered more severe and leads to neurotoxicity as cretinism or other human cognitive disorders, altered metabolism and underdeveloped organs. Medication and environmental exposures can result in hypothyroidism with changes in deiodinase enzyme activity. The drug iopanoic acid (IOP) decreased cutaneous cell proliferation by inhibition of deiodinase enzyme type 1 or 2 reducing the conversion of T$_4$ to T$_3$. The environmental chemical DE-71, a PBDE pentaBDE brominated flame retardant decreased hepatic deiodinase I transcription and enzyme activity in neonatal rats with hypothyroidism.

## Quantifying enzyme activity

In vitro, including cell culture experiments, deiodination activity is determined by incubating cells or homogenates with high amounts of labeled thyroxine (T$_4$) and required cosubstrates. As a measure of deiodination, the production of radioactive iodine and other physiological metabolites, in particular T$_3$ or reverse T$_3$, are determined and expressed (e.g. as fmol/mg protein/minute).
In vivo, deiodination activity is estimated from equilibrium levels of free T$_3$ and free T$_4$. A simple approximation is T$_3$/T$_4$ ratio, a more elaborate approach is calculating sum activity of peripheral deiodinases (SPINA-GD) from free T$_4$, free T$_3$ and parameters for protein binding, dissociation and hormone kinetics. In atypical cases, this last approach can benefit from measurements of TBG, but usually only requires measurement of TSH, fT3 and fT4, and as such has no added laboratory requirements besides the measurement of the same.