B, schematic representation of the membrane topology of the NIS polypeptide chain deduced from secondary structure prediction analyses C, transport of iodide from the extracellular fluid or plasma to the thyroid follicle lumen.
The uptake of iodide at the basolateral plasma membrane of thyrocytes must be active; it operates against an electrical gradient 0 - 50 mV and a concentration gradient, [ I- ]c being higher than extracellular [ I- ]. The transport of iodide from the cytoplasm to the follicle lumen should be a passive process, the electrical and concentration gradients being favorable.
Iodide that enters the thyroid remains in the free state only briefly before it is further metabolized and bound to tyrosyl residues in Tg. A major fraction of the intrathyroidal free iodide pool comes from deiodination of MIT and DIT; this iodide is either recycled within the thyroid or leaked into the circulation.
Some data suggest that iodide entering the gland by active transport segregates from that generated by deiodination of Tg within the gland 82; The proportion of an iodide load that is bound varies little, despite wide shifts in daily intake. In contrast, NIS activity is sensitive to both iodine availability and TSH stimulation, and transport rather than intrathyroidal binding is the controlling factor in making iodide available for hormonogenesis.
Ductal cells of the salivary glands express NIS In the mammary gland, NIS is processed differently after translation and subjected to regulation by lactogenic stimuli As it is absent in normal non-lactating tissue, NIS may represent a marker for breast malignancy and even a possible target for radioiodine therapy The thyroid, salivary glands, and gastric mucosa share a common embryologic derivation from the primitive alimentary tract and, in each of these tissues; iodide transport is inhibited by thiocyanate, perchlorate, and cardiac glycosides.
TSH stimulates transport only in the thyroid. An active transport for iodide in the gastric mucosa has an obvious value because it provides iodine to the circulation for use in the thyroid. Active concentration by the breast helps transfer iodide to milk.
Iodide concentration by the choroid plexus and salivary glands does not have any obvious physiologic benefit, but needs to be remembered for possible insights into pathways as yet undiscovered.
Iodine, particularly in the form of I2, may enter additional metabolic pathways outside the thyroid. Rats administered I2 orally showed much less circulating free iodide and much more iodine bound to proteins and lipids than did animals given iodide In another comparison of I2 versus iodide, administration of iodide to iodine-deficient rats eliminated thyroid hyperplasia much more efficiently than did I2.
Additionally, I2 decreased lobular hyperplasia and periductal fibrosis in the mammary glands, while iodide increased the former and had no effect on the latter Thyroperoxidase oxidizes iodide in the presence of H2O2. In crude thyroid homogenates, enzyme activity is associated to cell membranes.
It can be solubilized using detergents such as deoxycholate or digitonin. The enzyme activity is dependent on the association with a heme, the ferriprotoporphyrin IX or a closely related porphyrin 87; Chemical removal of the prosthetic group inactivates the enzyme, and recombination with the heme protein restores activity The apoprotein from human thyroid is not always fully saturated with its prosthetic group Some congenitally goitrous children have poor peroxidase function because the apoprotein has weak binding for the heme group Antibodies directed against the thyroid "microsomal antigen," which are present in the serum of patients with autoimmune thyroid disease AITD , led to identification of TPO.
These antibodies were found to react with proteins of kDa and to immunoprecipitate thyroid peroxidase TPO , thus identifying microsomal antigen as TPO A monoclonal antibody to purified microsomal antigen or antibodies directed againt thyroperoxidase were then used to clone human TPO Different laboratories then cloned TPO from various species: pig 99 , rat , and mouse Kimura et al. Both forms occur in normal and abnormal human thyroid tissue.
The C-terminal portion of the proteins exhibits a hydrophobic segment residues , likely corresponding to a transmembrane domain; thus, TPO has a short intracellular domain and most of the polypeptide chain is extracellular Fig. TPO1 is active, but TPO2 appears enzymatically inactive because it does not bind heme, degrades rapidly, and fails to reach the cell surface in transfected cell lines Different degradative pathways exist for the two forms Several other TPO variants resulting from exon skipping have been identified; they appear either active or inactive Pig TPO contains amino acids 99 ; mannose-rich oligosaccharide units occupy four of its five glycosylation sites The TPO gene resides on chromosome 2p13, spans over kbp, and has 17 exons Inactivating mutations in the TPO gene are responsible for a subtype of congenital hypothyroidism characterized by thyroid dyshormonogenesis due to iodide organification defect.
More than 60 annotated mutations have been reported; most of them result in total iodide organification defect with severe and permanent hypothyroidism ; TPO synthesized on polysomes is inserted in the membrane of the endoplasmic reticulum and undergoes core glycosylation.
TPO is then transported to the Golgi where it is subjected to terminal glycosylation and packaged into transport vesicles along with Tg Fig. These vesicles fuse with the apical plasma membrane in a process stimulated by TSH.
TPO delivered at the apical pole of thyrocytes exposes its catalytic site with the attached heme in the thyroid follicular lumen TPO activity is restricted to the apical membrane, but most of the thyroid TPO is intracellular, being located in the perinuclear part of the endoplasmic reticulum ; Most of this intracellular protein is incompletely or improperly folded; it contains only high mannose-type carbohydrate units, while the membrane TPO has complex carbohydrate units.
Glycosylation is essential for enzymatic activity C, hypothetical reaction scheme for TPO. H2O2 is presumed to oxidize the free enzyme with a loss of two electrons leading to the formation of complex I. A large body of older work reviewed in investigated possible sources using various in vitro models It was already suggested in that H2O2 would be produced at the apical plasma membrane of the thyrocyte by an enzyme that requires calcium and NADPH originating from the stimulation of the pentose phosphate pathway H2O2 produced by this NADPH-dependent protein is the limiting step of protein iodination and therefore of thyroid hormone synthesis when iodide supply is sufficient In human thyroid, the H2O2 production and iodination process are stimulated by the calcium-phosphatidylinositol pathway The quantity of H2O2 produced is important especially in stimulated thyrocytes; it is comparable to the ROS production of activated leukocytes.
While the activated leukocyte lives a few hours, the life of an adult thyrocyte is 7 yr ; Thus thyroid cells may be exposed to high doses of H2O2 and have to adapt to it by developing highly regulated generator and efficient protective systems. More than twenty years passed between the initial biochemical studies and the cloning of Duox as the catalytic enzymatic core of the H2O2 thyroid generating system. By two independent molecular strategies Duox enzymes were uncovered from the thyroid.
Dupuy isolated p Tox which turned out to be Duox2 lacking the first residues Simultaneously, De Deken et al cloned two cDNAs encoding NADPH oxidases using the strategy based on the functional similarities between H2O2 generation in the leukocytes and the thyroid according to the hypothesis that one of the components of the thyroid system would belong to the known gp91phox gene family and display sequence similarities with gp91phox, now called NOX2.
Duox1 and Duox2 genes are co-localized on chromosome 15q Duox1 gene is more telomeric, spans 36 kb and is composed of 35 exons; two first of them are non-coding. Duox2 spans In addition to thyroid, Duox expression is reported in several tissues: Duox1 is expressed in lung epithelia, in oocytes and Duox2 in gastrointestinal mucosa and salivary glands ; Multiple functions are attributed to Duox enzymes: airway fuid acidification , mucin secretion , wound healing ; and innate hoste defense Most of the time Duox activity is associated to a peculiar peroxidase activity like in oocyte with the ovoperoxidase involved in the fertilization process or with the lactoperoxidase in lung epithelia or in the gut ;;; Beside these killing mechanisms, Duox and H2O2 are certainly also involved in the interaction between host mucosa and bacteria to maintain mucosal homeostasis e.
In the thyroid, the specificity of the thyroid hormone machinery using Duox lays on TPO. Thus colocalization of Duox and TPO and their probable association at the apex of the thyrocyte would increase the efficiency of H2O2 producer-consumer system Onset of Duox expression study in thyroid embryonic development pointed Duox as a thyroid differentiation marker.
The proteins involved in the synthesis of thyroid hormones are expressed just after the thyroid precursor cells have completed their migration from the primitive pharynx and reached their final location around the trachea ; Until , the major obstacle for molecular studies of Duox was the lack of a suitable heterologous cell system for Duox correctly expressed at the plasma membrane in its active state.
HEK cells transfected with Duox2 generate rather small quantities of superoxide anions in a calcium-depnedent manner The reconstitution of a Duox-based functional H2O2 generating system requires a maturation factor called DuoxA.
The two human DuoxA paralogs were initially identified as thyroid specific expressed genes by in silico screenings of multiple parallel signature sequencing data bases DuoxA2 ORF spans 6 exons and encodes a amino acid protein predicted to compose five transmembrane segments, a large external loop presenting N-glycosylation sites between the second and third transmembrane helices and a C-terminal cytoplamic region Fig.
Four alternatively spliced DuoxA1 variants have been identified In heterologous systems DuoxA proteins in the absence of Duox are mainly retained in the endoplasmic reticulum. When co-transfected with Duox they cotransported with Duox to the plasma membrane where they probably form complexes. In addition it has been shown that the type of Duox-dependent ROS poduction is dictated by defined sequences in DuoxA This means that the Duox activators promote Duox maturation but also are parts of the H2O2 generating complex ; Mice deficient in DuoxA maturation factors present a maturation defect of Duox, lacking the N-glycan processing, and a loss of H2O2 production.
These mice develop severe goitrous congenital hypothyroidism with undetectable serum T4 and high serum TSH levels The reconstitution of this functional H2O2 producing system has been useful to measure and compare the intrinsic enzymatic activities of Duox1 and Duox2 in relationship with their expression at the plasma membrane under stimulation of the major signalling pathways active in the thyroid.
It has been shown that the basal activity of both isoenzymes is totally depending on calcium and functional EF-hands calcium binding motifs. However, the two oxidase enzymatic activities are differently regulated after activation of the two main signalling cascades in the thyroid. These results suggest that both Duox proteins could be involved in thyroid hormone synthesis by feeding H2O2 to TPO to oxidize iodide and couple iodotyrosines.
From in vitro and in vivo data it has been concluded that Duox-DuoxA constitutes the major if not the unique component of the hormonogenic thyroid H2O2 generating system.
The bidirectional promoter allows the coexpression of Duox and DuoxA in the same tissue but the mechanisms regulating their transcription are not well and definitely characterized ; It has been recently shown that Th2 cytokines, IL4 and IL13, up-regulate Duox2 and DuoxA2 genes in human thyrocytes through an activation of Jak-Stat pathway opening new perspectives for a better understanding of the eventual role of Duox in autoimmune diseases They were identified in the Netherlands by neonatal screening and followed up to determine the evolution of CH with the time.
One of the patients with total organification defect TIOD presented a permanent hypothyroidism and the 8 others presented a transient hypothyroidism with a partial organification defect PIOD. Of these last 8 patients 3 harboured heterozygous nonsense or frameshift mutations QX, RX, SfsX meaning that a single defective Duox2 allele can cause haploinsufficency resulting in mild transient CH. It is noteworthy that this hypothyroid status was limited to the neonatal period, when thyroid hormone requirement is the highest, and was not detectable in adulthood since adult heterozygotes in these families presented normal TSH serum levels.
No mutation was detected in Duox1. With the increasing number of reported Duox2 mutations in CH, it becomes more and more difficult to make the correlation between genotype and phenotype as initially described. Patients with compound heterozygous missense RW and a nonsense mutation RX , leading to a presumed non functional protein showed PIOD with mild and persistent hyperthyrotropinemia.
This suggests that Duox1 can compensate at least partially for the defect in Duox2 Varela et al.. The phenotype-genotype correlation suggested by the work of Moreno et al. Maruo et al. Three of them had low free T4 at birth, mild thyroid enlargement. The thyroid hormone replacement therapy ceased to be necessary by 9yr of age A French-Canadian patient with a transient CH initially detected by neonatal screening presented a compound heterozygozity for a hemizygous missense mutation GS inherited from the father and a deletion removing the part of the gene coding for the catalytic core of Duox2 inherited from the mother.
In vitro test proved that the missense mutant protein was totally inactive This case and others reported later provide further evidence that permanent or transient nature of CH is not directly related to the number of inactivated Duox2 alleles The first homozygous nonsense mutation in DuoxA2 YX that resulted in a non-functional protein tested in vitro has been found to be responsible of a permanent mild CH in a Chinese patient with a dyshormonogenic goiter ; The variety of observerd phenotypes associated with Duox2 and now DuoxA2 mutations suggest that the manifestation of Duox2 defects could likely be influenced by the environmental factors like iodine intake or by the activation of Duox1 or DuoxA1 in peculiar circumstances.
B, Schematic representation of the predicted structure of DuoxA from Its main function is to provide the polypeptide backbone for synthesis and storage of thyroid hormones It also offers a convenient depot for iodine storage and retrieval when external iodine availability is scarce or uneven. Neosynthesised Tg polypeptide chains entering the lumen of the rough endoplasmic reticulum RER are subjected to core glycosylation, dimerise and are transferred to the Golgi where they undergo terminal glycosylation Fig.
Iodination and hormone formation of Tg occur at the apical plasma membrane-lumen boundary and the mature hormone-containing molecules are stored in the follicular lumen, where they make up the bulk of the thyroid follicle colloid content. Inside the lumen of RER, newly-synthesized proteins undergo core glycosylation and by interacting with chaperones acquire their conformation.
Proteins are then transported to the Golgi apparatus G , where terminal glycosylation and other post-translational reactions take place. In the Trans-Golgi network TGN , mature proteins undergo sorting processes and are packed into transport vesicles. The thyroid hormones cross the follicular cell membrane towards the blood vessels by an unknown mechanism.
The thyroglobulin protein accounts for approximately half of the protein content of the thyroid gland. This introduces atoms of the element iodine , covalently bound, per tyrosine residue. Initiation of production in fetuses[ edit ] Thyrotropin-releasing hormone TRH is released from hypothalamus by 6 — 8 weeks, and thyroid-stimulating hormone TSH secretion from fetal pituitary is evident by 12 weeks of gestation , and fetal production of thyroxine T4 reaches a clinically significant level at 18—20 weeks.
Please help improve this section by adding citations to reliable sources. Unsourced material may be challenged and removed. April Learn how and when to remove this template message If there is a deficiency of dietary iodine , the thyroid will not be able to make thyroid hormone. Consequently, Iodide is highly concentrated in the thyroid gland compared to the rest of the body.
Thyroglobulin Synthesis Thyroglobulin is a protein that contains large numbers of tyrosine amino acids that go on to become individual thyroid hormone molecules. Thyroglobulin is synthesized within the follicular epithelial cell and secreted into the follicular lumen. Thyroid Peroxidase Thyroid Peroxidase is an enzyme present in the acellular colloid of the follicular lumen and performs several key reactions. Thyroid Peroxidase first generates I2 by oxidizing I- ions present in the follicular lumen.
Thyroid Peroxidase then "organifies" the generated I2 by covalently linking it with the tyrosine residues present in Thyroglobulin.Whatever the precise nature of the iodinating baseball, it is clear that iodide is oxidized by H2O2 and TPO, and filled to the tyrosyl groups of Tg. Whether a very small fraction of the personal hormone is free printable and biologically active, hence measuring Patagonia case study harvard of compulsory thyroid hormones is of great diagnostic standardization. Of particular interest is 2-iodohexadecanal ; Millions, nm. However, the two oxidase some activities are differently regulated after apple of the two biosynthesis idea cascades in the thyroid. Fluorescently-labeled Tg is thyroid inside thyrocytes.
The presence of a high number of cysteine residues in Tg, involved for most of them in disulfide bonds, probably gives rise to peculiar structural constraints.
The vesicles lose their coat and, through a complex fusion process, deliver their content into a first type of endocytic compartments, the early apical endosomes Fig The primary structure deduced from cDNA is also known for bovine, rat, and mouse Pseudopods representing extensions of the apical plasma membrane project into the follicle lumen and pinch off to form a resorption vacuole known as colloid droplet Identifying the donor tyrosyls has attracted considerable investigational interest over the past several decades.
Breast milk contains large amounts of iodide, mainly during the first 24 hours after ingestion Thyroid Peroxidase first generates I2 by oxidizing I- ions present in the follicular lumen. No mutation was detected in Duox1. In summary, contrary to NIS for which the anion selectivity 25 corresponds to what was expected, the ion selectivity of thyroid pendrin remains to be elucidated.
This introduces atoms of the element iodine , covalently bound, per tyrosine residue. This is an iodinated albumin, shown to be serum albumin that is iodinated in the thyroid Inside the lumen of RER, newly-synthesized proteins undergo core glycosylation and by interacting with chaperones acquire their conformation. Both forms occur in normal and abnormal human thyroid tissue.
The protein s insuring the second step is are not yet identified. Thyroid hormones T4 and T3 are produced by the follicular cells of the thyroid gland and are regulated by TSH made by the thyrotropes of the anterior pituitary gland. From Pitt-Rivers, R.
Patients with compound heterozygous missense RW and a nonsense mutation RX , leading to a presumed non functional protein showed PIOD with mild and persistent hyperthyrotropinemia.
The thyroid and kidneys remove most iodide from the plasma. Correction of this defect by site-directed mutagenesis returned Tg export to normal in transfected cells.
This case and others reported later provide further evidence that permanent or transient nature of CH is not directly related to the number of inactivated Duox2 alleles
Proteins are then transported to the Golgi apparatus G , where terminal glycosylation and other post-translational reactions take place.
T3 is 3- to 5- fold more active than T4. Iodide transport is energy-dependent and requires O2. Observations both in vivo and in vitro show an appreciable delay in coupling after initial formation of iodotyrosines.
Duox1 and Duox2 genes are co-localized on chromosome 15q Animals were given increasing doses of stable iodide. Of these last 8 patients 3 harboured heterozygous nonsense or frameshift mutations QX, RX, SfsX meaning that a single defective Duox2 allele can cause haploinsufficency resulting in mild transient CH. Onset of Duox expression study in thyroid embryonic development pointed Duox as a thyroid differentiation marker. Please help improve this section by adding citations to reliable sources.