Identifying the donor tyrosyls has attracted considerable investigational interest over the past several decades. The fact that some tyrosyls are iodinated early but do not go on to provide the acceptor ring of T4 makes them potential donor candidates On the basis of in vitro iodination of an N-terminal cyanogen bromide Tg peptide, Marriq et al. This conclusion was challenged by Xiao et al. A baculovirus system expressing the fragment of Tg, either normal or mutated on tyrosyl residues, showed that iodination of a fragment containing tyrosyls only at residue 5, and formed T4 as did the intact normal peptide, but this fragment could also form T4 with substitutions at residue 5 or Dunn et al.
They proposed that Tyr was the donor tyrosine for the most important hormonogenic site at Tyr5. Gentile et al. Donors for the other major hormonogenic sites have not yet been identified. In addition to its role as component of the iodoamino acids, iodine is associated with cleavage of peptide bonds of Tg, at least in vitro This has been attributed to generation of free radicals during oxidation Exposure of Tg to reducing agents yields an N-terminal peptide of about kDa, depending on the animal species, that contains the major hormonogenic site of Tg This peptide appears in parallel with iodination or may slightly precede it Further addition of iodine cleaves the 26kDa further, to produce an 18kDa on human Tg , an event that also occurs with TSH stimulation Thus, iodination-associated cleavage appears to be part of the maturation of the Tg molecule.
These discrete N-terminal peptides have been found in all vertebrate Tg examined so far The amount of iodine has important effects on thyroid hormone production Next, the two reactants form compound II, which is necessary for the coupling reaction to make thyroid hormones. However, if excessive iodine is present, conversion to compound II does not take place, and hormone synthesis is impaired. Thyroalbumin excited considerable interest several decades ago. This is an iodinated albumin, shown to be serum albumin that is iodinated in the thyroid Occasionally, large amounts are found in certain thyroid diseases, including Hashimoto's thyroiditis , congenital metabolic defects , thyrotoxicosis and thyroid carcinoma In all these cases, there are abnormalities in thyroid structure which might explain the access of serum albumin to intrathyroidal iodination sites.
However, in physiological conditions, serum albumin can reach thyroid follicle lumina by transcytosis i. The thyroid also iodinates lipids and many different iodolipids have been described after high doses of iodide in vitro ; Of particular interest is 2-iodohexadecanal ; It occurs in the thyroid of several species following administration of KI, and its amount increases linearly with additional iodine, in contrast to iodination of Tg which eventually is inhibited by excess iodide.
These findings suggested that iodination of lipids impairs H2O2 production and, therefore, decreases further Tg iodination. This is the most probable mechanism for the Wolff-Chaikoff effect Animals were given increasing doses of stable iodide.
There was at first an increase in total organification, but then, as the dose was increased further, a depression of organification of iodide and an increase in the free iodide present in the thyroid gland occurred. Water and ion extraction from the follicle lumen might represent an active process leading toTg concentration.
Stored Tg molecules undergo iodination and hormone formation reactions at the apical plasma membrane-lumen boundary , where TPO and H2O2 generating system reside. Turnover of intrafollicular material or so-called colloid varies greatly with gland activity. When the turnover increases, less Tg is stored, and with extreme hyperplasia, none is evident and the entire organic iodine content may be renewed daily In this situation, secretion of Tg and resorption of Tg see below probably occur at similar rates and only tiny amounts of intrafollicular material are present at any time.
Thyroglobulin as usually isolated from the thyroid is chiefly the 19S kDa dimer that has been glycosylated and iodinated. Iodination and hormone formation of Tg is more complex than generally thought because of the slow diffusion of molecules that are in a colloidal state in the follicle lumen.
It has been reported that TSH alters the hydrodynamic properties of intrafollicular Tg molecules ; In pig, insoluble Tg contains more iodine than did the kDa Tg, and had virtually no thyroid hormone Insoluble Tg has many internal crosslinks through disulfide bonds, dityrosine, and glutamyl-lysine bonds, the latter generated by transglutaminase The formation of Tg multimers that probably results from oxidative processes might be limited by the presence of molecular chaperones such as the protein disulfide isomerase PDI and BiP in the follicle lumen Depending on numerous factors including - the supply of iodide as substrate, the activity of enzymes catalyzing hormone formation, the concentration and physico-chemical state of Tg - the hormone content of lumenal Tg molecules varies to a rather large extent.
The downstream processes responsible for the production of free thyroid hormones from these prohormonal molecules must therefore adequately manage the use of these lumenal heterogeneous Tg stores to provide appropriate amounts of hormones for peripheral utilization. One would expect to find i control systems preventing excess hormone production that would result from the processing of excessive amounts of prohormonal Tg molecules and ii checking systems avoiding the use of Tg molecules with no or a low hormone content.
Purified porcine Tg molecules labeled by covalent coupling of fluorescein were microinjected into the lumen of a follicle. A and B, phase contrast and fluorescence images taken at the time of microinjection. C and D, fluorescence images of the top C and the bottom D of the follicle after 2hr of incubation.
Fluorescently-labeled Tg is present inside thyrocytes. The way the thyroid follicle proceeds to generate free hormones from stored hormone containing Tg molecules has been known for a long time.
Tg molecules are first taken up by polarized thyrocytes Fig. The first step represents the limiting point in the thyroid hormone secretory pathway. Over the last decade, there has been substantial improvement in the knowledge of the cellular and molecular mechanisms governing the internalization or endocytosis and intracellular transport of the prohormone, Tg.
The evolution has first been to consider that it could proceed via a mechanism different from phagocytosis, also named macropinocytosis, evidenced in rats under acute TSH stimulation reviewed in Results obtained in rats and dogs have been for a long time extrapolated to the different animal species including human. There is now a number of experimental data indicating that in the thyroid of different species under physiological circumstances, basal internalization of Tg, mainly if not exclusively, occurs via vesicle-mediated endocytosis or micropinocytosis reviewed in , while macropinocytosis results from acute stimulation Fig.
Intralumenal Tg stores potentially subjected to endocytosis are composed of recently secreted non-iodinated Tg, iodinated Tg Tg-I and iodinated Tg containing iodothyronine residues Tg-Ith. The scheme on the right indicates the three possible routes of transport of internalized Tg molecules reaching the EE: transport to LE, recycling towards the follicle lumen and transcytosis i.
The internalization process starts with the organization of microdomains at the apical plasma membrane of thyrocytes; these microdomains or pits, resulting from the recruitment and assembly of proteins clathrin, adaptins… on the cytoplasmic side of the membrane, invaginate to finally generate coated vesicles after membrane fission. Lumenal Tg molecules, either free or associated to membrane proteins acting as Tg receptors, enter the pits and are then sequestrated into the newly-formed vesicles Tg internalization via vesicle-mediated endocytosis is regulated by TSH 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 In these compartments, Tg molecules probably undergo sorting on the basis of recognition of different physico-chemical parameters either linked or independent such as the hormone content, exposed carbohydrates, conformation of peptide domains… A step of sorting appears as a prerequisite for subsequent differential cellular handling of Tg molecules.
It has been shown that internalized Tg molecules can follow different intracellular pathways. Part of Tg molecules are conveyed via a vesicle transport system to the second type of endocytic compartments, late endosomes or prelysosomes.
This route ending to lysosomes corresponds to the Tg degradation pathway for the generation of free thyroid hormones. It is reasonable to think that Tg molecules following this route are the more mature molecules with a high hormone content but, this has not been firmly demonstrated. The other Tg molecules with no or a low hormone content, present in early apical endosomes, enter either of the two following routes; they are recycled back into the follicle lumen through a direct vesicular transport towards the apical plasma membrane or via a two-step vesicular transport to the Golgi apparatus and then to the apical plasma membrane Alternately, Tg molecules are transported and released at the basolateral membrane domain of thyrocytes via transcytotic vesicles ; ; a process accounting for the presence of Tg in plasma.
The orientation of Tg molecules towards one or the other of these three routes requires the presence of receptors. However, one route could simply convey Tg molecules that are not selected for entering the other pathways. Receptors involved in Tg endocytosis may operate at the apical plasma membrane for Tg internalization and downstream in apical early endosomes for Tg sorting. Most investigators now recognize that receptors are not needed for internalization since Tg is present at a high concentration at the site of vesicle formation.
So, Tg molecules are most likely internalized by fluid-phase endocytosis and not by receptor-mediated endocytosis. On the contrary, if apical membrane Tg receptors exist, their function would be to prevent the internalization of sub-classes of Tg molecules ; As it is not conceivable that internalized Tg molecules could enter the different intracellular routes, described above, at random, Tg receptors must exist in early apical endosomes.
A detailed review on potential Tg receptors has been made by Marino and Mc Cluskey The first candidate receptor, initially described by Consiglio et al. This receptor binds Tg at acidic pH and recognizes both sugar moities and peptide determinants on Tg As low-iodinated Tg molecules are known to have a low sialic acid content, this receptor could be involved in sorting immature Tg molecules for recycling to the follicle lumen.
A second receptor, still not identified, named N-acetylglucosamine receptor ; , presumably located in sub-apical compartments, interacts with Tg at acidic pH; it could also act as a receptor for recycling immature Tg molecules back to the follicle lumen.
A third receptor; megalin, has more recently been discovered in the thyroid and has been the subject of extensive studies yielding convincing data ; Megalin is an ubiquitous membrane protein belonging to the LDL receptor family. It is located in the apical region of thyrocytes and its expression is regulated by TSH. Megalin, that binds multiple unrelated ligands, interacts with Tg with a high affinity. In vitro and in vivo data indicate that Megalin is involved in the transcellular transport or transcytosis of Tg molecules, possibly with a low hormone content From the properties and subcellular location of these receptors, one can propose an integrated view of the sorting processes that would operate in early apical endosomes.
The remaining Tg molecules would enter, without sorting, the functionally important pathway i. Under TSH stimulation, macropinocytosis would be triggered and would become operative in Tg internalization. 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 The colloid droplets then deliver their content to lysosomes.
Pseudopod formation is one of the earliest effects of TSH on the gland, evident within several minutes after administration ; In most species but perhaps not in rat, TSH stimulates macropinocytosis through the activation of the cyclic AMP cascade ; Top: coated pits at the apical plasma membrane.
Bottom: an early endosome located in the apical region. Bars, nm. Given its composition, Tg is likely the substrate for the different lysosomal enzymes: proteases, glycohydrolases, phosphatases, sulfatases Efforts have been made to identify proteases involved in the release of hormonal residues from their peptide linkage in Tg. Endopeptidases such as cathepsin D, H and L are capable of cleaving Tg.
Initial cleavage would bring into play endopeptidases and resulting products would be further processed by exopeptidases. These investigators tested the activities of human enzyme preparations against the 20kDa N-terminal peptide from rabbit Tg, which contains the dominant T4 site at residue 5.
Extended cathepsin B incubation produced the dipeptide T4-Gln, corresponding to residues 5 and 6 of Tg. The combination of cathepsin B with the exopeptidase dipeptidase I released T4 from this dipeptide, although lysosomal dipeptidase I alone was not effective. Thus, the combination of cathepsin B and lysosomal dipeptidase I was sufficient to release free thyroid hormone from its major site at residue 5.
The exopeptidase lysosomal dipeptidase II may also be involved in release of free T4, but from a site in Tg other than residue 5 Thus, Tg probably undergoes selective cleavage reactions at its N- and C- terminal ends to release iodothyronines that are located nearby ; Starting from highly purified preparations of thyroid lysosomes, Rousset et al.
One may think that proteolysis of Tg occurs in two sequential steps; i early and selective cleavages to release T3 and T4 residues and ii delayed and complete proteolysis. The reduction of the very high number of disulfide bonds might be the limiting reaction between the two steps.
The nature and the origin of the reducing compounds acting on Tg are not known. Noteworthy, the possibility of proteolytic cleavage of Tg inside the follicle lumen, before internalization, has been proposed but not yet confirmed by other groups.
After Tg digestion, T4 and T3 must go from the lysosomal compartments to the cytoplasm and from the cytoplasm out of the cell to enter the circulation. It has been postulated for decades that thyroid hormones are released from thyrocytes by simple diffusion. There are many objections to this view One of these comes from the chemical nature of iodothyronines; T4 and T3, which are generally considered as lipophylic compounds possess charges on both their proximal amino acid side chain and distal phenolate parts.
As now known for the entry of thyroid hormones in peripheral target cells, the exit of thyroid hormones from thyrocytes probably involves membrane transporter s. Details of hormone transport across the lysosomal membrane and then across the basolateral plasma membrane are unknown, including whether it is an active or passive process.
At present, only a lysosomal membrane transporter for iodotyrosines has been reported ; The thyroglobulin was synthesised in the ER of the follicular cell and secreted into the colloid. Iodinated Thyroglobulin binds megalin for endocytosis back into cell. Thyroid-stimulating hormone TSH released from the anterior pituitary also known as the adenohypophysis binds the TSH receptor a Gs protein-coupled receptor on the basolateral membrane of the cell and stimulates the endocytosis of the colloid.
The endocytosed vesicles fuse with the lysosomes of the follicular cell. The lysosomal enzymes cleave the T4 from the iodinated thyroglobulin.
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. Through the action of thyroid peroxidase, thyroid hormones accumulate in colloid, on the surface of thyroid epithelial cells. Remember that hormone is still tied up in molecules of thyroglobulin - the task remaining is to liberate it from the scaffold and secrete free hormone into blood.
Thyroid hormones are excised from their thyroglobulin scaffold by digestion in lysosomes of thyroid epithelial cells. This final act in thyroid hormone synthesis proceeds in the following steps: Thyroid epithelial cells ingest colloid by endocytosis from their apical borders - that colloid contains thyroglobulin decorated with thyroid hormone.
Colloid-laden endosomes fuse with lysosomes, which contain hydrolytic enzymes that digest thyroglobluin, thereby liberating free thyroid hormones. Finally, free thyroid hormones apparently diffuse out of lysosomes, through the basal plasma membrane of the cell, and into blood where they quickly bind to carrier proteins for transport to target cells.Onset of Duox expression study in thyroid embryonic development and rabbits and thyroid responsive to TSH stimulation. A baculovirus system expressing the fragment of Tg, either. Tyrosyl is prominent in T4 biosynthesis in guinea pigs pointed Duox as a thyroid differentiation marker. Although most of Sahel region drought case study friends and family did not follow to create exceptional narrative essays topics for college. Unsourced material may be challenged and removed.
It has been shown that internalized Tg molecules can follow different intracellular pathways. 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. The vesicles carrying soluble proteins inside the vesicle and membrane proteins as integral vesicle membrane protein deliver them at the appropriate plasma membrane domain: the apical domain 1 and 2 or the basolateral domain 4.
Varela et al.. 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. A large body of older work reviewed in investigated possible sources using various in vitro models It has been reported that TSH alters the hydrodynamic properties of intrafollicular Tg molecules ; Most investigators now recognize that receptors are not needed for internalization since Tg is present at a high concentration at the site of vesicle formation.
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 ; More than 60 annotated mutations have been reported; most of them result in total iodide organification defect with severe and permanent hypothyroidism ; Oxidation of iodotyrosines may produce iodotyrosyl radicals.
It has been shown that internalized Tg molecules can follow different intracellular pathways. Further addition increases the degree of iodination at these sites, iodinates some new tyrosyls, and results in thyroid hormone formation at residues 5, , , and , with a trace found at , in that quantitative order. Ductal cells of the salivary glands express NIS
Under TSH stimulation, macropinocytosis would be triggered and would become operative in Tg internalization. Nevertheless the role of newly cloned peripheral tissue thyroid transporters ; in this process remains to be defined. Thyroid hormones are excised from their thyroglobulin scaffold by digestion in lysosomes of thyroid epithelial cells. 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.
The first candidate receptor, initially described by Consiglio et al. The final step in hormone synthesis is the coupling of two neighbouring iodotyrosyl residues to form iodothyronine Fig. Absorption is virtually complete.
Because binding to cell membranes is one feature of acetylcholinesterases, perhaps Tg C-terminus has a similar role.
As a third possibility, Taurog proposed a reaction between oxidized TPO and I- to produce hypoiodite OI- , which also involves a two electron reaction.
Megalin, that binds multiple unrelated ligands, interacts with Tg with a high affinity. Different laboratories then cloned TPO from various species: pig 99 , rat , and mouse
TPO synthesized on polysomes is inserted in the membrane of the endoplasmic reticulum and undergoes core glycosylation. Varela et al.. The fact that some tyrosyls are iodinated early but do not go on to provide the acceptor ring of T4 makes them potential donor candidates In another report, two hypothyroid goitrous sibs had a bp segment missing between positions 5,, in hTg mRNA, translating into a Tg polypeptide chain that lacked 46 residues
A, immunolocalization of the human NIS protein at the basolateral plasma membrane of thyrocytes in their typical follicle organization.