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Defects in protein folding in congenital hypothyroidism
Journal Pre-proof Defects in protein folding in congenital hypothyroidism Héctor M. Targovnik, Karen G. Scheps, Carina M. Rivolta PII:
S0303-7207(19)30340-5
DOI:
https://doi.org/10.1016/j.mce.2019.110638
Reference:
MCE 110638
To appear in:
Molecular and Cellular Endocrinology
Received Date: 29 July 2019 Revised Date:
21 October 2019
Accepted Date: 1 November 2019
Please cite this article as: Targovnik, Hé.M., Scheps, K.G., Rivolta, C.M., Defects in protein folding in congenital hypothyroidism, Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/ j.mce.2019.110638. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Defects in protein folding in congenital hypothyroidism.
1 2 3
Héctor M. Targovnik1,2 *, Karen G. Scheps1,2, Carina M. Rivolta1,2
4 5 1
6
Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de
7
Microbiología, Inmunología, Biotecnología y Genética/Cátedra de Genética. Buenos Aires,
8
Argentina. 2
9 10
CONICET-Universidad de Buenos Aires. Instituto de Inmunología, Genética y Metabolismo (INIGEM). Buenos Aires, Argentina.
11 12
Short title: Protein folding in congenital hypothyroidism
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Keywords: Thyroid genes, Protein folding, ERAD, Congenital hypothyroidism.
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Word count of the full article:
15 16
*Address correspondence and requests for reprints to:
17
Dr. Héctor M. Targovnik
18
CONICET-Universidad de Buenos Aires. Instituto de Inmunología, Genética y Metabolismo
19
(INIGEM). Hospital de Clínicas “José de San Martín”, Av. Córdoba 2351, Cuarto Piso, Sala
20
5, C1120AAR - Buenos Aires, Argentina.
21
Tel.: 54-11-5950-8805 E-mail: [email protected], [email protected]
22
2
1
Abstract
2
Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and
3
one of the most common preventable causes of both cognitive and motor deficits. CH is a
4
heterogeneous group of thyroid disorders in which inadequate production of thyroid hormone
5
occurs due to defects in proteins involved in the gland organogenesis (dysembryogenesis) or in
6
multiple steps of thyroid hormone biosynthesis (dyshormonogenesis). Dysembryogenesis is
7
associated with genes responsible for the development or growth of thyroid cells: such as NKX2-1,
8
FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3 resulting in agenesis,
9
hypoplasia or ectopia of thyroid gland. Nevertheless, the etiology of the dysembryogenesis remains
10
unknown for most cases. In contrast, the majority of patients with dyshormonogenesis has been
11
linked to mutations in the SLC5A5, SLC26A4, SLC26A7, TPO, DUOX1, DUOX2, DUOXA1,
12
DUOXA2, IYD or TG genes, which usually originate goiter.
13
About 800 genetic mutations have been reported to cause CH in patients so far, including
14
missense, nonsense, in-frame deletion and splice-site variations. Many of these mutations are
15
implicated in specific domains, cysteine residues or glycosylation sites, affecting the maturation of
16
nascent proteins that go through the secretory pathway. Consequently, misfolded proteins are
17
permanently entrapped in the endoplasmic reticulum (ER) and are translocated to the cytosol for
18
proteasomal degradation by the ER- associated degradation (ERAD) machinery.
19
Despite of all these remarkable advances in the field of the CH pathogenesis, several points on
20
the development of this disease remain to be elucidated. The continuous study of thyroid gene
21
mutations with the application of new technologies will be useful for the understanding of the
22
intrinsic mechanisms related to CH. In this review we summarize the present status of knowledge
23
on the disorders in the protein folding caused by thyroid genes mutations.
24 25 26
3
1
1. Introduction
2
The thyroid hormone (TH) is essential for the growth and neural maturation, as well as
3
cardiovascular and renal functions. This hormone also regulates the lipid, carbohydrate and protein
4
metabolism [Mullur et al., 2014]. The thyroid produces predominantly the prohormone 3,5,3´,5´-
5
tetraiodothyronine (T4) and minority the 3,5,3´-triiodothyronine (T3), T3 is obtained by deiodination
6
of the outer phenolic ring of the T4 by means of type 1 iodothyronine deiodinases (D1) and type 2
7
iodothyronine deiodinases (D2) [Mullur et al., 2014]. TH action is mainly mediated by binding of
8
T3 to its nuclear receptor, a member of the superfamily of hormone-responsive nuclear transcription
9
factors [Cheng et al., 2010; Samuels et al., 1974].
10
The biosynthesis of TH takes place at the interface of the apical cell plasma membrane and the
11
colloid in polarized follicular thyroid cells and requires the presence of iodide, thyroid peroxidase
12
(TPO), a supply of hydrogen peroxide, an iodine acceptor protein, thyroglobulin (TG), proteolytic
13
enzymes and the rescue and recycling of iodide by action of iodotyrosine deiodinase-1 (IYD-1)
14
(Figure 1) [Degroot and Niepomniszcze, 1977; Gnidehou et al., 2004; Targovnik et al. 2010, 2011].
15
After the iodide is uptake in the basolateral membrane by the Na+/I- symporter (NIS) [Bizhanova
16
and Kopp, 2009; Dohan et al., 2003], it is transported at the apical membrane into the follicular
17
lumen by an anion exchange transmembrane protein called pendrin (Figure 1) [Bizhanova and
18
Kopp, 2009; Royaux et al., 2000; Yoshida et al., 2002]. Na+/I- symporter (NIS) cotransports one
19
iodide ion with two sodium ions against an electrochemical gradient. This sodium gradient is
20
generated by Na+/K+-ATPase. Roepke et al. [2009] and Purtell et al. [2012] demonstrated that
21
KCNQ1-KCNE2 potassium channel is required for NIS-mediated thyroid cell uptake of I- (Figure
22
1). It was later described that the Ca2+-activated chloride channel TMEM16A or anoctamin-1
23
(encoded by ANO1 gene) [Siveira and Kopp, 2015; Twyffels et al. 2014] and SLC26A7 also
24
mediate the iodide efflux through the apical membrane of thyroid cells [Ishii et al., 2019]. The
25
oxidized iodide is incorporated into selected tyrosyl residues of TG which results in formation of
26
monoiodotyrosines (MID) and diiodotyrosines (DIT). Coupling between either two DIT, or between
4
1
a DIT and a MIT, generates the formation of T4 or T3 respectively (Figure 1). The oxidition of I−
2
into I+, iodination and coupling reactions are mediated by TPO in presence of H2O2 [Degroot and
3
Niepomniszcze, 1977]. Hydrogen peroxide is generated by a metabolic pathway, involving two
4
members of the NADPH oxidase family: dual oxidase 1 (DUOX1) and dual oxidase 2 (DUOX2)
5
and two ER-resident proteins: DUOX maturation factor 1 (DUOXA1) and DUOX maturation factor
6
2 (DUOXA2) [De Deken et al., 2000, 2002; De Deken and Miot, 2019; Dupuy et al, 1999;
7
Grasberger and Refetoff, 2006]. TPO and the DUOX system are in close proximity, forming a
8
complex at the level of the apical plasma membrane of the follicular cell (Figure 1) [Song et al.,
9
2010].
10
The thyroid cells produce free T4 and T3 by endocytic internalization and proteolytic lysosomal
11
cleavage (cathepsin proteases and glutamate carboxypeptidase) of the TG, which are delivered to
12
the blood circulation for action at their peripheral target tissues [Jordans et al., 2009; Linke et al.,
13
2002; Suban et al., 2012]. The monocarboxylate transporter 8 (MCT8, encoded by SLC16A2 gene)
14
facilitates the efflux of TH from the thyrocytes (Figure 1) [Di Cosmo et al., 2010]. From TG, free
15
MIT and DIT are also released by the thyroid cells. Therefore, the rescue and recycling into thyroid
16
cell of iodide from MIT to DIT by the action of IYD-1 are essential to prevent loss of this rare
17
element, especially in iodine-deficient areas.
18
The thyroid stimulating hormone (TSH) and its receptor (TSHR), a member of the G- protein-
19
coupled receptor superfamily (GPCRs), constitute the main regulatory pathway of the thyroid
20
(Figure 1) [Vassart and Dumont, 1992]. The cAMP and Gq/phospholipase C cascade pathways
21
mediate most of its effects, including iodide uptake, expression of thyroid genes, biosynthesis of
22
TH, TPO activity, thyroid H2O2 generating system, endocytosis, proteolysis and hormone release.
23
[Vassart and Dumont, 1992].
24
In order to fulfill their functions, the specialized thyroid proteins described above must be
25
exported to the colloid or located at the basolateral and apical membranes through a series of way-
5
1
stations. Exported proteins enter at the endoplasmic reticulum (ER), the first station of this pathway.
2
The proper folding and assembly of new synthesized exportable proteins occur within the ER
3
compartment. Then, secretory proteins are translocated into the Golgi complex by carrier vesicles
4
and finally, they are exported to their final destination. Exportable protein folding is enhanced by
5
the actions of compartment-specific molecular chaperones and enzymes. Remarkably, unfolding or
6
misfolding of exportable proteins cause retention within the ER, induction of the synthesis of ER
7
chaperones and removal of misfolded proteins from the ER lumen by an anterograde traffic out of
8
the ER to be catabolized in the proteasomes, a phenomenon known as ER-associated degradation
9
(ERAD) mechanism. ERAD prevents the accumulation or secretion of the abnormal protein [Oi et
10
al., 2017].
11
Here, we review the latest contributions in the field of protein folding disorders in congenital
12
hypothyroidism caused by mutations in the main proteins that participate in the biosynthesis of TH
13
and in thyroid embryogenesis. We also provide a large references list of original publications.
14 15
2. Congenital hypothyroidism
16
Congenital hypothyroidism (CH) is the most frequent endocrine disease in infants, affects about
17
1 in 2,000-3000 newborns and is characterized by elevated levels of thyroid-stimulating hormone
18
(TSH) and low TH serum levels [Kwak, 2018; Park and Chatterjee, 2005; Rastogi and LaFranchi,
19
2010]. It is also one of the most common preventable causes of cognitive and motor deficits [Kwak,
20
2018, Park and Chatterjee, 2005; Rastogi and LaFranchi, 2010]. The genetic classification divides
21
CH into two main categories according to their causes: (a) disorders of thyroid gland development
22
(dysembriogenesis or thyroid dysgenesis group) or (b) defects in any of the steps of thyroid
23
hormone synthesis (dyshormonogenesis group) [Park and Chatterjee, 2005; Rastogi and LaFranchi,
24
2010; Targovnik et al. 2010, 2011, 2017]. The dysembryogenesis or thyroid dysgenesis group,
25
which accounts for the 80-85 % of the cases, results from a thyroid gland that is completely absent
6
1
in orthotopic or ectopic location (agenesis or athyreosis), severely reduced in size but in the proper
2
position in the neck (orthotopic hypoplasia) or located in an unusual position (thyroid ectopy) at the
3
base of the tongue or along the thyroglossal tract [Abu-Khudir et al., 2017; Targovnik et al. 2010,
4
2011, 2017]. In few patients, the CH has been associated with mutations in genes responsible for
5
the development or growth of thyroid cells: NKX2-1 (also known as TTF1), FOXE1 (also known as
6
TTF2 or FKHL15), PAX8 and NKX2-5 genes [Grasberger and Refetoff, 2011; Kwak, 2018;
7
Targovnik et al. 2010, 2011, 2017]. Recently, exome analysis identified DUOX2 mutations in
8
patients with ectopic thyroid suggesting that DUOX2 could also be involved in thyroid
9
embryogenesis [Kizys et al., 2017]. More recently, Zou et al. [2018] identified mutations in the
10
TBX1, CDCA8, HOXD3 and HOXB3 genes associated with dysgenesis. Loss-of-function mutations
11
in the TSHR gene have been shown to cause thyroid hypoplasia and TSH resistance in humans
12
[Grasberger and Refetoff, 2017]. In the majority of patients with a defect in thyroid organogenesis,
13
the genetic mechanism responsible remains to be elucidated.
14
Dyshormonogenesis, which accounts for the remaining 15-20% of the cases, has been linked to
15
mutations in the SLC5A5 [Santos-Silva et al., 2019; Spitzweg and Morris, 2010; Targovnik et al.,
16
2017], SLC26A4 [Bizhanova and Kopp, 2010; Chem et al., 2018; Dossena et al., 2009, 2011;
17
Makretskaya et al., 2018; Sun et al., 2018; Wémeau and Kopp, 2017, Zou et al., 2018], SLC26A7
18
[Cangul, et al., 2018; Ishii et al., 2019; Zou et al., 2018], TPO [Long et al., 2018; Makretskaya et
19
al., 2018; Ris-Stalpers and Bikker, 2010; Santos-Silva et al., 2019; Sun et al., 2018; Targovnik et
20
al., 2017; Zou et al., 2018], DUOX1 [Aycan, et al., 2017; Liu et al., 2019; Watanabe et al., 2019;
21
Zou et al., 2018], DUOX2 [Belforte et al., 2016a, 2016b; Chem et al., 2018; Grasberger, 2010; Long
22
et al., 2018; Makretskaya et al., 2018; Muzza and Fugazzola, 2017; Sun et al., 2018; Yu et al., 2018;
23
Zou et al., 2018], DUOXA1 [Liu et al., 2019], DUOXA2 [Chem et al., 2018; Muzza and Fugazzola,
24
2017 Sun et al., 2018], IYD [Moreno and Visser, 2010; Sun et al., 2018; Targovnik et al., 2017] and
25
TG [Chem et al., 2018; Citterio et al., 2019; Di Jeso and Arvan, 2016; Long et al., 2018;
7
1
Makretskaya et al., 2018; Santos-Silva et al., 2019; Sun et al., 2018; Targovnik et al., 2010, 2011,
2
2016, 2017; Yu et al., 2018; Zou et al., 2018] genes. In the last three decades about eight hundred
3
variants have been described in unrelated CH patients. These mutations produce a heterogeneous
4
spectrum of congenital hypothyroidism, characterized usually by the presence of congenital goiter
5
or goiter appearing shortly after birth with an autosomal recessive inheritance. Therefore, the
6
patients are typically homozygous or compound heterozygous for the gene mutations and the
7
parents, carriers of one mutation.
8
Iodide organification defects (IOD) are linked with inactivating mutations in the TPO, DUOX2,
9
DUOXA2 or SLC26A4 genes and probably associated with mutations in the DUOX1 and DUOXA1
10
genes. IOD is characterized by high levels of serum TG and a positive Perchlorate Discharge Test
11
(PDT) [El-Desouki et al., 1995], indicating that the iodide is taken up by thyroid cells but it is not
12
incorporated into the TG when the oxidation of iodine, iodination and coupling mechanisms fail.
13
PDT is used to distinguish Total Iodide Organification Defect (TIOD) from Partial Iodide
14
Organification Defect (PIOD, between 10 and 90%) [Kwak. 2018]. In patients with PIOD usually a
15
single allele is affected, whereas homozygous or compound-heterozygous mutations are associated
16
with TIOD. Most cases of CH associated with alterations in DUOX2 are caused by either biallelic or
17
monoallelic mutations which lead to extremely complex correlation between DUOX2 genotypes
18
and clinical phenotypes [De Marco et al., 2011; Moreno and Visser, 2007; Wang et al., 2014]. Both
19
biallelic and monoallelic DUOX2 mutations could be associated with transient CH (TCH) or
20
permanent CH (PCH) [Fu et al., 2016a; Tan et al., 2016]. Mutations in SLC26A4 gene cause
21
Pendred syndrome characterized by congenital sensorineural hearing loss, goiter with or without
22
hypothyroidism and usually PIOD [Bizhanova and Kopp, 2010; Wémeau and Kopp, 2017]. In
23
patients with IYD-1 deficiency, the organification process is not affected whereas the serum TG
24
levels are elevated [Moreno and Visser, 2010; Targovnik et al., 2017]. In contrast, the presence of
25
low or undetectable levels of TG and also negative PDT in a goitrous individual suggest a TG
8
1
defect [Citterio et al., 2019; Di Jeso and Arvan, 2016; Targovnik et al., 2010, 2011, 2017]. Patients
2
with an iodide transport defect by mutations in SLC5A5 gene have a normal-sized or somewhat
3
enlarged thyroid gland, elevated plasma TG levels and no radio-iodide uptake [Spitzweg and
4
Morris, 2010; Targovnik et al., 2017].
5
Recent technological advances in DNA sequencing through the implementation of next-
6
generation sequencing (NGS) platforms that allow the massive identification of sequence variants
7
have led to the identification of new mutations in the thyroid genes. Interestingly, Fu et al. [2016a,
8
2016b, 2016c] using NGS detected that most of the cases of CH with one or two DUOX2 gene
9
mutations are associated with subclinical or TCH, whereas patients with three or four (one in
10
homozygous) or five (two in homozygous) DUOX2 gene mutations are mostly associated with
11
PCH. The new technologies also showed the coexistence of multiple mutations in different thyroid
12
genes in the same patient with CH, for instances, mutations in DUOX2 associated with mutations in
13
DUOXA2 or TPO or TG or TSHR or SLC26A4 genes [Fu et al., 2016a, 2016b; Jiang 2016; Park et
14
al., 2016; Sun et al., 2018]. The identification of simultaneous mutations in the same gene or in
15
different thyroid specific genes could contribute to the accurate diagnosis and classification of the
16
defects.
17 18
3. Defects in protein folding of thyroglobulin
19
TG represents a highly specialized homodimeric glycoprotein which acts as a substrate for the
20
synthesis of T4 and T3 as well as the storage of the inactive forms of iodine. [Citterio et al., 2019;
21
Targovnik 2013; Targovnik et al., 2017]. Human TG gene is a single copy of 268 Kb long that maps
22
on chromosome 8 (8:132,866,958-133,134,903; GRCh38 assembly) and contains an 8,455 nt of
23
mRNA sequence (with a coding sequences of 8304, NCBI Accesion Number: NM_003235.5)
24
divided into 48 exons [Malthiéry and Lissitzky, 1987; Mendive et al., 2001; Mercken et al., 1985;
25
van de Graaf et al., 2001].
9
1
The monomeric human TG preprotein has a leader peptide of 19 amino acids followed by a
2
2749-amino-acid polypeptide (Figure 2). The most important T4-forming site couples donor DIT149
3
to acceptor DIT24 [Dunn et al., 1998; Lamas et al., 1989; Palumbo et al., 1990]; whereas the main
4
T3-forming site couples an MIT2766 at the antepenultimate residue of one TG monomer with the
5
antepenultimate DIT2766 in the opposite monomer of the TG dimer [Citterio et al., 2017].
6
The internal protein organization makes TG an example of gene evolution by intragenic
7
duplication events and gene fusions [Parma et al., 1987]. The N-terminal and the central parts of the
8
monomer include three types of repetitive motifs, called TG type 1, TG type 2, and TG type 3,
9
comprising cysteine-rich repeats covalently bound by disulfide bonds (Figure 2). The TG type 1
10
motif derives from an ancestral gene that was duplicated and modified during evolution by
11
mutations [Holzer et al., 2016; Malthiery and Lissitzky, 1987; Mercken et al., 1985; Molina et al.,
12
1996, Parma et al., 1987, van de Graaf et al., 2001] whereas TG type 2 repeats are distant members
13
of the GCC2/GCC3 domain superfamily [Lee et al, 2011] and TG type 3 repeats exhibit only
14
internal homology. Tg monomer contains eleven TG type 1 elements located between positions 31 -
15
358, 605 - 1210 and between 1511 - 1565; three TG type 2 elements located between amino acids
16
1456 and 1503, and five TG type 3 elements between residues 1603 and 2187 (Figure 2) [Holzer et
17
al., 2016; Malthiery and Lissitzky, 1987; Mercken et al., 1985; Molina et al., 1996, Parma et al.,
18
1987, van de Graaf et al., 2001]. TG is organized in four structural regions (I, II, III and IV) (Figure
19
2). Region I comprises 10 of the 11 TG type 1 repeats, two segments called linker (residues 359 to
20
604) and hinge (residues 1211 to 1455) plus the N-terminal T4 forming site. Region II contains 3
21
TG type 2 repeats and the eleventh TG type 1 repeat, whereas region III contains the five TG type 3
22
repeats. The fourth region is composed of the cholinesterase-like (ChEL) domain, a member of the
23
α/β-hydrolase fold family, located between residues 2211 to 2735 of the TG [Park and Arvan, 2004;
24
Swillens et al., 1986] and the C-terminal T3 forming site. The transitions between repetitive motifs
25
TG type 2-3 and TG type 1-11, between TG type 1-11 and TG type 3-a1, and between TG type 3-a3
10
1
and the ChEL domain correspond to non-repetitive sequences that could be called spacer 1 (residues
2
1504 to 1510), spacer 2 (1566 to 1602) and spacer 3 (2188 to 2210) segments, respectively (Figure
3
2).
4
During translation/translocation in the lumen of the ER, the newly synthesised TG starts to fold
5
and acquires its final three-dimensional glycoprotein structure as it passes through the Golgi
6
complex. TG is transferred to the follicle lumen as a noncovalent dimer, concentrated into secretory
7
vesicles and stored as colloid. Intensive post translational modifications take place in the ER, which
8
include glycosylation and formation of intrachain disulfide bonds.
9
All glycoproteins go through similar trimming in the ER and acquire their final glycoprotein
10
structure as they pass through the Golgi [Bedard et al, 2004]. The addition of carbohydrates
11
stabilizes the protein and increases its solubility. TG acquires the N-glycans during the process of
12
translocation and elongation of the polypeptide chain into the ER. First, two N-acetylglucosamines
13
(GlcNAc) and nine mannoses with three terminal glucose residues are assembled onto a core
14
oligosaccharide, which is then en bloc transferred to the consensus N-glycosylation sites
15
(Asparagine-X-Serine/Threonine, where X is any amino acid residue except proline) on nascent TG
16
chains by action of an oligosaccharyltransferase (Figure 3). Next, the three terminal glucoses of this
17
core are trimmed by sequential action of I and II α-glucosidases. Finally, a terminal mannose is
18
trimmed by α-mannosidase I with the formation of high-mannose type oligosaccharide [Helenius
19
and Aebi, 2004] (Figure 3).
20
Correct folding is assisted by interaction with chaperones and enzymes of the ER [Bedard et al.,
21
2005], such as calnexin (CNX), calreticulin (CRT,), GRP94, BiP, Protein Disulfide Isomerase
22
(PDI), ERp29, ERp57, ERp72 [Baryshev et al, 2004; Di Jeso et al., 2003, 2005; Kim et al., 1992;
23
Kim and Arvan, 1995; Kuznetsov et al., 1994; Menon et al., 2007; Muresan and Arvan, 1997;
24
Sargsyan et al., 2002]. Such interactions may also serve to prevent premature export of unfolded or
25
misfolded secretory proteins from the ER by a process known as ER-quality control [Bedard et al.,
11
1
2005; Qi el al., 2017], an adaptive cellular reaction that maintains ER homeostasis avoiding
2
perturbation by the accumulation of incorrectly folded proteins. GRP94 is associated with the
3
nascent TG [Muresan and Arvan, 1997], whereas BiP binds to unfolded polypeptides and prevents
4
protein aggregation through noncovalent associations [Chung et al, 2002]. The oxidative folding of
5
TG proceeds with the aid of two chaperone-oxidoreductase complexes, Bip/PDI and
6
CNX/CRT/ERp57 [Meunier et al., 2002]. The formation of intradomain disulfide bonds can be
7
catalyzed near BiP bound sites by PDI and near CNX/CRT bound sites by the ERp57
8
oxidoreductase [Di Jeso et al., 2005].
9
The last stage of TG conformational maturation in the ER is the non-covalent TG
10
homodimerization, without any interchain disulfide bridge, and only after TG has dissociated from
11
CNX/CRT system. ChEL domain is required for protein dimerization. Whereas correctly folded
12
proteins are exported from the ER to Golgi, misfolded proteins are retained in the ER and
13
retrotranslocated to the cytosol for proteasomal degradation by the ERAD machinery [Kim an
14
Arvan, 1998; Qi et al., 2017].
15
The next step in the post translational modifications of TG N-glycans occurs within the Golgi
16
and follows presumably the same general process described for other proteins. In brief, after
17
removal
18
glycosyltransferases (GnT) catalyze the branching and elongation of the carbohydrate chains,
19
producing hybrid-type or complex-type oligosaccharide [Vagin et al., 2009] (Figure 3).
of
mannose
residues
by
Golgi
mannosidases,
several
N-acetylglucosamine
20
The cDNA sequence of human TG contains 20 putative N-linked glycosylation sites, 16 of them
21
being glycosylated in the mature protein [Yang et al., 1996]. Eight sites (asparagines residues 76,
22
484, 529, 748, 816, 1716, 1774 and 2250) are linked to complex type oligosaccharide units
23
containing fucose and galactose in addition to mannose and glucosamine [Yang et al., 1996]. The
24
sites in positions 1220, 1349, 2013, 2295 and 2582 contain high-mannose type units (mannose and
25
glucosamine) and the sites in positions 198 and 1365 are linked to oligosaccharide units containing
12
1
galactose in addition to mannose and glucosamine but no fucose and may be either hybrid-type or
2
complex-type. [Yang et al., 1996]. Finally, very different oligosaccharide composition types were
3
found associated with position 947 (complex or high mannose) [Yang et al., 1996]. The human TG
4
also contains sulfated O-linked glycosaminoglycans [Fogelfeld and Schneider, 1990; Schneider et
5
al., 1988; Spiro and Bhoyroo, 1988]. Interestingly, non-glycosylated thyroglobulin loses the ability
6
to synthesize thyroid hormones [Mallet et al., 1995].
7
Phosphorylation of TG occurs in Golgi apparatus within carbohydrates and serine and tyrosine
8
residues [Consiglio et al., 1987], whereas sulfation is a late post translational modification step that
9
takes place in the trans-Golgi and contributes to the negative charge of the TG [Cauvi et al., 2003].
10
In mammals, newly synthesized TG is firstly glycosylated and folded via the formation of
11
numerous disulfide bonds [Citterio et al., 2019; Targovnik, 2013]. As indicated above, TG type 1,
12
TG type 2 and TG type 3 are cysteine-rich repeats that are covalently bound by intramolecular
13
disulfide bonds, which are involved in the conformation of the tertiary structure of TG and in the
14
hormonogenesis. The formation of disulfide bonds is catalyzed by endogenous ER oxidoreductases.
15
The TG type 1 domains are grouped into two subgroups, type 1A and type 1B. Type 1A repeats
16
(TG type 1–1 to TG type 1–8 and TG type 1–10) have a total of six Cys residues, whereas type 1B
17
repeats (TG type 1–9 and TG 1–11) contain only four Cys residues [Molina et al., 1996].
18
Conservative intradomain disulfide bonds patterns Cys1–Cys2, Cys3–Cys4, and Cys5–Cys6 prevail
19
in all type 1A repeats [Molina et al., 1996]. TG type 1B, TG type 2 and TG type 3 repeats and the
20
six cysteine residues of the ChEL domain include also intradomain disulfide bonding.
21
TG containing missense mutations in the cysteine residues or in the ChEL domain and splice site
22
mutations causing exon skipping could represent targets for intracellular retention and degradation
23
by ERAD pathway.
24
Sequencing analysis of the TG gene revealed missense mutations that involved cysteine residues:
25
p.C160S, p.C183Y, p.C194G, p.C726Y, p.C1077R, p.C1264R, p.C1281Y, p.C1476R, p.C1493Y,
13
1
p.C1510F, p.C1607F, p.C1897Y, p.C1904G, p.C1996S, p.C2000W, p.C2006Y and p.C2154Y
2
(Figure 2, Table 1) [Baryshev et al, 2004; Caputo et al., 2007; Citterio et al., 2013; Hishinuma et al,
3
1999, 2005, 2006; Hu et al., 2016; Kanou et al., 2007; Kitanaka et al., 2006; Narumi et al., 2011;
4
Nicholas et al., 2016; Zou et al. 2018]. The loss of cysteine residues can eliminate disulfide bonds
5
and alter the normal conformational structure of the TG, possibly preventing the interaction of
6
hormonogenic acceptor and donor sites, and causing a defect in the intracellular transport of the
7
protein. A detailed study of two unrelated patients with CH and two siblings with adenomatous
8
goiter revealed replacement of two conserved cysteines by arginine (C1264R) and serine (C1996S),
9
which resulted in the retention and aggregation of TG in the ER [Hishinuma et al. 1999]. Thyroid
10
tissue extracted from all patients exhibited higher expression levels of ER chaperones, BiP, GRP94,
11
PDI, ERp29 and ERp72 [Baryshev et al, 2004].
12
The ChEL domain is essential for the intracellular transport of TG to the site of its
13
hormonogenesis, via the secretory pathway [Lee et al., 2008, 2009, 2011; Lee and Arvan, 2011;
14
Park and Arvan, 2004; Wang et al., 2010]. It functions as an intramolecular chaperone and as a
15
molecular escort for TG regions I, II, and III [Lee et al., 2008]. The ChEL domain is also required
16
for protein dimerization and consequently plays a critical structural and functional role in the TG
17
protein. It is well documented that pathogenic missense mutations in the ChEL domain can result in
18
the intracellular retention of TG. The missense mutations located in this domain may cause TG
19
retention in the ER and premature degradation. Several missense mutations associated with CH
20
were reported to be present in the ChEL domain: p.A2234D [Caputo et al., 2007; Citterio et al.,
21
2013; Machiavelli et al., 2010; Pardo et al. 2008; 2009, Santos-Silva et al., 2019], p.R2242H [Caron
22
et al., 2003; Machiavelli et al., 2010; Raef et al., 2010, Zou et al., 2018], p.N2285D [Makretskaya et
23
al., 2018], p.G2319D [Hishinuma et al., 2006], p.R2336Q [Hishinuma et al., 2006; Kitanaka et al.,
24
2006 ; Siffo et al., 2018], p.G2341S [Watanabe et al. 2018], p.A2362P [Siffo et al., 2018],
25
p.W2365R [Siffo et al., 2018], p.G2374V [Hishinuma et al., 2006], p.G2375R [Hishinuma et al.,
14
1
2005; 2006; Kanou et al.; 2007; Long et al., 2018], p.I2394M [Sun et al., 2018], p.R2455H [Hu et
2
al., 2016; Long et al., 2018, Yu et al., 2018], p.A2471S [Sun et al., 2018], p.L2547Q [Nicholas et
3
al., 2016], p.R2585W [Hu et al., 2016, Jiang et al., 2016 ; Sun et al., 2018] p.N2616I [Hu et al.,
4
2016; Long et al., 2018, Sun et al. 2018; Yu et al., 2018], p.W2685L [Nicholas et al., 2016], and
5
p.A2713T [Long et al., 2018] (Figure 2, Table 2). Functional analysis suggests that the p.A2215D
6
mutation results in retention of the TG protein inside the ER and degradation via the proteasome
7
system [Pardo et al., 2009], as already observed in the cog/cog congenital goiter mouse [Kim et al,
8
1998] and the rdw/rdw non-goitrous CH rat [Kim et al, 2000]. The cog/cog mouse harboring the
9
p.L2263P TG mutation [Kim et al., 1998] and the WIC-rdw rat harboring the p.G2300R TG
10
mutation [Hishinuma et al., 2000; Kim et al., 2000] exhibit full-length synthesis of TG but impaired
11
transport from the ER.
12
Exon skipping in the TG gene can be caused by nucleotide substitutions or deletion in acceptor
13
or donor splice sites involving the -3/-2/-1 (c.275-3C>G, c.5042-2A>G, c.6563-2A>G, c.2762-
14
1G>A, c.6200-1G>C, c.7998-1G>A) or +1/+2/+3/+4/+5/+6 position (c.638+1G>A, c.745+1G>A,
15
c.2176+1G>A, c.4932+1G>C, c.5686+1G>T, c.5686+1G>A, c.5686+1G>C, c.6262+1delG,
16
c.6876+1delG, c.274+2T>G, c.5401+2T>C, c.7036+2T>A, c.7862+2T>A, c.4159+3_+4delAT;
17
c.3433+3_+6delGAGT, c.638+5G>A), respectively [Abdul-Hassan et al., 2013; Alzahrani et al.,
18
2006; Chen et al., 2018; Citterio et al., 2015, Fu et al., 2016c, Gutnisky et al., 2004; Hermanns et
19
al., 2013, Hishinuma et al., 2006; Hu et al., 2016, Ieiri et al. 1991; Makretskaya et al., 2018;
20
Medeiros-Neto et al., 1996; Narumi et al., 2011; Nicholas et al., 2016; Niu et al., 2009; Pardo et al.,
21
2008, 2009; Peteiro-Gonzalez et al., 2010; Rubio et al., 2008; Targovnik et al., 1995, 2001, 2012;
22
Watanabe et al., 2019; Zou et al., 2018]. Recently, two exonic cryptic 5' splicing sites in exons 6
23
(c.745+1G>A) [Citterio et al., 2015] and 19 (c.4159+3_4delAT) [Targovnik et al., 2012] of the TG
24
gene have been identified. The elimination of exons in the TG gene by aberrant splicing results in
25
an altered ability to transfer an iodophenoxyl group from the donor site to the acceptor iodotyrosine.
15
1
Interestingly, two affected patients with the c.5686+1G>T mutation which leads to the skipping of
2
exon 30 [Targovnik et al., 2001], showed a marked increase of the ER chaperones, GRP94 and BiP
3
[Medeiros-Neto et al., 1996]. Immunohistochemical localization of TG showed a notable decrease
4
of immunoreaction product in the follicular lumen of thyroid sections from affected patients. In
5
contrast, accumulation of intracellular immunoreactive TG was clearly demonstrable in the thyroid
6
epithelium, suggesting a defect in intracellular TG transport [Medeiros-Neto et al., 1996].
7 8
4. Defects in protein folding of thyroid peroxidase
9
TPO is a thyroid-specific enzyme that plays a key role in thyroid hormones biosynthesis and is
10
the major autoantigen in Hashimoto’s disease, the most common organ-specific autoimmune
11
disease (Czarnocka et al., 1985; Portmann et al., 1985). TPO is a member of the peroxidase family
12
which employs a heme group as part of their catalytic unit. The human TPO gene is located on
13
chromosome 2 (2:1,374,066-1,543,711; GRCh38 assembly). It comprises 17 exons, spands 170 kb
14
of genomic DNA and encodes a glycosylated hemoprotein of 933 amino acids (Figure 4). The
15
mRNA is 3,152 nucleotides long (NCBI Reference Sequence NM_000547.5) [Kimura et al., 1989]
16
and the pre-protein is composed of a putative 18 amino acids signal peptide followed by a 915
17
amino acids polypeptide which codifies a large extracellular domain (residues 19-846), a
18
transmembrane domain (residues 847-871), and a short intracellular tail (residues 872-933) (Figure
19
4) [https://www.uniprot.org/uniprot/P07202]. Baker et al. (1994) and Nishikawa et al. (1994)
20
suggested that human TPO may be a disulfide-linked dimer. TPO shows 42% sequence homology
21
with the myeloperoxidase (MPO) (Libert et al., 1987) and is part of the animal haem peroxidase
22
superfamily. The exons 13 and 14 encode the complement control protein (CCP)-like (residues 740-
23
795) and calcium-binding epidermal growth factor (EGF-Ca2+ binding)-like (residues 796-839)
24
domains, respectively (Figure 4) [https://www.uniprot.org/uniprot/P07202, Ruff and Carayon,
25
2006]. EGF-Ca2+ shows structural characteristics of a Sushi domain.
16
1
The TPO enzyme activity depends on both proper folding and membrane insertion, and an intact
2
catalytic site. After its synthesis, human TPO undergoes various postranslational modifications such
3
as N- and O-linked glycosylation and heme fixation [Ruff and Carayon, 2006]. Five potential
4
glycosylation sites are present at asparagine residues 129, 307, 342, 478, and 569 [Ruff and
5
Carayon, 2006]. N-glycans play an essential role in the correct folding, intracellular trafficking and
6
activity of TPO [Fayadat et al., 1998]. Exons 8, 9 and 10 encode the catalytic center of the TPO
7
protein (heme-binding region) [Rodrigues et al., 2005] which is crucial for the enzymatic activity
8
and contain the proximal (His239) and distal (His494) putative heme binding histidine residues
9
(Figure 4). According to the studies with MPO and lactoperoxidase, the heme group in TPO is a
10
bis-hydroxylated heme b covalently bound via ester linkages to Glu399 and Asp238 of the apoprotein
11
(Figure 4) [Ruff and Carayon, 2006].
12
The first description of a human TPO mutation was conducted in a boy with iodide
13
organification defect, who presented with hypothyroidism at the age of 4 months and developed a
14
compressive goiter at the age of 12 years [Abramowicz et al, 1992]. A homozygous duplication of a
15
tetranucleotide -GGCC- in exon 8 of the TPO gene was identified (c.1186_1187insGGCC or
16
c.1183_1186dupGGCC, also reported as 1187_1188insGCCG or c.1184_1187dup GCCG), 152 bp
17
upstream from the junction with intron 8. The resulting frameshift generates a stop codon in exon 9
18
(p.A397Pfs*76, Figure 4), which would result in a truncated protein with mutation of the proximal
19
and deletion of the distal putative heme binding histidine residues where by TPO activity is
20
expected to be absent. However, alternative splicing restores the normal reading frame
21
[Abramowicz et al, 1992]. The result is a nearly full-length protein with a 91-amino acid segment
22
replaced by a 51 residue unrelated segment. This translation product is expected to have an
23
unchanged distal putative heme binding His494, but to lack the proximal putative heme binding
24
His239 [Abramowicz et al, 1992]. The duplication GGCC in exon 8 is a common mutation of the
25
TPO gene in Caucasian population [Abramowicz et al., 1992; Altmann et al, 2013; Bakker et al.,
17
1
2000; Belforte et al., 2012; Bikker et al, 1995; Cangul et al., 2013; Cangul et al., 2015; Fugazzola et
2
al., 2005; Gruters et al.,1996; Nascimento et al., 2003, Neves et al., 2010; Rivolta et al 2003; Santos
3
et al., 1999].
4
Six cystein residues responsible for the formation of 3 disulfide bridges: Cys800-Cys814, Cys808-
5
Cys823 and Cys825-Cys838 are located in the EGF-Ca2+ binding domain of human TPO. The
6
substitution of cysteine for other amino acids affects the formation of the disulfide bond and
7
therefore can alter the tertiary structure of the EGF-Ca2+ binding domain of TPO [Rodrigues, et al.,
8
2005]. Disruption of the disulfide bonds, as result of p.C800R [Bӧrgel et al., 2005], p.C808R
9
[Rivolta et al., 2003] or p.C838S [Rodrigues, et al., 2005] mutations (Figure 4), would generate
10
significant conformational changes which could partially retain the TPO on the ER on the one hand,
11
and on the other hand, the proteins with conformational defects responsible for a lower net activity
12
of the enzyme might reach the membrane.
13
Interestingly, the p.S131P mutation (Figure 4) disrupts the potential glycosylation site at Asn129
14
residue [Rodrigues, et al., 2005]. The severity of this mutation is evident, the N-X-S/T site is
15
modified to N-X-P and consequently is very likely to induce major defects in the intracellular traffic
16
of the TPO.
17
Umeki et al. (2004) analyzed five mutated TPOs with alteration in expression probably due to
18
incorrect folding, four of them were missense mutations (p.R175Q, p.G533C, p.R665W, and
19
p.G771R, Figure 4) [Kotani et al., 2003, 2004; Umeki et al., 2002] and one corresponded to the
20
elimination of two contiguous amino acids (p.D574-L575del, Figure 4). The five mutated TPOs had
21
a pathogenic mutation on the extracellular domain but had no abnormality on the transmembrane
22
and cytoplasmic domains. The mutated mRNAs were transfected in Cho-Ki and NRK cells and then
23
they were studied by indirect immunofluorescence and flow cytometry to determine the intracellular
24
localization. In permeabilized condition the 5 mutated proteins were located in intracellular
25
structures like the nuclear envelope and ER in both cells line, whereas in nonpermeabilized
18
1
condition only p.D574-L575del was observed on the cell surface of Cho-KI and p.D574-L575del in
2
addition to p.G771R, on the cell surfase of NRK cells [Umeki et al. (2004]. The analysis of the
3
TPO protein expression on the cell surface by flow cytometry showed a positive fluorescense peak
4
in p.G533C, p.D574-L575del and p.G771R with less intensity compared with wild-type TPO. In
5
contrast, p.R175Q and p.R665W were not expressed on the cell surface. It has been reported that
6
calnexin and calreticulin binding to TPO are necessary for the initial folding steps [Fayadat et al.,
7
2000]. In immunoprecipitation by anti-TPO antibodies followed by Western blot using anti-TPO,
8
anti-calnexin and anti-calreticulin, p.G533C, p.D574-L575del and p.G771R exhibited increasing
9
interaction with calnexin. This suggests that some p.G533C, p.D574-L575del and p.G771R mutated
10
proteins would be fold properly in the calnexin cycle and secreted via Golgi to apical membrane of
11
the thyrocyte, while the remaining of these misfolded TPOs would be degraded in the ERAD
12
pathway. In p.R175Q and p.R665W missense mutations, an arginine was substituted for glutamine
13
or tryptophan, respectively. Arginine is a basic amino acid with positive charge, whereas glutamine
14
is uncharged and tryptophan is a hydrophobic amino acid. The loss of an arginine could cause
15
structural changes and as a consequence p.R175Q and p.R665W mutations would follow the
16
anterograde pathway and would be degraded in proteasomes.
17 18
5. Defects in protein folding of DUOX system
19
The DUOX1 and DUOX2 genes are located in opposite transcriptional orientation in an operon-
20
like unit on chromosome 15, encoding similar proteins that are inserted in the apical membrane of
21
thyroid cells [De Deken et al. 2000, 2002; De Deken and Miot, 2019; Dupuy et al, 1999;
22
Grasberger, 2010; Muzza and Fugazzola, 2017, Ohye and Sugawara, 2010]. The DUOX2 gene
23
spans 22 Kb of genomic DNA (15:45,092,650-45,114,344; reverse strand, GRCh38 assembly)
24
which includes 34 exons, being the first one non-coding. The mRNA (NCBI Reference Sequence:
25
NM_014080.4) is 6,428 nucleotides long and the preprotein is composed of a putative 21 amino
19
1
acids signal peptide followed by a 1,527 amino acids polypeptide (Figure 5) [De Deken et al. 2000,
2
2002; Dupuy et al, 1999]. DUOX1 gene encodes a homologous protein displaying 83 % sequence
3
similarity. The 36 kb DUOX1 gene (15:45,129,933-45,165,576; forward strand, GRCh38 assembly)
4
consists of 35 exons (being the first two noncoding) encoding a protein of 1551 amino acids
5
(mRNA: 5675 nucleotides long; NCBI Reference Sequence: NM_017434.5) in which the first 21
6
amino acids correspond to the putative signal peptide [De Deken et al. 2000, 2002; Dupuy et al,
7
1999]. The structure of DUOX1 and DUOX2 proteins contains three regions: 1) peroxidase
8
homology ectodomain (peroxidase-like domain): an N-terminal extracellular region, which has 43%
9
sequence homology with hemoperoxidases, which distinguishes DUOX from other family
10
NOX/DUOX, 2) a central region encompassing an intracellular loop containing potentially two EF-
11
hand calcium binding motifs, which would be involved in regulating the conformation and activity
12
of NADPH oxidase; 3) gp91phox/NOX2-like domain: a C-terminal region, comprising the last six
13
transmembrane domains (Figure 5) [De Deken et al., 2000; Dupuy et al., 1999; Morand et al., 2004;
14
Ohye and Sugawara, 2010]. The C-terminal domain includes four conserved histidines, which are
15
coordination sites for two heme prosthetic group [Muzza and Fugazzola, 2017]. The C-terminal
16
cytosolic orientation presents a binding site for FAD and NADPH (Figure 5) [De Deken et al.,
17
2000; Dupuy et al., 1999; Morand et al., 2004; Ohye and Sugawara, 2010]. It is believed that this
18
region is responsible for the transfer of electrons from NADPH through the membrane. DUOX2
19
exhibits higher expression and is also more efficient in the production of peroxide than DUOX1
20
[Muzza and Fugazzola, 2017]. DUOX1 is regulated by protein kinase A through Gs-PKA pathway,
21
while DUOX2 is stimulated by protein kinase C through Gq-phospholipase C pathway [Rigutto et
22
al., 2009]. Although both enzymes are expressed in the thyroid, DUOX1 is also expressed in the
23
respiratory and tongue epithelia, testis and cerebellum, and DUOX2, in respiratory epithelia, uterus,
24
gallbladder, pancreatic islets and digestive tract where these enzymes could provide H2O2 for
25
peroxidase-mediated antimicrobial defense processes [El Hassani et al., 2005; Forteza et al., 2005;
20
1
Geiszt et al, 2003]. DUOX1 and DUOX2 present two N-glycosylated states, the high mannose
2
glycosylated immature form of 180 kDa expressed in ER and the fully glycosylated mature form of
3
190 kDa which is translocated at the plasma membrane and generates H2O2 [Morand et al., 2004;
4
Muzza and Fugazzola, 2017]. Partially glycosylated DUOX2 remains in the ER [Grasberger and
5
Refetoff, 2006].
6
DUOXA1 and DUOXA2 are essential for DUOX maturation [Grasberger and Refetoff, 2006,
7
Muzza and Fugazzola, 2017]. The DUOXA1 and DUOXA2 genes are oriented head to head in the
8
intergenic region of 16 kb (DUOXA2: 15:45,114,321-45,118,421, forward strand; DUOXA1:
9
15:45,117,367-45,129,938, reverse strand, GRCh38 assembly) between both DUOX genes. The
10
DUOXA2 open reading frame spans 6 exons encoding a protein of 320 amino acids (mRNA: 1755
11
nucleotides long; NCBI Reference Sequence: NM_207581.4), whereas the DUOXA1 spans 11
12
exons encoding a protein of 483 amino acids (mRNA: 2021 nucleotides long; NCBI Reference
13
Sequence: NM_144565.4). The two DUOXA genes encode a N-terminal ectodomain, five
14
transmembrane domains and a C-terminal cytoplasmic region. The first extracellular loop presents
15
three N-glycosylation sites [Grasberger and Refetoff, 2006; Muzza and Fugazzola, 2017].
16
DUOXA1 and DUOXA2 enable the ER-to-Golgi transition, maturation, and surface expression of
17
functional DUOX1 and DUOX2 in a heterologous cell system (Figure 6) [De Deken and Miot,
18
2019; Grasberger and Refetoff, 2006]. A stable complex between DUOX2 and DUOXA2 is then
19
required for their enzymatic activity (Figure 6). DUOXA2 would be part of the DUOX2 quality
20
control system, indicating that DUOXA2 displays a chaperone-like function with respect to its partner.
21
[Carré et al., 2015; Grasberger et al. 2007].
22
DUOX2 alterations were first described in 2002 [Moreno et al.]. In the literature, Sanger
23
sequencing has been commonly used for DUOX2 mutations identification in both research and
24
clinical studies. Recently, high throughput sequencing has been reported to be a rapid and effective
25
tool for largest DUOX2 mutation screening for patients with or without family history [Chem et al.,
21
1
2018; Long et al., 2018; Makretskaya et al., 2018; Sun et al., 2018; Yu et al., 2018; Zou et al.,
2
2018]. The mutational spectrum shows heterogenous alterations dispersed in the exons and introns
3
of the entire gene (Figure 5) [Belforte et al., 2016a; De Deken and Miot, 2019]. To date, molecular
4
research has shown more than one hundred fifty pathogenic variations have been found to be
5
associated with TCH, PCH or subclinical congenital hypothyroidism [Belforte et al., 2016a; De
6
Deken and Miot, 2019; Fu et al., 2016a].
7
mutations were found to be altered proteins with a single amino acid substitution, followed by
8
deletions/insertion and nonsense mutations causing premature truncation of the DUOX2 protein
9
[Belforte et al., 2016a; Muzza and Fugazzola, 2017]. Premature termination codons can delete the
10
gp91phox/NOX2-like domain and the peroxidase-like domain. The functional consequence could
11
be the complete impossibility to generate H202 from the DUOX2 system. Only six DUOX2 splice
12
site mutations have been reported, c.514-2A>G [Tan et al., 2016], c.2335-1G>C [Belforte et al.,
13
2016b; Muzza et al., 2014], c.2655-2A>C [Varela et al., 2006], c.3516-1G>A [Tan et al., 2016]
14
c.3693+1G>T [Tan et al., 2016] and c.4240-1G>C [Matsuo et al., 2016]. The p.K530*, p.E879K,
15
p.R1110Q, p.L1160del, p.R1334W and p.L1343F mutations have been the most frequently
16
identified DUOX2 mutations in Asian population (Figure 5) [Belforte et al., 2016a; Muzza and
17
Fugazzola, 2017], whereas the frequent p.F965Sfs*29 (c.2895_2898delGTTC) mutation (Figure 5)
18
has proved to be the most frequent in the Caucasian population [Belforte et al., 2016a; Muzza and
19
Fugazzola, 2017].
The most frequent events of deleterious DUOX2
20
Grasberger et al. [2007] analyzed three missense mutations in DUOX2 (p.Q36H [Varela et al.,
21
2006], p.R376W [Cortinovis et al., 2008; Lü et al., 2011; Vigone et al., 2005] and p.D506N
22
[Cortinovis et al., 2008; Pfarr et al., 2006] involved in congenital hypothyroidism (Figure 5), in
23
terms of its impact on the generation of H2O2, folding, intracellular traffic and DUOXA2
24
interaction, in a reconstituted heterologous system. The three missense mutations displayed either
25
complete (p.Q36H and p.R376W) or partial loss (p.D506N) of H2O2-generating activity. The
22
1
mutations p.Q36H and p.R376W completely prevent the transit of DUOX2 to the cell surface and
2
showed a diminished oxidized-to-reduced ratio [Grasberger et al. 2007]. The mutant proteins are
3
present predominantly as core N-glycosylated (a thiol-reduced folding intermediate) and are
4
retained by the quality control system within the ER as indicated by a greater complexation with the
5
lectin calnexin. This interaction may be related to retrotranslocation and proteasomal degradation.
6
On the contrary, the p.D506N mutation showed a similar ratio of oxidized-to-reduced forms and a
7
diminished expression at the plasma membrane [Grasberger et al. 2007]. This showed a successful
8
route to the plasma membrane through of Golgi complex but with an abnormal maturation.
9
Oxidative folding of DUOX2 in the ER appears to be the rate-limiting step in the maturation of
10
DUOX2. ER-localized DUOX2 is almost exclusively in the reduced form (presumably inactive)
11
[Grasberger et al. 2007]. The oxidized form (presumably active) does not accumulate in the ER due
12
to rapid, DUOXA2-mediated export (Figure 6). DUOXA2 allows rapid ER exit of DUOX2 folded
13
and also enhances the degradation of DUOX2 protein mutants not suitable for ER output
14
[Grasberger et al. 2007]. However, DUOXA2 does not affect the reduced-to-oxidized transition,
15
this observation would indicate that DUOXA2 would act after the maturation of DUOX2, once
16
DUOX2 has acquired a stable disulfide-bonded conformation. Intramolecular (DUOX2) and
17
intermolecular (between DUOX2 and DUOXA2) disulfide bonds which are promoted and
18
maintained by the oxidizing environment of the ER seem to play an important role in the protein
19
folding, stability, function and protein-protein interaction. Carré et al. [2015] reported an
20
intramolecular bond in the DUOX2 between cysteine residues 124 (located in the N-terminal ectodomain)
21
and 1162 (located in the second extracellular loop), that provides structural support for the formation of
22
interdisulfide bridges between DUOX2 and DUOXA2 (Figure 6). The intermolecular bonds between
23
DUOX2 and DUOXA2 involve the N-terminal cysteine residues 568 and 582 of DUOX2 respectively and
24
cysteine residues 167 of the first extacellular loop and 233 of the second extacellular loop of DUOXA2
25
(Figure 6) [Carré et al., 2015]. It is importan to note that mutations of cysteine residues in the N-
26
terminal domain of human DUOX2 protein affect both targeting and extracellular H2O2 production
23
1
by human DUOX2 [Fortunato et al., 2010; Morand et al., 2004]. Recently, the p.C124* (dbSNP:
2
rs1197021807) and p.C568R (rs770603845) mutations (Figure 5) was reported in gnomAD data set
3
[https://gnomad.broadinstitute.org/gene/ENSG00000140279] in a heterozygous state, with an allele
4
frequency of 2/249972 (0,000008001) and 4/188376 (0.00002123) respectively.
5 6
6. Defects in protein folding of NIS
7
The SLC5A5 gene that encodes NIS, is located on chromosome 19, spanning 23 Kb
8
(19:17,871,945-17,895,174; GRCh38 assembly). The full-length human mRNA (NCBI Reference
9
Sequence: NM_000453.3) consists of 15 exons (3604 nucleotides) and encodes a 643 amino acids
10
peptide with 13 transmembrane segments, an extracelular N-terminal extreme and an intracellular
11
C-terminus (Figure 7) [De la Vieja et al.,2004, 2005; Spitzweg and Morris, 2010; Targovnik et al.,
12
2017]. This transporter has three N-glycosilation sites (Figure 7) [Spitzweg and Morris, 2010],
13
although their glycosilation is not essential for its function as iodine transporter [Levy et al., 1998]
14
or for reaching and inserting into the plasma membrane [Li et al., 2013].
15
The expression of NIS is regulated primarily by TSH levels [Carrasco, 2013], I- concentration in
16
plasma [Portulano et al., 2014] and by TG [Vono-Toniolo and Kopp, 2004]. Its trafficking to the
17
cell membrane is also regulated by the interaction with other proteins, such as pituitary tumor–
18
transforming gene (PTTG) binding factor (PBF) which promotes its internalization [Stratford et al.
19
2005].
20
The analysis of the effect of reported mutations affecting SLC5A5 and of different mutagenesis
21
studies allowed understanding the key role of certain residues for the folding and trafficking of the
22
NIS. Several loss-of-function mutations leading to I- transport defect (ITD) have been reported
23
[Martín et al., 2018a]; five of them lead to the intracellular retention of this transporter: p.R124H,
24
p.V270E, p.A439_P443del, p.S509Rfs*7 and p.G543E (Figure 7). The first three inhibit also the
25
transporter activity [Ravera et al., 2017]. The interaction between the δ-amino group at 124 and the
24
1
thiol in the Cys440, lost in p.R124H, is crucial for the folding of NIS and targeting to the plasma
2
membrane [Paroder et al, 2013]. The deleterious effect of p.A439_P443del is due to the loss of
3
Asn441 of the side chain which interacts with Gly444, capping the α-helix of transmembrane segment
4
XII [Li , et al., 2013]. The frameshift mutation p.S509Rfs*7 [Pohlenz, et al., 1998; 2000] leads to
5
the loss of the C-terminal portion of this transporter. Different studies [Martín et al., 2018b; Martín
6
et al., 2018c] have demonstrated the importance of this region (between residued Ile546 and Lys618),
7
which is subjected to post translational modifications, such as phosporilation for the migration to
8
the plasma membrane. It was shown that a putative tryptophan-acidic motif (Trp565-Asp566) is
9
required for leaving the ER and reaching the membrane. Furthermore, the region between Val580
10
and Lys618 is crucial for its basolateral location. Only alanine or serine are tolerated at position 543,
11
other substitutions as p.G543E lead to the intracellular retention of the altered peptide [De la Vieja
12
et al., 2005]. The mutation p.V270E alters the positive charge in the surface of the molecule, which
13
would alter the interaction with the proteins involved in the trafficking of NIS [Nicola et al, 2015].
14
The systematic assessment of sorting motifs in NIS, carried out by Darrouzet et al. [2016] revealed
15
an internal noncanonical PDZ-binding motif, that involves the residues Tyr118, Glu119, Tyr120 and
16
Leu121, necessary to reach the plasma membrane. This site was predicted bioinformatically and
17
assessed with functional assays, including directed mutagenesis which validated the role played by
18
Leu121.
19 20
7. Defects in protein folding of Pendrin
21
The passive transport of the iodine to the follicular lumen is mostly facilitated by pendrin [Bidart
22
et al., 2000], encoded by the SLC26A4 gene, that maps in chromosome 7 (7:107,660,828-
23
107,717,782; GRCh38 assembly). It comprises 21 exons (the first one is noncoding), covers 57 kb
24
of genomic DNA and encodes 780 amino acids (Figure 8). The mRNA is 4,737 nucleotides long
25
(NCBI Reference Sequence: NM_000441.2) and the canonical isoform codifies for a classic
25
1
predicted topology of 12 transmembrane domains with intracellular N- and C-terminal tails (Figure
2
8). Dossena et al., [2009] proposed another model of pendrin consisting of 15 transmembrane
3
segments, an extracelular N-terminal extreme and an intracellular C-terminus. It is interesting to
4
note that there is no experimental data proving any of the suggested models. Pendrin has a STAS
5
(sulfate transporter and antisigma factor antagonist) domain (Figure 8) [Aravind and Koonin, 2000].
6
Pendrin has affinity for chloride, iodide and bicarbonate, among other anions. The peptide is N-
7
glycosylated exclusively at Asn167 and Asn172 which are located in the second putative extracellular
8
loop of pendrin (Figure 8). N-glycosylation of membrane proteins plays a role in their correct
9
folding and targeting to the membrane [Azroyan et al., 2011].
10
As was previously mentioned, mutations in this gene can lead to nonsyndromic hearing loss
11
associated with enlarged vestibular aqueduct (EVA) or Pendred syndrome, an autosomal recessive
12
disorder characterized by sensorineural hearing impairment, thyroid goiter and a partial
13
organification problem detectable by a positive perchlorate test [Bizhanova and Kopp, 2009]. The
14
main molecular mechanism proposed for Pendred Syndrome has been SLC26A4 mutations causing
15
the retention of the mutants in the ER [Rotman-Pikielny et al, 2002]. Retention of misfolded
16
pendrin in the ER and subsequent degradation is a major pathogenic mechanism for Pendred
17
syndrome. There are more than 170 sequence variants associated with this phenotype [Campbell, et
18
al., 2001; Dossena et al., 2009, 2011; Tsukamoto et al., 2003], 64% of these are single nucleotide
19
changes leading to amino acid substitutions and at least eight of them (p.G102R; p.V138F; p.T193I;
20
p.L236P; p.T410M; p.L445W; p.Q446R and p.Y556C) [Coyle et al., 1998; de Moraes et al., 2016;
21
Taylor et al., 2002] (Figure 8) lead to diminished or abolished expression of the mutant pendrin in
22
the apical membrane. Although the specific molecular chaperones involved in the folding of this
23
protein have yet to be identified, it has been reported that Hsp90 is not involved, and furthermore,
24
its inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) increases the stability of Pendrin
25
by inducing the transcription of heat shock transcription factor 1 (Hsf1)-dependent chaperones [Lee,
26
1
et al., 2012]. The same authors also demonstrated that the E3 ubiquitin ligase Rma1 is involved in
2
ERAD Pendrin degradation.
3
Jung et al [2006] reported that the cytosolic heat shock cognate protein 70 (Hsc70) and the co-
4
chaperone DNAJC14 are involved in the unconventional Golgi-bypass trafficking pathway that can
5
be stimulated by ER stress-inducing agents. This can lead to cell surface expression of misfolded
6
variants, making them potential targets for treating Pendred syndrome.
7 8
8. Defects in protein folding of SLC26A7
9
In the recent years, the role of pendrin as the main apical iodine transporter has been challenged,
10
as consequence of the lack of a complete correlation between deleterious mutations affecting this
11
transporter and hypothyroidism [Bizhanova and Kopp, 2011] and the fact that apical iodide efflux is
12
also possible in the absence of pendrin [Silveira and Kopp, 2015]. Interestingly, 50% of patients
13
with Pendred syndrome are euthyroid. In the last two years, recessive loss-of-function mutations in
14
another transporter named SLC26A7 were identified associated with goitrous CH [Cangul, et al.,
15
2018; Ishii et al., 2019; Zou et al., 2018]. The SLC26A7 gene is located on chromosome 8, spanning
16
149 Kb (8:91,249,319-91,398,155; GRCh38 assembly) and including 19 exons, being the first one
17
non-coding. The full-length human mRNA (NCBI Reference Sequence:
18
of 5,237 nucleotides. Sequence analysis revealed an open reading frame of 1,971 nucleotides
19
encoding a 656-amino acid peptide [Lohi et al, 2002]. The structure of SLC26A7 is characterized
20
by citoplasmatic N- and C-terminal domains, 12 putative transmembrane segments and a C-terminal
21
cytoplasmic STAS consensus domain (Figure 9) [Alper and Sharma, 2013; Lohi et al, 2002]. In
22
addition, it contains two predicted N-glycosilation sites at Asn125 and Asn131 (Figure 9) [Lohi et al,
23
2002]. The SLC26A7 was first discovered as a chloride/anion exchanger in the kidney (expressed in
24
the basolateral membrane of the outer medullary collecting duct intercalated cells) and in the
25
stomach (expressed in the basolateral membrane of the gastric parietal cells) [Petrovic et al., 2003;
NM_052832.4) consists
27
1
2004]. Recently, immunohistochemical and RT-PCR analyses confirmed the expression of
2
SLC26A7 in human thyroid follicular cells, located predominantly on the apical side facing the
3
follicular lumen [Ishii et al., 2019].
4
In 2018, a homozygous mutation in the SLC26A7 gene (c.927_930delCACT, c.932_933delAA
5
[p.I309Mfs*9,p.E311Gfs*28] or c.927_933delinsG [p.I309_E311delinsM]) was firstly reported in a
6
family of Saudi origin with two CH patients [Zou et al., 2018] (Figure 9). The unaffected mother,
7
father and a sibling carried the mutation in a heterozygous state. Almost simultaneously, Cangul et
8
al. [2018] identified novel homozygous mutations in the SLC26A7 gene in six unrelated families
9
with goitrous CH. By whole exome sequencing and subsequent Sanger sequencing, it was identified
10
in three consanguineous families of Pakistani or Turkish origin a homozygote c.679C>T transition,
11
which replaces the arginine residue in position 227 with a stop codon (p.R227*) [Cangul et al.,
12
2018] (Figure 9). In the same paper, in other three non-consanguineous families of Finnish origin,
13
the authors reported a homozygous frameshift mutation (c.1893delT, p.F631Lfs*8) (Figure 9).
14
More recently, Ishii et al. [2019] described two Japanese siblings with goitrous CH caused by a
15
homozygous nonsense mutation (c.1498C>T; p.Q500*) (Figure 9). In vitro studies of the mutant
16
evidence a complete loss of iodide transport activity. The p.Q500* mutation, located in the STAS
17
domain, impaired the localization of SLC26A7 in the cell membrane. It is well documented that the
18
STAS domain is required for membrane localization and exchanger function [Dorwart et al., 2008;
19
Sharma,et al., 2011].
20
Remarkably, hypothyroidism caused by SLC26A7 mutants cannot be rescued by wild-type
21
pendrin, suggesting that SLC26A7 plays a critical function in thyroid hormone synthesis. [Zou et
22
al., 2018]. Ishii et al. [2019] hypothesized that the SLC26A7 may be expressed more abundantly
23
than pendrin in the human thyroid gland and that SLC26A7 may have another function in the
24
biosynthesis of thyroid hormones in addition to being an iodide transporter. These hypotheses
25
require further investigation.
28
1
9. Defects in protein folding of the proteins involved in disembryogenesis
2
In only 5-10% of the thyroid dysgenesis, the underlying genetic cause can be unequivocally
3
determined and linked to a specific sequence variant. As was mentioned earlier, the mutations
4
described affect mainly genes involved in the development, growth and differentiation of the
5
thyroid cells (NKX2-1, FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3).
6
The TSHR is member of the superfamily of G Protein-coupled receptors [Vassart and Dumont,
7
1992, Tao and Conn, 2014]. The canonical isoform of 764 amino acids consists of seven
8
transmembrane domains, with an N-terminal extracellular segment that interacts with the TSH and a
9
cytoplasmatic C-terminal domain, capable of interacting with the G protein [Corvilain, et al., 2001].
10
As other glycosylated transmembrane proteins, TSHR undergoes post translational modifications
11
and folding in the ER lumen where it interacts with the resident chaperones. Studies by Siffroi-
12
Fernandez et al., [2002] and Mizrachi and Segaloff [2004] showed the interaction of this nascent
13
peptide with calnexin and calreticulin. Siffroi-Fernandez et al., [2002] were also able to detect the
14
binding of BiP in K652 cells, whereas Mizrachi and Segaloff [2004] could not find this association
15
and demonstrated the interaction with the protein disulfide isomerase (PDI) in HEK293 cells.
16
Loss-of-function germline mutations in TSHR are expected to cause congenital hypothyroidism
17
with a syndrome of resistance to TSH [Corvilain et al., 2001]. Most of these variants lead to
18
misfolding of the receptor and its intracellular retention in the ER [Mizrachi and Segaloff, 2004].
19
Familial cases of congenital hypothiroidim due to mutations in NKX2-1 and PAX8, often show
20
incomplete penetrance, suggesting the presence of additional modifiers [De Felice and Di Lauro,
21
2004]. Molecular analyses of DHTP mice (Double Heterozygous for both Titf1- and Pax8-null
22
mutations) that presented thyroid hypoplasia during embryogenesis and high incidence of thyroid
23
hemiagenesis as adults [Amendola, et al., 2005], revealed the presence of a strain specific mutation
24
in DNAJC17 [Amendola, et al., 2010], making it a candidate locus for hypothyroidism
25
predisposition. The DNAJC17 human gene encodes 304 amino acids and is a member of the heat-
29
1
shock-protein-40 family (Hsp40), acting usually as cofactor of the Hsp70 chaperone, regulating
2
their ATPase activity, through the J domain that all members display. [Kampinga and Craig, 2010].
3
Nevertheless, many of the functions that this co-chaperon exercises are still unknown. Pascarella, et
4
al [2018] demonstrated its co-localization in the nuclear speckles (also known as interchromatin
5
granule clusters) and its interaction with spliceosomal components, suggesting a participation in
6
splicing processes. Germline loss-of-function mutations in a homozygous state in this gene were
7
also associated with the recessive inheritance of retinal dystrophy [Patel, et al., 2016] This gene was
8
screened in 89 patients with congenital hypothyroidism, but only a possible damaging rare variant
9
c.610G>C was identified in a single patient [Nettore, et al., 2018], showing that DNAJC17 is not an
10
initial candidate gene to be analyzed in thyroid dysgenesis.
11
In the recent years, it has been described that mutations affecting different genes lead to
12
congenital hypothirodism associated to other syndromes, such as the Krüppel-like zinc finger
13
transcription factor GLI-similar 3 (GLIS3) which causes neonatal diabetes, a large spectrum of
14
abnormalities and clinical presentations of congenital hypothyroidism, from thyroid agenesia to
15
dyshormogenesis [Dimitri, et al., 2015]. This transcription factor mediates the TSH/TSHR
16
proliferation of thyrocites and is required for the biosynthesis of thyroid hormones, since
17
experiment in mice showed it regulates the expression of NIS and pendrin [Kang, et al., 2017]. On
18
the other hand, loss-of-function mutations that affect the C-terminus of the immunoglobulin
19
superfamily member 1 and impair its trafficking to the plasma membrane, lead to X-linked
20
congenital central hypothyroidism, with prolactin and GH deficiency, delayed pubertal development
21
and testicular enlargement [Sun, et al., 2012; Turgeon, et al, 2017]. Deficient levels of
22
immunoglobulin superfamily member 1, decrease the pituitary response to TRH because the
23
expression of its receptor is downregulated.
24 25
10. Conclusions and future perspectives
30
1
Congenital hypothyroidism (CH) describes a hormonal state of insufficient thyroid hormone
2
secretion detected at birth or postnatally. Over the last 20 years, mutations in the candidate genes
3
were identified and functionally characterized providing an overwhelming wealth of new insights
4
into basic molecular mechanisms.
5
Mutations affecting the proteins involved in the cascade of biosynthesis of TH, thyroid
6
organogenesis and its regulating pathways have been classified as missense and nonsense
7
mutations, frameshift mutations by single or multiple nucleotide deletion or insertion and splicing
8
mutations in the exonic or intronic consensus sites. The mutations are located in specific domains or
9
cysteine residues or glycosylation sites affecting a crucial function or the formation of disulfide
10
bridges or conformational maturation, respectively. In the dishormonogenesis the thyroid gland
11
develops hypertrophy and hyperplasia by the proliferative effect as a consequence of the chronic
12
elevation of TSH. In contrast, the dysembryogenesis results agenesis or hypoplasia or ectopia.
13
Many of the mutations cause protein misfolding and accumulation in the ER, affecting the secretory
14
capacity of the thyroid cell. The retention of misfolded proteins alters the ER homeostasis and
15
activates the ERAD mechanism, responsible for their cytosolic degradation.
16
Despite all these remarkable advances numerous challenging aspects remain to be solved. The
17
recent generation of new potent approaches including new sequencing technology and the
18
generation of thyroid organoids provides additional tools that might be of great help in such studies.
19 20 21 22 23 24 25
31
1
Declaration of interest
2
The authors declare that there is no conflict of interest that could be perceived as prejudicing the
3
impartiality of the research reported.
4 5
Funding
6
This study was funded by grants from the FONCyT-ANPCyT-MINCyT (PICT 2014-1193 to CMR
7
and PICT 2015-1811 to HMT), CONICET (PIP 2015-11220150100499 to CMR) and Universidad
8
de Buenos Aires (UBACyT 2016-20020150100099BA to CMR).
9 10
Acknowledgements
11
C.M. Rivolta and H.M. Targovnik are established investigators of the Consejo Nacional de
12
Investigaciones Científicas y Técnicas (CONICET).
13
K.G. Scheps is a postdoctoral research fellow of the CONICET.
14
32
1
Legends of Figures
2
Figure 1. Biosynthesis of thyroid hormones and iodine recycling in the polarized follicular
3
thyroid cells. The major steps in iodide (I−) metabolism (uptake, efflux, organification, and
4
recycling) in the different compartments and thyroid proteins involved are shown. After I− uptake
5
at the basolateral membrane of thyrocytes by the Na+/I- symporter (NIS), it is transported at the
6
apical membrane into the follicular lumen through the pendrin, anoctamin-1 and SLC26A7
7
transporters. NIS is dependent on the sodium gradient created by the Na/K-ATPase and KCNQ1-
8
KCNE2 potassium channel is required for NIS-mediated thyroid cell uptake of I-. In the cell-colloid
9
interface, the I- is oxidized to iodinium (I+) and rapidly organified by incorporation into selected
10
tyrosyl residues of thyroglobulin (TG), which results in the formation of two intermediaries, 3-
11
monoiodotyrosine (MIT) and 3,5-diiodotyrosine (DIT). Subsecuently, iodotyrosine coupling results
12
in the synthesis of 3,5,3'-triiodothyronine (T3) and 3,5,3',5'-tretraiodothyronine (T4). The oxidizing,
13
iodination and coupling reactions are mediated by thyroid peroxidase (TPO) in presence of H2O2
14
generated by dual oxidase 1 and 2/dual oxidase maturation factor 1 and 2 (DUOX1 and
15
2/DUOXA1 and DUOXA2) complex. Iodinated TG is stored as colloid in the follicular lumen.
16
Once endocytosed into the thyrocytes, the TG is digested by lysosomal proteases, thus releasing
17
attached MIT, DIT, T4 and T3. T4 and T3 are then transported out of the follicular cells and into the
18
bloodstream, through the monocarboxylate transporter 8 (MCT8). Meanwhile, MITs and DITs are
19
deiodinated by the iodotyrosine deiodinase 1 (IYD-1), present at the apical plasma membrane and
20
in endocytic vesicles of the thyrocytes and the iodine atoms as I- are transported to the
21
intrathyroidal iodide pool. Simultaneously, free amino acids (aa) generated during proteolysis of Tg
22
are conducted to the intrathyroidal aa pool. The transcription of specific thyroid genes and thyroid
23
function is under the control of thyrotropin (TSH) via its protein G-coupled receptor (TSHR).
24
GDP: guanosine diphosphate.
33
1
Figure 2. Structural organization of the thyroglobulin homodimer. The repetitive units of
2
thyroglobulin (TG) TG type 1, TG type 2 and TG type 3 and the cholinesterase-like (ChEL)
3
domain, drawn to scale, are represented by boxes. N-terminal T4 (coupling of a donor DIT149 with
4
the acceptor DIT24) and N-terminal T3 (coupling of a MIT2766 at the antepenultimate residue of one
5
TG monomer with the antepenultimate DIT2766 in the opposite monomer) forming sites are shown.
6
The amino acid sequences (represented by single-letter code) of TG are indicated below of the
7
respective protein schematic diagrams. The mutated residues are shown in red and double
8
underlined (see Table 1 and 2). The amino acid position is designated according to the reference
9
sequences reported in NCBI accession number: NM_003235.5 The amino acid (aa) positions are
10
numbered including the 19 amino acid of the signal peptide (SP).
11 12
Figure 3. Schematic model of the N-glycosylation pathway of thyroglobulin. First, the nascent
13
polypeptide acquires the N-glycans by action of an oligosaccharyltransferase that transfers Glc3
14
(□), Man9 (○), GlcNAc2 (■) from dolichol phosphate precursor to the consensus N-glycosylation
15
sites (Asn, asparagine). Next, I and II α-glucosidases trim two glucose residues creating a
16
monoglucosylated N-glycan, later the third glucose is removed by α-glucosidase II. After removal
17
of one mannose residue in the ER, α-mannosidase I, the thyroglobulin is exported to the Golgi.
18
Finally, Golgi I and II α-mannosidases remove mannose residues and several N-acetylglucosamine
19
glycosyltransferases (GnT) may catalyze branching and elongation of the carbohydrate chains,
20
resulting in hybrid-type or complex-type N-glycans. N-glycans are elongated by addition of
21
galactose, fucose and sialic acid residues catalyzed by galactosyltransferases, fucosyltransferases
22
and sialyltransferases, respectively.
23 24
Figure 4. Structural organization of the thyroid peroxidase. The signal peptide (SP),
25
complement control protein (CCP) domain, calcium-binding EGF (EGF-Ca2+) domain, animal
34
1
haem peroxidase (An peroxidase) region and heme-binding sites (proximal histidine (H494), distal
2
histidine (H239), aspartic acid (D238), glutamic acid (E399), drawn to scale, are shown. The arrows
3
indicate where the mutated residues are located. The amino acid sequences (represented by single-
4
letter code) of thyroid peroxidase protein are indicated below of the respective protein schematic
5
diagrams. The mutated residues are shown in red and double underlined. The amino acid position is
6
designated according to the reference sequences reported in NCBI accession number:
7
NM_000547.5. The amino acid positions are numbered including the 18 amino acid of the signal
8
peptide (SP). ECD: extracellular domain, TM: transmembrane domain, ICD: intracellular domain,
9
aa: amino acid.
10 11
Figure 5. Structural organization of the DUOX2. The peroxidase-like and gp91phox/NOX2-like
12
domains, glycosilation sites (NXS/T site), EF-hand calcium binding motifs and FAD and NADPH
13
binding sites, drawn to scale, are shown. The seven alpha helice transmembrane domains are
14
represented by boxes (I, II, III, IV, V, VI and VII). The arrows indicate where the mutated residues
15
are located. The amino acid sequences (represented by single-letter code) of DUOX2 are indicated
16
below of the respective protein schematic diagrams. The mutated residues are shown in red and
17
double underlined. The amino acid position is designated according to the reference sequences
18
reported in NCBI accession number: NM_014080.4. The amino acid (aa) positions are numbered
19
including the 21 amino acid of the signal peptide (SP).
20 21
Figure 6. Folding model of DUOX2-DUOXA2. Formation of intermolecular disulfide bonds
22
between DUOX2 and DUOXA2 and trafficking of the DUOX2-DUOXA2 heterodimer to the
23
apical plasma membrane of the thyrocyte are shown. DUOX2 requires a DUOXA2 to exit from the
24
endoplasmic reticulum (ER) and reach the apical plasma membrane, through the Golgi apparatus.
25
The oxidative folding of DUOX2 and DUOXA2 takes place in the ER, generating an
35
1
intramolecular disulfide bridge in DUOX2 (between cysteine124 and cysteine1162) and
2
intermolecular disulfide bridges between the DUOX2 (cysteine568 and cysteine582) and DUOXA2
3
(cysteine167 and cysteine233). Both proteins form a stable heterodimer complex at the cell surface
4
which is fundamental for the reactive oxygen species production. N : N-glycosylation site,
5
cysteine, NADPH: binding site for the NADPH, FAD: binding site for the FAD.
:
6 7
Figure 7. Thirteen-TMS human Na+/I- symporter secondary structure model. Schematic
8
representation adapted from De la Vieja et al. [2004, 2005] and Spitzweg and Morris [2010] is
9
showed. The arrows indicate where the mutated residues are located. The amino acid sequences
10
(represented by single-letter code) of Na+/I- symporter are indicated below of the respective protein
11
schematic diagrams. The mutated residues are shown in red and double underlined.
12
glycosylation site, TM: transmembrane domain.
N : N-
13 14
Figure 8. Twelve-TMS human pendrin secondary structure model. Schematic representation
15
adapted from Dossena et al. [2009] is showed. The arrows indicate where the mutated residues are
16
located. The amino acid sequences (represented by single-letter code) of pendrin are indicated
17
below of the respective protein schematic diagrams. The mutated residues are shown in red and
18
double underlined. STAS (sulfate transporter and antisigma factor antagonist) domain is
19
underlined. N : N-glycosylation site, TM: transmembrane domain.
20 21
Figure 9. Twelve-TMS human SLC26A7 secondary structure model. Schematic representation
22
adapted from Ishii et al. [2019] is showed. The arrows indicate where the mutated residues are
23
located. The amino acid sequences (represented by single-letter code) of the SLC26A7 are
24
indicated below of the respective protein schematic diagrams. The mutated residues are shown in
36
1
red and double underlined. The STAS (sulfate transporter and antisigma factor antagonist) domain
2
is underlined. N : N-glycosylation site, TM: transmembrane domain.
3
37
1
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Table 1. Thyroglobulin missense mutations involved in the wild type cysteine residues Exon position
Nucleotide position
Amino acid position
References
Exon 5 Exon 5 Exon 5 Exon 10 Exon 14 Exon 17
c.479G>C c.548G>A c.580T>G c.2177G>A c.3229T>C c.3790T>C
p.C160S p.C183Y p.C194G p.C726Y p.C1077R p.C1264R
Exon 17 Exon 21 Exon 21 Exon 22 Exon 24 Exon 31 Exon 31 Exon 33
c.3842G>A c.4426T>C c.4478G>A c.4529G>T c.4820G>T c.5690G>A c.5986T>A
p.C1281Y p.C1476R p.C1493Y p.C1510F p.C1607F p.C1897Y p.C1904G p.C1996S
Exon 33 Exon 33 Exon 37
c.6000C>G c.6017G>A c.6461G>A
p.C2000W p.C2006Y p.C2154Y
Nicholas et al., 2016 Caputo et al., 2007 Hishinuma et al., 2006 Nicholas et al., 2016 Hishinuma et al., 2005; Hishinuma et al., 2006 Baryshev et al., 2004 ; Hishinuma et al., 1999, 2005, 2006; Kanou et al., 2007; Narumi et al., 2011 Citterio et al., 2013 Zou et al., 2018 Nicholas et al., 2016 Narumi et al., 2011 Hishinuma et al., 2006 Hishinuma et al., 2006; Kitanaka et al., 2006 Hu et al., 2016 Baryshev et al., 2004; Hishinuma et al., 1999, 2005, 2006 Citterio et al., 2013 Hishinuma et al., 2006 Hishinuma et al., 2006
The nucleotide position is designated according to TG mRNA reference sequences reported in NCBI Accession Number: NM_ 003235.5. The A of the ATG of the initiator methionine codon is denoted nucleotide +1. The amino acid positions are numbered including the 19 amino acid of the signal peptide.
Table 2. Thyroglobulin missense mutations in the ChEL domain Exon position
Nucleotide position
Amino acid position
References
Exon 38
c.6701C>A
p.A2234D
Exon 38
c.6725G>A
p.R2242H
Exon 39 Exon 40 Exon 40
c.6853A>G c.6956G>A c.7007G>A
p.N2285D p.G2319D p.R2336Q
Exon 40 Exon 41 Exon 41 Exon 41 Exon 41
c.7021G>A c.7084G>C c.7093T>C c.7121G>T c.7123G>A
p.G2341S p.A2362P p.W2365R p.G2374V p.G2375R
Exon 41 Exon 42
c.7182C>G c.7364G>A
p.I2394M p.R2455H
Exon 43 Exon 44 Exon 44
c.7411G>T c.7640T>A c.7753C>T
p.A2471S p.L2547Q p.R2585W
Exon 45
c.7847A>T
p.N2616I
Exon 47 Exon 47
c.8054G>T c.8137G>A
p.W2685L p.A2713T
Caputo et al., 2007; Citterio et al., 2013; Machiavelli et al., 2010; Pardo et al. 2008, 2009; Santos-Silva et al., 2019 Caron et al., 2003; Machiavelli et al., 2010; Raef et al., 2010; Zou et al., 2018 Makretskaya et al., 2018 Hishinuma et al., 2006 Hishinuma et al., 2006; Kitanaka et al., 2006; Siffo et al., 2018 Watanabe et al. 2018 Siffo et al., 2018 Siffo et al., 2018 Hishinuma et al., 2006 Hishinuma et al., 2005, 2006; Kanou et al.; 2007; Long et al., 2018 Sun et al., 2018 Hu et al., 2016; Long et al., 2018, Yu et al., 2018 Sun et al., 2018 Nicholas et al., 2016 Hu et al., 2016; Jiang et al., 2016; Sun et al., 2018 Hu et al., 2016; Long et al., 2018, Sun et al. 2018, Yu et al., 2018 Nicholas et al., 2016 Long et al., 2018
The nucleotide position is designated according to TG mRNA reference sequences reported in NCBI Accession Number: NM_ 003235.5. The A of the ATG of the initiator methionine codon is denoted nucleotide +1. The amino acid positions are numbered including the 19 amino acid of the signal peptide.
I-
TSH T3 T4 T T4 4 T4
TSHR Basolateral membrane
G protein
MCT8 T3 T
4
GDP
T4 T4 T4
T3 Tg
NIS IYD-1
KCNQ1-KCNE2 K+ channel
Thyroid transcription factors
T4
Na+/K+ ATPase
MIT
I-
DIT T4
Transcription
aa mRNA
T4
IYD-1
Traduction
Iodide recycled
I-
aa pool
TG
TG
Anoctamin-1
Pendrin TPO DUOX1/2
SLC26A7
Apical membrane
Folicular lumen
TG
I+
Figure 1
Iodination of tyrosines Coupling of iodotyrosines
Oxidation of
I-
IH2O2
DUOXA1/2
2130 2188 2211
1996
1893
1724
2736 2768 aa
Region IV
Region III
1511 1566 1603
1146 1211
1074
922
605 659 727
298
359
161
1 31 93
1456 1470 1487
Region II
Region I
ChEL domain Hinge
Linker
NH2
COOH
SP 1-1 1-2
1-3
1-4
1-5 1-6
1-7
1-8
1-9 1-10
TG type 1
2-1-2-3 1-11
3-a1
3-b1
3-a2
3-b2 3-a3
TG type 3
TG type 2
Spacer 3
Spacer 1
NH2
2736 2768
2130 2188 2211
1996
1893
1724
1511 1566 1603
1456 1470 1487
1146 1211
1074
922
605 659 727
298
359
161
1 31 93
Spacer 2
COOH
SP 1-1 1-2
1-3
1-4
1-5 1-6
1-7
1-8
SP
Figure 2
1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761
1-9 1-10
2-1-2-3 1-11
3-a1
3-b1
3-a2
3-b2 3-a3
T4
MALVLEIFTLLASICWVSANIFEYQVDAQPLRPCELQRETAFLKQADYVPQCAEDGSFQT VQCQNDGRSCWCVGANGSEVLGSRQPGRPVACLSFCQLQKQQILLSGYINSTDTSYLPQC QDSGDYAPVQCDVQQVQCWCVDAEGMEVYGTRQLGRPKRCPRSCEIRNRRLLHGVGDKSP PQCSAEGEFMPVQCKFVNTTDMMIFDLVHSYNRFPDAFVTFSSFQRRFPEVSGYCHCADS QGRELAETGLELLLDEIYDTIFAGLDLPSTFTETTLYRILQRRFLAVQSVISGRFRCPTK CEVERFTATSFGHPYVPSCRRNGDYQAVQCQTEGPCWCVDAQGKEMHGTRQQGEPPSCAE GQSCASERQQALSRLYFGTSGYFSQHDLFSSPEKRWASPRVARFATSCPPTIKELFVDSG LLRPMVEGQSQQFSVSENLLKEAIRAIFPSRGLARLALQFTTNPKRLQQNLFGGKFLVNV GQFNLSGALGTRGTFNFSQFFQQLGLASFLNGGRQEDLAKPLSVGLDSNSSTGTPEAAKK DGTMNKPTVGSFGFEINLQENQNALKFLASLLELPEFLLFLQHAISVPEDVARDLGDVME TVLSSQTCEQTPERLFVPSCTTEGSYEDVQCFSGECWCVNSWGKELPGSRVRGGQPRCPT DCEKQRARMQSLMGSQPAGSTLFVPACTSEGHFLPVQCFNSECYCVDAEGQAIPGTRSAI GKPKKCPTPCQLQSEQAFLRTVQALLSNSSMLPTLSDTYIPQCSTDGQWRQVQCNGPPEQ VFELYQRWEAQNKGQDLTPAKLLVKIMSYREAASGNFSLFIQSLYEAGQQDVFPVLSQYP SLQDVPLAALEGKRPQPRENILLEPYLFWQILNGQLSQYPGSYSDFSTPLAHFDLRNCWC VDEAGQELEGMRSEPSKLPTCPGSCEEAKLRVLQFIRETEEIVSASNSSRFPLGESFLVA KGIRLRNEDLGLPPLFPPREAFAEQFLRGSDYAIRLAAQSTLSFYQRRRFSPDDSAGASA LLRSGPYMPQCDAFGSWEPVQCHAGTGHCWCVDEKGGFIPGSLTARSLQIPQCPTTCEKS RTSGLLSSWKQARSQENPSPKDLFVPACLETGEYARLQASGAGTWCVDPASGEELRPGSS SSAQCPSLCNVLKSGVLSRRVSPGYVPACRAEDGGFSPVQCDQAQGSCWCVMDSGEEVPG TRVTGGQPACESPRCPLPFNASEVVGGTILCETISGPTGSAMQQCQLLCRQGSWSVFPPG PLICSLESGRWESQLPQPRACQRPQLWQTIQTQGHFQLQLPPGKMCSADYADLLQTFQVF ILDELTARGFCQIQVKTFGTLVSIPVCNNSSVQVGCLTRERLGVNVTWKSRLEDIPVASL PDLHDIERALVGKDLLGRFTDLIQSGSFQLHLDSKTFPAETIRFLQGDHFGTSPRTWFGC SEGFYQVLTSEASQDGLGCVKCPEGSYSQDEECIPCPVGFYQEQAGSLACVPCPVGRTTI SAGAFSQTHCVTDCQRNEAGLQCDQNGQYRASQKDRGSGKAFCVDGEGRRLPWWETEAPL EDSQCLMMQKFEKVPESKVIFDANAPVAVRSKVPDSEFPVMQCLTDCTEDEACSFFTVST TEPEISCDFYAWTSDNVACMTSDQKRDALGNSKATSFGSLRCQVKVRSHGQDSPAVYLKK GQGSTTTLQKRFEPTGFQNMLSGLYNPIVFSASGANLTDAHLFCLLACDRDLCCDGFVLT QVQGGAIICGLLSSPSVLLCNVKDWMDPSEAWANATCPGVTYDQESHQVILRLGDQEFIK SLTPLEGTQDTFTNFQQVYLWKDSDMGSRPESMGCRKDTVPRPASPTEAGLTTELFSPVD LNQVIVNGNQSLSSQKHWLFKHLFSAQQANLWCLSRCVQEHSFCQLAEITESASLYFTCT LYPEAQVCDDIMESNAQGCRLILPQMPKALFRKKVILEDKVKNFYTRLPFQKLMGISIRN KVPMSEKSISNGFFECERRCDADPCCTGFGFLNVSQLKGGEVTCLTLNSLGIQMCSEENG GAWRILDCGSPDIEVHTYPFGWYQKPIAQNNAPSFCPLVVLPSLTEKVSLDSWQSLALSS VVVDPSIRHFDVAHVSTAATSNFSAVRDLCLSECSQHEACLITTLQTQPGAVRCMFYADT QSCTHSLQGQNCRLLLREEATHIYRKPGISLLSYEASVPSVPISTHGRLLGRSQAIQVGT SWKQVDQFLGVPYAAPPLAERRFQAPEPLNWTGSWDASKPRASCWQPGTRTSTSPGVSED CLYLNVFIPQNVAPNASVLVFFHNTMDREESEGWPAIDGSFLAAVGNLIVVTASYRVGVF GFLSSGSGEVSGNWGLLDQVAALTWVQTHIRGFGGDPRRVSLAADRGGADVASIHLLTAR ATNSQLFRRAVLMGGSALSPAAVISHERAQQQAIALAKEVSCPMSSSQEVVSCLRQKPAN VLNDAQTKLLAVSGPFHYWGPVIDGHFLREPPARALKRSLWVEVDLLIGSSQDDGLINRA KAVKQFEESRGRTSSKTAFYQALQNSLGGEDSDARVEAAATWYYSLEHSTDDYASFSRAL ENATRDYFIICPIIDMASAWAKRARGNVFMYHAPENYGHGSLELLADVQFALGLPFYPAY EGQFSLEEKSLSLKIMQYFSHFIRSGNPNYPYEFSRKVPTFATPWPDFVPRAGGENYKEF SELLPNRQGLKKADCSFWSKYISSLKTSADGAKGGQSAESEEEELTAGSGLREDLLSLQE PGSKTYSK
T3
ChEL domain
Asn
GnT-II
NH2
NH2
α-mannosidase II
α-mannosidase I
NH2
SH
Asn
Asn
GnT-I
Complex-type
SH
SH
Asn
Asn
α-mannosidase I
NH2
Asn
α-glucosidase II
NH2
SH
SH
NH2
SH
Asn
Hybrid-type
NH2
High-mannose type
α-glucosidase II
GnT-IV
α-glucosidase I
GnT-IV
GnT-V GnT-VI
SH
Figure 3
Asn
Asn
NH2
NH2
NH2
Asn
SH
p.R665W p.D574-L575del
p.C800R
p.C838S
p.R175Q
933 aa
pH 494
dH 239
839 847 872
740
1 19
p.G771R
796
p.G533C
p.A397Pfs*76
p.S131P
NH2
COOH SP
D 238
E 399 ECD
An peroxidase
CCP EGF-Ca2+
TM
SP 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901
MRALAVLSVTLVMACTEAFFPFISRGKELLWGKPEESRVSSVLEESKRLVDTAMYATMQR NLKKRGILSPAQLLSFSKLPEPTSGVIARAAEIMETSIQAMKRKVNLKTQQSQHPTDALS EDLLSIIANMSGCLPYMLPPKCPNTCLANKYRPITGACNNRDHPRWGASNTALARWLPPV YEDGFSQPRGWNPGFLYNGFPLPPVREVTRHVIQVSNEVVTDDDRYSDLLMAWGQYIDHD IAFTPQSTSKAAFGGGADCQMTCENQNPCFPIQLPEEARPAAGTACLPFYRSSAACGTGD QGALFGNLSTANPRQQMNGLTSFLDASTVYGSSPALERQLRNWTSAEGLLRVHARLRDSG RAYLPFVPPRAPAACAPEPGIPGETRGPCFLAGDGRASEVPSLTALHTLWLREHNRLAAA LKALNAHWSADAVYQEARKVVGALHQIITLRDYIPRILGPEAFQQYVGPYEGYDSTANPT VSNVFSTAAFRFGHATIHPLVRRLDASFQEHPDLPGLWLHQAFFSPWTLLRGGGLDPLIR GLLARPAKLQVQDQLMNEELTERLFVLSNSSTLDLASINLQRGRDHGLPGYNEWREFCGL PRLETPADLSTAIASRSVADKILDLYKHPDNIDVWLGGLAENFLPRARTGPLFACLIGKQ MKALRDGDWFWWENSHVFTDAQRRELEKHSLSRVICDNTGLTRVPMDAFQVGKFPEDFES CDSITGMNLEAWRETFPQDDKCGFPESVENGDFVHCEESGRRVLVYSCRHGYELQGREQL TCTQEGWDFQPPLCKDVNECADGAHPPCHASARCRNTKGGFQCLCADPYELGDDGRTCVD SGRLPRVTWISMSLAALLIGGFAGLTSTVICRWTRTGTKSTLPISETGGGTPELRCGKHQ AVGTSPQRAAAQDSEQESAGMEGRDTHRLPRAL
Figure 4
ICD
SP
I
Peroxidase-like domain
EF-hand calcium biding motifs
heme
II III
1548 aa
1129 1186 1224 1245
855
899
1042 1077
heme
Ca2
Ca2
NH2
Ca Ca22
819
p.C568R 602
NXS/T site 537
NXS/T site 455
NXS/T site 100
1 22
p.Q36H
p.D506N p.K530* p.R1110Q p.L1160del p.R1334W p.F966Sfs*29 p.R376W p.L1343F p.E879K NXS/T site 348 NXS/T site 382
p.C124*
COOH
FAD NADPH IV V VI VII binding binding site site
Alpha helice transmembrane domain gp91phox/NOX2-like domain
SP 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501
MLRARPEALMLLGALLTGSLGPSGNQDALSLPWEVQRYDGWFNNLRHHERGAVGCRLQRR VPANYADGVYQALEEPQLPNPRRLSNAATRGIAGLPSLHNRTVLGVFFGYHVLSDVVSVE TPGCPAEFLNIRIPPGDPVFDPDQRGDVVLPFQRSRWDPETGRSPSNPRDLANQVTGWLD GSAIYGSSHSWSDALRSFSGGQLASGPDPAFPRDSQNPLLMWAAPDPATGQNGPRGLYAF GAERGNREPFLQALGLLWFRYHNLWAQRLARQHPDWEDEELFQHARKRVIATYQNIAVYE WLPSFLQKTLPEYTGYRPFLDPSISPEFVVASEQFFSTMVPPGVYMRNASCHFRKVLNKG FQSSQALRVCNNYWIRENPNLNSTQEVNELLLGMASQISELEDNIVVEDLRDYWPGPGKF SRTDYVASSIQRGRDMGLPSYSQALLAFGLDIPRNWSDLNPNVDPQVLEATAALYNQDLS QLELLLGGLLESHGDPGPLFSAIVLDQFVRLRDGDRYWFENTRNGLFSKKEIEDIRNTTL RDVLVAVINIDPSALQPNVFVWHKGAPCPQPKQLTTDGLPQCAPLTVLDFFEGSSPGFAI TIIALCCLPLVSLLLSGVVAYFRGREHKKLQKKLKESVKKEAAKDGVPAMEWPGPKERSS PIIIQLLSDRCLQVLNRHLTVLRVVQLQPLQQVNLILSNNRGCRTLLLKIPKEYDLVLLF SSEEERGAFVQQLWDFCVRWALGLHVAEMSEKELFRKAVTKQQRERILEIFFRHLFAQVL DINQADAGTLPLDSSQKVREALTCELSRAEFAESLGLKPQDMFVESMFSLADKDGNGYLS FREFLDILVVFMKGSPEDKSRLMFTMYDLDENGFLSKDEFFTMMRSFIEISNNCLSKAQL AEVVESMFRESGFQDKEELTWEDFHFMLRDHDSELRFTQLCVKGGGGGGNGIRDIFKQNI SCRVSFITRTPGERSHPQGLGPPAPEAPELGGPGLKKRFGKKAAVPTPRLYTEALQEKMQ RGFLAQKLQQYKRFVENYRRHIVCVAIFSAICVGVFADRAYYYGFASPPSDIAQTTLVGI ILSRGTAASVSFMFSYILLTMCRNLITFLRETFLNRYVPFDAAVDFHRWIAMAAVVLAIL HSAGHAVNVYIFSVSPLSLLACIFPNVFVNDGSKLPQKFYWWFFQTVPGMTGVLLLLVLA IMYVFASHHFRRRSFRGFWLTHHLYILLYALLIIHGSYALIQLPTFHIYFLVPAIIYGGD KLVSLSRKKVEISVVKAELLPSGVTYLQFQRPQGFEYKSGQWVRIACLALGTTEYHPFTL TSAPHEDTLSLHIRAVGPWTTRLREIYSSPKGNGCAGYPKLYLDGPFGEGHQEWHKFEVS VLVGGGIGVTPFASILKDLVFKSSLGSQMLCKKIYFIWVTRTQRQFEWLADIIQEVEEND HQDLVSVHIYVTQLAEKFDLRTTMLYICERHFQKVLNRSLFTGLRSITHFGRPPFEPFFN SLQEVHPQVRKIGVFSCGPPGMTKNVEKACQLVNRQDRAHFMHHYENF
Figure 5
N
Disulfide bridge
.
2 O 2-
2 O2
N
N
N
N
NH2
N NH2
Apical membrane
N
H 2 O2
N
Colloid
Cytosol COOH
COOH
DUOXA2/ DUOX2
NADP+ + H +
NADPH
N
N
NH2
Disulfide bridge
N
N
N
N
NH2
N N
Golgi Apparatus
DUOXA2/ DUOX2 COOH
COOH
N
N
NH2
Disulfide bridge
N
N
N
N
NH2
N N
Endoplasmic Reticulum COOH
COOH
N
N
NH2
N
N
N
NH2
N
N
N
DUOXA2
Figure 6
COOH
DUOX2
COOH
N N Extracellular N
NH2 1
p.S509Rfs*7 11
TM1
79
TM2
86
TM3
157
163
TM4
TM5
217
TM6
241
TM7
308
340
TM8
TM9
411
417
468
522
TM10 TM11 TM12
TM13
p.G543E 37
54
111
136
182
191
260
286
368
388
438
p.V270E
444 550
intracellular p.A439_P443del
p.R124H
COOH 643
1 61 121 181 241 301 361 421 481 541 601
MEAVETGERPTFGAWDYGVFALMLLVSTGIGLWVGLARGGQRSAEDFFTGGRRLAALPVG LSLSASFMSAVQVLGVPSEAYRYGLKFLWMCLGQLLNSVLTALLFMPVFYRLGLTSTYEY LEMRFSRAVRLCGTLQYIVATMLYTGIVIYAPALILNQVTGLDIWASLLSTGIICTFYTA VGGMKAVVWTDVFQVVVMLSGFWVVLARGVMLVGGPRQVLTLAQNHSRINLMDFNPDPRS RYTFWTFVVGGTLVWLSMYGVNQAQVQRYVACRTEKQAKLALLINQVGLFLIVSSAACCG IVMFVFYTDCDPLLLGRISAPDQYMPLLVLDIFEDLPGVPGLFLACAYSGTLSTASTSIN AMAAVTVEDLIKPRLRSLAPRKLVIISKGLSLIYGSACLTVAALSSLLGGGVLQGSFTVM GVISGPLLGAFILGMFLPACNTPGVLAGLGAGLALSLWVALGATLYPPSEQTMRVLPSSA ARCVALSVNASGLLDPALLPANDSSRAPSSGMDASRPALADSFYAISYLYYGALGTLTTV LCGALISCLTGPTKRSTLAPGLLWWDLARQTASVAPKEEVAILDDNLVKGPEELPTGNKK PPGFLPTNEDRLFFLGQKELEGAGSWTPCVGHDGGRDQQETNL
Figure 7
p.L236P p.T410M
p.V138F
N
N Extracellular
107
113 154
185
234
268
318
348
408
421
466
p.G102R TM1 87
TM2 131
TM3 136
TM4 207
TM5 213
TM6 286
TM7 295
TM8 368
TM9 384
478
TM10 TM11 TM12 443
448
504
intracellular 534
p.Y556C 1
NH2
p.T193I
p.L445W p.Q446R
STAS domain COOH 780
1 61 121 181 241 301 361 421 481 541 601 661 721
730
MAAPGGRSEPPQLPEYSCSYMVSRPVYSELAFQQQHERRLQERKTLRESLAKCCSCSRKR AFGVLKTLVPILEWLPKYRVKEWLLSDVISGVSTGLVATLQGMAYALLAAVPVGYGLYSA FFPILTYFIFGTSRHISVGPFPVVSLMVGSVVLSMAPDEHFLVSSSNGTVLNTTMIDTAA RDTARVLIASALTLLVGIIQLIFGGLQIGFIVRYLADPLVGGFTTAAAFQVLVSQLKIVL NVSTKNYNGVLSIIYTLVEIFQNIGDTNLADFTAGLLTIVVCMAVKELNDRFRHKIPVPI PIEVIVTIIATAISYGANLEKNYNAGIVKSIPRGFLPPELPPVSLFSEMLAASFSIAVVA YAIAVSVGKVYATKYDYTIDGNQEFIAFGISNIFSGFFSCFVATTALSRTAVQESTGGKT QVAGIISAAIVMIAILALGKLLEPLQKSVLAAVVIANLKGMFMQLCDIPRLWRQNKIDAV IWVFTCIVSIILGLDLGLLAGLIFGLLTVVLRVQFPSWNGLGSIPSTDIYKSTKNYKNIE EPQGVKILRFSSPIFYGNVDGFKKCIKSTVGFDAIRVYNKRLKALRKIQKLIKSGQLRAT KNGIISDAVSTNNAFEPDEDIEDLEELDIPTKEIEIQVDWNSELPVKVNVPKVPIHSLVL DCGAISFLDVVGVRSLRVIVKEFQRIDVNVYFASLQDYVIEKLEQCGFFDDNIRKDTFFL TVHDAILYLQNQVKSQEGQGSILETITLIQDCKDTLELIETELTEEELDVQDEAMRTLAS
Figure 8
p.I309_E311delinsM p.R227*
N 66
71 121
TM1
TM2
46
91
TM3 101
Extracellular
N 142
TM4 162
198
223
TM5 178
TM6 243
279
TM7 259
307
TM8 327
363
TM9 343
375
427
448
TM10 TM11 TM12 395
407
468
intracellular 1
NH2
p.Q500*
495
STAS domain
p.F631Lfs*8
631 656
1 61 121 181 241 301 361 421 481 541 601
COOH
MTGAKRKKKSMLWSKMHTPQCEDIIQWCRRRLPILDWAPHYNLKENLLPDTVSGIMLAVQ QVTQGLAFAVLSSVHPVFGLYGSLFPAIIYAIFGMGHHVATGTFALTSLISANAVERIVP QNMQNLTTQSNTSVLGLSDFEMQRIHVAAAVSFLGGVIQVAMFVLQLGSATFVVTEPVIS AMTTGAATHVVTSQVKYLLGMKMPYISGPLGFFYIYAYVFENIKSVRLEALLLSLLSIVV LVLVKELNEQFKRKIKVVLPVDLVLIIAASFACYCTNMENTYGLEVVGHIPQGIPSPRAP PMNILSAVITEAFGVALVGYVASLALAQGSAKKFKYSIDDNQEFLAHGLSNIVSSFFFCI PSAAAMGRTAGLYSTGAKTQVACLISCIFVLIVIYAIGPLLYWLPMCVLASIIVVGLKGM LIQFRDLKKYWNVDKIDWGIWVSTYVFTICFAANVGLLFGVVCTIAIVIGRFPRAMTVSI KNMKEMEFKVKTEMDSETLQQVKIISINNPLVFLNAKKFYTDLMNMIQKENACNQPLDDI SKCEQNTLLNSLSNGNCNEEASQSCPNEKCYLILDCSGFTFFDYSGVSMLVEVYMDCKGR SVDVLLAHCTASLIKAMTYYGNLDSEKPIFFESVSAAISHIHSNKNLSKLSDHSEV
Figure 9
Highlights
• Congenital hypothyroidism is the most frequent endocrine disease in children. • Mutations in the candidate genes were identified and functionally characterized. • Protein folding disorders are caused by mutations in thyroid genes. • Misfolded of thyroid proteins causes retention within the endoplasmic reticulum. • The retention of misfolded proteins activates the ERAD mechanism.
S0303-7207(19)30340-5
DOI:
https://doi.org/10.1016/j.mce.2019.110638
Reference:
MCE 110638
To appear in:
Molecular and Cellular Endocrinology
Received Date: 29 July 2019 Revised Date:
21 October 2019
Accepted Date: 1 November 2019
Please cite this article as: Targovnik, Hé.M., Scheps, K.G., Rivolta, C.M., Defects in protein folding in congenital hypothyroidism, Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/ j.mce.2019.110638. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Defects in protein folding in congenital hypothyroidism.
1 2 3
Héctor M. Targovnik1,2 *, Karen G. Scheps1,2, Carina M. Rivolta1,2
4 5 1
6
Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de
7
Microbiología, Inmunología, Biotecnología y Genética/Cátedra de Genética. Buenos Aires,
8
Argentina. 2
9 10
CONICET-Universidad de Buenos Aires. Instituto de Inmunología, Genética y Metabolismo (INIGEM). Buenos Aires, Argentina.
11 12
Short title: Protein folding in congenital hypothyroidism
13
Keywords: Thyroid genes, Protein folding, ERAD, Congenital hypothyroidism.
14
Word count of the full article:
15 16
*Address correspondence and requests for reprints to:
17
Dr. Héctor M. Targovnik
18
CONICET-Universidad de Buenos Aires. Instituto de Inmunología, Genética y Metabolismo
19
(INIGEM). Hospital de Clínicas “José de San Martín”, Av. Córdoba 2351, Cuarto Piso, Sala
20
5, C1120AAR - Buenos Aires, Argentina.
21
Tel.: 54-11-5950-8805 E-mail: [email protected], [email protected]
22
2
1
Abstract
2
Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and
3
one of the most common preventable causes of both cognitive and motor deficits. CH is a
4
heterogeneous group of thyroid disorders in which inadequate production of thyroid hormone
5
occurs due to defects in proteins involved in the gland organogenesis (dysembryogenesis) or in
6
multiple steps of thyroid hormone biosynthesis (dyshormonogenesis). Dysembryogenesis is
7
associated with genes responsible for the development or growth of thyroid cells: such as NKX2-1,
8
FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3 resulting in agenesis,
9
hypoplasia or ectopia of thyroid gland. Nevertheless, the etiology of the dysembryogenesis remains
10
unknown for most cases. In contrast, the majority of patients with dyshormonogenesis has been
11
linked to mutations in the SLC5A5, SLC26A4, SLC26A7, TPO, DUOX1, DUOX2, DUOXA1,
12
DUOXA2, IYD or TG genes, which usually originate goiter.
13
About 800 genetic mutations have been reported to cause CH in patients so far, including
14
missense, nonsense, in-frame deletion and splice-site variations. Many of these mutations are
15
implicated in specific domains, cysteine residues or glycosylation sites, affecting the maturation of
16
nascent proteins that go through the secretory pathway. Consequently, misfolded proteins are
17
permanently entrapped in the endoplasmic reticulum (ER) and are translocated to the cytosol for
18
proteasomal degradation by the ER- associated degradation (ERAD) machinery.
19
Despite of all these remarkable advances in the field of the CH pathogenesis, several points on
20
the development of this disease remain to be elucidated. The continuous study of thyroid gene
21
mutations with the application of new technologies will be useful for the understanding of the
22
intrinsic mechanisms related to CH. In this review we summarize the present status of knowledge
23
on the disorders in the protein folding caused by thyroid genes mutations.
24 25 26
3
1
1. Introduction
2
The thyroid hormone (TH) is essential for the growth and neural maturation, as well as
3
cardiovascular and renal functions. This hormone also regulates the lipid, carbohydrate and protein
4
metabolism [Mullur et al., 2014]. The thyroid produces predominantly the prohormone 3,5,3´,5´-
5
tetraiodothyronine (T4) and minority the 3,5,3´-triiodothyronine (T3), T3 is obtained by deiodination
6
of the outer phenolic ring of the T4 by means of type 1 iodothyronine deiodinases (D1) and type 2
7
iodothyronine deiodinases (D2) [Mullur et al., 2014]. TH action is mainly mediated by binding of
8
T3 to its nuclear receptor, a member of the superfamily of hormone-responsive nuclear transcription
9
factors [Cheng et al., 2010; Samuels et al., 1974].
10
The biosynthesis of TH takes place at the interface of the apical cell plasma membrane and the
11
colloid in polarized follicular thyroid cells and requires the presence of iodide, thyroid peroxidase
12
(TPO), a supply of hydrogen peroxide, an iodine acceptor protein, thyroglobulin (TG), proteolytic
13
enzymes and the rescue and recycling of iodide by action of iodotyrosine deiodinase-1 (IYD-1)
14
(Figure 1) [Degroot and Niepomniszcze, 1977; Gnidehou et al., 2004; Targovnik et al. 2010, 2011].
15
After the iodide is uptake in the basolateral membrane by the Na+/I- symporter (NIS) [Bizhanova
16
and Kopp, 2009; Dohan et al., 2003], it is transported at the apical membrane into the follicular
17
lumen by an anion exchange transmembrane protein called pendrin (Figure 1) [Bizhanova and
18
Kopp, 2009; Royaux et al., 2000; Yoshida et al., 2002]. Na+/I- symporter (NIS) cotransports one
19
iodide ion with two sodium ions against an electrochemical gradient. This sodium gradient is
20
generated by Na+/K+-ATPase. Roepke et al. [2009] and Purtell et al. [2012] demonstrated that
21
KCNQ1-KCNE2 potassium channel is required for NIS-mediated thyroid cell uptake of I- (Figure
22
1). It was later described that the Ca2+-activated chloride channel TMEM16A or anoctamin-1
23
(encoded by ANO1 gene) [Siveira and Kopp, 2015; Twyffels et al. 2014] and SLC26A7 also
24
mediate the iodide efflux through the apical membrane of thyroid cells [Ishii et al., 2019]. The
25
oxidized iodide is incorporated into selected tyrosyl residues of TG which results in formation of
26
monoiodotyrosines (MID) and diiodotyrosines (DIT). Coupling between either two DIT, or between
4
1
a DIT and a MIT, generates the formation of T4 or T3 respectively (Figure 1). The oxidition of I−
2
into I+, iodination and coupling reactions are mediated by TPO in presence of H2O2 [Degroot and
3
Niepomniszcze, 1977]. Hydrogen peroxide is generated by a metabolic pathway, involving two
4
members of the NADPH oxidase family: dual oxidase 1 (DUOX1) and dual oxidase 2 (DUOX2)
5
and two ER-resident proteins: DUOX maturation factor 1 (DUOXA1) and DUOX maturation factor
6
2 (DUOXA2) [De Deken et al., 2000, 2002; De Deken and Miot, 2019; Dupuy et al, 1999;
7
Grasberger and Refetoff, 2006]. TPO and the DUOX system are in close proximity, forming a
8
complex at the level of the apical plasma membrane of the follicular cell (Figure 1) [Song et al.,
9
2010].
10
The thyroid cells produce free T4 and T3 by endocytic internalization and proteolytic lysosomal
11
cleavage (cathepsin proteases and glutamate carboxypeptidase) of the TG, which are delivered to
12
the blood circulation for action at their peripheral target tissues [Jordans et al., 2009; Linke et al.,
13
2002; Suban et al., 2012]. The monocarboxylate transporter 8 (MCT8, encoded by SLC16A2 gene)
14
facilitates the efflux of TH from the thyrocytes (Figure 1) [Di Cosmo et al., 2010]. From TG, free
15
MIT and DIT are also released by the thyroid cells. Therefore, the rescue and recycling into thyroid
16
cell of iodide from MIT to DIT by the action of IYD-1 are essential to prevent loss of this rare
17
element, especially in iodine-deficient areas.
18
The thyroid stimulating hormone (TSH) and its receptor (TSHR), a member of the G- protein-
19
coupled receptor superfamily (GPCRs), constitute the main regulatory pathway of the thyroid
20
(Figure 1) [Vassart and Dumont, 1992]. The cAMP and Gq/phospholipase C cascade pathways
21
mediate most of its effects, including iodide uptake, expression of thyroid genes, biosynthesis of
22
TH, TPO activity, thyroid H2O2 generating system, endocytosis, proteolysis and hormone release.
23
[Vassart and Dumont, 1992].
24
In order to fulfill their functions, the specialized thyroid proteins described above must be
25
exported to the colloid or located at the basolateral and apical membranes through a series of way-
5
1
stations. Exported proteins enter at the endoplasmic reticulum (ER), the first station of this pathway.
2
The proper folding and assembly of new synthesized exportable proteins occur within the ER
3
compartment. Then, secretory proteins are translocated into the Golgi complex by carrier vesicles
4
and finally, they are exported to their final destination. Exportable protein folding is enhanced by
5
the actions of compartment-specific molecular chaperones and enzymes. Remarkably, unfolding or
6
misfolding of exportable proteins cause retention within the ER, induction of the synthesis of ER
7
chaperones and removal of misfolded proteins from the ER lumen by an anterograde traffic out of
8
the ER to be catabolized in the proteasomes, a phenomenon known as ER-associated degradation
9
(ERAD) mechanism. ERAD prevents the accumulation or secretion of the abnormal protein [Oi et
10
al., 2017].
11
Here, we review the latest contributions in the field of protein folding disorders in congenital
12
hypothyroidism caused by mutations in the main proteins that participate in the biosynthesis of TH
13
and in thyroid embryogenesis. We also provide a large references list of original publications.
14 15
2. Congenital hypothyroidism
16
Congenital hypothyroidism (CH) is the most frequent endocrine disease in infants, affects about
17
1 in 2,000-3000 newborns and is characterized by elevated levels of thyroid-stimulating hormone
18
(TSH) and low TH serum levels [Kwak, 2018; Park and Chatterjee, 2005; Rastogi and LaFranchi,
19
2010]. It is also one of the most common preventable causes of cognitive and motor deficits [Kwak,
20
2018, Park and Chatterjee, 2005; Rastogi and LaFranchi, 2010]. The genetic classification divides
21
CH into two main categories according to their causes: (a) disorders of thyroid gland development
22
(dysembriogenesis or thyroid dysgenesis group) or (b) defects in any of the steps of thyroid
23
hormone synthesis (dyshormonogenesis group) [Park and Chatterjee, 2005; Rastogi and LaFranchi,
24
2010; Targovnik et al. 2010, 2011, 2017]. The dysembryogenesis or thyroid dysgenesis group,
25
which accounts for the 80-85 % of the cases, results from a thyroid gland that is completely absent
6
1
in orthotopic or ectopic location (agenesis or athyreosis), severely reduced in size but in the proper
2
position in the neck (orthotopic hypoplasia) or located in an unusual position (thyroid ectopy) at the
3
base of the tongue or along the thyroglossal tract [Abu-Khudir et al., 2017; Targovnik et al. 2010,
4
2011, 2017]. In few patients, the CH has been associated with mutations in genes responsible for
5
the development or growth of thyroid cells: NKX2-1 (also known as TTF1), FOXE1 (also known as
6
TTF2 or FKHL15), PAX8 and NKX2-5 genes [Grasberger and Refetoff, 2011; Kwak, 2018;
7
Targovnik et al. 2010, 2011, 2017]. Recently, exome analysis identified DUOX2 mutations in
8
patients with ectopic thyroid suggesting that DUOX2 could also be involved in thyroid
9
embryogenesis [Kizys et al., 2017]. More recently, Zou et al. [2018] identified mutations in the
10
TBX1, CDCA8, HOXD3 and HOXB3 genes associated with dysgenesis. Loss-of-function mutations
11
in the TSHR gene have been shown to cause thyroid hypoplasia and TSH resistance in humans
12
[Grasberger and Refetoff, 2017]. In the majority of patients with a defect in thyroid organogenesis,
13
the genetic mechanism responsible remains to be elucidated.
14
Dyshormonogenesis, which accounts for the remaining 15-20% of the cases, has been linked to
15
mutations in the SLC5A5 [Santos-Silva et al., 2019; Spitzweg and Morris, 2010; Targovnik et al.,
16
2017], SLC26A4 [Bizhanova and Kopp, 2010; Chem et al., 2018; Dossena et al., 2009, 2011;
17
Makretskaya et al., 2018; Sun et al., 2018; Wémeau and Kopp, 2017, Zou et al., 2018], SLC26A7
18
[Cangul, et al., 2018; Ishii et al., 2019; Zou et al., 2018], TPO [Long et al., 2018; Makretskaya et
19
al., 2018; Ris-Stalpers and Bikker, 2010; Santos-Silva et al., 2019; Sun et al., 2018; Targovnik et
20
al., 2017; Zou et al., 2018], DUOX1 [Aycan, et al., 2017; Liu et al., 2019; Watanabe et al., 2019;
21
Zou et al., 2018], DUOX2 [Belforte et al., 2016a, 2016b; Chem et al., 2018; Grasberger, 2010; Long
22
et al., 2018; Makretskaya et al., 2018; Muzza and Fugazzola, 2017; Sun et al., 2018; Yu et al., 2018;
23
Zou et al., 2018], DUOXA1 [Liu et al., 2019], DUOXA2 [Chem et al., 2018; Muzza and Fugazzola,
24
2017 Sun et al., 2018], IYD [Moreno and Visser, 2010; Sun et al., 2018; Targovnik et al., 2017] and
25
TG [Chem et al., 2018; Citterio et al., 2019; Di Jeso and Arvan, 2016; Long et al., 2018;
7
1
Makretskaya et al., 2018; Santos-Silva et al., 2019; Sun et al., 2018; Targovnik et al., 2010, 2011,
2
2016, 2017; Yu et al., 2018; Zou et al., 2018] genes. In the last three decades about eight hundred
3
variants have been described in unrelated CH patients. These mutations produce a heterogeneous
4
spectrum of congenital hypothyroidism, characterized usually by the presence of congenital goiter
5
or goiter appearing shortly after birth with an autosomal recessive inheritance. Therefore, the
6
patients are typically homozygous or compound heterozygous for the gene mutations and the
7
parents, carriers of one mutation.
8
Iodide organification defects (IOD) are linked with inactivating mutations in the TPO, DUOX2,
9
DUOXA2 or SLC26A4 genes and probably associated with mutations in the DUOX1 and DUOXA1
10
genes. IOD is characterized by high levels of serum TG and a positive Perchlorate Discharge Test
11
(PDT) [El-Desouki et al., 1995], indicating that the iodide is taken up by thyroid cells but it is not
12
incorporated into the TG when the oxidation of iodine, iodination and coupling mechanisms fail.
13
PDT is used to distinguish Total Iodide Organification Defect (TIOD) from Partial Iodide
14
Organification Defect (PIOD, between 10 and 90%) [Kwak. 2018]. In patients with PIOD usually a
15
single allele is affected, whereas homozygous or compound-heterozygous mutations are associated
16
with TIOD. Most cases of CH associated with alterations in DUOX2 are caused by either biallelic or
17
monoallelic mutations which lead to extremely complex correlation between DUOX2 genotypes
18
and clinical phenotypes [De Marco et al., 2011; Moreno and Visser, 2007; Wang et al., 2014]. Both
19
biallelic and monoallelic DUOX2 mutations could be associated with transient CH (TCH) or
20
permanent CH (PCH) [Fu et al., 2016a; Tan et al., 2016]. Mutations in SLC26A4 gene cause
21
Pendred syndrome characterized by congenital sensorineural hearing loss, goiter with or without
22
hypothyroidism and usually PIOD [Bizhanova and Kopp, 2010; Wémeau and Kopp, 2017]. In
23
patients with IYD-1 deficiency, the organification process is not affected whereas the serum TG
24
levels are elevated [Moreno and Visser, 2010; Targovnik et al., 2017]. In contrast, the presence of
25
low or undetectable levels of TG and also negative PDT in a goitrous individual suggest a TG
8
1
defect [Citterio et al., 2019; Di Jeso and Arvan, 2016; Targovnik et al., 2010, 2011, 2017]. Patients
2
with an iodide transport defect by mutations in SLC5A5 gene have a normal-sized or somewhat
3
enlarged thyroid gland, elevated plasma TG levels and no radio-iodide uptake [Spitzweg and
4
Morris, 2010; Targovnik et al., 2017].
5
Recent technological advances in DNA sequencing through the implementation of next-
6
generation sequencing (NGS) platforms that allow the massive identification of sequence variants
7
have led to the identification of new mutations in the thyroid genes. Interestingly, Fu et al. [2016a,
8
2016b, 2016c] using NGS detected that most of the cases of CH with one or two DUOX2 gene
9
mutations are associated with subclinical or TCH, whereas patients with three or four (one in
10
homozygous) or five (two in homozygous) DUOX2 gene mutations are mostly associated with
11
PCH. The new technologies also showed the coexistence of multiple mutations in different thyroid
12
genes in the same patient with CH, for instances, mutations in DUOX2 associated with mutations in
13
DUOXA2 or TPO or TG or TSHR or SLC26A4 genes [Fu et al., 2016a, 2016b; Jiang 2016; Park et
14
al., 2016; Sun et al., 2018]. The identification of simultaneous mutations in the same gene or in
15
different thyroid specific genes could contribute to the accurate diagnosis and classification of the
16
defects.
17 18
3. Defects in protein folding of thyroglobulin
19
TG represents a highly specialized homodimeric glycoprotein which acts as a substrate for the
20
synthesis of T4 and T3 as well as the storage of the inactive forms of iodine. [Citterio et al., 2019;
21
Targovnik 2013; Targovnik et al., 2017]. Human TG gene is a single copy of 268 Kb long that maps
22
on chromosome 8 (8:132,866,958-133,134,903; GRCh38 assembly) and contains an 8,455 nt of
23
mRNA sequence (with a coding sequences of 8304, NCBI Accesion Number: NM_003235.5)
24
divided into 48 exons [Malthiéry and Lissitzky, 1987; Mendive et al., 2001; Mercken et al., 1985;
25
van de Graaf et al., 2001].
9
1
The monomeric human TG preprotein has a leader peptide of 19 amino acids followed by a
2
2749-amino-acid polypeptide (Figure 2). The most important T4-forming site couples donor DIT149
3
to acceptor DIT24 [Dunn et al., 1998; Lamas et al., 1989; Palumbo et al., 1990]; whereas the main
4
T3-forming site couples an MIT2766 at the antepenultimate residue of one TG monomer with the
5
antepenultimate DIT2766 in the opposite monomer of the TG dimer [Citterio et al., 2017].
6
The internal protein organization makes TG an example of gene evolution by intragenic
7
duplication events and gene fusions [Parma et al., 1987]. The N-terminal and the central parts of the
8
monomer include three types of repetitive motifs, called TG type 1, TG type 2, and TG type 3,
9
comprising cysteine-rich repeats covalently bound by disulfide bonds (Figure 2). The TG type 1
10
motif derives from an ancestral gene that was duplicated and modified during evolution by
11
mutations [Holzer et al., 2016; Malthiery and Lissitzky, 1987; Mercken et al., 1985; Molina et al.,
12
1996, Parma et al., 1987, van de Graaf et al., 2001] whereas TG type 2 repeats are distant members
13
of the GCC2/GCC3 domain superfamily [Lee et al, 2011] and TG type 3 repeats exhibit only
14
internal homology. Tg monomer contains eleven TG type 1 elements located between positions 31 -
15
358, 605 - 1210 and between 1511 - 1565; three TG type 2 elements located between amino acids
16
1456 and 1503, and five TG type 3 elements between residues 1603 and 2187 (Figure 2) [Holzer et
17
al., 2016; Malthiery and Lissitzky, 1987; Mercken et al., 1985; Molina et al., 1996, Parma et al.,
18
1987, van de Graaf et al., 2001]. TG is organized in four structural regions (I, II, III and IV) (Figure
19
2). Region I comprises 10 of the 11 TG type 1 repeats, two segments called linker (residues 359 to
20
604) and hinge (residues 1211 to 1455) plus the N-terminal T4 forming site. Region II contains 3
21
TG type 2 repeats and the eleventh TG type 1 repeat, whereas region III contains the five TG type 3
22
repeats. The fourth region is composed of the cholinesterase-like (ChEL) domain, a member of the
23
α/β-hydrolase fold family, located between residues 2211 to 2735 of the TG [Park and Arvan, 2004;
24
Swillens et al., 1986] and the C-terminal T3 forming site. The transitions between repetitive motifs
25
TG type 2-3 and TG type 1-11, between TG type 1-11 and TG type 3-a1, and between TG type 3-a3
10
1
and the ChEL domain correspond to non-repetitive sequences that could be called spacer 1 (residues
2
1504 to 1510), spacer 2 (1566 to 1602) and spacer 3 (2188 to 2210) segments, respectively (Figure
3
2).
4
During translation/translocation in the lumen of the ER, the newly synthesised TG starts to fold
5
and acquires its final three-dimensional glycoprotein structure as it passes through the Golgi
6
complex. TG is transferred to the follicle lumen as a noncovalent dimer, concentrated into secretory
7
vesicles and stored as colloid. Intensive post translational modifications take place in the ER, which
8
include glycosylation and formation of intrachain disulfide bonds.
9
All glycoproteins go through similar trimming in the ER and acquire their final glycoprotein
10
structure as they pass through the Golgi [Bedard et al, 2004]. The addition of carbohydrates
11
stabilizes the protein and increases its solubility. TG acquires the N-glycans during the process of
12
translocation and elongation of the polypeptide chain into the ER. First, two N-acetylglucosamines
13
(GlcNAc) and nine mannoses with three terminal glucose residues are assembled onto a core
14
oligosaccharide, which is then en bloc transferred to the consensus N-glycosylation sites
15
(Asparagine-X-Serine/Threonine, where X is any amino acid residue except proline) on nascent TG
16
chains by action of an oligosaccharyltransferase (Figure 3). Next, the three terminal glucoses of this
17
core are trimmed by sequential action of I and II α-glucosidases. Finally, a terminal mannose is
18
trimmed by α-mannosidase I with the formation of high-mannose type oligosaccharide [Helenius
19
and Aebi, 2004] (Figure 3).
20
Correct folding is assisted by interaction with chaperones and enzymes of the ER [Bedard et al.,
21
2005], such as calnexin (CNX), calreticulin (CRT,), GRP94, BiP, Protein Disulfide Isomerase
22
(PDI), ERp29, ERp57, ERp72 [Baryshev et al, 2004; Di Jeso et al., 2003, 2005; Kim et al., 1992;
23
Kim and Arvan, 1995; Kuznetsov et al., 1994; Menon et al., 2007; Muresan and Arvan, 1997;
24
Sargsyan et al., 2002]. Such interactions may also serve to prevent premature export of unfolded or
25
misfolded secretory proteins from the ER by a process known as ER-quality control [Bedard et al.,
11
1
2005; Qi el al., 2017], an adaptive cellular reaction that maintains ER homeostasis avoiding
2
perturbation by the accumulation of incorrectly folded proteins. GRP94 is associated with the
3
nascent TG [Muresan and Arvan, 1997], whereas BiP binds to unfolded polypeptides and prevents
4
protein aggregation through noncovalent associations [Chung et al, 2002]. The oxidative folding of
5
TG proceeds with the aid of two chaperone-oxidoreductase complexes, Bip/PDI and
6
CNX/CRT/ERp57 [Meunier et al., 2002]. The formation of intradomain disulfide bonds can be
7
catalyzed near BiP bound sites by PDI and near CNX/CRT bound sites by the ERp57
8
oxidoreductase [Di Jeso et al., 2005].
9
The last stage of TG conformational maturation in the ER is the non-covalent TG
10
homodimerization, without any interchain disulfide bridge, and only after TG has dissociated from
11
CNX/CRT system. ChEL domain is required for protein dimerization. Whereas correctly folded
12
proteins are exported from the ER to Golgi, misfolded proteins are retained in the ER and
13
retrotranslocated to the cytosol for proteasomal degradation by the ERAD machinery [Kim an
14
Arvan, 1998; Qi et al., 2017].
15
The next step in the post translational modifications of TG N-glycans occurs within the Golgi
16
and follows presumably the same general process described for other proteins. In brief, after
17
removal
18
glycosyltransferases (GnT) catalyze the branching and elongation of the carbohydrate chains,
19
producing hybrid-type or complex-type oligosaccharide [Vagin et al., 2009] (Figure 3).
of
mannose
residues
by
Golgi
mannosidases,
several
N-acetylglucosamine
20
The cDNA sequence of human TG contains 20 putative N-linked glycosylation sites, 16 of them
21
being glycosylated in the mature protein [Yang et al., 1996]. Eight sites (asparagines residues 76,
22
484, 529, 748, 816, 1716, 1774 and 2250) are linked to complex type oligosaccharide units
23
containing fucose and galactose in addition to mannose and glucosamine [Yang et al., 1996]. The
24
sites in positions 1220, 1349, 2013, 2295 and 2582 contain high-mannose type units (mannose and
25
glucosamine) and the sites in positions 198 and 1365 are linked to oligosaccharide units containing
12
1
galactose in addition to mannose and glucosamine but no fucose and may be either hybrid-type or
2
complex-type. [Yang et al., 1996]. Finally, very different oligosaccharide composition types were
3
found associated with position 947 (complex or high mannose) [Yang et al., 1996]. The human TG
4
also contains sulfated O-linked glycosaminoglycans [Fogelfeld and Schneider, 1990; Schneider et
5
al., 1988; Spiro and Bhoyroo, 1988]. Interestingly, non-glycosylated thyroglobulin loses the ability
6
to synthesize thyroid hormones [Mallet et al., 1995].
7
Phosphorylation of TG occurs in Golgi apparatus within carbohydrates and serine and tyrosine
8
residues [Consiglio et al., 1987], whereas sulfation is a late post translational modification step that
9
takes place in the trans-Golgi and contributes to the negative charge of the TG [Cauvi et al., 2003].
10
In mammals, newly synthesized TG is firstly glycosylated and folded via the formation of
11
numerous disulfide bonds [Citterio et al., 2019; Targovnik, 2013]. As indicated above, TG type 1,
12
TG type 2 and TG type 3 are cysteine-rich repeats that are covalently bound by intramolecular
13
disulfide bonds, which are involved in the conformation of the tertiary structure of TG and in the
14
hormonogenesis. The formation of disulfide bonds is catalyzed by endogenous ER oxidoreductases.
15
The TG type 1 domains are grouped into two subgroups, type 1A and type 1B. Type 1A repeats
16
(TG type 1–1 to TG type 1–8 and TG type 1–10) have a total of six Cys residues, whereas type 1B
17
repeats (TG type 1–9 and TG 1–11) contain only four Cys residues [Molina et al., 1996].
18
Conservative intradomain disulfide bonds patterns Cys1–Cys2, Cys3–Cys4, and Cys5–Cys6 prevail
19
in all type 1A repeats [Molina et al., 1996]. TG type 1B, TG type 2 and TG type 3 repeats and the
20
six cysteine residues of the ChEL domain include also intradomain disulfide bonding.
21
TG containing missense mutations in the cysteine residues or in the ChEL domain and splice site
22
mutations causing exon skipping could represent targets for intracellular retention and degradation
23
by ERAD pathway.
24
Sequencing analysis of the TG gene revealed missense mutations that involved cysteine residues:
25
p.C160S, p.C183Y, p.C194G, p.C726Y, p.C1077R, p.C1264R, p.C1281Y, p.C1476R, p.C1493Y,
13
1
p.C1510F, p.C1607F, p.C1897Y, p.C1904G, p.C1996S, p.C2000W, p.C2006Y and p.C2154Y
2
(Figure 2, Table 1) [Baryshev et al, 2004; Caputo et al., 2007; Citterio et al., 2013; Hishinuma et al,
3
1999, 2005, 2006; Hu et al., 2016; Kanou et al., 2007; Kitanaka et al., 2006; Narumi et al., 2011;
4
Nicholas et al., 2016; Zou et al. 2018]. The loss of cysteine residues can eliminate disulfide bonds
5
and alter the normal conformational structure of the TG, possibly preventing the interaction of
6
hormonogenic acceptor and donor sites, and causing a defect in the intracellular transport of the
7
protein. A detailed study of two unrelated patients with CH and two siblings with adenomatous
8
goiter revealed replacement of two conserved cysteines by arginine (C1264R) and serine (C1996S),
9
which resulted in the retention and aggregation of TG in the ER [Hishinuma et al. 1999]. Thyroid
10
tissue extracted from all patients exhibited higher expression levels of ER chaperones, BiP, GRP94,
11
PDI, ERp29 and ERp72 [Baryshev et al, 2004].
12
The ChEL domain is essential for the intracellular transport of TG to the site of its
13
hormonogenesis, via the secretory pathway [Lee et al., 2008, 2009, 2011; Lee and Arvan, 2011;
14
Park and Arvan, 2004; Wang et al., 2010]. It functions as an intramolecular chaperone and as a
15
molecular escort for TG regions I, II, and III [Lee et al., 2008]. The ChEL domain is also required
16
for protein dimerization and consequently plays a critical structural and functional role in the TG
17
protein. It is well documented that pathogenic missense mutations in the ChEL domain can result in
18
the intracellular retention of TG. The missense mutations located in this domain may cause TG
19
retention in the ER and premature degradation. Several missense mutations associated with CH
20
were reported to be present in the ChEL domain: p.A2234D [Caputo et al., 2007; Citterio et al.,
21
2013; Machiavelli et al., 2010; Pardo et al. 2008; 2009, Santos-Silva et al., 2019], p.R2242H [Caron
22
et al., 2003; Machiavelli et al., 2010; Raef et al., 2010, Zou et al., 2018], p.N2285D [Makretskaya et
23
al., 2018], p.G2319D [Hishinuma et al., 2006], p.R2336Q [Hishinuma et al., 2006; Kitanaka et al.,
24
2006 ; Siffo et al., 2018], p.G2341S [Watanabe et al. 2018], p.A2362P [Siffo et al., 2018],
25
p.W2365R [Siffo et al., 2018], p.G2374V [Hishinuma et al., 2006], p.G2375R [Hishinuma et al.,
14
1
2005; 2006; Kanou et al.; 2007; Long et al., 2018], p.I2394M [Sun et al., 2018], p.R2455H [Hu et
2
al., 2016; Long et al., 2018, Yu et al., 2018], p.A2471S [Sun et al., 2018], p.L2547Q [Nicholas et
3
al., 2016], p.R2585W [Hu et al., 2016, Jiang et al., 2016 ; Sun et al., 2018] p.N2616I [Hu et al.,
4
2016; Long et al., 2018, Sun et al. 2018; Yu et al., 2018], p.W2685L [Nicholas et al., 2016], and
5
p.A2713T [Long et al., 2018] (Figure 2, Table 2). Functional analysis suggests that the p.A2215D
6
mutation results in retention of the TG protein inside the ER and degradation via the proteasome
7
system [Pardo et al., 2009], as already observed in the cog/cog congenital goiter mouse [Kim et al,
8
1998] and the rdw/rdw non-goitrous CH rat [Kim et al, 2000]. The cog/cog mouse harboring the
9
p.L2263P TG mutation [Kim et al., 1998] and the WIC-rdw rat harboring the p.G2300R TG
10
mutation [Hishinuma et al., 2000; Kim et al., 2000] exhibit full-length synthesis of TG but impaired
11
transport from the ER.
12
Exon skipping in the TG gene can be caused by nucleotide substitutions or deletion in acceptor
13
or donor splice sites involving the -3/-2/-1 (c.275-3C>G, c.5042-2A>G, c.6563-2A>G, c.2762-
14
1G>A, c.6200-1G>C, c.7998-1G>A) or +1/+2/+3/+4/+5/+6 position (c.638+1G>A, c.745+1G>A,
15
c.2176+1G>A, c.4932+1G>C, c.5686+1G>T, c.5686+1G>A, c.5686+1G>C, c.6262+1delG,
16
c.6876+1delG, c.274+2T>G, c.5401+2T>C, c.7036+2T>A, c.7862+2T>A, c.4159+3_+4delAT;
17
c.3433+3_+6delGAGT, c.638+5G>A), respectively [Abdul-Hassan et al., 2013; Alzahrani et al.,
18
2006; Chen et al., 2018; Citterio et al., 2015, Fu et al., 2016c, Gutnisky et al., 2004; Hermanns et
19
al., 2013, Hishinuma et al., 2006; Hu et al., 2016, Ieiri et al. 1991; Makretskaya et al., 2018;
20
Medeiros-Neto et al., 1996; Narumi et al., 2011; Nicholas et al., 2016; Niu et al., 2009; Pardo et al.,
21
2008, 2009; Peteiro-Gonzalez et al., 2010; Rubio et al., 2008; Targovnik et al., 1995, 2001, 2012;
22
Watanabe et al., 2019; Zou et al., 2018]. Recently, two exonic cryptic 5' splicing sites in exons 6
23
(c.745+1G>A) [Citterio et al., 2015] and 19 (c.4159+3_4delAT) [Targovnik et al., 2012] of the TG
24
gene have been identified. The elimination of exons in the TG gene by aberrant splicing results in
25
an altered ability to transfer an iodophenoxyl group from the donor site to the acceptor iodotyrosine.
15
1
Interestingly, two affected patients with the c.5686+1G>T mutation which leads to the skipping of
2
exon 30 [Targovnik et al., 2001], showed a marked increase of the ER chaperones, GRP94 and BiP
3
[Medeiros-Neto et al., 1996]. Immunohistochemical localization of TG showed a notable decrease
4
of immunoreaction product in the follicular lumen of thyroid sections from affected patients. In
5
contrast, accumulation of intracellular immunoreactive TG was clearly demonstrable in the thyroid
6
epithelium, suggesting a defect in intracellular TG transport [Medeiros-Neto et al., 1996].
7 8
4. Defects in protein folding of thyroid peroxidase
9
TPO is a thyroid-specific enzyme that plays a key role in thyroid hormones biosynthesis and is
10
the major autoantigen in Hashimoto’s disease, the most common organ-specific autoimmune
11
disease (Czarnocka et al., 1985; Portmann et al., 1985). TPO is a member of the peroxidase family
12
which employs a heme group as part of their catalytic unit. The human TPO gene is located on
13
chromosome 2 (2:1,374,066-1,543,711; GRCh38 assembly). It comprises 17 exons, spands 170 kb
14
of genomic DNA and encodes a glycosylated hemoprotein of 933 amino acids (Figure 4). The
15
mRNA is 3,152 nucleotides long (NCBI Reference Sequence NM_000547.5) [Kimura et al., 1989]
16
and the pre-protein is composed of a putative 18 amino acids signal peptide followed by a 915
17
amino acids polypeptide which codifies a large extracellular domain (residues 19-846), a
18
transmembrane domain (residues 847-871), and a short intracellular tail (residues 872-933) (Figure
19
4) [https://www.uniprot.org/uniprot/P07202]. Baker et al. (1994) and Nishikawa et al. (1994)
20
suggested that human TPO may be a disulfide-linked dimer. TPO shows 42% sequence homology
21
with the myeloperoxidase (MPO) (Libert et al., 1987) and is part of the animal haem peroxidase
22
superfamily. The exons 13 and 14 encode the complement control protein (CCP)-like (residues 740-
23
795) and calcium-binding epidermal growth factor (EGF-Ca2+ binding)-like (residues 796-839)
24
domains, respectively (Figure 4) [https://www.uniprot.org/uniprot/P07202, Ruff and Carayon,
25
2006]. EGF-Ca2+ shows structural characteristics of a Sushi domain.
16
1
The TPO enzyme activity depends on both proper folding and membrane insertion, and an intact
2
catalytic site. After its synthesis, human TPO undergoes various postranslational modifications such
3
as N- and O-linked glycosylation and heme fixation [Ruff and Carayon, 2006]. Five potential
4
glycosylation sites are present at asparagine residues 129, 307, 342, 478, and 569 [Ruff and
5
Carayon, 2006]. N-glycans play an essential role in the correct folding, intracellular trafficking and
6
activity of TPO [Fayadat et al., 1998]. Exons 8, 9 and 10 encode the catalytic center of the TPO
7
protein (heme-binding region) [Rodrigues et al., 2005] which is crucial for the enzymatic activity
8
and contain the proximal (His239) and distal (His494) putative heme binding histidine residues
9
(Figure 4). According to the studies with MPO and lactoperoxidase, the heme group in TPO is a
10
bis-hydroxylated heme b covalently bound via ester linkages to Glu399 and Asp238 of the apoprotein
11
(Figure 4) [Ruff and Carayon, 2006].
12
The first description of a human TPO mutation was conducted in a boy with iodide
13
organification defect, who presented with hypothyroidism at the age of 4 months and developed a
14
compressive goiter at the age of 12 years [Abramowicz et al, 1992]. A homozygous duplication of a
15
tetranucleotide -GGCC- in exon 8 of the TPO gene was identified (c.1186_1187insGGCC or
16
c.1183_1186dupGGCC, also reported as 1187_1188insGCCG or c.1184_1187dup GCCG), 152 bp
17
upstream from the junction with intron 8. The resulting frameshift generates a stop codon in exon 9
18
(p.A397Pfs*76, Figure 4), which would result in a truncated protein with mutation of the proximal
19
and deletion of the distal putative heme binding histidine residues where by TPO activity is
20
expected to be absent. However, alternative splicing restores the normal reading frame
21
[Abramowicz et al, 1992]. The result is a nearly full-length protein with a 91-amino acid segment
22
replaced by a 51 residue unrelated segment. This translation product is expected to have an
23
unchanged distal putative heme binding His494, but to lack the proximal putative heme binding
24
His239 [Abramowicz et al, 1992]. The duplication GGCC in exon 8 is a common mutation of the
25
TPO gene in Caucasian population [Abramowicz et al., 1992; Altmann et al, 2013; Bakker et al.,
17
1
2000; Belforte et al., 2012; Bikker et al, 1995; Cangul et al., 2013; Cangul et al., 2015; Fugazzola et
2
al., 2005; Gruters et al.,1996; Nascimento et al., 2003, Neves et al., 2010; Rivolta et al 2003; Santos
3
et al., 1999].
4
Six cystein residues responsible for the formation of 3 disulfide bridges: Cys800-Cys814, Cys808-
5
Cys823 and Cys825-Cys838 are located in the EGF-Ca2+ binding domain of human TPO. The
6
substitution of cysteine for other amino acids affects the formation of the disulfide bond and
7
therefore can alter the tertiary structure of the EGF-Ca2+ binding domain of TPO [Rodrigues, et al.,
8
2005]. Disruption of the disulfide bonds, as result of p.C800R [Bӧrgel et al., 2005], p.C808R
9
[Rivolta et al., 2003] or p.C838S [Rodrigues, et al., 2005] mutations (Figure 4), would generate
10
significant conformational changes which could partially retain the TPO on the ER on the one hand,
11
and on the other hand, the proteins with conformational defects responsible for a lower net activity
12
of the enzyme might reach the membrane.
13
Interestingly, the p.S131P mutation (Figure 4) disrupts the potential glycosylation site at Asn129
14
residue [Rodrigues, et al., 2005]. The severity of this mutation is evident, the N-X-S/T site is
15
modified to N-X-P and consequently is very likely to induce major defects in the intracellular traffic
16
of the TPO.
17
Umeki et al. (2004) analyzed five mutated TPOs with alteration in expression probably due to
18
incorrect folding, four of them were missense mutations (p.R175Q, p.G533C, p.R665W, and
19
p.G771R, Figure 4) [Kotani et al., 2003, 2004; Umeki et al., 2002] and one corresponded to the
20
elimination of two contiguous amino acids (p.D574-L575del, Figure 4). The five mutated TPOs had
21
a pathogenic mutation on the extracellular domain but had no abnormality on the transmembrane
22
and cytoplasmic domains. The mutated mRNAs were transfected in Cho-Ki and NRK cells and then
23
they were studied by indirect immunofluorescence and flow cytometry to determine the intracellular
24
localization. In permeabilized condition the 5 mutated proteins were located in intracellular
25
structures like the nuclear envelope and ER in both cells line, whereas in nonpermeabilized
18
1
condition only p.D574-L575del was observed on the cell surface of Cho-KI and p.D574-L575del in
2
addition to p.G771R, on the cell surfase of NRK cells [Umeki et al. (2004]. The analysis of the
3
TPO protein expression on the cell surface by flow cytometry showed a positive fluorescense peak
4
in p.G533C, p.D574-L575del and p.G771R with less intensity compared with wild-type TPO. In
5
contrast, p.R175Q and p.R665W were not expressed on the cell surface. It has been reported that
6
calnexin and calreticulin binding to TPO are necessary for the initial folding steps [Fayadat et al.,
7
2000]. In immunoprecipitation by anti-TPO antibodies followed by Western blot using anti-TPO,
8
anti-calnexin and anti-calreticulin, p.G533C, p.D574-L575del and p.G771R exhibited increasing
9
interaction with calnexin. This suggests that some p.G533C, p.D574-L575del and p.G771R mutated
10
proteins would be fold properly in the calnexin cycle and secreted via Golgi to apical membrane of
11
the thyrocyte, while the remaining of these misfolded TPOs would be degraded in the ERAD
12
pathway. In p.R175Q and p.R665W missense mutations, an arginine was substituted for glutamine
13
or tryptophan, respectively. Arginine is a basic amino acid with positive charge, whereas glutamine
14
is uncharged and tryptophan is a hydrophobic amino acid. The loss of an arginine could cause
15
structural changes and as a consequence p.R175Q and p.R665W mutations would follow the
16
anterograde pathway and would be degraded in proteasomes.
17 18
5. Defects in protein folding of DUOX system
19
The DUOX1 and DUOX2 genes are located in opposite transcriptional orientation in an operon-
20
like unit on chromosome 15, encoding similar proteins that are inserted in the apical membrane of
21
thyroid cells [De Deken et al. 2000, 2002; De Deken and Miot, 2019; Dupuy et al, 1999;
22
Grasberger, 2010; Muzza and Fugazzola, 2017, Ohye and Sugawara, 2010]. The DUOX2 gene
23
spans 22 Kb of genomic DNA (15:45,092,650-45,114,344; reverse strand, GRCh38 assembly)
24
which includes 34 exons, being the first one non-coding. The mRNA (NCBI Reference Sequence:
25
NM_014080.4) is 6,428 nucleotides long and the preprotein is composed of a putative 21 amino
19
1
acids signal peptide followed by a 1,527 amino acids polypeptide (Figure 5) [De Deken et al. 2000,
2
2002; Dupuy et al, 1999]. DUOX1 gene encodes a homologous protein displaying 83 % sequence
3
similarity. The 36 kb DUOX1 gene (15:45,129,933-45,165,576; forward strand, GRCh38 assembly)
4
consists of 35 exons (being the first two noncoding) encoding a protein of 1551 amino acids
5
(mRNA: 5675 nucleotides long; NCBI Reference Sequence: NM_017434.5) in which the first 21
6
amino acids correspond to the putative signal peptide [De Deken et al. 2000, 2002; Dupuy et al,
7
1999]. The structure of DUOX1 and DUOX2 proteins contains three regions: 1) peroxidase
8
homology ectodomain (peroxidase-like domain): an N-terminal extracellular region, which has 43%
9
sequence homology with hemoperoxidases, which distinguishes DUOX from other family
10
NOX/DUOX, 2) a central region encompassing an intracellular loop containing potentially two EF-
11
hand calcium binding motifs, which would be involved in regulating the conformation and activity
12
of NADPH oxidase; 3) gp91phox/NOX2-like domain: a C-terminal region, comprising the last six
13
transmembrane domains (Figure 5) [De Deken et al., 2000; Dupuy et al., 1999; Morand et al., 2004;
14
Ohye and Sugawara, 2010]. The C-terminal domain includes four conserved histidines, which are
15
coordination sites for two heme prosthetic group [Muzza and Fugazzola, 2017]. The C-terminal
16
cytosolic orientation presents a binding site for FAD and NADPH (Figure 5) [De Deken et al.,
17
2000; Dupuy et al., 1999; Morand et al., 2004; Ohye and Sugawara, 2010]. It is believed that this
18
region is responsible for the transfer of electrons from NADPH through the membrane. DUOX2
19
exhibits higher expression and is also more efficient in the production of peroxide than DUOX1
20
[Muzza and Fugazzola, 2017]. DUOX1 is regulated by protein kinase A through Gs-PKA pathway,
21
while DUOX2 is stimulated by protein kinase C through Gq-phospholipase C pathway [Rigutto et
22
al., 2009]. Although both enzymes are expressed in the thyroid, DUOX1 is also expressed in the
23
respiratory and tongue epithelia, testis and cerebellum, and DUOX2, in respiratory epithelia, uterus,
24
gallbladder, pancreatic islets and digestive tract where these enzymes could provide H2O2 for
25
peroxidase-mediated antimicrobial defense processes [El Hassani et al., 2005; Forteza et al., 2005;
20
1
Geiszt et al, 2003]. DUOX1 and DUOX2 present two N-glycosylated states, the high mannose
2
glycosylated immature form of 180 kDa expressed in ER and the fully glycosylated mature form of
3
190 kDa which is translocated at the plasma membrane and generates H2O2 [Morand et al., 2004;
4
Muzza and Fugazzola, 2017]. Partially glycosylated DUOX2 remains in the ER [Grasberger and
5
Refetoff, 2006].
6
DUOXA1 and DUOXA2 are essential for DUOX maturation [Grasberger and Refetoff, 2006,
7
Muzza and Fugazzola, 2017]. The DUOXA1 and DUOXA2 genes are oriented head to head in the
8
intergenic region of 16 kb (DUOXA2: 15:45,114,321-45,118,421, forward strand; DUOXA1:
9
15:45,117,367-45,129,938, reverse strand, GRCh38 assembly) between both DUOX genes. The
10
DUOXA2 open reading frame spans 6 exons encoding a protein of 320 amino acids (mRNA: 1755
11
nucleotides long; NCBI Reference Sequence: NM_207581.4), whereas the DUOXA1 spans 11
12
exons encoding a protein of 483 amino acids (mRNA: 2021 nucleotides long; NCBI Reference
13
Sequence: NM_144565.4). The two DUOXA genes encode a N-terminal ectodomain, five
14
transmembrane domains and a C-terminal cytoplasmic region. The first extracellular loop presents
15
three N-glycosylation sites [Grasberger and Refetoff, 2006; Muzza and Fugazzola, 2017].
16
DUOXA1 and DUOXA2 enable the ER-to-Golgi transition, maturation, and surface expression of
17
functional DUOX1 and DUOX2 in a heterologous cell system (Figure 6) [De Deken and Miot,
18
2019; Grasberger and Refetoff, 2006]. A stable complex between DUOX2 and DUOXA2 is then
19
required for their enzymatic activity (Figure 6). DUOXA2 would be part of the DUOX2 quality
20
control system, indicating that DUOXA2 displays a chaperone-like function with respect to its partner.
21
[Carré et al., 2015; Grasberger et al. 2007].
22
DUOX2 alterations were first described in 2002 [Moreno et al.]. In the literature, Sanger
23
sequencing has been commonly used for DUOX2 mutations identification in both research and
24
clinical studies. Recently, high throughput sequencing has been reported to be a rapid and effective
25
tool for largest DUOX2 mutation screening for patients with or without family history [Chem et al.,
21
1
2018; Long et al., 2018; Makretskaya et al., 2018; Sun et al., 2018; Yu et al., 2018; Zou et al.,
2
2018]. The mutational spectrum shows heterogenous alterations dispersed in the exons and introns
3
of the entire gene (Figure 5) [Belforte et al., 2016a; De Deken and Miot, 2019]. To date, molecular
4
research has shown more than one hundred fifty pathogenic variations have been found to be
5
associated with TCH, PCH or subclinical congenital hypothyroidism [Belforte et al., 2016a; De
6
Deken and Miot, 2019; Fu et al., 2016a].
7
mutations were found to be altered proteins with a single amino acid substitution, followed by
8
deletions/insertion and nonsense mutations causing premature truncation of the DUOX2 protein
9
[Belforte et al., 2016a; Muzza and Fugazzola, 2017]. Premature termination codons can delete the
10
gp91phox/NOX2-like domain and the peroxidase-like domain. The functional consequence could
11
be the complete impossibility to generate H202 from the DUOX2 system. Only six DUOX2 splice
12
site mutations have been reported, c.514-2A>G [Tan et al., 2016], c.2335-1G>C [Belforte et al.,
13
2016b; Muzza et al., 2014], c.2655-2A>C [Varela et al., 2006], c.3516-1G>A [Tan et al., 2016]
14
c.3693+1G>T [Tan et al., 2016] and c.4240-1G>C [Matsuo et al., 2016]. The p.K530*, p.E879K,
15
p.R1110Q, p.L1160del, p.R1334W and p.L1343F mutations have been the most frequently
16
identified DUOX2 mutations in Asian population (Figure 5) [Belforte et al., 2016a; Muzza and
17
Fugazzola, 2017], whereas the frequent p.F965Sfs*29 (c.2895_2898delGTTC) mutation (Figure 5)
18
has proved to be the most frequent in the Caucasian population [Belforte et al., 2016a; Muzza and
19
Fugazzola, 2017].
The most frequent events of deleterious DUOX2
20
Grasberger et al. [2007] analyzed three missense mutations in DUOX2 (p.Q36H [Varela et al.,
21
2006], p.R376W [Cortinovis et al., 2008; Lü et al., 2011; Vigone et al., 2005] and p.D506N
22
[Cortinovis et al., 2008; Pfarr et al., 2006] involved in congenital hypothyroidism (Figure 5), in
23
terms of its impact on the generation of H2O2, folding, intracellular traffic and DUOXA2
24
interaction, in a reconstituted heterologous system. The three missense mutations displayed either
25
complete (p.Q36H and p.R376W) or partial loss (p.D506N) of H2O2-generating activity. The
22
1
mutations p.Q36H and p.R376W completely prevent the transit of DUOX2 to the cell surface and
2
showed a diminished oxidized-to-reduced ratio [Grasberger et al. 2007]. The mutant proteins are
3
present predominantly as core N-glycosylated (a thiol-reduced folding intermediate) and are
4
retained by the quality control system within the ER as indicated by a greater complexation with the
5
lectin calnexin. This interaction may be related to retrotranslocation and proteasomal degradation.
6
On the contrary, the p.D506N mutation showed a similar ratio of oxidized-to-reduced forms and a
7
diminished expression at the plasma membrane [Grasberger et al. 2007]. This showed a successful
8
route to the plasma membrane through of Golgi complex but with an abnormal maturation.
9
Oxidative folding of DUOX2 in the ER appears to be the rate-limiting step in the maturation of
10
DUOX2. ER-localized DUOX2 is almost exclusively in the reduced form (presumably inactive)
11
[Grasberger et al. 2007]. The oxidized form (presumably active) does not accumulate in the ER due
12
to rapid, DUOXA2-mediated export (Figure 6). DUOXA2 allows rapid ER exit of DUOX2 folded
13
and also enhances the degradation of DUOX2 protein mutants not suitable for ER output
14
[Grasberger et al. 2007]. However, DUOXA2 does not affect the reduced-to-oxidized transition,
15
this observation would indicate that DUOXA2 would act after the maturation of DUOX2, once
16
DUOX2 has acquired a stable disulfide-bonded conformation. Intramolecular (DUOX2) and
17
intermolecular (between DUOX2 and DUOXA2) disulfide bonds which are promoted and
18
maintained by the oxidizing environment of the ER seem to play an important role in the protein
19
folding, stability, function and protein-protein interaction. Carré et al. [2015] reported an
20
intramolecular bond in the DUOX2 between cysteine residues 124 (located in the N-terminal ectodomain)
21
and 1162 (located in the second extracellular loop), that provides structural support for the formation of
22
interdisulfide bridges between DUOX2 and DUOXA2 (Figure 6). The intermolecular bonds between
23
DUOX2 and DUOXA2 involve the N-terminal cysteine residues 568 and 582 of DUOX2 respectively and
24
cysteine residues 167 of the first extacellular loop and 233 of the second extacellular loop of DUOXA2
25
(Figure 6) [Carré et al., 2015]. It is importan to note that mutations of cysteine residues in the N-
26
terminal domain of human DUOX2 protein affect both targeting and extracellular H2O2 production
23
1
by human DUOX2 [Fortunato et al., 2010; Morand et al., 2004]. Recently, the p.C124* (dbSNP:
2
rs1197021807) and p.C568R (rs770603845) mutations (Figure 5) was reported in gnomAD data set
3
[https://gnomad.broadinstitute.org/gene/ENSG00000140279] in a heterozygous state, with an allele
4
frequency of 2/249972 (0,000008001) and 4/188376 (0.00002123) respectively.
5 6
6. Defects in protein folding of NIS
7
The SLC5A5 gene that encodes NIS, is located on chromosome 19, spanning 23 Kb
8
(19:17,871,945-17,895,174; GRCh38 assembly). The full-length human mRNA (NCBI Reference
9
Sequence: NM_000453.3) consists of 15 exons (3604 nucleotides) and encodes a 643 amino acids
10
peptide with 13 transmembrane segments, an extracelular N-terminal extreme and an intracellular
11
C-terminus (Figure 7) [De la Vieja et al.,2004, 2005; Spitzweg and Morris, 2010; Targovnik et al.,
12
2017]. This transporter has three N-glycosilation sites (Figure 7) [Spitzweg and Morris, 2010],
13
although their glycosilation is not essential for its function as iodine transporter [Levy et al., 1998]
14
or for reaching and inserting into the plasma membrane [Li et al., 2013].
15
The expression of NIS is regulated primarily by TSH levels [Carrasco, 2013], I- concentration in
16
plasma [Portulano et al., 2014] and by TG [Vono-Toniolo and Kopp, 2004]. Its trafficking to the
17
cell membrane is also regulated by the interaction with other proteins, such as pituitary tumor–
18
transforming gene (PTTG) binding factor (PBF) which promotes its internalization [Stratford et al.
19
2005].
20
The analysis of the effect of reported mutations affecting SLC5A5 and of different mutagenesis
21
studies allowed understanding the key role of certain residues for the folding and trafficking of the
22
NIS. Several loss-of-function mutations leading to I- transport defect (ITD) have been reported
23
[Martín et al., 2018a]; five of them lead to the intracellular retention of this transporter: p.R124H,
24
p.V270E, p.A439_P443del, p.S509Rfs*7 and p.G543E (Figure 7). The first three inhibit also the
25
transporter activity [Ravera et al., 2017]. The interaction between the δ-amino group at 124 and the
24
1
thiol in the Cys440, lost in p.R124H, is crucial for the folding of NIS and targeting to the plasma
2
membrane [Paroder et al, 2013]. The deleterious effect of p.A439_P443del is due to the loss of
3
Asn441 of the side chain which interacts with Gly444, capping the α-helix of transmembrane segment
4
XII [Li , et al., 2013]. The frameshift mutation p.S509Rfs*7 [Pohlenz, et al., 1998; 2000] leads to
5
the loss of the C-terminal portion of this transporter. Different studies [Martín et al., 2018b; Martín
6
et al., 2018c] have demonstrated the importance of this region (between residued Ile546 and Lys618),
7
which is subjected to post translational modifications, such as phosporilation for the migration to
8
the plasma membrane. It was shown that a putative tryptophan-acidic motif (Trp565-Asp566) is
9
required for leaving the ER and reaching the membrane. Furthermore, the region between Val580
10
and Lys618 is crucial for its basolateral location. Only alanine or serine are tolerated at position 543,
11
other substitutions as p.G543E lead to the intracellular retention of the altered peptide [De la Vieja
12
et al., 2005]. The mutation p.V270E alters the positive charge in the surface of the molecule, which
13
would alter the interaction with the proteins involved in the trafficking of NIS [Nicola et al, 2015].
14
The systematic assessment of sorting motifs in NIS, carried out by Darrouzet et al. [2016] revealed
15
an internal noncanonical PDZ-binding motif, that involves the residues Tyr118, Glu119, Tyr120 and
16
Leu121, necessary to reach the plasma membrane. This site was predicted bioinformatically and
17
assessed with functional assays, including directed mutagenesis which validated the role played by
18
Leu121.
19 20
7. Defects in protein folding of Pendrin
21
The passive transport of the iodine to the follicular lumen is mostly facilitated by pendrin [Bidart
22
et al., 2000], encoded by the SLC26A4 gene, that maps in chromosome 7 (7:107,660,828-
23
107,717,782; GRCh38 assembly). It comprises 21 exons (the first one is noncoding), covers 57 kb
24
of genomic DNA and encodes 780 amino acids (Figure 8). The mRNA is 4,737 nucleotides long
25
(NCBI Reference Sequence: NM_000441.2) and the canonical isoform codifies for a classic
25
1
predicted topology of 12 transmembrane domains with intracellular N- and C-terminal tails (Figure
2
8). Dossena et al., [2009] proposed another model of pendrin consisting of 15 transmembrane
3
segments, an extracelular N-terminal extreme and an intracellular C-terminus. It is interesting to
4
note that there is no experimental data proving any of the suggested models. Pendrin has a STAS
5
(sulfate transporter and antisigma factor antagonist) domain (Figure 8) [Aravind and Koonin, 2000].
6
Pendrin has affinity for chloride, iodide and bicarbonate, among other anions. The peptide is N-
7
glycosylated exclusively at Asn167 and Asn172 which are located in the second putative extracellular
8
loop of pendrin (Figure 8). N-glycosylation of membrane proteins plays a role in their correct
9
folding and targeting to the membrane [Azroyan et al., 2011].
10
As was previously mentioned, mutations in this gene can lead to nonsyndromic hearing loss
11
associated with enlarged vestibular aqueduct (EVA) or Pendred syndrome, an autosomal recessive
12
disorder characterized by sensorineural hearing impairment, thyroid goiter and a partial
13
organification problem detectable by a positive perchlorate test [Bizhanova and Kopp, 2009]. The
14
main molecular mechanism proposed for Pendred Syndrome has been SLC26A4 mutations causing
15
the retention of the mutants in the ER [Rotman-Pikielny et al, 2002]. Retention of misfolded
16
pendrin in the ER and subsequent degradation is a major pathogenic mechanism for Pendred
17
syndrome. There are more than 170 sequence variants associated with this phenotype [Campbell, et
18
al., 2001; Dossena et al., 2009, 2011; Tsukamoto et al., 2003], 64% of these are single nucleotide
19
changes leading to amino acid substitutions and at least eight of them (p.G102R; p.V138F; p.T193I;
20
p.L236P; p.T410M; p.L445W; p.Q446R and p.Y556C) [Coyle et al., 1998; de Moraes et al., 2016;
21
Taylor et al., 2002] (Figure 8) lead to diminished or abolished expression of the mutant pendrin in
22
the apical membrane. Although the specific molecular chaperones involved in the folding of this
23
protein have yet to be identified, it has been reported that Hsp90 is not involved, and furthermore,
24
its inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) increases the stability of Pendrin
25
by inducing the transcription of heat shock transcription factor 1 (Hsf1)-dependent chaperones [Lee,
26
1
et al., 2012]. The same authors also demonstrated that the E3 ubiquitin ligase Rma1 is involved in
2
ERAD Pendrin degradation.
3
Jung et al [2006] reported that the cytosolic heat shock cognate protein 70 (Hsc70) and the co-
4
chaperone DNAJC14 are involved in the unconventional Golgi-bypass trafficking pathway that can
5
be stimulated by ER stress-inducing agents. This can lead to cell surface expression of misfolded
6
variants, making them potential targets for treating Pendred syndrome.
7 8
8. Defects in protein folding of SLC26A7
9
In the recent years, the role of pendrin as the main apical iodine transporter has been challenged,
10
as consequence of the lack of a complete correlation between deleterious mutations affecting this
11
transporter and hypothyroidism [Bizhanova and Kopp, 2011] and the fact that apical iodide efflux is
12
also possible in the absence of pendrin [Silveira and Kopp, 2015]. Interestingly, 50% of patients
13
with Pendred syndrome are euthyroid. In the last two years, recessive loss-of-function mutations in
14
another transporter named SLC26A7 were identified associated with goitrous CH [Cangul, et al.,
15
2018; Ishii et al., 2019; Zou et al., 2018]. The SLC26A7 gene is located on chromosome 8, spanning
16
149 Kb (8:91,249,319-91,398,155; GRCh38 assembly) and including 19 exons, being the first one
17
non-coding. The full-length human mRNA (NCBI Reference Sequence:
18
of 5,237 nucleotides. Sequence analysis revealed an open reading frame of 1,971 nucleotides
19
encoding a 656-amino acid peptide [Lohi et al, 2002]. The structure of SLC26A7 is characterized
20
by citoplasmatic N- and C-terminal domains, 12 putative transmembrane segments and a C-terminal
21
cytoplasmic STAS consensus domain (Figure 9) [Alper and Sharma, 2013; Lohi et al, 2002]. In
22
addition, it contains two predicted N-glycosilation sites at Asn125 and Asn131 (Figure 9) [Lohi et al,
23
2002]. The SLC26A7 was first discovered as a chloride/anion exchanger in the kidney (expressed in
24
the basolateral membrane of the outer medullary collecting duct intercalated cells) and in the
25
stomach (expressed in the basolateral membrane of the gastric parietal cells) [Petrovic et al., 2003;
NM_052832.4) consists
27
1
2004]. Recently, immunohistochemical and RT-PCR analyses confirmed the expression of
2
SLC26A7 in human thyroid follicular cells, located predominantly on the apical side facing the
3
follicular lumen [Ishii et al., 2019].
4
In 2018, a homozygous mutation in the SLC26A7 gene (c.927_930delCACT, c.932_933delAA
5
[p.I309Mfs*9,p.E311Gfs*28] or c.927_933delinsG [p.I309_E311delinsM]) was firstly reported in a
6
family of Saudi origin with two CH patients [Zou et al., 2018] (Figure 9). The unaffected mother,
7
father and a sibling carried the mutation in a heterozygous state. Almost simultaneously, Cangul et
8
al. [2018] identified novel homozygous mutations in the SLC26A7 gene in six unrelated families
9
with goitrous CH. By whole exome sequencing and subsequent Sanger sequencing, it was identified
10
in three consanguineous families of Pakistani or Turkish origin a homozygote c.679C>T transition,
11
which replaces the arginine residue in position 227 with a stop codon (p.R227*) [Cangul et al.,
12
2018] (Figure 9). In the same paper, in other three non-consanguineous families of Finnish origin,
13
the authors reported a homozygous frameshift mutation (c.1893delT, p.F631Lfs*8) (Figure 9).
14
More recently, Ishii et al. [2019] described two Japanese siblings with goitrous CH caused by a
15
homozygous nonsense mutation (c.1498C>T; p.Q500*) (Figure 9). In vitro studies of the mutant
16
evidence a complete loss of iodide transport activity. The p.Q500* mutation, located in the STAS
17
domain, impaired the localization of SLC26A7 in the cell membrane. It is well documented that the
18
STAS domain is required for membrane localization and exchanger function [Dorwart et al., 2008;
19
Sharma,et al., 2011].
20
Remarkably, hypothyroidism caused by SLC26A7 mutants cannot be rescued by wild-type
21
pendrin, suggesting that SLC26A7 plays a critical function in thyroid hormone synthesis. [Zou et
22
al., 2018]. Ishii et al. [2019] hypothesized that the SLC26A7 may be expressed more abundantly
23
than pendrin in the human thyroid gland and that SLC26A7 may have another function in the
24
biosynthesis of thyroid hormones in addition to being an iodide transporter. These hypotheses
25
require further investigation.
28
1
9. Defects in protein folding of the proteins involved in disembryogenesis
2
In only 5-10% of the thyroid dysgenesis, the underlying genetic cause can be unequivocally
3
determined and linked to a specific sequence variant. As was mentioned earlier, the mutations
4
described affect mainly genes involved in the development, growth and differentiation of the
5
thyroid cells (NKX2-1, FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3).
6
The TSHR is member of the superfamily of G Protein-coupled receptors [Vassart and Dumont,
7
1992, Tao and Conn, 2014]. The canonical isoform of 764 amino acids consists of seven
8
transmembrane domains, with an N-terminal extracellular segment that interacts with the TSH and a
9
cytoplasmatic C-terminal domain, capable of interacting with the G protein [Corvilain, et al., 2001].
10
As other glycosylated transmembrane proteins, TSHR undergoes post translational modifications
11
and folding in the ER lumen where it interacts with the resident chaperones. Studies by Siffroi-
12
Fernandez et al., [2002] and Mizrachi and Segaloff [2004] showed the interaction of this nascent
13
peptide with calnexin and calreticulin. Siffroi-Fernandez et al., [2002] were also able to detect the
14
binding of BiP in K652 cells, whereas Mizrachi and Segaloff [2004] could not find this association
15
and demonstrated the interaction with the protein disulfide isomerase (PDI) in HEK293 cells.
16
Loss-of-function germline mutations in TSHR are expected to cause congenital hypothyroidism
17
with a syndrome of resistance to TSH [Corvilain et al., 2001]. Most of these variants lead to
18
misfolding of the receptor and its intracellular retention in the ER [Mizrachi and Segaloff, 2004].
19
Familial cases of congenital hypothiroidim due to mutations in NKX2-1 and PAX8, often show
20
incomplete penetrance, suggesting the presence of additional modifiers [De Felice and Di Lauro,
21
2004]. Molecular analyses of DHTP mice (Double Heterozygous for both Titf1- and Pax8-null
22
mutations) that presented thyroid hypoplasia during embryogenesis and high incidence of thyroid
23
hemiagenesis as adults [Amendola, et al., 2005], revealed the presence of a strain specific mutation
24
in DNAJC17 [Amendola, et al., 2010], making it a candidate locus for hypothyroidism
25
predisposition. The DNAJC17 human gene encodes 304 amino acids and is a member of the heat-
29
1
shock-protein-40 family (Hsp40), acting usually as cofactor of the Hsp70 chaperone, regulating
2
their ATPase activity, through the J domain that all members display. [Kampinga and Craig, 2010].
3
Nevertheless, many of the functions that this co-chaperon exercises are still unknown. Pascarella, et
4
al [2018] demonstrated its co-localization in the nuclear speckles (also known as interchromatin
5
granule clusters) and its interaction with spliceosomal components, suggesting a participation in
6
splicing processes. Germline loss-of-function mutations in a homozygous state in this gene were
7
also associated with the recessive inheritance of retinal dystrophy [Patel, et al., 2016] This gene was
8
screened in 89 patients with congenital hypothyroidism, but only a possible damaging rare variant
9
c.610G>C was identified in a single patient [Nettore, et al., 2018], showing that DNAJC17 is not an
10
initial candidate gene to be analyzed in thyroid dysgenesis.
11
In the recent years, it has been described that mutations affecting different genes lead to
12
congenital hypothirodism associated to other syndromes, such as the Krüppel-like zinc finger
13
transcription factor GLI-similar 3 (GLIS3) which causes neonatal diabetes, a large spectrum of
14
abnormalities and clinical presentations of congenital hypothyroidism, from thyroid agenesia to
15
dyshormogenesis [Dimitri, et al., 2015]. This transcription factor mediates the TSH/TSHR
16
proliferation of thyrocites and is required for the biosynthesis of thyroid hormones, since
17
experiment in mice showed it regulates the expression of NIS and pendrin [Kang, et al., 2017]. On
18
the other hand, loss-of-function mutations that affect the C-terminus of the immunoglobulin
19
superfamily member 1 and impair its trafficking to the plasma membrane, lead to X-linked
20
congenital central hypothyroidism, with prolactin and GH deficiency, delayed pubertal development
21
and testicular enlargement [Sun, et al., 2012; Turgeon, et al, 2017]. Deficient levels of
22
immunoglobulin superfamily member 1, decrease the pituitary response to TRH because the
23
expression of its receptor is downregulated.
24 25
10. Conclusions and future perspectives
30
1
Congenital hypothyroidism (CH) describes a hormonal state of insufficient thyroid hormone
2
secretion detected at birth or postnatally. Over the last 20 years, mutations in the candidate genes
3
were identified and functionally characterized providing an overwhelming wealth of new insights
4
into basic molecular mechanisms.
5
Mutations affecting the proteins involved in the cascade of biosynthesis of TH, thyroid
6
organogenesis and its regulating pathways have been classified as missense and nonsense
7
mutations, frameshift mutations by single or multiple nucleotide deletion or insertion and splicing
8
mutations in the exonic or intronic consensus sites. The mutations are located in specific domains or
9
cysteine residues or glycosylation sites affecting a crucial function or the formation of disulfide
10
bridges or conformational maturation, respectively. In the dishormonogenesis the thyroid gland
11
develops hypertrophy and hyperplasia by the proliferative effect as a consequence of the chronic
12
elevation of TSH. In contrast, the dysembryogenesis results agenesis or hypoplasia or ectopia.
13
Many of the mutations cause protein misfolding and accumulation in the ER, affecting the secretory
14
capacity of the thyroid cell. The retention of misfolded proteins alters the ER homeostasis and
15
activates the ERAD mechanism, responsible for their cytosolic degradation.
16
Despite all these remarkable advances numerous challenging aspects remain to be solved. The
17
recent generation of new potent approaches including new sequencing technology and the
18
generation of thyroid organoids provides additional tools that might be of great help in such studies.
19 20 21 22 23 24 25
31
1
Declaration of interest
2
The authors declare that there is no conflict of interest that could be perceived as prejudicing the
3
impartiality of the research reported.
4 5
Funding
6
This study was funded by grants from the FONCyT-ANPCyT-MINCyT (PICT 2014-1193 to CMR
7
and PICT 2015-1811 to HMT), CONICET (PIP 2015-11220150100499 to CMR) and Universidad
8
de Buenos Aires (UBACyT 2016-20020150100099BA to CMR).
9 10
Acknowledgements
11
C.M. Rivolta and H.M. Targovnik are established investigators of the Consejo Nacional de
12
Investigaciones Científicas y Técnicas (CONICET).
13
K.G. Scheps is a postdoctoral research fellow of the CONICET.
14
32
1
Legends of Figures
2
Figure 1. Biosynthesis of thyroid hormones and iodine recycling in the polarized follicular
3
thyroid cells. The major steps in iodide (I−) metabolism (uptake, efflux, organification, and
4
recycling) in the different compartments and thyroid proteins involved are shown. After I− uptake
5
at the basolateral membrane of thyrocytes by the Na+/I- symporter (NIS), it is transported at the
6
apical membrane into the follicular lumen through the pendrin, anoctamin-1 and SLC26A7
7
transporters. NIS is dependent on the sodium gradient created by the Na/K-ATPase and KCNQ1-
8
KCNE2 potassium channel is required for NIS-mediated thyroid cell uptake of I-. In the cell-colloid
9
interface, the I- is oxidized to iodinium (I+) and rapidly organified by incorporation into selected
10
tyrosyl residues of thyroglobulin (TG), which results in the formation of two intermediaries, 3-
11
monoiodotyrosine (MIT) and 3,5-diiodotyrosine (DIT). Subsecuently, iodotyrosine coupling results
12
in the synthesis of 3,5,3'-triiodothyronine (T3) and 3,5,3',5'-tretraiodothyronine (T4). The oxidizing,
13
iodination and coupling reactions are mediated by thyroid peroxidase (TPO) in presence of H2O2
14
generated by dual oxidase 1 and 2/dual oxidase maturation factor 1 and 2 (DUOX1 and
15
2/DUOXA1 and DUOXA2) complex. Iodinated TG is stored as colloid in the follicular lumen.
16
Once endocytosed into the thyrocytes, the TG is digested by lysosomal proteases, thus releasing
17
attached MIT, DIT, T4 and T3. T4 and T3 are then transported out of the follicular cells and into the
18
bloodstream, through the monocarboxylate transporter 8 (MCT8). Meanwhile, MITs and DITs are
19
deiodinated by the iodotyrosine deiodinase 1 (IYD-1), present at the apical plasma membrane and
20
in endocytic vesicles of the thyrocytes and the iodine atoms as I- are transported to the
21
intrathyroidal iodide pool. Simultaneously, free amino acids (aa) generated during proteolysis of Tg
22
are conducted to the intrathyroidal aa pool. The transcription of specific thyroid genes and thyroid
23
function is under the control of thyrotropin (TSH) via its protein G-coupled receptor (TSHR).
24
GDP: guanosine diphosphate.
33
1
Figure 2. Structural organization of the thyroglobulin homodimer. The repetitive units of
2
thyroglobulin (TG) TG type 1, TG type 2 and TG type 3 and the cholinesterase-like (ChEL)
3
domain, drawn to scale, are represented by boxes. N-terminal T4 (coupling of a donor DIT149 with
4
the acceptor DIT24) and N-terminal T3 (coupling of a MIT2766 at the antepenultimate residue of one
5
TG monomer with the antepenultimate DIT2766 in the opposite monomer) forming sites are shown.
6
The amino acid sequences (represented by single-letter code) of TG are indicated below of the
7
respective protein schematic diagrams. The mutated residues are shown in red and double
8
underlined (see Table 1 and 2). The amino acid position is designated according to the reference
9
sequences reported in NCBI accession number: NM_003235.5 The amino acid (aa) positions are
10
numbered including the 19 amino acid of the signal peptide (SP).
11 12
Figure 3. Schematic model of the N-glycosylation pathway of thyroglobulin. First, the nascent
13
polypeptide acquires the N-glycans by action of an oligosaccharyltransferase that transfers Glc3
14
(□), Man9 (○), GlcNAc2 (■) from dolichol phosphate precursor to the consensus N-glycosylation
15
sites (Asn, asparagine). Next, I and II α-glucosidases trim two glucose residues creating a
16
monoglucosylated N-glycan, later the third glucose is removed by α-glucosidase II. After removal
17
of one mannose residue in the ER, α-mannosidase I, the thyroglobulin is exported to the Golgi.
18
Finally, Golgi I and II α-mannosidases remove mannose residues and several N-acetylglucosamine
19
glycosyltransferases (GnT) may catalyze branching and elongation of the carbohydrate chains,
20
resulting in hybrid-type or complex-type N-glycans. N-glycans are elongated by addition of
21
galactose, fucose and sialic acid residues catalyzed by galactosyltransferases, fucosyltransferases
22
and sialyltransferases, respectively.
23 24
Figure 4. Structural organization of the thyroid peroxidase. The signal peptide (SP),
25
complement control protein (CCP) domain, calcium-binding EGF (EGF-Ca2+) domain, animal
34
1
haem peroxidase (An peroxidase) region and heme-binding sites (proximal histidine (H494), distal
2
histidine (H239), aspartic acid (D238), glutamic acid (E399), drawn to scale, are shown. The arrows
3
indicate where the mutated residues are located. The amino acid sequences (represented by single-
4
letter code) of thyroid peroxidase protein are indicated below of the respective protein schematic
5
diagrams. The mutated residues are shown in red and double underlined. The amino acid position is
6
designated according to the reference sequences reported in NCBI accession number:
7
NM_000547.5. The amino acid positions are numbered including the 18 amino acid of the signal
8
peptide (SP). ECD: extracellular domain, TM: transmembrane domain, ICD: intracellular domain,
9
aa: amino acid.
10 11
Figure 5. Structural organization of the DUOX2. The peroxidase-like and gp91phox/NOX2-like
12
domains, glycosilation sites (NXS/T site), EF-hand calcium binding motifs and FAD and NADPH
13
binding sites, drawn to scale, are shown. The seven alpha helice transmembrane domains are
14
represented by boxes (I, II, III, IV, V, VI and VII). The arrows indicate where the mutated residues
15
are located. The amino acid sequences (represented by single-letter code) of DUOX2 are indicated
16
below of the respective protein schematic diagrams. The mutated residues are shown in red and
17
double underlined. The amino acid position is designated according to the reference sequences
18
reported in NCBI accession number: NM_014080.4. The amino acid (aa) positions are numbered
19
including the 21 amino acid of the signal peptide (SP).
20 21
Figure 6. Folding model of DUOX2-DUOXA2. Formation of intermolecular disulfide bonds
22
between DUOX2 and DUOXA2 and trafficking of the DUOX2-DUOXA2 heterodimer to the
23
apical plasma membrane of the thyrocyte are shown. DUOX2 requires a DUOXA2 to exit from the
24
endoplasmic reticulum (ER) and reach the apical plasma membrane, through the Golgi apparatus.
25
The oxidative folding of DUOX2 and DUOXA2 takes place in the ER, generating an
35
1
intramolecular disulfide bridge in DUOX2 (between cysteine124 and cysteine1162) and
2
intermolecular disulfide bridges between the DUOX2 (cysteine568 and cysteine582) and DUOXA2
3
(cysteine167 and cysteine233). Both proteins form a stable heterodimer complex at the cell surface
4
which is fundamental for the reactive oxygen species production. N : N-glycosylation site,
5
cysteine, NADPH: binding site for the NADPH, FAD: binding site for the FAD.
:
6 7
Figure 7. Thirteen-TMS human Na+/I- symporter secondary structure model. Schematic
8
representation adapted from De la Vieja et al. [2004, 2005] and Spitzweg and Morris [2010] is
9
showed. The arrows indicate where the mutated residues are located. The amino acid sequences
10
(represented by single-letter code) of Na+/I- symporter are indicated below of the respective protein
11
schematic diagrams. The mutated residues are shown in red and double underlined.
12
glycosylation site, TM: transmembrane domain.
N : N-
13 14
Figure 8. Twelve-TMS human pendrin secondary structure model. Schematic representation
15
adapted from Dossena et al. [2009] is showed. The arrows indicate where the mutated residues are
16
located. The amino acid sequences (represented by single-letter code) of pendrin are indicated
17
below of the respective protein schematic diagrams. The mutated residues are shown in red and
18
double underlined. STAS (sulfate transporter and antisigma factor antagonist) domain is
19
underlined. N : N-glycosylation site, TM: transmembrane domain.
20 21
Figure 9. Twelve-TMS human SLC26A7 secondary structure model. Schematic representation
22
adapted from Ishii et al. [2019] is showed. The arrows indicate where the mutated residues are
23
located. The amino acid sequences (represented by single-letter code) of the SLC26A7 are
24
indicated below of the respective protein schematic diagrams. The mutated residues are shown in
36
1
red and double underlined. The STAS (sulfate transporter and antisigma factor antagonist) domain
2
is underlined. N : N-glycosylation site, TM: transmembrane domain.
3
37
1
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Table 1. Thyroglobulin missense mutations involved in the wild type cysteine residues Exon position
Nucleotide position
Amino acid position
References
Exon 5 Exon 5 Exon 5 Exon 10 Exon 14 Exon 17
c.479G>C c.548G>A c.580T>G c.2177G>A c.3229T>C c.3790T>C
p.C160S p.C183Y p.C194G p.C726Y p.C1077R p.C1264R
Exon 17 Exon 21 Exon 21 Exon 22 Exon 24 Exon 31 Exon 31 Exon 33
c.3842G>A c.4426T>C c.4478G>A c.4529G>T c.4820G>T c.5690G>A c.5986T>A
p.C1281Y p.C1476R p.C1493Y p.C1510F p.C1607F p.C1897Y p.C1904G p.C1996S
Exon 33 Exon 33 Exon 37
c.6000C>G c.6017G>A c.6461G>A
p.C2000W p.C2006Y p.C2154Y
Nicholas et al., 2016 Caputo et al., 2007 Hishinuma et al., 2006 Nicholas et al., 2016 Hishinuma et al., 2005; Hishinuma et al., 2006 Baryshev et al., 2004 ; Hishinuma et al., 1999, 2005, 2006; Kanou et al., 2007; Narumi et al., 2011 Citterio et al., 2013 Zou et al., 2018 Nicholas et al., 2016 Narumi et al., 2011 Hishinuma et al., 2006 Hishinuma et al., 2006; Kitanaka et al., 2006 Hu et al., 2016 Baryshev et al., 2004; Hishinuma et al., 1999, 2005, 2006 Citterio et al., 2013 Hishinuma et al., 2006 Hishinuma et al., 2006
The nucleotide position is designated according to TG mRNA reference sequences reported in NCBI Accession Number: NM_ 003235.5. The A of the ATG of the initiator methionine codon is denoted nucleotide +1. The amino acid positions are numbered including the 19 amino acid of the signal peptide.
Table 2. Thyroglobulin missense mutations in the ChEL domain Exon position
Nucleotide position
Amino acid position
References
Exon 38
c.6701C>A
p.A2234D
Exon 38
c.6725G>A
p.R2242H
Exon 39 Exon 40 Exon 40
c.6853A>G c.6956G>A c.7007G>A
p.N2285D p.G2319D p.R2336Q
Exon 40 Exon 41 Exon 41 Exon 41 Exon 41
c.7021G>A c.7084G>C c.7093T>C c.7121G>T c.7123G>A
p.G2341S p.A2362P p.W2365R p.G2374V p.G2375R
Exon 41 Exon 42
c.7182C>G c.7364G>A
p.I2394M p.R2455H
Exon 43 Exon 44 Exon 44
c.7411G>T c.7640T>A c.7753C>T
p.A2471S p.L2547Q p.R2585W
Exon 45
c.7847A>T
p.N2616I
Exon 47 Exon 47
c.8054G>T c.8137G>A
p.W2685L p.A2713T
Caputo et al., 2007; Citterio et al., 2013; Machiavelli et al., 2010; Pardo et al. 2008, 2009; Santos-Silva et al., 2019 Caron et al., 2003; Machiavelli et al., 2010; Raef et al., 2010; Zou et al., 2018 Makretskaya et al., 2018 Hishinuma et al., 2006 Hishinuma et al., 2006; Kitanaka et al., 2006; Siffo et al., 2018 Watanabe et al. 2018 Siffo et al., 2018 Siffo et al., 2018 Hishinuma et al., 2006 Hishinuma et al., 2005, 2006; Kanou et al.; 2007; Long et al., 2018 Sun et al., 2018 Hu et al., 2016; Long et al., 2018, Yu et al., 2018 Sun et al., 2018 Nicholas et al., 2016 Hu et al., 2016; Jiang et al., 2016; Sun et al., 2018 Hu et al., 2016; Long et al., 2018, Sun et al. 2018, Yu et al., 2018 Nicholas et al., 2016 Long et al., 2018
The nucleotide position is designated according to TG mRNA reference sequences reported in NCBI Accession Number: NM_ 003235.5. The A of the ATG of the initiator methionine codon is denoted nucleotide +1. The amino acid positions are numbered including the 19 amino acid of the signal peptide.
I-
TSH T3 T4 T T4 4 T4
TSHR Basolateral membrane
G protein
MCT8 T3 T
4
GDP
T4 T4 T4
T3 Tg
NIS IYD-1
KCNQ1-KCNE2 K+ channel
Thyroid transcription factors
T4
Na+/K+ ATPase
MIT
I-
DIT T4
Transcription
aa mRNA
T4
IYD-1
Traduction
Iodide recycled
I-
aa pool
TG
TG
Anoctamin-1
Pendrin TPO DUOX1/2
SLC26A7
Apical membrane
Folicular lumen
TG
I+
Figure 1
Iodination of tyrosines Coupling of iodotyrosines
Oxidation of
I-
IH2O2
DUOXA1/2
2130 2188 2211
1996
1893
1724
2736 2768 aa
Region IV
Region III
1511 1566 1603
1146 1211
1074
922
605 659 727
298
359
161
1 31 93
1456 1470 1487
Region II
Region I
ChEL domain Hinge
Linker
NH2
COOH
SP 1-1 1-2
1-3
1-4
1-5 1-6
1-7
1-8
1-9 1-10
TG type 1
2-1-2-3 1-11
3-a1
3-b1
3-a2
3-b2 3-a3
TG type 3
TG type 2
Spacer 3
Spacer 1
NH2
2736 2768
2130 2188 2211
1996
1893
1724
1511 1566 1603
1456 1470 1487
1146 1211
1074
922
605 659 727
298
359
161
1 31 93
Spacer 2
COOH
SP 1-1 1-2
1-3
1-4
1-5 1-6
1-7
1-8
SP
Figure 2
1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761
1-9 1-10
2-1-2-3 1-11
3-a1
3-b1
3-a2
3-b2 3-a3
T4
MALVLEIFTLLASICWVSANIFEYQVDAQPLRPCELQRETAFLKQADYVPQCAEDGSFQT VQCQNDGRSCWCVGANGSEVLGSRQPGRPVACLSFCQLQKQQILLSGYINSTDTSYLPQC QDSGDYAPVQCDVQQVQCWCVDAEGMEVYGTRQLGRPKRCPRSCEIRNRRLLHGVGDKSP PQCSAEGEFMPVQCKFVNTTDMMIFDLVHSYNRFPDAFVTFSSFQRRFPEVSGYCHCADS QGRELAETGLELLLDEIYDTIFAGLDLPSTFTETTLYRILQRRFLAVQSVISGRFRCPTK CEVERFTATSFGHPYVPSCRRNGDYQAVQCQTEGPCWCVDAQGKEMHGTRQQGEPPSCAE GQSCASERQQALSRLYFGTSGYFSQHDLFSSPEKRWASPRVARFATSCPPTIKELFVDSG LLRPMVEGQSQQFSVSENLLKEAIRAIFPSRGLARLALQFTTNPKRLQQNLFGGKFLVNV GQFNLSGALGTRGTFNFSQFFQQLGLASFLNGGRQEDLAKPLSVGLDSNSSTGTPEAAKK DGTMNKPTVGSFGFEINLQENQNALKFLASLLELPEFLLFLQHAISVPEDVARDLGDVME TVLSSQTCEQTPERLFVPSCTTEGSYEDVQCFSGECWCVNSWGKELPGSRVRGGQPRCPT DCEKQRARMQSLMGSQPAGSTLFVPACTSEGHFLPVQCFNSECYCVDAEGQAIPGTRSAI GKPKKCPTPCQLQSEQAFLRTVQALLSNSSMLPTLSDTYIPQCSTDGQWRQVQCNGPPEQ VFELYQRWEAQNKGQDLTPAKLLVKIMSYREAASGNFSLFIQSLYEAGQQDVFPVLSQYP SLQDVPLAALEGKRPQPRENILLEPYLFWQILNGQLSQYPGSYSDFSTPLAHFDLRNCWC VDEAGQELEGMRSEPSKLPTCPGSCEEAKLRVLQFIRETEEIVSASNSSRFPLGESFLVA KGIRLRNEDLGLPPLFPPREAFAEQFLRGSDYAIRLAAQSTLSFYQRRRFSPDDSAGASA LLRSGPYMPQCDAFGSWEPVQCHAGTGHCWCVDEKGGFIPGSLTARSLQIPQCPTTCEKS RTSGLLSSWKQARSQENPSPKDLFVPACLETGEYARLQASGAGTWCVDPASGEELRPGSS SSAQCPSLCNVLKSGVLSRRVSPGYVPACRAEDGGFSPVQCDQAQGSCWCVMDSGEEVPG TRVTGGQPACESPRCPLPFNASEVVGGTILCETISGPTGSAMQQCQLLCRQGSWSVFPPG PLICSLESGRWESQLPQPRACQRPQLWQTIQTQGHFQLQLPPGKMCSADYADLLQTFQVF ILDELTARGFCQIQVKTFGTLVSIPVCNNSSVQVGCLTRERLGVNVTWKSRLEDIPVASL PDLHDIERALVGKDLLGRFTDLIQSGSFQLHLDSKTFPAETIRFLQGDHFGTSPRTWFGC SEGFYQVLTSEASQDGLGCVKCPEGSYSQDEECIPCPVGFYQEQAGSLACVPCPVGRTTI SAGAFSQTHCVTDCQRNEAGLQCDQNGQYRASQKDRGSGKAFCVDGEGRRLPWWETEAPL EDSQCLMMQKFEKVPESKVIFDANAPVAVRSKVPDSEFPVMQCLTDCTEDEACSFFTVST TEPEISCDFYAWTSDNVACMTSDQKRDALGNSKATSFGSLRCQVKVRSHGQDSPAVYLKK GQGSTTTLQKRFEPTGFQNMLSGLYNPIVFSASGANLTDAHLFCLLACDRDLCCDGFVLT QVQGGAIICGLLSSPSVLLCNVKDWMDPSEAWANATCPGVTYDQESHQVILRLGDQEFIK SLTPLEGTQDTFTNFQQVYLWKDSDMGSRPESMGCRKDTVPRPASPTEAGLTTELFSPVD LNQVIVNGNQSLSSQKHWLFKHLFSAQQANLWCLSRCVQEHSFCQLAEITESASLYFTCT LYPEAQVCDDIMESNAQGCRLILPQMPKALFRKKVILEDKVKNFYTRLPFQKLMGISIRN KVPMSEKSISNGFFECERRCDADPCCTGFGFLNVSQLKGGEVTCLTLNSLGIQMCSEENG GAWRILDCGSPDIEVHTYPFGWYQKPIAQNNAPSFCPLVVLPSLTEKVSLDSWQSLALSS VVVDPSIRHFDVAHVSTAATSNFSAVRDLCLSECSQHEACLITTLQTQPGAVRCMFYADT QSCTHSLQGQNCRLLLREEATHIYRKPGISLLSYEASVPSVPISTHGRLLGRSQAIQVGT SWKQVDQFLGVPYAAPPLAERRFQAPEPLNWTGSWDASKPRASCWQPGTRTSTSPGVSED CLYLNVFIPQNVAPNASVLVFFHNTMDREESEGWPAIDGSFLAAVGNLIVVTASYRVGVF GFLSSGSGEVSGNWGLLDQVAALTWVQTHIRGFGGDPRRVSLAADRGGADVASIHLLTAR ATNSQLFRRAVLMGGSALSPAAVISHERAQQQAIALAKEVSCPMSSSQEVVSCLRQKPAN VLNDAQTKLLAVSGPFHYWGPVIDGHFLREPPARALKRSLWVEVDLLIGSSQDDGLINRA KAVKQFEESRGRTSSKTAFYQALQNSLGGEDSDARVEAAATWYYSLEHSTDDYASFSRAL ENATRDYFIICPIIDMASAWAKRARGNVFMYHAPENYGHGSLELLADVQFALGLPFYPAY EGQFSLEEKSLSLKIMQYFSHFIRSGNPNYPYEFSRKVPTFATPWPDFVPRAGGENYKEF SELLPNRQGLKKADCSFWSKYISSLKTSADGAKGGQSAESEEEELTAGSGLREDLLSLQE PGSKTYSK
T3
ChEL domain
Asn
GnT-II
NH2
NH2
α-mannosidase II
α-mannosidase I
NH2
SH
Asn
Asn
GnT-I
Complex-type
SH
SH
Asn
Asn
α-mannosidase I
NH2
Asn
α-glucosidase II
NH2
SH
SH
NH2
SH
Asn
Hybrid-type
NH2
High-mannose type
α-glucosidase II
GnT-IV
α-glucosidase I
GnT-IV
GnT-V GnT-VI
SH
Figure 3
Asn
Asn
NH2
NH2
NH2
Asn
SH
p.R665W p.D574-L575del
p.C800R
p.C838S
p.R175Q
933 aa
pH 494
dH 239
839 847 872
740
1 19
p.G771R
796
p.G533C
p.A397Pfs*76
p.S131P
NH2
COOH SP
D 238
E 399 ECD
An peroxidase
CCP EGF-Ca2+
TM
SP 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901
MRALAVLSVTLVMACTEAFFPFISRGKELLWGKPEESRVSSVLEESKRLVDTAMYATMQR NLKKRGILSPAQLLSFSKLPEPTSGVIARAAEIMETSIQAMKRKVNLKTQQSQHPTDALS EDLLSIIANMSGCLPYMLPPKCPNTCLANKYRPITGACNNRDHPRWGASNTALARWLPPV YEDGFSQPRGWNPGFLYNGFPLPPVREVTRHVIQVSNEVVTDDDRYSDLLMAWGQYIDHD IAFTPQSTSKAAFGGGADCQMTCENQNPCFPIQLPEEARPAAGTACLPFYRSSAACGTGD QGALFGNLSTANPRQQMNGLTSFLDASTVYGSSPALERQLRNWTSAEGLLRVHARLRDSG RAYLPFVPPRAPAACAPEPGIPGETRGPCFLAGDGRASEVPSLTALHTLWLREHNRLAAA LKALNAHWSADAVYQEARKVVGALHQIITLRDYIPRILGPEAFQQYVGPYEGYDSTANPT VSNVFSTAAFRFGHATIHPLVRRLDASFQEHPDLPGLWLHQAFFSPWTLLRGGGLDPLIR GLLARPAKLQVQDQLMNEELTERLFVLSNSSTLDLASINLQRGRDHGLPGYNEWREFCGL PRLETPADLSTAIASRSVADKILDLYKHPDNIDVWLGGLAENFLPRARTGPLFACLIGKQ MKALRDGDWFWWENSHVFTDAQRRELEKHSLSRVICDNTGLTRVPMDAFQVGKFPEDFES CDSITGMNLEAWRETFPQDDKCGFPESVENGDFVHCEESGRRVLVYSCRHGYELQGREQL TCTQEGWDFQPPLCKDVNECADGAHPPCHASARCRNTKGGFQCLCADPYELGDDGRTCVD SGRLPRVTWISMSLAALLIGGFAGLTSTVICRWTRTGTKSTLPISETGGGTPELRCGKHQ AVGTSPQRAAAQDSEQESAGMEGRDTHRLPRAL
Figure 4
ICD
SP
I
Peroxidase-like domain
EF-hand calcium biding motifs
heme
II III
1548 aa
1129 1186 1224 1245
855
899
1042 1077
heme
Ca2
Ca2
NH2
Ca Ca22
819
p.C568R 602
NXS/T site 537
NXS/T site 455
NXS/T site 100
1 22
p.Q36H
p.D506N p.K530* p.R1110Q p.L1160del p.R1334W p.F966Sfs*29 p.R376W p.L1343F p.E879K NXS/T site 348 NXS/T site 382
p.C124*
COOH
FAD NADPH IV V VI VII binding binding site site
Alpha helice transmembrane domain gp91phox/NOX2-like domain
SP 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501
MLRARPEALMLLGALLTGSLGPSGNQDALSLPWEVQRYDGWFNNLRHHERGAVGCRLQRR VPANYADGVYQALEEPQLPNPRRLSNAATRGIAGLPSLHNRTVLGVFFGYHVLSDVVSVE TPGCPAEFLNIRIPPGDPVFDPDQRGDVVLPFQRSRWDPETGRSPSNPRDLANQVTGWLD GSAIYGSSHSWSDALRSFSGGQLASGPDPAFPRDSQNPLLMWAAPDPATGQNGPRGLYAF GAERGNREPFLQALGLLWFRYHNLWAQRLARQHPDWEDEELFQHARKRVIATYQNIAVYE WLPSFLQKTLPEYTGYRPFLDPSISPEFVVASEQFFSTMVPPGVYMRNASCHFRKVLNKG FQSSQALRVCNNYWIRENPNLNSTQEVNELLLGMASQISELEDNIVVEDLRDYWPGPGKF SRTDYVASSIQRGRDMGLPSYSQALLAFGLDIPRNWSDLNPNVDPQVLEATAALYNQDLS QLELLLGGLLESHGDPGPLFSAIVLDQFVRLRDGDRYWFENTRNGLFSKKEIEDIRNTTL RDVLVAVINIDPSALQPNVFVWHKGAPCPQPKQLTTDGLPQCAPLTVLDFFEGSSPGFAI TIIALCCLPLVSLLLSGVVAYFRGREHKKLQKKLKESVKKEAAKDGVPAMEWPGPKERSS PIIIQLLSDRCLQVLNRHLTVLRVVQLQPLQQVNLILSNNRGCRTLLLKIPKEYDLVLLF SSEEERGAFVQQLWDFCVRWALGLHVAEMSEKELFRKAVTKQQRERILEIFFRHLFAQVL DINQADAGTLPLDSSQKVREALTCELSRAEFAESLGLKPQDMFVESMFSLADKDGNGYLS FREFLDILVVFMKGSPEDKSRLMFTMYDLDENGFLSKDEFFTMMRSFIEISNNCLSKAQL AEVVESMFRESGFQDKEELTWEDFHFMLRDHDSELRFTQLCVKGGGGGGNGIRDIFKQNI SCRVSFITRTPGERSHPQGLGPPAPEAPELGGPGLKKRFGKKAAVPTPRLYTEALQEKMQ RGFLAQKLQQYKRFVENYRRHIVCVAIFSAICVGVFADRAYYYGFASPPSDIAQTTLVGI ILSRGTAASVSFMFSYILLTMCRNLITFLRETFLNRYVPFDAAVDFHRWIAMAAVVLAIL HSAGHAVNVYIFSVSPLSLLACIFPNVFVNDGSKLPQKFYWWFFQTVPGMTGVLLLLVLA IMYVFASHHFRRRSFRGFWLTHHLYILLYALLIIHGSYALIQLPTFHIYFLVPAIIYGGD KLVSLSRKKVEISVVKAELLPSGVTYLQFQRPQGFEYKSGQWVRIACLALGTTEYHPFTL TSAPHEDTLSLHIRAVGPWTTRLREIYSSPKGNGCAGYPKLYLDGPFGEGHQEWHKFEVS VLVGGGIGVTPFASILKDLVFKSSLGSQMLCKKIYFIWVTRTQRQFEWLADIIQEVEEND HQDLVSVHIYVTQLAEKFDLRTTMLYICERHFQKVLNRSLFTGLRSITHFGRPPFEPFFN SLQEVHPQVRKIGVFSCGPPGMTKNVEKACQLVNRQDRAHFMHHYENF
Figure 5
N
Disulfide bridge
.
2 O 2-
2 O2
N
N
N
N
NH2
N NH2
Apical membrane
N
H 2 O2
N
Colloid
Cytosol COOH
COOH
DUOXA2/ DUOX2
NADP+ + H +
NADPH
N
N
NH2
Disulfide bridge
N
N
N
N
NH2
N N
Golgi Apparatus
DUOXA2/ DUOX2 COOH
COOH
N
N
NH2
Disulfide bridge
N
N
N
N
NH2
N N
Endoplasmic Reticulum COOH
COOH
N
N
NH2
N
N
N
NH2
N
N
N
DUOXA2
Figure 6
COOH
DUOX2
COOH
N N Extracellular N
NH2 1
p.S509Rfs*7 11
TM1
79
TM2
86
TM3
157
163
TM4
TM5
217
TM6
241
TM7
308
340
TM8
TM9
411
417
468
522
TM10 TM11 TM12
TM13
p.G543E 37
54
111
136
182
191
260
286
368
388
438
p.V270E
444 550
intracellular p.A439_P443del
p.R124H
COOH 643
1 61 121 181 241 301 361 421 481 541 601
MEAVETGERPTFGAWDYGVFALMLLVSTGIGLWVGLARGGQRSAEDFFTGGRRLAALPVG LSLSASFMSAVQVLGVPSEAYRYGLKFLWMCLGQLLNSVLTALLFMPVFYRLGLTSTYEY LEMRFSRAVRLCGTLQYIVATMLYTGIVIYAPALILNQVTGLDIWASLLSTGIICTFYTA VGGMKAVVWTDVFQVVVMLSGFWVVLARGVMLVGGPRQVLTLAQNHSRINLMDFNPDPRS RYTFWTFVVGGTLVWLSMYGVNQAQVQRYVACRTEKQAKLALLINQVGLFLIVSSAACCG IVMFVFYTDCDPLLLGRISAPDQYMPLLVLDIFEDLPGVPGLFLACAYSGTLSTASTSIN AMAAVTVEDLIKPRLRSLAPRKLVIISKGLSLIYGSACLTVAALSSLLGGGVLQGSFTVM GVISGPLLGAFILGMFLPACNTPGVLAGLGAGLALSLWVALGATLYPPSEQTMRVLPSSA ARCVALSVNASGLLDPALLPANDSSRAPSSGMDASRPALADSFYAISYLYYGALGTLTTV LCGALISCLTGPTKRSTLAPGLLWWDLARQTASVAPKEEVAILDDNLVKGPEELPTGNKK PPGFLPTNEDRLFFLGQKELEGAGSWTPCVGHDGGRDQQETNL
Figure 7
p.L236P p.T410M
p.V138F
N
N Extracellular
107
113 154
185
234
268
318
348
408
421
466
p.G102R TM1 87
TM2 131
TM3 136
TM4 207
TM5 213
TM6 286
TM7 295
TM8 368
TM9 384
478
TM10 TM11 TM12 443
448
504
intracellular 534
p.Y556C 1
NH2
p.T193I
p.L445W p.Q446R
STAS domain COOH 780
1 61 121 181 241 301 361 421 481 541 601 661 721
730
MAAPGGRSEPPQLPEYSCSYMVSRPVYSELAFQQQHERRLQERKTLRESLAKCCSCSRKR AFGVLKTLVPILEWLPKYRVKEWLLSDVISGVSTGLVATLQGMAYALLAAVPVGYGLYSA FFPILTYFIFGTSRHISVGPFPVVSLMVGSVVLSMAPDEHFLVSSSNGTVLNTTMIDTAA RDTARVLIASALTLLVGIIQLIFGGLQIGFIVRYLADPLVGGFTTAAAFQVLVSQLKIVL NVSTKNYNGVLSIIYTLVEIFQNIGDTNLADFTAGLLTIVVCMAVKELNDRFRHKIPVPI PIEVIVTIIATAISYGANLEKNYNAGIVKSIPRGFLPPELPPVSLFSEMLAASFSIAVVA YAIAVSVGKVYATKYDYTIDGNQEFIAFGISNIFSGFFSCFVATTALSRTAVQESTGGKT QVAGIISAAIVMIAILALGKLLEPLQKSVLAAVVIANLKGMFMQLCDIPRLWRQNKIDAV IWVFTCIVSIILGLDLGLLAGLIFGLLTVVLRVQFPSWNGLGSIPSTDIYKSTKNYKNIE EPQGVKILRFSSPIFYGNVDGFKKCIKSTVGFDAIRVYNKRLKALRKIQKLIKSGQLRAT KNGIISDAVSTNNAFEPDEDIEDLEELDIPTKEIEIQVDWNSELPVKVNVPKVPIHSLVL DCGAISFLDVVGVRSLRVIVKEFQRIDVNVYFASLQDYVIEKLEQCGFFDDNIRKDTFFL TVHDAILYLQNQVKSQEGQGSILETITLIQDCKDTLELIETELTEEELDVQDEAMRTLAS
Figure 8
p.I309_E311delinsM p.R227*
N 66
71 121
TM1
TM2
46
91
TM3 101
Extracellular
N 142
TM4 162
198
223
TM5 178
TM6 243
279
TM7 259
307
TM8 327
363
TM9 343
375
427
448
TM10 TM11 TM12 395
407
468
intracellular 1
NH2
p.Q500*
495
STAS domain
p.F631Lfs*8
631 656
1 61 121 181 241 301 361 421 481 541 601
COOH
MTGAKRKKKSMLWSKMHTPQCEDIIQWCRRRLPILDWAPHYNLKENLLPDTVSGIMLAVQ QVTQGLAFAVLSSVHPVFGLYGSLFPAIIYAIFGMGHHVATGTFALTSLISANAVERIVP QNMQNLTTQSNTSVLGLSDFEMQRIHVAAAVSFLGGVIQVAMFVLQLGSATFVVTEPVIS AMTTGAATHVVTSQVKYLLGMKMPYISGPLGFFYIYAYVFENIKSVRLEALLLSLLSIVV LVLVKELNEQFKRKIKVVLPVDLVLIIAASFACYCTNMENTYGLEVVGHIPQGIPSPRAP PMNILSAVITEAFGVALVGYVASLALAQGSAKKFKYSIDDNQEFLAHGLSNIVSSFFFCI PSAAAMGRTAGLYSTGAKTQVACLISCIFVLIVIYAIGPLLYWLPMCVLASIIVVGLKGM LIQFRDLKKYWNVDKIDWGIWVSTYVFTICFAANVGLLFGVVCTIAIVIGRFPRAMTVSI KNMKEMEFKVKTEMDSETLQQVKIISINNPLVFLNAKKFYTDLMNMIQKENACNQPLDDI SKCEQNTLLNSLSNGNCNEEASQSCPNEKCYLILDCSGFTFFDYSGVSMLVEVYMDCKGR SVDVLLAHCTASLIKAMTYYGNLDSEKPIFFESVSAAISHIHSNKNLSKLSDHSEV
Figure 9
Highlights
• Congenital hypothyroidism is the most frequent endocrine disease in children. • Mutations in the candidate genes were identified and functionally characterized. • Protein folding disorders are caused by mutations in thyroid genes. • Misfolded of thyroid proteins causes retention within the endoplasmic reticulum. • The retention of misfolded proteins activates the ERAD mechanism.