RESUMEN
The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones-the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient1,2. For nearly 50 years before its molecular identification3, NIS was the molecule at the centre of the single most effective internal radiation cancer therapy: radioiodide (131I-) treatment for thyroid cancer2. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency2. Here we report three structures of rat NIS, determined by single-particle cryo-electron microscopy: one with no substrates bound; one with two Na+ and one I- bound; and one with one Na+ and the oxyanion perrhenate bound. Structural analyses, functional characterization and computational studies show the substrate-binding sites and key residues for transport activity. Our results yield insights into how NIS selects, couples and translocates anions-thereby establishing a framework for understanding NIS function-and how it transports different substrates with different stoichiometries and releases substrates from its substrate-binding cavity into the cytosol.
Asunto(s)
Yoduros , Sodio , Simportadores , Animales , Ratas , Microscopía por Crioelectrón , Yoduros/metabolismo , Sodio/metabolismo , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestructura , Sitios de Unión , Especificidad por Sustrato , Transporte IónicoRESUMEN
Obesity is a major health problem worldwide, given its growing incidence and its association with a variety of comorbidities. Weight gain results from an increase in energy intake without a concomitant increase in energy expenditure. To combat the obesity epidemic, many studies have focused on the pathways underlying satiety and hunger signaling, while other studies have concentrated on the mechanisms involved in energy expenditure, most notably adaptive thermogenesis. Hypothyroidism in humans is typically associated with a decreased basal metabolic rate, lower energy expenditure, and weight gain. However, hypothyroid mouse models have been reported to have a leaner phenotype than euthyroid controls. To elucidate the mechanism underlying this phenomenon, we used a drug-free mouse model of hypothyroidism: mice lacking the sodium/iodide symporter (NIS), the plasma membrane protein that mediates active iodide uptake in the thyroid. In addition to being leaner than euthyroid mice, owing in part to reduced food intake, these hypothyroid mice show signs of compensatory up-regulation of the skeletal-muscle adaptive thermogenic marker sarcolipin, with an associated increase in fatty acid oxidation (FAO). Neither catecholamines nor thyroid-stimulating hormone (TSH) are responsible for sarcolipin expression or FAO stimulation; rather, thyroid hormones are likely to negatively regulate both processes in skeletal muscle. Our findings indicate that hypothyroidism in mice results in a variety of metabolic changes, which collectively lead to a leaner phenotype. A deeper understanding of these changes may make it possible to develop new strategies against obesity.
Asunto(s)
Hipotiroidismo/metabolismo , Músculo Esquelético/metabolismo , Termogénesis/fisiología , Animales , Modelos Animales de Enfermedad , Ingestión de Alimentos/fisiología , Masculino , Ratones , Ratones Noqueados , Proteínas Musculares/metabolismo , Fenotipo , Proteolípidos/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
Active iodide (I-) transport in both the thyroid and some extrathyroidal tissues is mediated by the Na+/I- symporter (NIS). In the thyroid, NIS-mediated I- uptake plays a pivotal role in thyroid hormone (TH) biosynthesis. THs are key during embryonic and postembryonic development and critical for cell metabolism at all stages of life. The molecular characterization of NIS in 1996 and the use of radioactive I- isotopes have led to significant advances in the diagnosis and treatment of thyroid cancer and provide the molecular basis for studies aimed at extending the use of radioiodide treatment in extrathyroidal malignancies. This review focuses on the most recent findings on I- homeostasis and I- transport deficiency-causing NIS mutations, as well as current knowledge of the structure/function properties of NIS and NIS regulatory mechanisms. We also discuss employing NIS as a reporter gene using viral vectors and stem cells in imaging, diagnostic, and therapeutic procedures.
Asunto(s)
Yoduros/metabolismo , Simportadores/metabolismo , Animales , Genes Reporteros/genética , Humanos , Simportadores/genética , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Glándula Tiroides/fisiología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/fisiopatologíaRESUMEN
Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergistically with lowered insulin secretion, increases serum glucose levels, which stimulates de novo lipogenesis (DNL) in the liver. Up-regulation of DNL also contributes to NAFLD. In contrast, severely hypothyroid mice show down-regulation of TH signaling in their livers and profound suppression of adipose tissue lipolysis, which decreases delivery of FAs to the liver. The resulting lack of substrates for triglyceride esterification protects severely hypothyroid mice against NAFLD. Our findings demonstrate that NAFLD occurs when TH levels are mildly reduced, but, paradoxically, not when they are severely reduced. Our results show that the pathogenesis of hypothyroidism-induced NAFLD is both intra- and extrahepatic; they also reveal key metabolic differences between mild and severe hypothyroidism.
Asunto(s)
Hipotiroidismo/complicaciones , Resistencia a la Insulina , Células Secretoras de Insulina/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Tejido Adiposo/metabolismo , Tejido Adiposo/fisiopatología , Animales , Modelos Animales de Enfermedad , Hipotiroidismo/etiología , Insulina/metabolismo , Secreción de Insulina , Metabolismo de los Lípidos , Lipólisis/fisiología , Hígado/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/complicaciones , Simportadores/genéticaRESUMEN
The sodium/iodide symporter (NIS) mediates active I(-) transport in the thyroid-the first step in thyroid hormone biosynthesis-with a 2 Na(+): 1 I(-) stoichiometry. The two Na(+) binding sites (Na1 and Na2) and the I(-) binding site interact allosterically: when Na(+) binds to a Na(+) site, the affinity of NIS for the other Na(+) and for I(-) increases significantly. In all Na(+)-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na(+) ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues-S66, D191, Q194, and Q263-are also involved in Na(+) coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I(-) These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance.
Asunto(s)
Yodo/metabolismo , Sodio/metabolismo , Simportadores/metabolismo , Glándula Tiroides/metabolismo , Aminoácidos/química , Aminoácidos/genética , Sitios de Unión , Humanos , Yodo/química , Transporte Iónico/genética , Iones/química , Ligandos , Modelos Moleculares , Simportadores/genética , Glándula Tiroides/química , Hormonas Tiroideas/biosíntesis , Hormonas Tiroideas/metabolismoRESUMEN
BACKGROUND: Structured educative programs have demonstrated their usefulness as a strategy to improve metabolic control in diabetic patients. AIM: To evaluate the effectiveness of a structured educative program for Chilean diabetic patients. MATERIAL AND METHODS: A randomized clinical trial in diabetic patients with glycosylated hemoglobin over 7.5%. One hundred fifteen patients were studied, 59 patients participated in the structured educative program (experimental group) and 56 patients received no structured education (control group). Patients were followed for 12 months. RESULTS: Between baseline and 12 months of follow-up, glycosylated hemoglobin changed from 10.05 to 9.11% in experimental patients and from 9.86 to 9.25% in controls. No significant differences between experimental and control groups in other clinical and metabolic parameters were observed. In the experimental group, glycosylated hemoglobin reductions differed among the different educators who carried out the program. CONCLUSIONS: A structured educative program resulted in a 35% greater reduction in glycosylated hemoglobin levels, compared with a control group. Metabolic control improvement differed between the educators who carried out the program.
Asunto(s)
Diabetes Mellitus/sangre , Hemoglobina Glucada/metabolismo , Educación del Paciente como Asunto/normas , Chile , Femenino , Humanos , Masculino , Persona de Mediana Edad , Educación del Paciente como Asunto/métodos , Evaluación de Programas y Proyectos de SaludRESUMEN
Na(+)/I(-) symporter (NIS)-mediated active accumulation of I(-) in thyrocytes is a key step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report novel findings derived from the thorough characterization of the ITD-causing mutation R124H, located in the second intracellular loop (IL-2). R124H NIS is incompletely glycosylated and colocalizes with endoplasmic reticulum (ER)-resident protein markers. As a result, R124H NIS is not targeted to the plasma membrane and therefore does not mediate any I(-) transport in transfected COS-7 cells. Strikingly, however, the mutant is intrinsically active, as revealed by its ability to mediate I(-) transport in membrane vesicles. Of all the amino acid substitutions we carried out at position 124 (K, D, E, A, W, N and Q), only Gln restored targeting of NIS to the plasma membrane and NIS activity, suggesting a key structural role for the δ-amino group of R124 in the transporter's maturation and cell surface targeting. Using our NIS homology model based on the structure of the Vibrio parahaemolyticus Na(+)/galactose symporter, we propose an interaction between the δ-amino group of either R or Q124 and the thiol group of C440, located in IL-6. We conclude that the interaction between IL-2 and IL-6 is critical for the local folding required for NIS maturation and plasma membrane trafficking.
Asunto(s)
Yoduros/metabolismo , Mutación , Simportadores/genética , Simportadores/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Perros , Humanos , Células de Riñón Canino Madin Darby , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Transporte de Proteínas , Relación Estructura-Actividad , Simportadores/química , TransfecciónRESUMEN
The Na(+)/I(-) symporter (NIS) is a plasma membrane glycoprotein that mediates active I(-) transport in the thyroid, the first step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report a thorough characterization of the ITD-causing NIS mutation in which the sixth intracellular loop residues 439-443 are missing. This mutant protein was intracellularly retained, incompletely glycosylated, and intrinsically inactive. Engineering 5 Ala at positions 439-443 partially recovered cell surface targeting and activity (â¼15%). Strikingly, NIS with the sequence 439-AANAA-443, in which Asn was restored at position 441, was targeted to the plasma membrane and exhibited â¼95% the transport activity of WT NIS. Based on our NIS homology model, we propose that the side chain of N441, a residue conserved throughout most of the SLC5 family, interacts with the main chain amino group of G444, capping the α-helix of transmembrane segment XII and thus stabilizing the structure of the molecule. Our data provide insight into a critical interhelical interaction required for NIS folding and activity.
Asunto(s)
Asparagina/metabolismo , Pliegue de Proteína , Simportadores/química , Simportadores/metabolismo , Secuencia de Aminoácidos , Animales , Asparagina/genética , Sitios de Unión/genética , Transporte Biológico/genética , Células COS , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Immunoblotting , Yodo/metabolismo , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Simportadores/genética , Vesículas Transportadoras/metabolismoRESUMEN
I(-) uptake in the thyroid, the first step in thyroid hormone biosynthesis, is mediated by the Na(+)/I(-) symporter (NIS) with an electrogenic 2Na(+):1I(-) stoichiometry. We have obtained mechanistic information on NIS by characterizing the congenital I(-) transport defect-causing NIS mutant G93R. This mutant is targeted to the plasma membrane but is inactive. Substitutions at position 93 show that the longer the side chain of the neutral residue at this position, the higher the K(m) for the anion substrates. Unlike WT NIS, which mediates symport of Na(+) and the environmental pollutant perchlorate electroneutrally, G93T/N/Q/E/D NIS, strikingly, do it electrogenically with a 21 stoichiometry. Furthermore, G93E/Q NIS discriminate between anion substrates, a discovery with potential clinical relevance. A 3D homology model of NIS based on the structure of the bacterial Na(+)/galactose transporter identifies G93 as a critical player in the mechanism of the transporter: the changes from an outwardly to an inwardly open conformation during the transport cycle use G93 as a pivot.
Asunto(s)
Yoduros/metabolismo , Sodio/metabolismo , Simportadores/metabolismo , Animales , Aniones , Células COS , Línea Celular , Chlorocebus aethiops , Humanos , Modelos Moleculares , Simportadores/químicaRESUMEN
The KCNQ1 α subunit and the KCNE2 ß subunit form a potassium channel in thyroid epithelial cells. Genetic disruption of KCNQ1-KCNE2 causes hypothyroidism in mice, resulting in cardiac hypertrophy, dwarfism, alopecia, and prenatal mortality. Here, we investigated the mechanistic requirement for KCNQ1-KCNE2 in thyroid hormone biosynthesis, utilizing whole-animal dynamic positron emission tomography. The KCNQ1-specific antagonist (-)-[3R,4S]-chromanol 293B (C293B) significantly impaired thyroid cell I(-) uptake, which is mediated by the Na(+)/I(-) symporter (NIS), in vivo (dSUV/dt: vehicle, 0.028 ± 0.004 min(-1); 10 mg/kg C293B, 0.009 ± 0.006 min(-1)) and in vitro (EC(50): 99 ± 10 µM C293B). Na(+)-dependent nicotinate uptake by SMCT, however, was unaffected. Kcne2 deletion did not alter the balance of free vs. thyroglobulin-bound I(-) in the thyroid (distinguished using ClO(4)(-), a competitive inhibitor of NIS), indicating that KCNQ1-KCNE2 is not required for Duox/TPO-mediated I(-) organification. However, Kcne2 deletion doubled the rate of free I(-) efflux from the thyroid following ClO(4)(-) injection, a NIS-independent process. Thus, KCNQ1-KCNE2 is necessary for adequate thyroid cell I(-) uptake, the most likely explanation being that it is prerequisite for adequate NIS activity.
Asunto(s)
Yoduros/metabolismo , Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Glándula Tiroides/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Hipotiroidismo/genética , Canal de Potasio KCNQ1/antagonistas & inhibidores , Canal de Potasio KCNQ1/genética , Ratones , Tomografía de Emisión de Positrones , Canales de Potasio con Entrada de Voltaje/genética , Simportadores/antagonistas & inhibidores , Simportadores/metabolismo , Glándula Tiroides/efectos de los fármacosRESUMEN
Dietary I(-) absorption in the gastrointestinal tract is the first step in I(-) metabolism. Given that I(-) is an essential constituent of the thyroid hormones, its concentrating mechanism is of significant physiological importance. We recently described the expression of the Na(+)/I(-) symporter (NIS) on the apical surface of the intestinal epithelium as a central component of the I(-) absorption system and reported reduced intestinal NIS expression in response to an I(-)-rich diet in vivo. Here, we evaluated the mechanism involved in the regulation of NIS expression by I(-) itself in enterocytes. Excess I(-) reduced NIS-mediated I(-) uptake in IEC-6 cells in a dose- and time-dependent fashion, which was correlated with a reduction of NIS expression at the plasma membrane. Perchlorate, a competitive inhibitor of NIS, prevented these effects, indicating that an increase in intracellular I(-) regulates NIS. Iodide induced rapid intracellular recruitment of plasma membrane NIS molecules and NIS protein degradation. Lower NIS mRNA levels were detected in response to I(-) treatment, although no transcriptional effect was observed. Interestingly, I(-) decreased NIS mRNA stability, affecting NIS translation. Heterologous green fluorescent protein-based reporter constructs revealed a significant repressive effect of the I(-)-targeting NIS mRNA 3 untranslated region. In conclusion, excess I(-) downregulates NIS expression in enterocytes by virtue of a complex mechanism. Our data suggest that I(-) regulates intestinal NIS mRNA expression at the post-transcriptional level as part of an autoregulatory effect of I(-) on its own metabolism.
Asunto(s)
Yoduro de Potasio/farmacología , Simportadores/fisiología , Animales , Línea Celular , Dieta , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Transcripción GenéticaRESUMEN
BACKGROUND & AIMS: Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate gut development and differentiation of intestinal epithelial cells; they are candidate tumor suppressors of colorectal carcinomas. Because the functions of CDX1 and CDX2 in the colonic epithelium are not fully understood, we sought to identify genes that they target. METHODS: We conducted a chromatin immunoprecipitation (ChIP) screen to identify genes that bind the CDX transcription factors. Expression of target genes was analyzed in colon cells and tissues from Cdx1(-/-), Cdx2(+/-), Apc(+/Delta716), and wild-type (control) mice. RESULTS: Using the ChIP screen, we identified solute carrier family 5, member 8 (SLC5A8, also known as SMCT1) as a direct target of CDX1 and CDX2. CDX transcription factors bind to the promoter region of SLC5A8 and transactivate SLC5A8 reporter constructs. Overexpression of Cdx1 or Cdx2 in human colon cancer cell lines induced expression of endogenous SLC5A8, whereas CDX1 and CDX2 knockdowns reduced its level. Consistently, Slc5a8 expression was significantly reduced in colons of Cdx1(-/-) or Cdx2(+/-) mice compared with wild-type mice. Slc5a8 levels were also reduced in colonic adenomatous polyps and hamartomas from Apc(+/Delta716) and Cdx2(+/-) mutant mice, respectively, compared with adjacent normal colon tissues. CONCLUSIONS: CDX1 and CDX2 bind the promoter region of SLC5A8 and up-regulate its expression in cultured cells and in colonic epithelium. SLC5A8 transports monocarboxylates such as pyruvate, lactate, and butyrate; CDX1 and CDX2 might therefore regulate the uptake of these substances in the colon.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Colon/metabolismo , Células Epiteliales/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Factor de Transcripción CDX2 , Proteínas de Transporte de Catión/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Colon/patología , Células Epiteliales/patología , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Transportadores de Ácidos Monocarboxílicos , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Background: The sodium/iodide (Na+/I-) symporter (NIS) mediates active transport of I- into the thyroid gland. Mutations in the SLC5A5 gene, which encodes NIS, cause I- transport defects (ITDs)-which, if left untreated, lead to congenital hypothyroidism and consequent cognitive and developmental deficiencies. The ITD-causing NIS mutation Y348D, located in transmembrane segment (TMS) 9, was reported in three Sudanese patients. Methods: We generated cDNAs coding for Y348D NIS and mutants with other hydrophilic and hydrophobic amino acid substitutions at position 348 and transfected them into cells. The activity of the resulting mutants was quantitated by radioiodide transport assays. NIS glycosylation was investigated by Western blotting after endoglycosidase H (Endo H) and PNGase-F glycosidase treatment. Subcellular localization of the mutant proteins was ascertained by flow cytometry analysis, cell surface biotinylation, and immunofluorescence. The intrinsic activity of Y348D was studied by measuring radioiodide transport in membrane vesicles prepared from Y348D-NIS-expressing cells. Our NIS homology models and molecular dynamics simulations were used to identify residues that interact with Y348 and investigate possible interactions between Y348 and the membrane. The sequences of several Slc5 family transporters were aligned, and a phylogenetic tree was generated in ClustalX. Results: Cells expressing Y348D NIS transport no I-. Furthermore, Y348D NIS is only partially glycosylated, is retained intracellularly, and is intrinsically inactive. Hydrophilic residues other than Asp at position 348 also yield NIS proteins that fail to be targeted to the plasma membrane (PM), whereas hydrophobic residues at this position, which we show do not interact with the membrane, rescue PM targeting and function. Conclusions: Y348D NIS does not reach the PM and is intrinsically inactive. Hydrophobic amino acid substitutions at position 348, however, preserve NIS activity. Our findings are consistent with our homology model's prediction that Y348 should face the side opposite the TMS9 residues that coordinate Na+ and participate in Na+ transport, and with the notion that Y348 interacts only with hydrophobic residues. Hydrophilic or charged residues at position 348 have deleterious effects on NIS PM targeting and activity, whereas a hydrophobic residue at this position rescues NIS activity.
Asunto(s)
Membrana Celular/metabolismo , Yodo/metabolismo , Mutación/genética , Simportadores/genética , Glándula Tiroides/metabolismo , Sustitución de Aminoácidos , Transporte Biológico/genética , Biotinilación , Hipotiroidismo Congénito/genética , ADN/genética , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Radioisótopos de Yodo , Mutagénesis Sitio-Dirigida , Fracciones Subcelulares/metabolismoRESUMEN
CONTEXT: Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. OBJECTIVE: This work aims to determine the molecular basis of a patient's ITD clinical phenotype. METHODS: The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. RESULTS: Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif toward a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. CONCLUSION: We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.
Asunto(s)
Hipotiroidismo Congénito/genética , Errores Innatos del Metabolismo/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Simportadores/genética , Hormonas Tiroideas/metabolismo , Animales , Humanos , Recién Nacido , Yoduros/metabolismo , Cinesinas , Masculino , Mutación Missense , Fenotipo , Ratas , Glándula Tiroides/metabolismoRESUMEN
The Na(+)/I(-) symporter (NIS) is a key plasma membrane protein that mediates active I(-) uptake in the thyroid, lactating breast, and other tissues with an electrogenic stoichiometry of 2 Na(+) per I(-). In the thyroid, NIS-mediated I(-) uptake is the first step in the biosynthesis of the iodine-containing thyroid hormones, which are essential early in life for proper CNS development. In the lactating breast, NIS mediates the translocation of I(-) to the milk, thus supplying this essential anion to the nursing newborn. Perchlorate (ClO(4)(-)) is a well known competitive inhibitor of NIS. Exposure to food and water contaminated with ClO(4)(-) is common in the U.S. population, and the public health impact of such exposure is currently being debated. To date, it is still uncertain whether ClO(4)(-) is a NIS blocker or a transported substrate of NIS. Here we show in vitro and in vivo that NIS actively transports ClO(4)(-), including ClO(4)(-) translocation to the milk. A simple mathematical fluxes model accurately predicts the effect of ClO(4)(-) transport on the rate and extent of I(-) accumulation. Strikingly, the Na(+)/ ClO(4)(-) transport stoichiometry is electroneutral, uncovering that NIS translocates different substrates with different stoichiometries. That NIS actively concentrates ClO(4)(-) in maternal milk suggests that exposure of newborns to high levels of ClO(4)(-) may pose a greater health risk than previously acknowledged because ClO(4)(-) would thus directly inhibit the newborns' thyroidal I(-) uptake.
Asunto(s)
Contaminantes Ambientales/metabolismo , Yodo/metabolismo , Percloratos/metabolismo , Simportadores/metabolismo , Animales , Transporte Biológico , Línea Celular , Perros , Yodo/análisis , Cinética , Leche/química , Modelos Teóricos , Percloratos/análisis , Ratas , TransfecciónRESUMEN
The Na+/I- symporter (NIS), the plasma membrane protein that actively transports I- (stoichiometry 2Na+:1I-) in thyroid physiology and radioiodide-based thyroid cancer treatment, also transports the environmental pollutant perchlorate (stoichiometry 1Na+:1ClO4-), which competes with I- for transport. Until now, the mechanism by which NIS transports different anion substrates with different stoichiometries has remained unelucidated. We carried out transport measurements and analyzed these using a statistical thermodynamics-based equation and electrophysiological experiments to show that the different stoichiometry of ClO4- transport is due to ClO4- binding to a high-affinity non-transport allosteric site that prevents Na+ from binding to one of its two sites. Furthermore, low concentrations of ClO4- inhibit I- transport not only by competition but also, critically, by changing the stoichiometry of I- transport to 1:1, which greatly reduces the driving force. The data reveal that ClO4- pollution in drinking water is more dangerous than previously thought.
Asunto(s)
Percloratos/metabolismo , Simportadores/química , Simportadores/metabolismo , Regulación Alostérica , Sitio Alostérico , Animales , Aniones/química , Aniones/metabolismo , Sitios de Unión , Transporte Biológico , Perros , Electrofisiología/métodos , Femenino , Humanos , Yodo/metabolismo , Células de Riñón Canino Madin Darby , Mutación , Oocitos/metabolismo , Oocitos/fisiología , Percloratos/química , Ratas , Sodio/metabolismo , Simportadores/genética , Termodinámica , Xenopus laevisRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The Na+/iodide (I-) symporter (NIS), a glycoprotein expressed at the basolateral plasma membrane of thyroid follicular cells, mediates I- accumulation for thyroid hormonogenesis and radioiodide therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit lower I- transport than normal thyroid tissue (or even undetectable I- transport). Paradoxically, the majority of differentiated thyroid cancers show intracellular NIS expression, suggesting abnormal targeting to the plasma membrane. Therefore, a thorough understanding of the mechanisms that regulate NIS plasma membrane transport would have multiple implications for radioiodide therapy. In this study, we show that the intracellularly facing carboxy-terminus of NIS is required for the transport of the protein to the plasma membrane. Moreover, the carboxy-terminus contains dominant basolateral information. Using internal deletions and site-directed mutagenesis at the carboxy-terminus, we identified a highly conserved monoleucine-based sorting motif that determines NIS basolateral expression. Furthermore, in clathrin adaptor protein (AP)-1B-deficient cells, NIS sorting to the basolateral plasma membrane is compromised, causing the protein to also be expressed at the apical plasma membrane. Computer simulations suggest that the AP-1B subunit σ1 recognizes the monoleucine-based sorting motif in NIS carboxy-terminus. Although the mechanisms by which NIS is intracellularly retained in thyroid cancer remain elusive, our findings may open up avenues for identifying molecular targets that can be used to treat radioiodide-refractory thyroid tumors that express NIS intracellularly.
Asunto(s)
Membrana Celular/metabolismo , Simportadores/química , Simportadores/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Transporte Biológico , Membrana Celular/genética , Humanos , Yoduros/metabolismo , Leucina/genética , Leucina/metabolismo , Transporte de Proteínas , Ratas , Alineación de Secuencia , Simportadores/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismoRESUMEN
BACKGROUND: We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. METHODS: AAV9-hNIS (2×1011 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (99mTcO4-), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99mTcO4- ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification. RESULTS: As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001). CONCLUSIONS: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.