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Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and has traditionally been thought to begin with the uptake of the Sec carrier selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP undergoes metabolisation via selenocysteine lyase (SCLY), producing selenide, a substrate used by selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor - selenophosphate - for the biosynthesis of the selenocysteine tRNA. Here, we report the discovery of an alternative pathway mediating Sec metabolisation that is independent of SCLY and mediated by peroxiredoxin 6 (PRDX6). Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the presence and functional significance of this alternative route in cancer cells where we reveal a notable association between elevated expression of PRDX6 with a highly aggressive neuroblastoma subtype. Altogether, our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering new avenues for therapeutic exploitation.
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CONTEXT: Monocarboxylate transporter 8 (MCT8) deficiency is a rare X-chromosomal inherited disease leading to severe cognitive impairment, muscular hypotonia and symptoms of peripheral thyrotoxicosis. Experimental approaches aiming to functionally rescue mutant MCT8 activity by the chemical chaperone phenylbutyrate (PB) demonstrated promising effects in vitro for several MCT8 missense mutations. OBJECTIVE: The objective was to evaluate biochemical and clinical effects of PB in doses equivalent to those approved for the treatment of urea cycle disorders in a boy with MCT8 deficiency due to a novel MCT8 missense mutation c.703G > T (p.V235L). RESULTS: During a treatment period of 13 months, PB led to a significant decrease of elevated TSH and T3 serum concentrations, while fT4 increased. Weight z-score of the toddler remained remarkably stable during the treatment period. Neurodevelopmental assessments (BSID-III) revealed a slight increase of gross motor skills from developmental age 4 to 6 months. However, increasing liver enzyme serum activities and accumulation of phenylacetate (PAA) in urine led to treatment interruptions and dose alterations. In vitro analyses in MDCK1 cells confirmed the pathogenicity of MCT8 p.V235L. However, while PB increased expression of the mutant protein, it did not rescue T3 transport, suggesting a PB effect on thyroid function tests independent of restoring MCT8 activity. CONCLUSION: In a clinical attempt of PB treatment in MCT8 deficiency we observed a significant improvement of thyroid hormone function tests, tendencies towards body weight stabilization and slight neurodevelopmental improvement. Hepatotoxicity of PB may be a limiting factor in MCT8 deficiency and requires further investigation.
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Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid ß (Aß) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aß generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.
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Colesterol , Fibroblastos , Lisosomas , Proteína Niemann-Pick C1 , Presenilina-1 , Presenilina-2 , Animales , Ratones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Colesterol/metabolismo , Fibroblastos/metabolismo , Glicosilación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Lisosomas/metabolismo , Ratones Noqueados , Neuronas/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Presenilina-2/genéticaRESUMEN
Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.
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Transportadores de Ácidos Monocarboxílicos , Simportadores , Triyodotironina , Humanos , Triyodotironina/metabolismo , Triyodotironina/farmacología , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Animales , Simportadores/genética , Simportadores/metabolismo , Perros , Células de Riñón Canino Madin Darby , Células Hep G2 , Interferencia de ARN , Transporte Biológico , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genéticaRESUMEN
CONTEXT: Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disease that leads to severe global developmental delay. MCT8 facilitates thyroid hormone (TH) transport across the cell membrane, and the serum TH profile is characterized by high T3 and low T4 levels. Recent studies have shown that the chemical chaperone sodium phenylbutyrate (NaPB) restored mutant MCT8 function and increased TH content in patient-derived induced pluripotent stem cells, making it a potential treatment for MCT8 deficiency. OBJECTIVE: We aimed to assess the efficacy and safety of glycerol phenylbutyrate (GPB) in MCT8 deficiency. METHODS: We treated two monozygotic twins aged 14.5 years with MCT8 deficiency due to P321L mutation with escalating doses of GPB over 13 months. We recorded TH, Vital signs, anthropometric measurements and neurocognitive functions. Resting metabolic rate (RMR) was measured by indirect calorimetry. Serum metabolites of GPB were monitored as a safety measure. In-vitro effects of NaPB were evaluated in MDCK1 cells stably expressing the MCT8P321L mutation. The effects of GPB were compared to the effects of DITPA and TRIAC, thyromimetic medications that the patients received in the past. RESULTS: NaPB restored mutant MCT8 expression in MDCK1 cells and increased T3 transport into cells carrying the P321L mutation. GPB treatment reduced high T3 and increased low T4 levels. The patients showed a significant weight gain simultaneously with a reduction in RMR. Only minor neuro-cognitive improvement was observed, in hyperreflexia score and in cognitive functions. Serum metabolites did not exceed the toxic range but elevated liver transaminases were observed. CONCLUSIONS: In the first report of GPB treatment in MCT8 deficiency we found an improvement in TH profile and body-mass index, with minor neuro-developmental changes.
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Endoscopy training models (ETM) using artificial organs are practical, hygienic and comfortable for trainees. However, few models exist for training endoscopic retrograde cholangiopancreatography (ERCP) in patients with surgically altered anatomy. This training is necessary as the number of bariatric surgeries performed worldwide increases. ETM with human-like anatomy were developed to represent the postoperative anatomy after Billroth II (BII) reconstruction for a standard duodenoscope and the situs of a long-limbed Roux-en-Y (RY) for device-assisted enteroscopy (DAE). In three independent workshops, the models were evaluated by international ERCP experts. In RY model, a simulation for small bowel behavior in endoscopy was created. Thirty-three experts rated the ETM in ERCP expert courses. The BII model was evaluated as suitable for training (school grades 1.36), with a haptic and visual impression rating of 1.73. The RY model was rated 1.50 for training suitability and 2.06 for overall impression. Animal tissue-free ETMs for ERCP in surgically altered anatomy were successfully created. Evaluation by experienced endoscopists indicated that the models are suitable for hands-on ERCP training, including device-assisted endoscopy. It is expected that patient care will improve with appropriate training in advanced procedures.
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Órganos Artificiales , Colangiopancreatografia Retrógrada Endoscópica , Humanos , Colangiopancreatografia Retrógrada Endoscópica/métodos , Intestino Delgado , Endoscopía Gastrointestinal , Anastomosis en-Y de Roux/métodos , Estudios RetrospectivosRESUMEN
Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.
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Ferroptosis , Neuroblastoma , Humanos , Línea Celular Tumoral , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/tratamiento farmacológico , Selenocisteína/uso terapéutico , AnimalesRESUMEN
BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is crucial to the treatment of biliopancreatic diseases with iatrogenic perforation as a potential complication. As of yet, the wall load during ERCP is unknown, as it is not directly measurable during an ERCP in patients. METHODS: In a life-like, animal-free model, a sensor system consisting of five load cells was attached to the artificial intestines (sensors 1 + 2: pyloric canal-pyloric antrum, sensor 3: duodenal bulb, sensor 4: descending part of the duodenum, sensor 5: distal to the papilla). Measurements were made with five duodenoscopes (n = 4 reusable and n = 1 single use). RESULTS: Fifteen standardized duodenoscopies were performed. Peak stresses were found at the antrum during the gastrointestinal transit (sensor 1 max. 8.95 N, sensor 2 max. 2.79 N). The load reduced from the proximal to the distal duodenum and the greatest load in the duodenum was discovered at the level of the papilla in 80.0% (sensor 3 max. 2.06 N). CONCLUSIONS: For the first time, intraprocedural load measurements and exerting forces obtained during a duodenoscopy for ERCP in an artificial model were recorded. None of the tested duodenoscopes were classified as dangerous for patient safety.
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BACKGROUND: For endoscopists, knowledge of the available hemotherapeutic devices and materials as well as competence in using them is a life-saving expertise in the treatment of patients with acute gastrointestinal bleeding. These competences can be acquired in training on live animals, animal organs, or simulators. We present an animal tissue-free training model of the upper gastrointestinal tract for bleeding therapy. METHODS: An artificial, animal tissue-free mucosa and submucosa with the opportunity of injection and clipping therapy were created first. Patches with this artificial mucosa and submucosa were placed into silicone and latex organs with human-like anatomy. Esophageal bleeding situations were imitated as variceal bleeding and bleeding of a reflux esophagitis in latex organs. Finally, a modular training model with human anatomy and replaceable bleeding sources was created. Evaluation of the novel model for gastroscopic training was performed in a multicentric setting with endoscopic beginners and experts. RESULTS: Evaluation was carried out by 38 physicians with different levels of education in endoscopy. Evaluation of the model was made with grades from one (excellent) to six (bad): suitability for endoscopic training was 1.4, relevance of the endoscopic training was 1.6, and grading for haptic and optic impression of the model was 1.7. CONCLUSIONS: The creation of a gastroscopic model for the training of hemostatic techniques without animal tissues was possible and multiple endoscopic bleeding skills could be trained in it. Evaluation showed good results for this new training option, which could be used in every endoscopic unit or other places without hygienic doubts.
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Background and study aim: Endoscopic negative pressure therapy (ENPT) is well established in the treatment of perforations of various etiologies in the upper and lower gastrointestinal tract. For duodenal perforations exist only case reports and series. Different indications are possible for ENPT in duodenal position: primary therapy for leaks, preemptive therapy after surgery for example, after ulcer suturing or resection with anastomoses, or as second line therapy in cases of recurrent anastomotic insufficiencies with leakage of duodenal secretion. Methods: A retrospective 4-year case series of negative pressure therapy in duodenal position indicated by different etiologies and a comprehensive review of current literature on endoscopic negative pressure duodenal therapy are presented. Results: Patients with primary duodenal leaks n= 6 and with duodenal stump insufficiencies n = 4 were included. In seven patients ENPT was the first line and sole therapy. Primary surgery for duodenal leak was performed in n = 3 patients. Mean duration of ENPT was 11.0 days, mean hospital stay was 30.0 days. Re-operation after start of ENPT was necessary in two patients with duodenal stump insufficiencies. Surgery after termination of the ENPT was not necessary in any patient. Discussion: In our case series and in the literature, ENPT has been shown to be very successful in the therapy of duodenal leaks. A challenge in ENPT for duodenal leaks is the appropriate length of the probe to safely reach the leak and keep the open pore element at the end of the probe in place despite intestinal motility.
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The development and maturation of ovarian follicles is a complex and highly regulated process, which is essential for successful ovulation. During recent decades, several mouse models provided insights into the regulation of folliculogenesis. In contrast to the commonly used transgenic or knockout mouse models, the Dummerstorf high-fertility mouse line 1 (FL1) is a worldwide unique selection experiment for increased female reproductive performance and extraordinary high fertility. Interactions of cycle-related alterations of parameters of the hypothalamic pituitary gonadal axis and molecular factors in the ovary lead to improved follicular development and therefore increased ovulation rates in FL1 mice. FL1 females almost doubled the number of ovulated oocytes compared to the unselected control mouse line. To gain insights into the cellular mechanisms leading to the high fertility phenotype we used granulosa cells isolated from antral follicles for mRNA sequencing. Based on the results of the transcriptome analysis we additionally measured hormones and growth factors associated with follicular development to complement the picture of how the signaling pathways are regulated. While IGF1 levels are decreased in FL1 mice in estrus, we found no differences in insulin, prolactin and oxytocin levels in FL1 mice compared to the control line. The results of the mRNA sequencing approach revealed that the actions of insulin, prolactin and oxytocin are restricted local to the granulosa cells, since hormonal receptor expression is differentially regulated in FL1 mice. Additionally, numerous genes, which are involved in important gonadotropin, apoptotic and metabolic signaling pathways in granulosa cells, are differentially regulated in granulosa cells of FL1 mice.We showed that an overlap of different signaling pathways reflects the crosstalk between gonadotropin and growth factor signaling pathways, follicular atresia in FL1 mice is decreased due to improved granulosa cell survival and by improving the efficiency of intracellular signaling, glucose metabolism and signal transduction, FL1 mice have several advantages in reproductive performance and therefore increased the ovulation rate. Therefore, this worldwide unique high fertility model can provide new insights into different factors leading to improved follicular development and has the potential to improve our understanding of high fertility.
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Insulinas , Prolactina , Femenino , Ratones , Animales , Prolactina/metabolismo , Oxitocina/metabolismo , Atresia Folicular/genética , Atresia Folicular/metabolismo , Células de la Granulosa/metabolismo , Gonadotropinas/metabolismo , Fertilidad , Redes y Vías Metabólicas , ARN Mensajero/metabolismo , Insulinas/metabolismoRESUMEN
Proteolytic cleavage of thyroglobulin (Tg) for thyroid hormone (TH) liberation is followed by TH release from thyroid follicles into the circulation, enabled by TH transporters. The existence of a functional link between Tg-processing cathepsin proteases and TH transporters has been shown to be independent of the hypothalamus-pituitary-thyroid axis. Thus, lack of cathepsin K, combined with genetic defects in the TH transporters Mct8 and Mct10, that is the Ctsk-/-/Mct8-/y/Mct10-/- genotype, results in persistent Tg proteolysis due to autophagy induction. Because amino acid transport by L-type amino acid transporter 2 (Lat2) has been described to regulate autophagy, we asked whether Lat2 availability is affected in Ctsk-/-/Mct8-/y/Mct10-/- thyroid glands. Our data revealed that while mRNA amounts and subcellular localization of Lat2 remained unaltered in thyroid tissue of Ctsk-/-/Mct8-/y/Mct10-/- mice in comparison to WT controls, the Lat2 protein amounts were significantly reduced. These data suggest a direct link between Lat2 function and autophagy induction in Ctsk-/-/Mct8-/y/Mct10-/- mice. Indeed, thyroid tissue of Lat2-/- mice showed enhanced endo-lysosomal cathepsin activities, increased autophagosome formation, and enhanced autophagic flux. Collectively, these results suggest a mechanistic link between insufficient Lat2 protein function and autophagy induction in the thyroid gland of male mice.
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Sistemas de Transporte de Aminoácidos , Autofagia , Glándula Tiroides , Animales , Masculino , Ratones , Autofagia/genética , Catepsinas , GenotipoRESUMEN
Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 (SECISBP2) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2, we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNASec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3' of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2-mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2R543Q-mutant brains.
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Elementos Transponibles de ADN , Selenocisteína , Selenocisteína/genética , Selenocisteína/metabolismo , Codón de Terminación/genética , Selenoproteínas/genética , Selenoproteínas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismoRESUMEN
Mice with constitutive disruption of the Selenop gene have been key to delineate the importance of selenoproteins in neurobiology. However, the phenotype of this mouse model is exquisitely dependent on selenium supply and timing of selenium supplementation. Combining biochemical, histological, and behavioral methods, we tested the hypothesis that parvalbumin-expressing interneurons in the primary somatosensory cortex and hippocampus depend on dietary selenium availability in Selenop-/- mice. Selenop-deficient mice kept on adequate selenium diet (0.15 mg/kg, i.e. the recommended dietary allowance, RDA) developed ataxia, tremor, and hyperexcitability between the age of 4-5 weeks. Video-electroencephalography demonstrated epileptic seizures in Selenop-/- mice fed the RDA diet, while Selenop± heterozygous mice behaved normally. Both neurological phenotypes, hyperexcitability/seizures and ataxia/dystonia were successfully prevented by selenium supplementation from birth or transgenic expression of human SELENOP under a hepatocyte-specific promoter. Selenium supplementation with 10 µM selenite in the drinking water on top of the RDA diet increased the activity of glutathione peroxidase in the brains of Selenop-/- mice to control levels. The effects of selenium supplementation on the neurological phenotypes were dose- and time-dependent. Selenium supplementation after weaning was apparently too late to prevent ataxia/dystonia, while selenium withdrawal from rescued Selenop-/- mice eventually resulted in ataxia. We conclude that SELENOP expression is essential for preserving interneuron survival under limiting Se supply, while SELENOP appears dispensable under sufficiently high Se status.
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Expression of selenoproteins is widespread in neurons of the central nervous system. There is continuous evidence presented over decades that low levels of selenium or selenoproteins are linked to seizures and epilepsy indicating a failure of the inhibitory system. Many developmental processes in the brain depend on the thyroid hormone T3. T3 levels can be locally increased by the action of iodothyronine deiodinases on the prohormone T4. Since deiodinases are selenoproteins, it is expected that selenoprotein deficiency may affect development of the central nervous system. Studies in genetically modified mice or clinical observations of patients with rare diseases point to a role of selenoproteins in brain development and degeneration. In particular selenoprotein P is central to brain function by virtue of its selenium transport function into and within the brain. We summarize which selenoproteins are essential for the brain, which processes depend on selenoproteins, and what is known about genetic deficiencies of selenoproteins in humans. This review is not intended to cover the potential influence of selenium or selenoproteins on major neurodegenerative disorders in human.
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Selenio , Animales , Encéfalo/metabolismo , Humanos , Ratones , Selenio/metabolismo , Selenoproteína P/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Hormonas Tiroideas/metabolismoRESUMEN
The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin-fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C) or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3.
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Yoduro Peroxidasa , Animales , Células COS , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Isoenzimas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Yodotironina Deyodinasa Tipo IIRESUMEN
The Dummerstorf high-fertility mouse line FL1 is a worldwide unique selection experiment for increased female reproductive performance. After more than 190 generations of selection, these mice doubled the amount of offspring per litter compared to the unselected control line. FL1 females have a superior lifetime fecundity and the highest Silver fecundity index that has been described in mice, while their offspring show no signs of growth retardation. The reasons for the increased reproductive performance remained unclear. Thus, this study aims to characterize the Dummerstorf high-fertility mouse line FL1 on endocrine and molecular levels on the female side. We analyzed parameters of the hypothalamic pituitary gonadal axis on both hormonal and transcriptional levels. Gonadotropin-releasing hormone and follicle-stimulating hormone (FSH) concentrations were decreased in FL1 throughout the whole estrous cycle. Luteinizing hormone (LH) was increased in FL1 mice in estrus. Progesterone concentrations were decreased in estrus in FL1 mice and not affected in diestrus. We used a holistic gene expression approach in the ovary to obtain a global picture of how the high-fertility phenotype is achieved. We found several differentially expressed genes in the ovaries of FL1 mice that are associated with different female fertility traits. Our results indicate that ovulation rates in mice can be increased despite decreased FSH levels. Cycle-related alterations of progesterone and LH levels have the potential to improve follicular maturation, and interactions of endocrine and molecular factors lead to enhanced follicular survival, more successful folliculogenesis and therefore higher ovulation rates in female FL1 mice.
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Fertilidad , Progesterona , Animales , Femenino , Fertilidad/genética , Hormona Folículo Estimulante , Hormona Luteinizante , Ratones , Reproducción/genéticaRESUMEN
Background: Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disease leading to a severe developmental delay due to a lack of thyroid hormones (THs) during critical stages of human brain development. Some MCT8-deficient patients are not as severely affected as others. Previously, we hypothesized that these patients' mutations do not affect the functionality but destabilize the MCT8 protein, leading to a diminished number of functional MCT8 molecules at the cell surface. Methods: We have already demonstrated that the chemical chaperone sodium phenylbutyrate (NaPB) rescues the function of these mutants by stabilizing their protein expression in an overexpressing cell system. Here, we expanded our previous work and used iPSC (induced pluripotent stem cell)-derived brain microvascular endothelial-like cells (iBMECs) as a physiologically relevant cell model of human origin to test for NaPB responsiveness. The effects on mutant MCT8 expression and function were tested by Western blotting and radioactive uptake assays. Results: We found that NaPB rescues decreased mutant MCT8 expression and restores transport function in iBMECs carrying patient's mutation MCT8-P321L. Further, we identified MCT10 as an alternative TH transporter in iBMECs that contributes to triiodothyronine uptake, the biological active TH. Our results indicate an upregulation of MCT10 after NaPB treatment. In addition, we detected an increase in thyroxine (T4) uptake after NaPB treatment that was not mediated by rescued MCT8 but an unidentified T4 transporter. Conclusions: We demonstrate that NaPB is suitable to stabilize a pathogenic missense mutation in a human-derived cell model. Further, it activates TH transport independent of MCT8. Both options fuel future studies to investigate repurposing the Food and Drug Administration-approved drug NaPB in selected cases of MCT8 deficiency.
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Transportadores de Ácidos Monocarboxílicos , Simportadores , Transporte Biológico , Encéfalo/metabolismo , Humanos , Discapacidad Intelectual Ligada al Cromosoma X , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Hipotonía Muscular , Atrofia Muscular , Fenilbutiratos , Simportadores/genética , Simportadores/metabolismo , Hormonas Tiroideas/metabolismo , Triyodotironina/metabolismo , Triyodotironina/farmacologíaRESUMEN
BACKGROUND: Esophageal cancer (EC) is the sixth-leading cause of cancer-related deaths in the world. Esophagectomy is the most effective treatment for patients without invasion of adjacent organs or distant metastasis. Complications and relevant problems may occur in the early post-operative course or in a delayed fashion. Here, innovative endoscopic techniques for the treatment of postsurgical problems were developed during the past 20 years. METHODS: Endoscopic treatment strategies for the following postoperative complications are presented: anastomotic bleeding, anastomotic insufficiency, delayed gastric passage and anastomotic stenosis. Based on a literature review covering the last two decades, therapeutic procedures are presented and analyzed. RESULTS: Addressing the four complications mentioned, clipping, stenting, injection therapy, dilatation, and negative pressure therapy are successfully utilized as endoscopic treatment techniques today. CONCLUSION: Endoscopic treatment plays a major role in both early-postoperative and long-term aftercare. During the past 20 years, essential therapeutic measures have been established. A continuous development of these techniques in the field of endoscopy can be expected.
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Transporter-dependent steroid hormone uptake into target cells was demonstrated in genetically engineered mice and fruit flies. We hypothesized that mutations in such transporters may cause differences in sex development (DSD) in humans. Exome sequencing was performed in 16 genetically unsolved cases of 46,XY DSD selected from an anonymized collection of 708 lines of genital fibroblasts (GF) that were taken from individuals with incomplete virilization. Selection criteria were based on available biochemical characterization of GF compatible with reduced androgen uptake. Two unrelated individuals were identified with mutations in LDL receptor-related protein 2 (LRP2), a gene previously associated with partial sex steroid insensitivity in mice. Like Lrp2-/- mice, affected individuals had non-descended testes. Western blots on GF confirmed reduced LRP2 expression, and endocytosis of sex hormone-binding globulin was reduced. In three unrelated individuals, two with undescended testes, mutations in another endocytic receptor gene, limb development membrane protein 1 like (LMBR1L), were detected. Two of these individuals had mutations affecting the same codon. In a transfected cell model, mutated LMBR1L showed reduced cell surface expression. Our findings suggest that endocytic androgen uptake in complex with sex hormone-binding globulin is relevant in human. LMBR1L may play a similar role in androgen uptake.