Allan-Herndon-Dudley syndrome is an X-linked rare genetic disease caused by mutations in the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). Subjects with this syndrome suffer from severe psychomotor retardation in combination with symptoms related to altered thyroid hormone (TH) levels in different tissues like tachycardia and low body weight. In AHDS, there is a reduced presence of mutated MCT8 at the plasma membrane and this phenomenon could be the result of accumulation of the protein inside the cytoplasm or its degradation by endoplasmic reticulum-associated degradation (ERAD) pathway. The aim of this thesis work was to analyse MCT8 functionality after the recovery of its membrane expression by small molecules, called CFTR correctors. These compounds are protein folding correctors useful to recover folding defective proteins, first developed for cystic fibrosis, but showing efficacy also in sarcoglycanopathies. We performed in vitro experiments using Madin-Darby canine kidney cells (MDCK1) constitutively expressing the WT, as positive control, or mutated (L291R and L568P) MCT8 cDNAs. Once evaluated the protein recovery at the plasma membrane, after the treatment of cells with some correctors (alone or in combination), we analysed the transporter functionality. To assess the functionality of the MCT8, the conventional method is based on the uptake of 125I-T3 by the cells. To overcome the need of using radioactive compounds, we have developed an indirect method based on a biosensor sensitive to T3 entrance. The biosensor relies on a vector expressing the firefly luciferase reporter, controlled by the minimal SV40 promoter and the thyroid response element (TRE) enhancer. Only when the T3 hormone enters the cells, the thyroid hormone receptor can bind the TRE, luciferase is expressed and cells become luminescent. The setup of this system is described in this thesis. We expect high expression of luciferase, because of high T3 entrance, when the wild type MCT8 is expressed by the cells, or when a mutated, but functional, form has been recovered at the cell surface. This system could be of benefit for a first quick screening and evaluation of the compounds effective in inducing the rescue of the TH transporter.
La sindrome Allan-Herndon-Dudley è una malattia genetica rara legata al cromosoma X causata da mutazioni al trasportatore dell’ormone tiroideo MCT8 (monocarboxylate transporter 8). I soggetti affetti da questa sindrome soffrono di un severo ritardo psicomotorio in combinazione a sintomi legati a livelli alterati dell’ormone tiroideo in diversi tessuti, come tachicardia e basso peso corporeo. Nella AHDS, vi è una ridotta presenza dell’MCT8 mutato nella membrana plasmatica e questo fenomeno potrebbe essere il risultato dell’accumulo della proteina nel citoplasma o la degradazione di essa da parte del sistema di degradazione associato al reticolo endoplasmatico chiamato ERAD. Lo scopo di questo lavoro di tesi era quello di analizzare la funzionalità dell’MCT8 dopo il recupero della sua espressione in membrana attraverso piccole molecole, chiamate correttori del CFTR. Questi composti sono correttori del protein folding utili per il recupero del ripiegamento in proteine difettose, precedentemente sviluppati per la fibrosi cistica, ma che mostrano efficacia anche nel caso delle sarcoglicanopatie. Noi abbiamo effettuato esperimenti in vitro usando cellule Madin-Darby di rene canino (MDCK1) che esprimono costitutivamente il WT, utilizzato come controllo positivo, o cDNA mutati (L291R e L568P). Una volta valutato il recupero della proteina nella membrana plasmatica, dopo il trattamento delle cellule con alcuni correttori (da soli o in combinazione), abbiamo analizzato la funzionalità del trasportatore. Per valutare la funzionalità dell’MCT8, il metodo convenzionale è basato sull’assorbimento di 125I-T3 da parte delle cellule. Per evitare l’utilizzo di composti radioattivi, abbiamo sviluppato un metodo indiretto basato su un biosensore sensibile all’ingresso di T3. Il biosensore consiste in un vettore che esprime la firefly Luciferasi utilizzata come reporter, la cui espressione sarà controllata dal promotore minimale SV40 e come enhancer l’elemento responsivo tiroideo (TRE). Solo quando l’ormone entra nelle cellule, il recettore dell’ormone tiroideo potrà legare il TRE, la Luciferasi sarà espressa e le cellule diventeranno luminescenti. Il meccanismo di questo sistema viene descritto in questa tesi. Noi ci aspettiamo un’alta espressione della Luciferasi, a causa di un importante ingresso di T3, quando le cellule esprimono l’MCT8 wild type, o nel caso in cui il trasportatore mutato viene recuperato sulla superficie cellulare. Il vantaggio di questo sistema potrebbe essere un primo veloce screening e valutazione dell’efficacia dei composti efficaci nell’indurre il recupero del trasportatore.
Sviluppo di un biosensore per verificare la funzionalità del trasportatore dell'ormone tiroideo in modelli cellulari della sindrome di Allan-Herndon-Dudley
ZEMA, MARCO
2022/2023
Abstract
Allan-Herndon-Dudley syndrome is an X-linked rare genetic disease caused by mutations in the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). Subjects with this syndrome suffer from severe psychomotor retardation in combination with symptoms related to altered thyroid hormone (TH) levels in different tissues like tachycardia and low body weight. In AHDS, there is a reduced presence of mutated MCT8 at the plasma membrane and this phenomenon could be the result of accumulation of the protein inside the cytoplasm or its degradation by endoplasmic reticulum-associated degradation (ERAD) pathway. The aim of this thesis work was to analyse MCT8 functionality after the recovery of its membrane expression by small molecules, called CFTR correctors. These compounds are protein folding correctors useful to recover folding defective proteins, first developed for cystic fibrosis, but showing efficacy also in sarcoglycanopathies. We performed in vitro experiments using Madin-Darby canine kidney cells (MDCK1) constitutively expressing the WT, as positive control, or mutated (L291R and L568P) MCT8 cDNAs. Once evaluated the protein recovery at the plasma membrane, after the treatment of cells with some correctors (alone or in combination), we analysed the transporter functionality. To assess the functionality of the MCT8, the conventional method is based on the uptake of 125I-T3 by the cells. To overcome the need of using radioactive compounds, we have developed an indirect method based on a biosensor sensitive to T3 entrance. The biosensor relies on a vector expressing the firefly luciferase reporter, controlled by the minimal SV40 promoter and the thyroid response element (TRE) enhancer. Only when the T3 hormone enters the cells, the thyroid hormone receptor can bind the TRE, luciferase is expressed and cells become luminescent. The setup of this system is described in this thesis. We expect high expression of luciferase, because of high T3 entrance, when the wild type MCT8 is expressed by the cells, or when a mutated, but functional, form has been recovered at the cell surface. This system could be of benefit for a first quick screening and evaluation of the compounds effective in inducing the rescue of the TH transporter.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/48064