Trichothiodystrophy (TTD) is a rare hereditary disorder which features broad skin, neurological and growth abnormalities. Several causative genes are known for TTD, whose products include subunits of the DNA repair/transcription factor TFIIH, the beta subunit of the transcription factor TFIIE, the TTDN1 and RNF113A spliceosomal factors and four recently identified aminoacyl-tRNA synthetases (ARSs) that are essential in protein synthesis. Based on functions of the known defective factors, TTD has been classified as DNA repair disease or as transcription syndrome and, following identification of ARSs as disease genes, redefined as a gene expression syndrome. ARSs are the enzymes responsible for charging of proper (cognate) amino acids on tRNAs. The four ARS associated with TTD are cysteinyl-tRNA synthetase (CysRS, encoded by CARS1), threonyl-tRNA synthetase (ThrRS, encoded by TARS1), alanyl-tRNA synthetase (AlaRS, encoded by AARS1) and methionyl-tRNA synthetase (MetRS, encoded by MARS1). Pathogenic alterations in these ARSs have been shown to cause protein destabilization and aminoacylation deficiency. However, how these alterations may lead to the TTD disease has not been completely resolved yet. This study was aimed at contributing to the understanding of ARS-related TTD clinical outcome. To this purpose, two activities were carried out: 1) evaluation of translation fidelity in ARS-defective TTD patient cells; 2) computational search in the human proteome of proteins enriched in the cognate amino acids of the mutated ARSs, followed by experimental analysis in TTD cells. 1) Previous work of the laboratory had suggested that TTD associated ARS variants might affect the charging accuracy of the encoded enzymes, thus resulting in impaired translation fidelity. To further explore this aspect, the work of this thesis was focused on evaluation of translation fidelity in ARS-defective TTD primary fibroblasts. To this purpose, luciferase-based assays were performed. Although different experimental conditions were used, we could not detect any alteration in patient cells. We came up with the demonstration that sensitivity of these assays in primary fibroblasts was very low, thus highlighting the need for other model cell systems and more sensitive experimental approaches to detect translation infidelity. 2) A logical expectation is that impaired activity of the mutated ARSs would preferentially affect proteins with high content of the respective cognate amino acids. Therefore, Cys, Thr, Ala or Met enriched proteins were sorted by a computational analysis of the human proteome. Functional enrichment analyses to identify over-represented (enriched) pathways revealed that keratinization was a common pathway for proteins enriched in Cys, Thr, Ala or Met. Keratinization refers to the processes that occur in epidermal keratinocytes during their terminal differentiation. Of note, hair and nails whose alteration is a defining feature of TTD, are highly keratinized structures. Further computational analysis was carried out to identify proteins with high content of all four amino acids. Through this work, the secreted LY6/PLAUR domain containing 1 (SLURP1) protein was identified. SLURP1 is expressed in epidermal keratinocytes and is essential in skin homeostasis. Interestingly, reduced levels of SLURP1 were observed by immnunoblot analysis in TTD keratinocytes suggesting that cellular content of SLURP1 might be affected by TTD mutations.
Caratterizzazione funzionale di varianti patogenetiche in geni delle aminoacil-tRNA sintetasi associate alla tricotiodistrofia. La tricotiodistrofia (TTD) è una rara malattia ereditaria caratterizzata da un ampio spettro di anomalie cutanee, neurologiche e dello sviluppo. Sono noti diversi geni associati alla TTD, i cui prodotti comprendono subunità del fattore di riparazione/trascrizione TFIIH, la subunità beta del fattore di trascrizione TFIIE, i fattori TTDN1 e RNF113A coinvolti nella maturazione dei trascritti e quattro distinte aminoacil-tRNA sintetasi (ARS) essenziali nella sintesi proteica. Sulla base delle funzioni dei fattori coinvolti, la TTD è stata classificata come malattia da difetti nella riparazione del DNA o come sindrome da difetti trascrizionali. In seguito alla recente identificazione delle ARS come geni malattia, la TTD è stata ridefinita come sindrome da difetti nell'espressione genica. Le ARS sono gli enzimi responsabili del caricamento di specifici aminoacidi (definiti “cognati”) sui tRNA. Le 4 ARS associate alla TTD sono la cisteinil-tRNA sintetasi, la treonil-tRNA sintetasi, l'alanil-tRNA sintetasi e la metionil-tRNA sintetasi. È stato dimostrato che le alterazioni patogeniche in queste ARS destabilizzano le corrispondenti proteine e causano un deficit di aminoacilazione. Tuttavia, il modo in cui queste alterazioni possono portare alla TTD non è stato ancora completamente chiarito. Questo studio si propone di contribuire alla comprensione di come il fenotipo clinico della TTD possa essere correlato a difetti nelle ARS. A tal fine, sono state svolte due attività: 1) valutazione della fedeltà del processo di traduzione in cellule di pazienti TTD mutati nelle ARS; 2) ricerca computazionale nel proteoma umano delle proteine arricchite negli aminoacidi caricati dalle ARS mutate, seguita da analisi sperimentale in cellule TTD. 1) Un precedente lavoro svolto nel laboratorio aveva suggerito che le varianti ARS associate alla TTD potessero influenzare la precisione di caricamento dei corrispondenti enzimi, alterando così la fedeltà della traduzione. Il lavoro di questa tesi si è concentrato sulla valutazione della fedeltà della traduzione in fibroblasti primari TTD mutati nelle ARS. A questo scopo, sono stati eseguiti saggi basati sulla luciferasi. Sebbene siano state utilizzate condizioni sperimentali diverse, non siamo riusciti a rilevare alterazioni nelle cellule dei pazienti. Abbiamo quindi dimostrato che la sensibilità di questi saggi nei fibroblasti primari è molto bassa, evidenziando così la necessità di altri sistemi cellulari modello e di approcci sperimentali più sensibili per rilevare infedeltà della traduzione. 2) Una aspettativa logica è che l'attività compromessa delle ARS mutate colpisca preferenzialmente le proteine con un elevato contenuto degli aminoacidi “cognati”. Pertanto, le proteine arricchite in Cys, Thr, Ala o Met sono state selezionate mediante un'analisi computazionale del proteoma umano. Successive analisi di “functional enrichment”, utilizzate per identificare i pathways sovra-rappresentati (arricchiti), hanno messo in evidenza che la cheratinizzazione è un pathway comune per le proteine arricchite in Cys Thr, Ala o Met. È interessante notare che i capelli e le unghie, la cui alterazione è una caratteristica distintiva della TTD, sono strutture altamente cheratinizzate. Un'ulteriore analisi computazionale è stata infine eseguita per identificare le proteine che hanno un elevato contenuto di tutti e quattro gli aminoacidi. Grazie a questa analisi, è stata identificata la proteina secreted LY6/PLAUR domain containing 1 (SLURP1). SLURP1 è espressa nei cheratinociti dell’epidermide ed è essenziale per l'omeostasi della pelle. Mediante analisi di immunoblot sono stati osservati ridotti livelli di SLURP1 in cheratinociti TTD, suggerendo che il contenuto cellulare di SLURP1 potrebbe essere influenzato dalle mutazioni TTD.
Functional characterization of pathogenic variants in aminoacyl-tRNA synthetase genes associated with trichothiodystrophy
BREVI, FRANCESCA
2022/2023
Abstract
Trichothiodystrophy (TTD) is a rare hereditary disorder which features broad skin, neurological and growth abnormalities. Several causative genes are known for TTD, whose products include subunits of the DNA repair/transcription factor TFIIH, the beta subunit of the transcription factor TFIIE, the TTDN1 and RNF113A spliceosomal factors and four recently identified aminoacyl-tRNA synthetases (ARSs) that are essential in protein synthesis. Based on functions of the known defective factors, TTD has been classified as DNA repair disease or as transcription syndrome and, following identification of ARSs as disease genes, redefined as a gene expression syndrome. ARSs are the enzymes responsible for charging of proper (cognate) amino acids on tRNAs. The four ARS associated with TTD are cysteinyl-tRNA synthetase (CysRS, encoded by CARS1), threonyl-tRNA synthetase (ThrRS, encoded by TARS1), alanyl-tRNA synthetase (AlaRS, encoded by AARS1) and methionyl-tRNA synthetase (MetRS, encoded by MARS1). Pathogenic alterations in these ARSs have been shown to cause protein destabilization and aminoacylation deficiency. However, how these alterations may lead to the TTD disease has not been completely resolved yet. This study was aimed at contributing to the understanding of ARS-related TTD clinical outcome. To this purpose, two activities were carried out: 1) evaluation of translation fidelity in ARS-defective TTD patient cells; 2) computational search in the human proteome of proteins enriched in the cognate amino acids of the mutated ARSs, followed by experimental analysis in TTD cells. 1) Previous work of the laboratory had suggested that TTD associated ARS variants might affect the charging accuracy of the encoded enzymes, thus resulting in impaired translation fidelity. To further explore this aspect, the work of this thesis was focused on evaluation of translation fidelity in ARS-defective TTD primary fibroblasts. To this purpose, luciferase-based assays were performed. Although different experimental conditions were used, we could not detect any alteration in patient cells. We came up with the demonstration that sensitivity of these assays in primary fibroblasts was very low, thus highlighting the need for other model cell systems and more sensitive experimental approaches to detect translation infidelity. 2) A logical expectation is that impaired activity of the mutated ARSs would preferentially affect proteins with high content of the respective cognate amino acids. Therefore, Cys, Thr, Ala or Met enriched proteins were sorted by a computational analysis of the human proteome. Functional enrichment analyses to identify over-represented (enriched) pathways revealed that keratinization was a common pathway for proteins enriched in Cys, Thr, Ala or Met. Keratinization refers to the processes that occur in epidermal keratinocytes during their terminal differentiation. Of note, hair and nails whose alteration is a defining feature of TTD, are highly keratinized structures. Further computational analysis was carried out to identify proteins with high content of all four amino acids. Through this work, the secreted LY6/PLAUR domain containing 1 (SLURP1) protein was identified. SLURP1 is expressed in epidermal keratinocytes and is essential in skin homeostasis. Interestingly, reduced levels of SLURP1 were observed by immnunoblot analysis in TTD keratinocytes suggesting that cellular content of SLURP1 might be affected by TTD mutations.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/16492