Contribution of TRNA modifications for proteostasis in human cells
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10773/24859 |
Resumo: | Mutations in transfer RNAs (tRNA), key molecules in the translation process, lead to loss of accuracy of messenger RNA (mRNA) translation. This may induce changes in protein synthesis rate, protein misfolding and accumulation of aggregated proteins which, in turn, leads to proteotoxic stress and UPR activation, some of the characteristics of conformational protein diseases. To be fully active, tRNA molecules undergo post-transcriptional modifications catalyzed by different tRNA modifying enzymes. Deregulation of some modifications of tRNAs and some of the modifying enzymes has already been correlated with changes in translation fidelity and efficiency, especially in yeast. However, a comprehensive study on the impact of all tRNA-modifying enzymes and modifications in mammals has not been performed yet. In order to identify the key modifying enzymes for proteostasis preservation, our team developed a protein aggregation reporter system (pcDNA3.1 Hsp27-GFP) that allows the detection of the production of protein aggregates in vivo at the cellular level. After establishing a stable cell line expressing this reporter, a phenotypic screening was performed, identifying ELP1, ELP3, ELP6, URM1 and TRMT2A enzymes as essential for proteostasis. The UPS pathway was activated in the absence of the ELP1, ELP3, ELP6 enzymes and proteomic analyzes of ELP3 silencing cells revealed that the most altered pathways are concerned to transcription and translation processes, which are generally dysregulated in neurological diseases, such as Amyotrophic Lateral Sclerosis. In this thesis, the proteostasis relevant tRNA modifying enzymes are identified and characterized in human cells. This group of enzymes may represent promising therapeutic targets for conformational diseases |
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Contribution of TRNA modifications for proteostasis in human cellsTransfer RNAtRNA modifying enzymesProteostasisProtein aggregationMutations in transfer RNAs (tRNA), key molecules in the translation process, lead to loss of accuracy of messenger RNA (mRNA) translation. This may induce changes in protein synthesis rate, protein misfolding and accumulation of aggregated proteins which, in turn, leads to proteotoxic stress and UPR activation, some of the characteristics of conformational protein diseases. To be fully active, tRNA molecules undergo post-transcriptional modifications catalyzed by different tRNA modifying enzymes. Deregulation of some modifications of tRNAs and some of the modifying enzymes has already been correlated with changes in translation fidelity and efficiency, especially in yeast. However, a comprehensive study on the impact of all tRNA-modifying enzymes and modifications in mammals has not been performed yet. In order to identify the key modifying enzymes for proteostasis preservation, our team developed a protein aggregation reporter system (pcDNA3.1 Hsp27-GFP) that allows the detection of the production of protein aggregates in vivo at the cellular level. After establishing a stable cell line expressing this reporter, a phenotypic screening was performed, identifying ELP1, ELP3, ELP6, URM1 and TRMT2A enzymes as essential for proteostasis. The UPS pathway was activated in the absence of the ELP1, ELP3, ELP6 enzymes and proteomic analyzes of ELP3 silencing cells revealed that the most altered pathways are concerned to transcription and translation processes, which are generally dysregulated in neurological diseases, such as Amyotrophic Lateral Sclerosis. In this thesis, the proteostasis relevant tRNA modifying enzymes are identified and characterized in human cells. This group of enzymes may represent promising therapeutic targets for conformational diseasesMutações nas moléculas de RNA de transferência (tRNAs), moléculas chave no processo de tradução, levam à perda da precisão da tradução do RNA mensageiro (mRNA). Isto traduz-se em alterações na taxa de síntese proteica, misfolding de proteínas e acumulação de proteínas agregadas que, por sua vez, levam a stress proteotóxico e ativação da UPR, algumas das características de doenças conformacionais de proteínas. Para serem totalmente ativos, os tRNA sofrem modificações pós-transcricionais catalisadas por diferentes enzimas modificadoras de tRNA. A desregulação de algumas modificações de tRNA e de algumas das enzimas modificadoras foram já correlacionadas com alterações ao nível da fidelidade e eficiência da tradução, especialmente em leveduras. No entanto, um estudo exaustivo sobre o impacto de todas as modificações e enzimas modificadoras de tRNA em mamíferos não foi ainda realizado. Para identificar quais as enzimas modificadoras essenciais para a manutenção da proteostase, a nossa equipa desenvolveu um sistema repórter de agregação de proteínas (pcDNA3.1 Hsp27-GFP) que nos permite detetar in vivo a nível celular a produção de agregados proteicos. Após o estabelecimento de uma linha celular estável a expressar este repórter, foi realizado um screening fenotípico que identificou as enzimas ELP1, ELP3, ELP6, URM1 e TRMT2A como essenciais para a proteostase. Verificou-se que a via UPS foi ativada na ausência das enzimas ELP1, ELP3, ELP6 e análises de proteómica de células com silenciamento da ELP3 revelaram que as vias mais alteradas dizem respeito à tradução e transcrição, entre outras, que estão geralmente desreguladas em doenças neurológicas, como a Esclerose Lateral Amiotrófica. Os resultados desta tese identificam e demonstram que um grupo particular de enzimas modificadoras de tRNA afetam a proteostase em células humanas e que as mesmas podem ser novos alvos terapêuticos para doenças conformacionais2020-12-03T00:00:00Z2018-11-27T00:00:00Z2018-11-27info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/24859TID:202235475engPaulo, Cristiana Gonçalvesinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-22T11:48:35Zoai:ria.ua.pt:10773/24859Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:58:23.244585Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Contribution of TRNA modifications for proteostasis in human cells |
title |
Contribution of TRNA modifications for proteostasis in human cells |
spellingShingle |
Contribution of TRNA modifications for proteostasis in human cells Paulo, Cristiana Gonçalves Transfer RNA tRNA modifying enzymes Proteostasis Protein aggregation |
title_short |
Contribution of TRNA modifications for proteostasis in human cells |
title_full |
Contribution of TRNA modifications for proteostasis in human cells |
title_fullStr |
Contribution of TRNA modifications for proteostasis in human cells |
title_full_unstemmed |
Contribution of TRNA modifications for proteostasis in human cells |
title_sort |
Contribution of TRNA modifications for proteostasis in human cells |
author |
Paulo, Cristiana Gonçalves |
author_facet |
Paulo, Cristiana Gonçalves |
author_role |
author |
dc.contributor.author.fl_str_mv |
Paulo, Cristiana Gonçalves |
dc.subject.por.fl_str_mv |
Transfer RNA tRNA modifying enzymes Proteostasis Protein aggregation |
topic |
Transfer RNA tRNA modifying enzymes Proteostasis Protein aggregation |
description |
Mutations in transfer RNAs (tRNA), key molecules in the translation process, lead to loss of accuracy of messenger RNA (mRNA) translation. This may induce changes in protein synthesis rate, protein misfolding and accumulation of aggregated proteins which, in turn, leads to proteotoxic stress and UPR activation, some of the characteristics of conformational protein diseases. To be fully active, tRNA molecules undergo post-transcriptional modifications catalyzed by different tRNA modifying enzymes. Deregulation of some modifications of tRNAs and some of the modifying enzymes has already been correlated with changes in translation fidelity and efficiency, especially in yeast. However, a comprehensive study on the impact of all tRNA-modifying enzymes and modifications in mammals has not been performed yet. In order to identify the key modifying enzymes for proteostasis preservation, our team developed a protein aggregation reporter system (pcDNA3.1 Hsp27-GFP) that allows the detection of the production of protein aggregates in vivo at the cellular level. After establishing a stable cell line expressing this reporter, a phenotypic screening was performed, identifying ELP1, ELP3, ELP6, URM1 and TRMT2A enzymes as essential for proteostasis. The UPS pathway was activated in the absence of the ELP1, ELP3, ELP6 enzymes and proteomic analyzes of ELP3 silencing cells revealed that the most altered pathways are concerned to transcription and translation processes, which are generally dysregulated in neurological diseases, such as Amyotrophic Lateral Sclerosis. In this thesis, the proteostasis relevant tRNA modifying enzymes are identified and characterized in human cells. This group of enzymes may represent promising therapeutic targets for conformational diseases |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-11-27T00:00:00Z 2018-11-27 2020-12-03T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10773/24859 TID:202235475 |
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http://hdl.handle.net/10773/24859 |
identifier_str_mv |
TID:202235475 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
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info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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