Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes

Detalhes bibliográficos
Autor(a) principal: Lopes, Beatriz Rasgado
Data de Publicação: 2022
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10451/59197
Resumo: Tese de mestrado, Bioquímica (Bioquímica Médica), 2022, Universidade de Lisboa, Faculdade de Ciências
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spelling Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromesdoenças mitocondriaisaminoacil-tRNA sintetaseshEARS2Teses de mestrado - 2022Departamento de Química e BioquímicaTese de mestrado, Bioquímica (Bioquímica Médica), 2022, Universidade de Lisboa, Faculdade de CiênciasMitochondrial diseases (MD) are a group of complex metabolic disorders defined by genetic defects that predominantly affect the mitochondrial oxidative phosphorylation pathway (OXPHOS)1 . Pathologies that result in combined OXPHOS defects are often associated with the impairment of processes such as replication, transcription, or translation of mtDNA2 . This leads to a clinical and therapeutic heterogeneity due to the ubiquitous nature of mitochondria. A group of enzymes that have been identified as key components of the mitochondrial translation apparatus are the mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs)2 . These enzymes are responsible for the addition of the corresponding amino acid into the correct tRNA molecule during protein translation in the mitochondria 2 . Recently, an increasing number of mitochondrial protein synthesis deficiencies have been identified and associated to mutations in mt-aaRS genes. These deficiencies have been linked with diverse clinical presentations, mainly affecting the central nervous system. Although reports of mutations in these enzymes seem to be increasing, there is still limited information regarding the structural and conformational consequences of disease-causing mutations. This project strives to fill that gap by employing a variety of biochemical and biophysical approaches to perform a structural and conformational characterization of the human mitochondrial glutamyl-tRNA synthetase (hEARS2). We have conducted expression tests and showed that the ideal condition for recombinant protein expression in E.coli is to grow cells at 22ºC with overnight induction. The purification process was optimized to include an ion exchange (IEX) column to enrich the extract with the hEARS2 protein, prior to its application on the His-Trap affinity column. This purification protocol resulted in a hEARS2 fraction more than 95% pure. Circular dichroism and fluorescence spectroscopy were used to characterize the protein’s secondary and tertiary structure, and to measure hEARS2 thermal stability. Spectra acquired suggested that the purified protein presented a typical /β fold, with dispersed Trp residues that seem to be partially buried in the interior of the protein. Thermal stability studies showed that hEARS2 contains a cooperative unfolding curve with an apparent Tm app of around 50℃. These studies bring important new information to the field and will help to study disease-related variants that could permit to establish how genotypic changes of the hEARS2 variants lead to the phenotypic heterogeneity associated with mt-aaRS associated disorders.Henriques, Bárbara J.Gomes, Cláudio MRepositório da Universidade de LisboaLopes, Beatriz Rasgado202220222025-10-29T00:00:00Z2022-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10451/59197enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-08T17:08:13Zoai:repositorio.ul.pt:10451/59197Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T22:09:10.186890Repositório 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 Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
title Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
spellingShingle Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
Lopes, Beatriz Rasgado
doenças mitocondriais
aminoacil-tRNA sintetases
hEARS2
Teses de mestrado - 2022
Departamento de Química e Bioquímica
title_short Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
title_full Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
title_fullStr Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
title_full_unstemmed Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
title_sort Human Mitochondrial Glutamyl-tRNA Synthetase: Biochemical Studies and Role in Neurological Syndromes
author Lopes, Beatriz Rasgado
author_facet Lopes, Beatriz Rasgado
author_role author
dc.contributor.none.fl_str_mv Henriques, Bárbara J.
Gomes, Cláudio M
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Lopes, Beatriz Rasgado
dc.subject.por.fl_str_mv doenças mitocondriais
aminoacil-tRNA sintetases
hEARS2
Teses de mestrado - 2022
Departamento de Química e Bioquímica
topic doenças mitocondriais
aminoacil-tRNA sintetases
hEARS2
Teses de mestrado - 2022
Departamento de Química e Bioquímica
description Tese de mestrado, Bioquímica (Bioquímica Médica), 2022, Universidade de Lisboa, Faculdade de Ciências
publishDate 2022
dc.date.none.fl_str_mv 2022
2022
2022-01-01T00:00:00Z
2025-10-29T00:00:00Z
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/10451/59197
url http://hdl.handle.net/10451/59197
dc.language.iso.fl_str_mv eng
language eng
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dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
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