Aberrant protein synthesis in human HEK293FT cells

Detalhes bibliográficos
Autor(a) principal: Varanda, Ana Sofia Paulo
Data de Publicação: 2011
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10773/7287
Resumo: Protein synthesis is tightly regulated and fidelity in this process is essential for maintenance of essential cellular functions. Under normal physiological conditions errors can occur, at frequencies around 10-4 errors per codon decoded. This level of mistranslation can be tolerated by cells. When the frequencies of errors increase mechanisms of protein quality control can fail leading to accumulation of misfolded proteins, which are more prone to form toxic aggregates, can compromise cell viability, disrupt cellular homeostasis and lead to the development of diseases. In order to elucidate how cells respond to mistranslation and understand how protein accumulation can cause disease and cell degeneration, we exposed human HEK293FT cells to canavanine and azetidine-2-carboxylic acid (AZC) which are analogues of arginine and proline, respectively. Their misincorporation into proteins leads to erroneous protein synthesis, misfolding and likely to protein aggregation. Such mistranslation event decreased slightly cellular viability and increased the number of cells arrested in G2/M and S phases of cell cycle. In HEK293FT cells was detected an increase in proteins conjugated with ubiquitin, without altering proteasome activity, which may indicate that at this level of mistranslation, protein quality control mechanisms are active to counteract the formation of protein aggregates. Transcriptional analysis showed that down-regulated genes were mainly associated with extracellular matrix and cell adhesion and up-regulated genes were involved in negative regulation of transcription and response to unfolded proteins. This study provides new insights into the response of human cells to mistranslation by giving a global analysis of transcriptional alterations that occur in response to proteotoxic stress. HEK293FT cells can be a good model to understand the molecular basis of human diseases caused by mRNA mistranslation.
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spelling Aberrant protein synthesis in human HEK293FT cellsBiologia molecularProteínas - SínteseFisiologia celularProtein synthesis is tightly regulated and fidelity in this process is essential for maintenance of essential cellular functions. Under normal physiological conditions errors can occur, at frequencies around 10-4 errors per codon decoded. This level of mistranslation can be tolerated by cells. When the frequencies of errors increase mechanisms of protein quality control can fail leading to accumulation of misfolded proteins, which are more prone to form toxic aggregates, can compromise cell viability, disrupt cellular homeostasis and lead to the development of diseases. In order to elucidate how cells respond to mistranslation and understand how protein accumulation can cause disease and cell degeneration, we exposed human HEK293FT cells to canavanine and azetidine-2-carboxylic acid (AZC) which are analogues of arginine and proline, respectively. Their misincorporation into proteins leads to erroneous protein synthesis, misfolding and likely to protein aggregation. Such mistranslation event decreased slightly cellular viability and increased the number of cells arrested in G2/M and S phases of cell cycle. In HEK293FT cells was detected an increase in proteins conjugated with ubiquitin, without altering proteasome activity, which may indicate that at this level of mistranslation, protein quality control mechanisms are active to counteract the formation of protein aggregates. Transcriptional analysis showed that down-regulated genes were mainly associated with extracellular matrix and cell adhesion and up-regulated genes were involved in negative regulation of transcription and response to unfolded proteins. This study provides new insights into the response of human cells to mistranslation by giving a global analysis of transcriptional alterations that occur in response to proteotoxic stress. HEK293FT cells can be a good model to understand the molecular basis of human diseases caused by mRNA mistranslation.A síntese proteica é um mecanismo sujeito a uma apertada regulação, pois a manutenção de funções celulares essenciais está dependente da fidelidade deste processo. Em condições fisiológicas normais podem ocorrer erros, a uma frequência de aproximadamente 10-4 erros por codão descodificado. Este nível de erro é tolerado pelas células, mas quando a frequência destes aumenta, os mecanismos de controlo de qualidade das proteínas podem falhar levando à acumulação de proteínas aberrantes (misfolded) que tendem a formar agregados tóxicos, podem comprometer a viabilidade celular, alterar a homeostasia e levar ao desenvolvimento de doenças. Com o objectivo de elucidar os mecanismos de resposta à acumulação de erros durante a síntese proteica e entender como o aumento de proteínas aberrantes pode levar ao desenvolvimento de doenças e degeneração celular, foram utilizadas células HEK293FT expostas a canavanina e azetidine-2- carboxylic acid (AZC), análogos da arginina e prolina respectivamente. A incorporação destes análogos de aminoácidos leva a uma síntese proteica aberrante e origina proteínas que tendem a agregar e ter consequências tóxicas para as células. No nosso estudo observámos que a incorporação de análogos de aminoácidos levou a uma diminuição ligeira da viabilidade celular, assim como levou ao aumento do número de células nas fases G2/M e S do ciclo celular. Também foi detectado um aumento de proteínas conjugadas com ubiquitina, não se observando alteração na actividade do proteasoma. Isto pode indicar que a este nível de erro a que as células estão sujeitas, os mecanismos de controlo de qualidade de proteínas estão a ser activados de forma a evitarem a agregação proteica. Através das análises ao transcriptoma observou-se que os genes cuja expressão diminuiu estão relacionados com a matriz extracelular e adesão celular e os genes cuja expressão aumentou, estão envolvidos na regulação negativa da transcrição e na resposta a proteínas aberrantes. Este estudo permitiu adquirir novos conhecimentos acerca da resposta celular à incorporação de erros durante a síntese proteica, através da análise de alterações transcripcionais causadas pelo stress proteotóxico. As células HEK293FT são um bom modelo de estudo para compreender as bases moleculares de doenças humanas originadas por uma síntese proteica aberrante.Universidade de Aveiro2013-12-06T15:48:56Z2011-12-06T00:00:00Z2011-12-06info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/7287engVaranda, Ana Sofia Pauloinfo: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:RCAAP2024-02-22T11:12:34Zoai:ria.ua.pt:10773/7287Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:45:00.643930Repositório 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 Aberrant protein synthesis in human HEK293FT cells
title Aberrant protein synthesis in human HEK293FT cells
spellingShingle Aberrant protein synthesis in human HEK293FT cells
Varanda, Ana Sofia Paulo
Biologia molecular
Proteínas - Síntese
Fisiologia celular
title_short Aberrant protein synthesis in human HEK293FT cells
title_full Aberrant protein synthesis in human HEK293FT cells
title_fullStr Aberrant protein synthesis in human HEK293FT cells
title_full_unstemmed Aberrant protein synthesis in human HEK293FT cells
title_sort Aberrant protein synthesis in human HEK293FT cells
author Varanda, Ana Sofia Paulo
author_facet Varanda, Ana Sofia Paulo
author_role author
dc.contributor.author.fl_str_mv Varanda, Ana Sofia Paulo
dc.subject.por.fl_str_mv Biologia molecular
Proteínas - Síntese
Fisiologia celular
topic Biologia molecular
Proteínas - Síntese
Fisiologia celular
description Protein synthesis is tightly regulated and fidelity in this process is essential for maintenance of essential cellular functions. Under normal physiological conditions errors can occur, at frequencies around 10-4 errors per codon decoded. This level of mistranslation can be tolerated by cells. When the frequencies of errors increase mechanisms of protein quality control can fail leading to accumulation of misfolded proteins, which are more prone to form toxic aggregates, can compromise cell viability, disrupt cellular homeostasis and lead to the development of diseases. In order to elucidate how cells respond to mistranslation and understand how protein accumulation can cause disease and cell degeneration, we exposed human HEK293FT cells to canavanine and azetidine-2-carboxylic acid (AZC) which are analogues of arginine and proline, respectively. Their misincorporation into proteins leads to erroneous protein synthesis, misfolding and likely to protein aggregation. Such mistranslation event decreased slightly cellular viability and increased the number of cells arrested in G2/M and S phases of cell cycle. In HEK293FT cells was detected an increase in proteins conjugated with ubiquitin, without altering proteasome activity, which may indicate that at this level of mistranslation, protein quality control mechanisms are active to counteract the formation of protein aggregates. Transcriptional analysis showed that down-regulated genes were mainly associated with extracellular matrix and cell adhesion and up-regulated genes were involved in negative regulation of transcription and response to unfolded proteins. This study provides new insights into the response of human cells to mistranslation by giving a global analysis of transcriptional alterations that occur in response to proteotoxic stress. HEK293FT cells can be a good model to understand the molecular basis of human diseases caused by mRNA mistranslation.
publishDate 2011
dc.date.none.fl_str_mv 2011-12-06T00:00:00Z
2011-12-06
2013-12-06T15:48:56Z
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url http://hdl.handle.net/10773/7287
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dc.publisher.none.fl_str_mv Universidade de Aveiro
publisher.none.fl_str_mv Universidade de Aveiro
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
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