Aberrant protein synthesis in human HEK293FT cells

• 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
• topic: Biologia molecular; Proteínas - Síntese; Fisiologia celular

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.