Aspergillus nidulans como modelo para manipulação de genes envolvidos no processo de unfolded protein response

Author(s)

Mariane Paludetti Zubieta

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-07-13

Examining board members

André Ricardo de Lima Damásio; Iran Malavazi; Gabriela Felix Persinoti; Fernando Segato; Marcelo Mendes Brandão

• Advisor: André Ricardo de Lima Damásio; Fabio Marcio Squina

Abstract

In eukaryotes, the unfolded protein response (UPR) positively regulates genes responsible for restoring homeostasis in the endoplasmic reticulum (ER) during accumulation of misfolded proteins. The homeostasis is restored due to the activation of genes related to proteins secretion such as those encoding for chaperones and foldases, which, in turn, increase protein folding capacity by RE. In fact, some systems for heterologous protein production have been developed by the individual overexpression of chaperones and foldases in the hosting cells. However, the success rate of this strategy usually is quite low. Studies on the manipulation of genes that respond to UPR in fungal strains are interesting aiming a higher production of proteins. In this work, we initially identified the profile of proteins that are recruited to express and produce heterologous proteins in A. nidulans by mass spectrometry. Subsequently, we proceeded the identification of genes that respond to UPR-activating chemicals such as dithiothreitol and tunicamycin. Finally, we selected 12 genes with a predicted function in the A. nidulans secretion pathway. These 12 genes were deleted in an A. nidulans recombinant strain producing an homologous xylanase (xlnE, 5B3 strain) and another recombinant strain producing an heterologous xylanase (tpet_0854, 854 strain). The deletion of cyclophilin and a molecular chaperone Hsp40 resulted in an increase around 1.25 and 1.70-fold in the xlnE activity, respectively. Similarly, the deletion of thioredoxin and glycosyl phosphatidyl inositol-mannosyltransferase also increased the xlnE secretion even at lower levels. The results also showed a decreased production of total proteins production in these deleted strains. Thus, our results suggest that proteins such as glycosyl phosphatidyl inositol-mannosyltransferase, chaperone Hsp40, cyclophilin and thioredoxin play an important role in the regulation of proteins production by A. nidulans. However, we still do not understand the mechanism involved in increased secretion of 5B3 after the deletions. We suggest that the increased production of enzymes in the deleted strains is related to the activation of the UPR and a "less stringent" protein folding by the cell, resulting in a mild quality control and higher secretion of proteins into the extracellular medium (AU)

• FAPESP's process: 14/15403-6 - Aspergillus nidulans as a model to manipulate unfolded protein response-related genes
• Grantee: Mariane Paludetti Zubieta
• Support Opportunities: Scholarships in Brazil - Doctorate