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Title: The role of BAG6 in protein quality control
Author: Koay, Yee Hui
ISNI:       0000 0004 7656 5649
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2017
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Proteins must be folded into their correct three-dimensional structure for functions. Failure of proteins to achieve their native conformation is harmful to cells because these proteins are prone to aggregate and engage in non-native interactions. Cells possess extensive protein quality control systems to deal with misfolded proteins, either by refolding the proteins or degrading terminally misfolded proteins. BAG6 has been implicated in cellular protein quality control systems, specifically by recognising stretches of hydrophobic amino acids, recruiting E3 ubiquitin ligase(s) to promote substrate ubiquitination and preventing substrate aggregation. Such stretches of hydrophobic amino acids are typically found in transmembrane domains and endoplasmic reticulum targeting sequences, and BAG6 has been shown to play a role in the quality control and degradation of these proteins. In addition, cytoplasmic proteins may also contain hydrophobic sequences but these would normally be buried within the native structure, and only become exposed if the proteins misfold. However, the precise role of BAG6 in these pathways and the wider contribution of BAG6 to the handling of other aggregation-prone proteins in the cytosol are yet to be studied. Hence, the aim of this project was to determine BAG6 substrate specificity and to gain a better understanding of BAG6 role by identifying endogenous BAG6 substrates and co-factors through targeted and unbiased approaches. UBR4 has been identified as a protein that interacts with a BAG6 substrate (Sec61β) and interacting factor (SGTA) in two independent experiments. UBR4 was found to play a role in endoplasmic reticulum-associated degradation of a BAG6 substrate (opsin-degron), possibly by affecting BAG6-substrate and BAG6-E3 ligase interactions. The unspliced form of XBP1 was predicted to be a BAG6 substrate since XBP1 is a cytoplasmic protein with hydrophobic domain likely to be exposed in the cytoplasm for BAG6 recognition. BAG6 was shown to interact with XBP1 through the hydrophobic domain and affect XBP1 turnover through ubiquitination. Unspliced XBP1 acts as a negative regulator of the spliced XBP1, which is an important transcription factor in the unfolded protein response. BioID was performed as an unbiased approach for BAG6 substrates and co-factors identification. Biotin ligase fused to BAG6 biotinylated proteins coming into close proximity to BAG6, and biotinylated proteins were isolated and analysed with mass spectrometry. Based on bioinformatic analysis of the mass spectrometry data, hydrophobicity of BAG6 'substrates' varied greatly. Biochemical analysis is needed in the future to validate these BAG6-substrate interactions. Together, these results further our understanding of BAG6 role in the protein quality control, especially in the unfolded protein response which was reported for the first time.
Supervisor: Hubbard, Simon ; High, Stephen ; Swanton, Eileithyia ; Pickering-Brown, Stuart Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available