Functional characterisation of the role of the Small Glutamine Rich Tetratricopeptide Repeat Containing Protein Alpha (SGTA) in protein quality control

Hill, Jake (2020) Functional characterisation of the role of the Small Glutamine Rich Tetratricopeptide Repeat Containing Protein Alpha (SGTA) in protein quality control. Masters thesis, University of Lincoln.

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Functional characterisation of the role of the Small Glutamine Rich Tetratricopeptide Repeat Containing Protein Alpha (SGTA) in protein quality control
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Hill, Jake - Biochemistry - August 2020.pdf - Whole Document

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Item Type:Thesis (Masters)
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Abstract

Proteins which mislocalise or misfold must be effectively dealt with by the cell, if left to accumulate these proteins aggregate, which has been linked to neurodegeneration. Therefore, cells employ multifaceted quality control processes in order to deal with aberrant proteins. The Small Glutamine Rich Tetratricopeptide Repeat Containing Protein Alpha (SGTA) is a major component of these quality control pathways. SGTA is thought to be the ‘first responder’ in a molecular triage reaction, recognising aberrant membrane proteins and deliver these clients to a multiprotein complex which decides the fate of the client protein, either directing them for degradation or rescuing them for refolding. However, the molecular details about how SGTA functions in quality control are poorly understood. In studying the functions of quality control components, depleting a protein usually provides insights about their function. A novel technique (‘Trim-Away’) which utilises an antibody receptor TRIM21, and an antibody targeting a protein of interest to enable rapid depletion of target proteins was attempted.

In this study the role of SGTA in protein quality control was investigated with emphasis on the fate of client proteins in the presence of a panel of mutants. Mutants of SGTA were created targeting conserved motifs in the C-terminus, namely the Q-rich region and the NNP repeat motifs. These mutants when expressed in a HeLa cell culture system increased the steady state levels of a model mislocalised protein, OP91 beyond the WT levels. Pulse-chase analysis showed that the mutants delayed degradation of OP91 which was rationalised by a reduction in ubiquitination. Immunofluorescence confocal microscopy revealed formation of punctate inclusions where SGTA mutants and OP91 colocalised, which was confirmed by co-immunoprecipitation. Further investigations into the fate of OP91 and nature of these structures using fluorescence recovery after bleaching (FRAP), eludes to them being dynamic phase-separated structures with a possible role in spatial quality control. As part of this spatial quality control mechanism, immunoprecipitation on cell culture media from SGTA and OP91 expressing cells revealed the export of OP91 and SGTA into the media and a novel ability of SGTA to transfer between cells in a possibly prion-like manner. Further to this, a variation of ‘Trim-Away’, utilising a cell-penetrating peptide tag was developed to attempt compartment-specific depletion of SGTA in order to study the resultant effects. Therefore, the SGTA mutants developed in this study are valuable tools which will enable an in-depth study of the role of SGTA in protein quality control.

Divisions:College of Science > School of Life Sciences
ID Code:47813
Deposited On:18 Jan 2022 10:17

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