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    The aggregation of alpha-synuclein and its disaggregation by the Hsp70 disaggregation machinery

    Monistrol, Jim Emilien Jacques (2022) The aggregation of alpha-synuclein and its disaggregation by the Hsp70 disaggregation machinery. PhD thesis, Birkbeck, University of London.

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    Abstract

    Neurodegenerative disorders are characterised by the deposition of proteinaceous aggregates in the brain. In the case of synucleinopathies, such as Parkinson’s disease, the aggregates are mainly composed of α-synuclein (αSyn). The aggregation process of αSyn is toxic for neurons but the cause of the toxicity remains elusive. In vitro, the constitutively expressed cytosolic heat shock 70 protein, Hsc70, together with two cochaperones, the J-domain protein DNAJB1 and the nucleotide exchange factor Apg2, can disassemble αSyn amyloid fibres and small oligomers. However, the model is incomplete, lacking structural as well as mechanistic details. To address these two issues (αSyn aggregation in cells and the incomplete model of αSyn disaggregation in vitro), I have used electron and fluorescence microscopy and biochemical assays. Correlating fluorescence images and cryo tomograms, I have investigated the structure of αSyn aggregates in a yeast model. αSyn colocalises at the plasma membrane and forms cytosolic inclusions rich in vesicles and lipid droplets. Using cryo electron microscopy (cryo-EM) and tomography, I have shown that recombinant αSyn does not form large oligomers on the surface of tubulated liposomes. Using single particle cryo-EM, I have studied the complex formed of αSyn amyloid fibres and a mutant of DNAJB1 lacking the J-domain and G/F linker (ΔJ-DNAJB1). A low-resolution reconstruction of the complex was generated in which horseshoe-like densities, identified as ΔJ-DNAJB1, surrounded the fibre. I also produced a high-resolution map of the αSyn fibre showing a new conformation. Lastly, using cryo-EM, fluorescence microscopy and biochemical assays, I have investigated the function of Apg2 in disaggregation. Unexpectedly, Apg2 enhances Hsc70 recruitment to the fibres, preferentially at one end. This Apg2-induced recruitment regulates the disaggregation activity. The work in this thesis provides new information about αSyn aggregation, its toxicity and its disaggregation by the Hsc70 system.

    Metadata

    Item Type: Thesis
    Copyright Holders: The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted.
    Depositing User: Acquisitions And Metadata
    Date Deposited: 01 Jul 2022 15:46
    Last Modified: 01 Nov 2023 15:37
    URI: https://eprints.bbk.ac.uk/id/eprint/48576
    DOI: https://doi.org/10.18743/PUB.00048576

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