Structural and functional characterisation of nuclear import complexes in human health and disease

Kate Smith

    Research output: ThesisDoctoral Thesis

    84 Downloads (Pure)

    Abstract

    Nuclear transport is a critical process in eukaryotes that has important implications in growth and development, cancer progression, disease pathogenesis, and viral infection. The classical nuclear import pathway involves the use of the import receptor importin alpha (IMPa) and the adapter molecule importin beta (IMPb). These form a complex in the cytoplasm which is able to recognise cargo proteins that contain nuclear localisation signals (NLSs) to translocate them into the nucleus.

    This PhD aimed to characterise the molecular binding determinants between IMPa and cargo molecules that are important in human health and disease. To better understand how NLSs are recognised by IMPa, the hitherto poorly appreciated binding determinant position 4 (P4) and the role it plays in the IMPa binding interface was explored. The findings revealed that P4 can modulate both interaction strength and nuclear targeting ability for proteins that bear a minimal NLS, advancing our knowledge of NLS function. Important viral-host interfaces were structurally characterised for both DNA viruses (Human immunodeficiency virus-1) and RNA viruses (Hendra virus, Nipah virus, Dengue virus, and Zika virus), as well as an important protein involved in chromatin remodelling and breast cancer disease progression (LSD1). These structures provided important information about the binding determinants in the cargo proteins recognised by IMPa.

    It is important to note that the human genome encodes for seven isoforms of IMPa (a1,a3-8), which display wide varieties of isoform and tissue specific specificity to cargo proteins. However, the cargo binding region of IMPa is highly conserved across all isoforms and we do not have a comprehensive understanding of the biophysical and molecular determinants that dictate the observed cargo specificity by IMPa isoforms. Previous studies have elucidated important functional determinants for cargo binding, auto-inhibition, regulation and recycling of IMP, although not all seven isoforms have been fully characterised. From the Nipah virus (NiV) and Hendra virus (HeV) W protein study a novel isoform specificity mechanism was identified for the IMPa3 isoform in the positioning of the C-terminus. Findings from this thesis also validated a previously described N-terminus mechanism of IMPa3.

    Overall, this thesis delineated important cargo recognition mechanisms for IMPa, described critical host-pathogen interfaces, and interfaces required for cancer progression. Additionally, the described specificity mechanisms for IMPa3 were confirmed with the cargo free IMPa3 structure. As more attention has been placed on inhibition of nuclear import pathways for treating infections and cancers, these findings can potentially be used as a platform in the development of novel therapeutics.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • Charles Sturt University
    Supervisors/Advisors
    • Forwood, Jade, Principal Supervisor
    • Aragao, David, Co-Supervisor, External person
    • Raidal, Shane, Co-Supervisor
    Place of PublicationAustralia
    Publisher
    Publication statusPublished - 2019

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