Structural Determination of Protein Complexes in Nucleocytoplasmic Transport

Research output: ThesisDoctoral Thesis

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Abstract

All eukaryotic cells contain a nucleus, an organelle which is physically separated from the cytoplasm by a double nuclear membrane. Segregation of the nuclear material from the cytoplasm allows a high level of regulation of gene expression, signal transduction and cell cycle progression. Transport across the nuclear envelope occurs through nuclear pore complexes, whereby proteins larger than 40 kDa are actively transported across the nuclear envelope. These larger sized proteins utilise a family of transporter proteins known as the β-karyopherins. There are a number of known of nuclear import pathways, with the main pathways that mediate translocation known as: the classical nuclear import pathway, which involves the nuclear importin alpha receptor and importin beta docking protein. The interaction of the importin complex and the nuclear localisation signal within the protein initiates translocation through the nuclear pore complex in an energy dependent fashion. The other type of nuclear import pathway is the non-classical pathway, and occurs in the absence of the import receptor, whereby nuclear localisation sequences bind directly to importin beta.

The localisation of proteins from the nucleus to the cytoplasm is mediated by both classical and non-classical pathways. The interaction between the nuclear import receptor and the nuclear localisation sequence within the cargo protein is the first critical step in this nuclear localisation mechanism.

This study examines the structural characterisation of proteins utilising both classical and non-classical pathway, and their interactions with nuclear import receptors. The interaction between importins and their cargo proteins can provide an enhanced molecular understanding of genetic disorders or diseases. To elucidate these interactions at a structural and molecular level, target cargo proteins were recombinantly expressed, and where necessary, a target sequence, containing the NLS, was cloned into expression vectors using ligation independent cloning. Importins and cargo proteins were expressed recombinantly and purified to homogeneity in complex with their binding partners. Proteins characterised in this study, including structural characterisation in complex with importin were; SRY, involved in genetic sex determination; SOX9, upregulated by SRY and functions as a transcription factor involved in a number of the genetic sex determination pathways; SOX2, important for maintenance and self-renewal of embryonic stem cells; PrP20, a regulatory protein for the nuclear localisation cycle; and Dengue virus non-structural protein 5, the polymerase responsible for replicating the viral genome. This latter structure led to further work involving a nuclear transport inhibitor and resulted in a high resolution structure that will be of high impact in the development of a viable vaccine for dengue. These structures have revealed features in the binding interface that will form the basis for future molecular work in the relevant fields.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Charles Sturt University
Supervisors/Advisors
  • Forwood, Jade, Principal Supervisor
  • Cowieson, Nathan P., Principal Supervisor, External person
Publisher
Publication statusPublished - 2017

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