Electronic Theses and Dissertations (Masters)

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Browsing Electronic Theses and Dissertations (Masters) by Author "Fanucchi, Sylvia"

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    A Clot to Uncover: FOXP3 and SARS-CoV-2 Nucleocapsid Interactions and Their Effect on DNA Binding
    (University of the Witwatersrand, Johannesburg, 2024) Mcinnes, Keiran; Fanucchi, Sylvia
    During COVID-19, systemic coagulopathy, which can lead to strokes and embolisms, is often observed in COVID-19 patients and may also contribute to long COVID. This coagulopathy is the result of overactivated platelets in circulation that leads to inappropriate clot formation. FOXP3 is a transcription factor involved in platelet development and loss of FOXP3 function leads to platelets that resemble those seen during COVID-19. Thus, FOXP3 may be dysregulated in COVID-19. The SARS-CoV- 2 nucleocapsid (NC) is a multifunctional protein typically associated with viral genome packaging and virion assembly. However, it is also capable of binding DNA and is potentially able to alter regulation of host protein expression. Here, potential interactions between the DNA-binding forkhead domain (FHD) of FOXP3 and the SARS-CoV-2 NC were investigated. Identification of a novel interaction between FOXP3 and SARS CoV-2 NC may provide new clues as to the pathophysiology of COVID-19. To address this aim, both proteins were overexpressed in T7 E. coli, purified via immobilised metal affinity chromatography, and monitored for potential interactions in the absence and presence of DNA using pull-down assays and fluorescence anisotropy. A direct interaction was identified between the two proteins in the absence of DNA. Additionally, it was found that both proteins are capable of binding to DNA at the same time, but excess NC was found to cause FHD dissociation from the FHD- NC-DNA complex. This result implicates NC in FOXP3 dysfunction which may be associated with the coagulopathy and other symptoms seen during COVID-19. Additionally, NC DNA binding does not appear to be driven by the FOXP3 consensus sequence, indicating that FOXP3 may not be the only transcription factor potentially dysregulated by NC
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    Probing the Protein-Protein Interactions of the FOXP1, FOXP2 and FOXP3 Forkhead Domains
    (University of the Witwatersrand, Johannesburg, 2023-07) Mhlongo, Paulina; Fanucchi, Sylvia
    The FOXP proteins are classified amongst the forkhead box superfamily of transcription factors due to their highly conserved forkhead winged-helix domain (FHD). It is through this domain that FOX transcription factors are able to bind DNA in order to perform crucial roles in the regulation of gene transcription from development through adulthood. The FHD of FOXP1, FOXP2 and FOXP3 is remarkably unique in its ability to establish domain-swapped dimerization, postulated to drive interchromosomal interactions to regulate the transcription of distal genetic material. The FOXP1 and FOXP2 proteins are co-expressed and have also been demonstrated to directly interact in vivo and in vitro via a domain upstream of the FHD. Similarly, the FOXP1 and FOXP3 proteins have been established to form direct heterotypic interactions to function in regulatory T-cells (Treg). Provided that the FOXP FHD has the exceptional capability for dimerization, the study of the heterodimerization of the FHDs of these FOXP transcription factors may provide insight into the role of FHD-dimerization and its importance in the physiological roles of these proteins. Therefore, the aim of this work was to investigate whether a FOXP FHD heterodimerization event can occur between the FOXP1 and FOXP2 FHDs as well as between the FOXP1 and FOXP3 FHDs. The three FHD proteins were expressed and isolated for downstream interaction studies. The activity of the purified FHDs was studied using basic electrophoretic mobility shift assay. Their secondary and tertiary structures were characterized with circular dichroism and intrinsic fluorescence spectroscopies. Concentration-dependent size exclusion chromatography was employed to study their propensity for dimerization and fluorescence anisotropy was used to investigate both the homo and heterodimerization of the FHDs. It was revealed that FOXP3 FHD showed the highest propensity to homodimerize, whilst FOXP2 FHD showed the weakest propensity. Despite this, the homodimers of FOXP1 FHD appear to be less stable than that of the FOXP2 FHD. The heterodimerization studies suggest that FOXP1 FHD has preference in forming heterotypic associations with FOXP3 FHD rather than FOXP2 FHD.