School of Molecular & Cell Biology (ETDs)

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Browsing School of Molecular & Cell Biology (ETDs) 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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    Identification and characterisation of the interaction between FOXP2 and the ligand binding domain of oestrogen receptor α
    (University of the Witwatersrand, Johannesburg, 2023-10) Lakhi, Aasiya Yakub; Fanucchi, Sylvia
    Forkhead box P2 (FOXP2) regulates the expression of various genes and is associated with language and speech, neural development and outgrowth, and cancer. As transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation and cancer progression and outcome. The association between ER1 and FOXP2 was first identified in MCF-7 cells using co-immunoprecipitation. Thereafter, the interaction was characterised biophysically by overexpressing the FOXP2’s DNA-binding forkhead domain (FHD) and N-terminal region (NT), and ER1’s ligand-binding domain (LBD) in E. coli cells. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the thermodynamic parameters and regulation of interaction between FOXP2 FHD and ER1 LBD, respectively. Electrophoretic mobility shift assay was used to investigate the effect of the interaction on FOXP2’s DNA binding ability. Following the successful overexpression and purification of all three proteins, ER1 LBD was found to interact with FOXP2 FHD but not with FOXP2 NT. The affinity of the ER1 LBD for FOXP2 FHD increases with an increase in salt concentration. ITC shows a similar trend and reveals that the interaction is enthalpically favoured at lower salt concentrations but enthalpically opposed at higher salt concentrations. Additionally, the FOXP2-ER1 LBD interaction remains unaffected by the inclusion of oestrogen, but addition of FOXP2 cognate DNA results in inhibition of the formation of the complex. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the DNA simultaneously suggesting a probable role of this interaction in regulating the transcriptional pathway of FOXP2. This study serves as a foundation for further investigation into the interaction between FOXP2 and ER1 in different cell lines and its relevance in FOXP2-mediated outcomes in cancer and neurodevelopmental disorders.
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    Investigating FOXP2 dynamics, stability, and DNA-binding capabilities
    (University of the Witwatersrand, Johannesburg, 2024) Perumal, Cardon; Fanucchi, Sylvia
    All forkhead box (FOX) transcription factors share a conserved DNA-binding domain called the forkhead domain. They regulate gene expression in different organisms and have widespread biological roles, ranging from embryogenesis to immune regulation. The FOXP subfamily, unlike other FOX transcription factors, have the capacity for dimerisation, an evolutionary fate yet to be fully understood. In particular, the FOXP2 forkhead domain (FHD) has been shown in vitro to form domain-swapped dimers. Additionally, the FOXP2 leucine zipper domain forms heterotypic associations with FOXP1, 2, and 4. These somewhat isolated structural characterisations of FOXP2 have informed the domain-specific functionality of the DNA-binding forkhead domain but the leucine zipper domain has been characterized to a lesser extent, although it is thought to be implicated in FOXP2 DNA-binding as well. Elucidating the structural and functional impact of the leucine zipper on FOXP2 DNA-binding remains challenging, as it is unclear how these motifs work together to achieve binding and whether complexation is a requirement for DNA-binding. Owing to this, the cooperative structural contributions of both the leucine zipper and forkhead domain and the effect of DNA on the structure and stability of these domains have not been considered. Consequently, the aim of this study was to gain a comprehensive understanding of the FOXP2 DNA-binding mechanism by comparing the structure, stability, and dynamics of both the leucine zipper domain and the forkhead domain in the presence and absence of DNA. Assessed here, for the first time, is the conformational dynamics of the FOXP2 leucine zipper domain and FHD flanking disordered regions using hydrogen-deuterium exchange mass spectrometry. The results confirm the binding of DNA to the FHD recognition helix as well as the change in dynamics of the interlinking loop region. Additionally, the FOXP2 folding mechanisms and stability for each domain was characterised, revealing a 2-state unfolding mechanism for the forkhead domain and a 3-state mechanism for the longer LeuZip variant. Fluorescence anisotropy studies revealed that the LeuZip variant bound DNA with a higher affinity than the forkhead domain. The findings of this study highlight the structural significance of the leucine zipper domain and unstructured regions and the functional cooperativity of the FOXP2 domains investigated
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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.