3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Structural and functional characterisation of FOXP1 and FOXP2 near-full length variants and their protein-protein interactions(2019) Thulo, MonareFOXP1 and FOXP2 are members of the FOXP subfamily of transcription factors characterised by multiple domains, including a highly conserved C-terminal DNA binding domain (DBD). Additionally, the FOXP proteins are characterised by two functional dimerisation interfaces, comprising of the DBD, the forkhead domain (FHD) and an N-terminal leucine zipper domain. Sequence analysis also shows several regions of intrinsic disorder in the sequence, including the polyglutamine region, the domain linkers and a C-terminal acid rich region, conserved in FOXP1, FOXP2 and FOXP4. FOXP1 and FOXP2 are expressed in several organs including the lungs and brain and are important during embryonic development. FOXP1 is associated with cognition and retardation while FOXP2 has been implicated in language development. The similarities in structure and function between FOXP1 and FOXP2 and the partial overlap that has been reported in their expression patterns in brain regions important for language may indicate a possible interaction between them. More recently, disruptions in FOXP1 have been implicated in speech delay, intellectual disability, and autism, suggesting that just like FOXP2, FOXP1 has a significant involvement in speech and language development. Because of multiple domains, the expression and purification of the full length FOXP protein is relatively difficult to achieve in vitro. In this study, the structural and functional characteristics of FOXP1 and FOXP2 variants were determined using biophysical techniques. Four variants, two of FOXP1 and two of FOXP2 were constructed containing the FHD and the leucine zipper domain and differing in whether or not they additionally contained the C-terminal acid rich tail. The four variants were expressed in the soluble Escherichia coli cell fraction and were purified to relative homogeneity using chromatography. Although it has been suggested that FOXP1 and FOXP2 regulate transcription as dimers, we cannot rule out the possibility of both proteins performing their functions as higher oligomers. Indeed, the formation of higher order oligomers was observed in this study through SEC, DLS and native PAGE measurements. Higher order oligomers-DNA complexes were observed in EMSA, therefore suggesting that formation of oligomers may also occur during DNA binding. The significance of the disordered C-terminal tail, ART, to structural stability was studied in the presence of a simple alcohol, ethanol. Simple alcohols are enough to destabilise the tertiary structure of a protein and to stabilise the helical structure, leading to a possible quaternary conformational state change. FOXP1 and FOXP2 variants exist in solution predominantly as higher order oligomers at different concentrations. However, for FOXP1 and FOXP2 LZ-FHD, decreases in solvent dielectric result in dissociation of the higher oligomers to form a mixture of monomer and dimer. Although there is also a decrease in quaternary state in the presence of ethanol for FOXP1 and FOXP2 LZ-End, the ART seem to be enough to maintain dimeric and higher oligomeric state. In agreement with other studies, the use of ethanol was able to destabilise the tertiary structure, shown by a red shift in fluorescence emission for each variant, and to a characteristic increase in helical content. The folding of FOXP1 and FOXP2 LZ-End in mixtures of water with a simple alcohol directly relate to decrease in the dielectric constant of the solution. The hetero-association of FOXP1 with FOXP2 was defined using pull down assays on purified FOXP1 and FOXP2 truncated variants encompassing the leucine zipper, FHD and ART and fluorescence anisotropy to study the binding affinity of FOXP2 variants for FOXP1 variants in vitro. Size exclusion chromatography showed that the hetero-associated proteins exist mainly as dimers and fluorescence anisotropy revealed relatively weak association, both in the presence and absence of DNA in comparison to the isolated FHD of both proteins. The loose assembly of FOXP1 and FOXP2 near full-length variants native structure suggest that this is a mechanism that is needed for both interaction with binding partners such as the other FOXP proteins during heterodimerisation and during DNA binding. These results also suggest a possibility of regulation by a dynamic equilibrium between different states which leads to a ‘partial occupancy’ upon DNA binding. The existence of different quaternary states and conformation suggests that both FOXP1 and FOXP2 might also control transcription as components of supramolecular regulatory complexes stabilised by different subdomains, including the C-terminal acid rich region. This, therefore, suggests that local folding of the proteins must be frequently coupled to DNA binding. Consequently, hetero-oligomerisation of the full-length protein could be a transient event, that occurs for limited time.Item Molecular evaluation of ribosomal protein L9 and lipoic acid synthetase genes and in lung and apoptosis(2012-09-05) Mphahlele, Raesibe PaulinahBackground: A human ribosomal protein L9 (RPL9) encodes a protein that is a component of the 60S subunit. RPL9 is located on chromosome 4p14 and is approximately 5.5 kb in length and contains 8 exons. The message for human RPL9 is 712 nucleotides long. Some of the functions of RPL9 documented so far include the crucial involvement of the gene product in cell proliferation and protein biosynthesis. Lipoic acid synthetase (LIAS) is a 1.73 kb gene also located at chromosome 4p14. Alternative splicing occurs at these locus and two transcript variants encoding distinct isoforms have been identified but in this study the results represents both isoforms together. The protein encoded by LIAS gene belongs to the biotin and lipoic acid synthetases family and localizes in the mitochondrion. Function of lipoic acid synthetase is not yet well documented. Some studies have attempted to characterise its function by looking at the biological pathways at which LIAS gene product plays a crucial role, for example the biosynthesis of alpha-lipoic acid. Alpha lipoic acid is a natural antioxidant and it is also naturally-occurring enzyme co-factor found in a number of multi-enzyme complexes regulating oxidative metabolism. Motivation for study: RPL9 and LIAS were previously found to be mutated in CHO (Chinese Hamster Ovary) cell lines and these mutant lines had gained resistance to apoptosis. Aim: The main objective of this study was to evaluate the expression pattern of RPL9 and LIAS in lung cancer and to characterise their role in apoptosis and also to determine if the expression pattern of this genes varies between normal and diseased state of the tissue. Methods: In Situ hybridization, quantitative Real Time PCR, TUNEL and Bio-informatics have been employed in order to attain the objectives of this study. Results: In Situ hybridization showed that RPL9 localises in the cytoplasm and it is up-regulated in lung cancer relative to normal lung. LIAS localises in the cytoplasm and it is also up-regulated in lung cancer. The expression of RPL9 was relatively higher than that of LIAS determined by the intensity of localisation. Quantitative real time PCR confirmed the up-regulation of RPL9 and LIAS in lung cancer. RPL9 and LIAS were found to be up-regulated 8 and 4 fold respectively in lung A549 lung adenocarcinoma relative to MRC5 normal lung fibroblast cell lines. TUNEL showed the highest DNA fragmentation in adenocarcinoma, followed by squamous cell lung carcinoma then large cell lung carcinoma which is the same pattern observed in RPL9 and LIAS mRNA localisation by In Situ hybridization. To further characterise the role of RPL9 and LIAS in human, Bio-informatics tools were used and the results revealed that RPL9 is highly conserved through evolution, up-to 100 % identical to chimpanzee and 98 % to mouse. LIAS was found to be 91 % identical to rat and 90 % identical to mouse. It has been documented that the rate of conservation of a gene in evolution is believed to be correlated with its biological importance and its number of protein–protein interactions. Conclusion: All these discoveries coupled with resistance to apoptosis of CHO cell line in which RPL9 and LIAS were found to be mutated following promoter-trap mutagenesis, strongly suggests that RPL9 might be playing a role in cell cycle and apoptosis. RPL9 has been highly conserved through evolution. Manipulation of this gene can lead to greater biological discoveries in cancer research and the elevated expression of RPL9 can be used as a molecular marker for early detection of cancer.Item The role of the domain interface in the stability, folding and function of CLIC1(2008-09-08T08:36:18Z) Stoychev, Stoyan HristovChloride intracellular channel protein 1 (CLIC1) is a dual-state protein existing in both soluble monomeric conformation as well as integral-membrane form. The role of the domain interface in the conversion between these species was investigated. Bioinformatics-based analysis was undertaken to compare and contrast the domain interfaces of dimeric GSTs with their monomeric homologues CLIC1 and CLIC4. The mutants CLIC1-M32A and CLIC1-E81M were used as experimental case studies on the role of domain-domain interactions in the stability and folding of CLIC family proteins. A consensus interface was revealed with the prominent interaction being a conserved inter-domain lock-and-key type motif previously studied in class Alpha GSTs (Wallace et al., 2000). A number of domain-interface interactions were found to be unique to the CLIC family and as such thought to play a role in the conversion of these proteins from their soluble form to an integral membrane form. Overall the domain interfaces of monomeric CLIC1 and CLIC4 did not differ significantly from the domain interfaces of dimeric GSTs. The removal of the unique CLIC family salt-bridges between Arg29 and Glu81 and the cavity forming domain interface mutation Met32Ala did not induce significant changes in the conformational flexibility of the native state. The true role of the Arg29-Glu81 salt-bridges was masked by the introduction of stabilizing hydrophobic contacts. Removal of the inter-domain lock-and-key interaction destabilized CLIC1 significantly with concomitant loss in cooperative folding that resulted in the stabilization of a molten globule-like species. This intermediate state was less stable and less structured than the equilibrium intermediate of wtCLIC1 at pH 5.5. However the bulk of the structures found to unfold during intermediate-species formation was the same in mutant and wild-type proteins. It was concluded that formation of the membrane-competent form of CLIC1 involves re-structuring of the N-terminal thioredoxin domain that takes place after destabilization of the salt bridges connecting h1 and h3 and uncoupling of the inter-domain lock-and-key motif.