ETD Collection
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Item The conformational stability of a detoxification enzyme widely used as a fusion-protein affinity tag.(1997) Kaplan, Warren HA glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is widely used as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an ad titional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible for-ration of significant amounts of 160 -kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, ultraviolet melting, differential scanning micro calorimetry , and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the umolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration with a ~GO(H20) = 26 ±1.7 kcal/mol. The conformational stability was unchanged in the presence of the leading antischistosomal drug Praziquantel, which bound the protein with a Kd = 9 ±1.8 p,M. The strong relationship observed between the m-v,llue and the size of the protein indicates that the amount of protem. surface exposed to solvent upon unfolding is the major structural de.erminant for the dependence of the protein's free energy of unfolding on urea concentration. 'Ihermograms obtained by differential scanning calorimetry also fitted to a two-state irreversible unfolding transition, both in the presence and absence of Praziquantel, with values of ~Cp = 1779 cal mol-IK-I , ~HcaI = 227 kcal/mol, AHVH ::::::233 kcal/mol (r :::::~:HVHIAlIcal = 1.02) and AS = 354 cal mol''K". The low ~Cp and ~S, when compared with the theoretically determined values, implied that the thermal denaturation of Sj26GST did not result in complete unfolding of the protein,Item Temporal expression of Dmp53 and SNAMA isoforms and their relation to genotoxic stress.(2015) Nweke, Ekene EmmanuelRBBP6 is an E3 Ubiquitin ligase protein with a U-box motif. It interacts with p53 and Rb and is linked to several cellular functions. SNAMA is the Drosophila RBBP6 homolog, but is less characterized than its vertebrate counterparts. Gene expression studies on Drosophila have a potential to advance the knowledge on molecular mechanism underlying genotoxic stress. Previous studies have shown that SNAMA plays a critical role as an apoptosis suppressor and possibly in responses to genotoxic stress. The molecular basis for this is, however, unknown. Initially, two isoforms were identified by bioinformatics and one (Snama A) experimentally as well. Here, we confirm experimentally the existence of the second isoform (Snama B). We also show that these are differentially expressed during development and when the organism undergoes genotoxic stress. Total RNA samples were used to demonstrate gene expression by using Reverse Transcriptase Polymerase Chain Reaction. Using samples collected at different stages of development and from adult flies treated with the DNA damaging agent, irinotecan, it is shown that these isoforms are differentially expressed throughout development and upon genotoxic stress. This knowledge may help to understand the functional role SNAMA plays in normal physiology and in response to genotoxic stress. Furthermore, the results show that SNAMA is involved in a potentially beneficial intervention whereby the glycolytic pathway is bypassed by the addition of methyl pyruvate.Item Protein interactions with drosophila p53(2014-09-23) Cajee, Umar-FaruqDrosophila melanogaster, a key model organism, has cognates of over 70% of human disease genes. This has created opportunities in the development of treatments for life threatening illnesses like cancer. Mutations on the p53 tumour suppressor protein, which is an activator of apoptosis, are common in many cancers. In mammals, p53 interacts with the Retinoblastoma Binding Protein 6 (RBBP6) which enhances the activity of MDM2, the prototypical negative regulator of p53, that is absent in invertebrates. In the absence of MDM2 the Drosophila RBBP6 homolog, SNAMA, through its DWNN Catalytic Module (DCM), is suspected to play an important role in the regulation of p53, probably via the ubiquitin proteasome pathway. Through bioinformatics analyses, and experimental analysis of transcripts, this study has shown the existence of two isoforms of SNAMA named here SNAMA A and SNAMA B for the long and short isoforms, respectively. SNAMA B appears to be expressed after genotoxic stress (DNA damage) in adults as well as during embryonic development. Recombinant protein expression in bacterial and yeast systems as well as HIS-tag chromatography and Western blot analyses were used to investigate interactions with Dmp53. Due to poor expression of recombinant Dmp53 protein in both prokaryotic and eukaryotic systems and unreliable commercial antibodies, it was impossible to complete interaction studies. Overall, these studies show that the SNAMA isoforms may play important roles during development and in response to DNA damage.Item Silencing RBBP6 (Retinoblastoma Binding Protein 6) sensitizes breast cancer cells to staurosporine and camptothecin-induced cell death(2014-09-02) Moela, PontshoRetinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown. Nonetheless, it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity (Ntwasa, 2008). Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer. This study is intended to investigate the potential applications of RNA interference (RNAi) to block RBBP6 expression as well as its subsequent effect on cell growth and apoptosis. To achieve these methodologies, the following techniques were used: RT-PCR, western blotting, xCELLigence system and flow cytometry. Our studies indicate that the knockdown of RBBP6 expression by siRNA modulates p53 gene involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6siRNA significantly reduced cell index (CI) compared to the control samples and we observed an inhibition of cellular proliferation in the interval of between 24 and 48 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. These results were further confirmed by flow cytometry which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and Staurosporine treated there was only 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote staurosporine- and camptothecin-induced apoptosis in breast cancer cells. Keywords: Retinoblastoma Binding Protein 6, staurosporine, camptothecinItem The role of Tyr540 in dimerisation of the FOXP forkhead domain(2014-07-02) Perumal, KershiaThe forkhead box (FOX) proteins are a family of transcription factors that interact with DNA via a winged helix motif that forms part of the forkhead domain. The FOXP (FOXP1-4) subfamily is unique in the family in that the forkhead domains of these proteins exhibit domain swapping where structural elements are exchanged via extension of the hinge-loop region. The FOXP subfamily members have high sequence homology, yet wild-type FOXP3 is a stable domain-swapped dimer in solution whereas FOXP1 and FOXP2 exist in a monomer/domain-swapped dimer equilibrium. A single amino acid difference is observed in the hinge region of the FOXP subfamily. This corresponds to Tyr540 in FOXP2 and Phe373 in FOXP3. We propose that it is the phenylalanine residue in FOXP3 that shifts the equilibrium towards dimer. Here we use FOXP2 to investigate the effect of a mutation, Y540F, on the structure and dimerisation propensity of the FOXP subfamily. Crystals for the Y540F variant in the presence of DNA have been obtained to demonstrate conclusively that domain swapping occurs. Size-exclusion chromatography indicates that the wild type FOXP2 forkhead domain is almost entirely monomeric at concentrations less than 100 μM. The Y540F variant is shown to stabilise the dimer and the ratio between monomer and dimer is concentration-dependent. DNA binding assays suggest that the Y540F variant binds less favourably to the cognate binding sequence than does the WT FOXP2 forkhead domain. Taken together, these findings suggest that domain swapping may modulate DNA binding.Item Tumour suppressor proteins in proliferating and differentiating cells(2014-03-05) Bodalina, Umesh MadanCells have evolved the ability to change continuously and adapt to their environment. An important way in which this dynamic modulation is achieved is by reversible phosphorylation, mediated by protein kinases and phosphatases. This thesis focuses on the temporal variations in expression of the proteins protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and p53 tumour suppressor protein in proliferating and hexamethylene bisacetamide (HMBA) induced differentiating murine erythroleukaemic (MEL) cells. The study included analysis of variations of p53 mRNA in these cells. Protein variations were analysed in cell extracts using western immunoblotting. The p53 variations were evaluated further using enzyme-linked immunosorbent assay (ELISA); p53 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Dynamic variations in protein expression and mRNA were detected in both the untreated and HMBA treated MEL cell preparations. For PP1, an immunospecific band of molecular mass 36 kDa, corresponding to the catalytic subunit, was detected, while for PP2A, two immunospecific bands of 32 and 36 kDa were observed. For the PP2A, the 36 kDa band corresponded to the catalytic subunit of this protein and the 32 kDa band was believed to be a proteolytically cleaved form of the catalytic subunit. The mean values of results showed little significant difference between proliferating and differentiating MEL cells, emphasising that single time-point studies give incomplete and probably misleading information. Multiple time analysis for expression clearly showed evidence of oscillatory behaviour and modulation by the differentiating agent. The influence of HMBA on PP1, PP2A and p53 expression was variable for the different experiments and affected both the frequency and phasing of rhythms. The results add support to the view that dynamic oscillatory control processes play an important role in regulating cellular behaviour. Modulation of the dynamics of key proteins in the cell, such as PP1, PP2A and p53, may be an important mechanism of controlling cellular function and reversing neoplastic transformation.Item The role of the domain linker in the stability of Glutaredoxin-2(2013-02-01) Ntshudisane, Obakeng MolebogengThe three dimensional native structure of multi domain proteins is only achieved when the adjacent domains recognise each other through the domain-domain interface. The domain-domain interface of the Glutathione S-transferase (GST) family has been studied extensively; however, no studies have been conducted on the role of the linker regions in the domain-domain interactions. Glutaredoxin 2 (Grx2) protein, from the GST family was chosen as model to investigate the possible role of linkers in protein stability by mutational analysis. Bioinformatics data revealed a conserved residue within the linker region (Leu78 in Grx2). A Grx2 mutant was created by replacing the conserved residue (Leu78) within the linker region with an alanine. This mutation (Leu to Ala) was performed in order to assess the role of the conserved residue leucine; whilst maintaining Grx2 function. A previous Grx2 mutant (Grx2 Y58W) was utilised because it incorporates tryptophan into domain 1; therefore it was possible to follow tertiary structural changes in this domain. Grx2 Y58W was compared against the mutant created within the linker Grx2 Y58W/L78A. Far-UV CD spectrum indicated that there was an increase of (~30 %) in ellipticity of Grx2 Y58W/L78A protein whereas; tryptophan fluorescence probes indicated no change in tertiary structure. Conformational stability studies showed a decrease of ΔΔG (H2O) = 3.8 kcal.mol-1 due to the impact of the Y58W/L78A mutation. The m-value which is indicative of the co-operativity between the two domains has decreased slightly by ~0.4 kcal.mol-1 M-1. This reduction in the m-value suggested the formation of intermediate however; it was not evident when using ANS as a probe. This study indicates that replacing a leucine with an alanine in the linker region causes a reduction in domain co-operativity. Therefore, the linker region in addition to separating the two domains plays a role in interdomain co-operativity.