List of Titles

Characterization of Trypanosoma brucei Sti1 and its interactions with Trypanosoma brucei Hsp83 and human Hsp90

Authors: Jamabo, Miebaka
Date: 2023-03-31
Subjects: Trypanosoma brucei , Heat shock proteins , HSP90 , HSP83 , Molecular chaperones
Language: English
Type: Academic theses , Doctoral theses , text
Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
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Date Issued: 2023-03-31

Fucoidans from South African brown seaweeds: establishing the link between their structure and biological properties (anti-diabetic and anti-cancer activities)

- Mabate, Blessing

Authors: Mabate, Blessing
Date: 2022-10-14
Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
Language: English
Type: Academic theses , Doctoral theses , text
Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
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Date Issued: 2022-10-14

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Repurposing a polymer precursor scaffold for medicinal application: Synthesis, characterization and biological evaluation of ferrocenyl 1,3-benzoxazine derivatives as potential antiprotozoal and anticancer agents

- Mbaba, Mziyanda

Authors: Mbaba, Mziyanda
Date: 2020
Subjects: Uncatalogued
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/164502 , vital:41124 , DOI 10.21504/10962/164502
Description: The benzoxazines are a prominent class of heterocyclic compounds that possess a multitude of properties. To this end, benzoxazine derivatives have been used as versatile compounds for various utilities ranging from biological applications to the fabrication of polymers. Particularly, the 1,3-benzoxazine scaffold has featured in several bioactive compounds showing antimalarial, anticancer and antibacterial activities. Traditionally, it has been employed as a substrate in the synthesis of polymers with appealing physical and chemical properties. Due to the increasing interest in the polymer application of 1,3-benzoxazines, research of the 1,3-benzoxazine motif for polymer synthesis has been prioritized over other applications including its medicinal potential. The continuous development of resistance to clinical anticancer and antimalarial drugs has necessitated the need for the search of innovative bioactive compounds as potential alternative medicinal agents. To address this, the field of medicinal chemistry is adapting new approaches to counter resistance by incorporating nonconventional chemical moieties such as organometallic complexes, like ferrocene, into bioactive chemical motifs to serve as novel compounds with medicinal benefits. Incorporation of ferrocene into known bioactive chemical moieties has been shown to impart beneficial biological effects into the resultant compounds, which include the introduction of novel, and sometimes varied, mechanistic modalities and enhanced potency. Presented with the benefits of this strategy, the current work aims to design and evaluate the pharmaceutical capacity of novel derivatives containing 1,3-benzoxazine scaffold (traditionally applied in polymer synthesis) hybridized with the organometallic ferrocene unit as bioactive agents. Using a combination of expedient synthetic procedures such as the Burke three-component Mannich-type condensation, Vilsmeier-Haack formylation and reductive amination, four series of ferrocenyl 1,3-benzoxazine derivatives were synthesized and their structures confirmed by common spectroscopic techniques: nuclear magnetic resonance (NMR), infrared spectroscopy (IR) and high-resolution mass spectrometry (HRMS). The target compounds were evaluated in vitro for potential antimalarial and anticancer activities against strains of the malaria parasite (Plasmodium falciparum 3D7 and Dd2) and the triple-negative breast cancer cell line HCC70. Compounds exhibited higher potency towards the Plasmodium falciparum strains with IC50 values in the low and sub-micromolar range in comparison to the breast cancer cell line against for which mid-molar activities were observed. To gain insight into the possible mode of action of ferrocenyl 1,3-benzoxazines, representative compounds showing most efficacy from each series were assessed for DNA binding affinity by employing UV-Vis and fluorescence DNA titration experiments. The selected compounds were found to interact with the DNA by binding to the minor groove, and these findings were confirmed by in silico ligand docking studies using a B-DNA structure as the receptor. Compound 3.16c (IC50: 0.261 μM [3D7], 0.599 μM [Dd2], 11.0 μM [HCC70]), which emerged as the most promising compound, was found to induce DNA damage in HCC70 cancer cells when investigated for effects of DNA interaction. Additionally, compound 3.16c displayed a higher binding constant (Kb) against DNA isolated from 3D7 Plasmodium falciparum trophozoites (Kb = 1.88×106 M-1) than the mammalian DNA (Kb = 6.33×104 M-1) from calf thymus, thus explaining the preferred selectivity of the compounds for the malaria parasite. Moreover, the investigated compounds demonstrated binding affinity for synthetic hemozoin, β-hematin. Collectively, these data suggest that the compounds possess a dual mode of action for antimalarial activity involving DNA interaction and hemozoin inhibition. , Thesis (PhD) -- Faculty of Science, Chemistry, 2020
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Date Issued: 2020

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The Role of HSP70/HSP90 Organizing Protein (Hop) in the Heat Shock Factor 1 (HSF1)-mediated Stress Response

- Chakraborty, Abantika

Authors: Chakraborty, Abantika
Date: 2020
Subjects: Uncatalogued
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/163204 , vital:41018 , doi:10.21504/10962/163204
Description: Molecular chaperones regulate cellular proteostasis. They control protein conformation and prevent misfolding and aggregation under both normal and stressful environments, ultimately resulting in cell survival. The project aimed to understand the role of the HSP70 – HSP90 Organizing Protein (Hop/STIP1) in the survival of stressed cells and the function of the stress-responsive transcription factor, Heat Shock Factor 1 (HSF1). HSF1 protein levels were significantly reduced in Hop-depleted HEK293T cells compared to controls by ELISA, western blot, and mass spectrometry. HSF1 transcriptional activity at the HSP70 promoter, and binding of a biotinylated HSE oligonucleotide under basal conditions were significantly reduced, consistent with the reduced levels of HSF1. In response to heat shock, HSF1 levels in Hop-depleted cells increased to that of controls, but there was still significantly lowerHSF1 transcriptional activity and HSE binding. Hop-depleted HEK293T cells were more sensitive than controls to the HSF1 inhibitor KRIBB11 and showed reduced short-term and long-term proliferation. Unlike the HSP90 inhibitor 17-DMAG, which had no effect, the HSP70 inhibitor JG98, further decreased the levels of HSF1 in Hop-depleted cells, suggesting a role for HSP70 in the Hop-mediated effects. There was punctate nuclear staining for HSF1 in Hop-depleted cells under both basal and heat shock conditions, as well as reduced nuclear localization and increased cytoplasmic accumulation of HSF1 in response to heat shock. Hop and HSF1 colocalized in cells, and HSF1 could be isolated in complex with Hop and HSP70. Loss of Hop reduced HSF1 in HSP70complexes but did not affect HSF1 abundance in HSP90 complexes. Hop-depleted cells showed reduced short-term and long-term survival compared to controls, an effect that was potentiated by the JG98 HSP70 inhibitor. Taken together, these data suggest that Hop regulation of HSF1activity is via a mechanism involving reductions in HSP70 interaction, as well as reduced nuclear localization, and DNA binding, and is consistent with reduced cellular fitness under basal and stress conditions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
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Date Issued: 2020

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Towards a biological profile for South African perinatal remains: osteological and genetic perspectives

- Thornton, Roxanne

Authors: Thornton, Roxanne
Date: 2019
Subjects: Identification , Forensic osteology , Methylation , RNA , Autopsy
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/68102 , vital:29198 , DOI 10.21504/10962/68102
Description: Forensic identification of abandoned and suspected infanticide cases admitted to the South African Forensic Pathology Services is often impossible due to decomposition of the remains. In these cases, investigation of suspected criminal activity is almost never pursued. Ancillary tests in the form of anthropological and molecular analyses can assist with the forensic identification of perinatal remains. To provide fundamental information about bone development of perinatal skeleton, osteological and genetic techniques focusing on the pars basilaris, pars lateralis, sternal rib and left femur were used. Samples were obtained from unidentified and unclaimed remains originating from the Johannesburg Forensic Pathology Service (JFPS). To provide a biological age to individuals in the collection, dental aging was used to categorize remains for comparisons with anthropological and molecular data. A molecular protocol was designed to sex individuals using the X-linked G6PD and Y-linked SRY genes. Bone development was studied using osteometric and morphological data of dry bone remains coupled with bone mineral density analysis (Micro-CT). The methylation levels of CpG rich sites within the promoter region of selected bone-associated genes were incorporated to examine silencing of genes during development. Osteological results support the use of the pars basilaris, pars lateralis and femur for age-at-death estimations as well as provide the foundation for dry bone aging criteria for South African individuals. Data compared with established skeletal aging standards indicated developmental differences between populations. Through the use of animal models and the perinatal sternal rib tissue, insights and precautions into the use of post mortem bone derived RNA for forensic applications is communicated. The methylation status of CpG rich sites within the promoter regions support the hypothesis for interdependent machinery involving selected genes during early bone development. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
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Date Issued: 2019

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Evaluating metabolism-induced toxicity using a non-hepatic cell line

- Weyers, Carli

Authors: Weyers, Carli
Date: 2018
Subjects: Cytochrome P-450 , Drugs Metabolism , Drugs Design
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/61950 , vital:28087
Description: The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
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Date Issued: 2018

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Synthesis, characterisation and evaluation of ferrocene-containing Novobiocin analogues for anticancer and antiplasmodial activity through inhibition of Hsp90

- Mbaba, Mziyanda

Authors: Mbaba, Mziyanda
Date: 2017
Subjects: Antibiotics Synthesis , Ferrocene , Heat shock proteins , Antimalarials , Cancer Chemotherapy
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/65111 , vital:28690
Description: Novobiocin (Nb) is a coumarin type antibiotic isolated from the bacterium species of Streptomyces and possesses modest anticancer and antimalarial activities. Nb and analogues have been extensively explored as potential anticancer agents through inhibition of the C- terminal domain of heat shock protein 90 (Hsp90), which plays a pivotal role in the proteinfolding machinery of cells. There has been little effort in the exploration of Nb and derivatives for antimalarial activity. Incorporation of organometallic units, such as ferrocene (Fc), into bioactive chemical scaffolds remains an attractive approach for developing new therapeutic agents for treatment of several ailments. The current study sought to investigate the anticancer and antiplasmodial effects of incorporating ferrocene (Fc) into Nb scaffold presumably through inhibition of Hsp90. The ferrocenyl Nb analogues containing simplified structural motifs such as phenyl, benzyl, and piperidine were synthesized in six to nine steps employing conventional synthetic organic protocols adapted from literature, and the compounds were accessed in reasonable yields. For comparison purposes, a selection of organic Nb analogues were also included in the study. The target compounds were characterized by spectroscopic techniques including 1-dimensional nuclear magnetic resonance (1D NMR) and high-resolution mass spectroscopy. The synthesized compounds were evaluated in vitro for potential anticancer and antiplasmodial activities using the breast cancer cell line (HCC38) and chloroquine-sensitive strain (3D7) of the malaria parasite, Plasmodium falciparum. The presence of the Fc unit was found to enhance both anticancer and antiplasmodial activities of the resultant ferrocenyl Nb compounds with IC50 values in the low to mid micromolar range. Hsp90 inhibitory studies of the ferrocenyl Nb analogues possessing superior activities (2.13a and 2.20c) were also conducted using different yeast strains expressing both human and malarial Hsp90 isoforms: hHsp90a/p and PfHsp90, respectively. The results of Hsp90 inhibitory studies suggested no direct correlation between the observed activities of the analogues and Hsp90 inhibition. However, since the conditions of the assay were not optimised due to time constrains of the project, these observed data remained to be confirmed. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
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Date Issued: 2017

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The Role of HOP in Emerin-Mediated Nuclear Structure

- Kituyi, Sarah Naulikha

Authors: Kituyi, Sarah Naulikha
Date: 2017
Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
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Date Issued: 2017

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Investigating the role of Hsp90 and LRP1 in FN matrix dynamics

- Boël, Natasha Marie-Eraine

Authors: Boël, Natasha Marie-Eraine
Date: 2016
Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
Language: English
Type: Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/2713 , vital:20319
Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
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Date Issued: 2016

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Establishment of human OCT4 as a putative HSP90 client protein: a case for HSP90 chaperoning pluripotency

- Sterrenberg, Jason Neville

Authors: Sterrenberg, Jason Neville
Date: 2015
Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
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Date Issued: 2015

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The role of Stress Inducible Protein 1 (STI1) in the regulation of actin dynamics

- Beckley, Samantha Joy

Authors: Beckley, Samantha Joy
Date: 2015
Subjects: Heat shock proteins , Molecular chaperones , Actin , Microfilament proteins , Cell migration , Adenosine triphosphatase , Metastasis
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/193941 , vital:45409
Description: Stress-inducible protein 1 (STI1) otherwise known as Hop (Hsp70/Hsp90 organising protein) is a highly conserved abundant co-chaperone of the Hsp70 and Hsp90 chaperones. STI1 acts as an adapter protein, where it regulates the transfer of protein substrates from Hsp70 to Hsp90 during the assembly of a number of chaperone-client protein complexes. The role of STI1 associating independently with non-chaperone proteins has become increasingly prominent. Recent data from colocalisation and co-sedimentation analyses in our laboratory suggested a direct interaction between STI1 and the cytoskeletal protein, actin. However, there was a lack of information on the motifs which mediated this interaction, as well as the exact role of STI1 in the regulation of cytoskeletal dynamics. Two putative actin binding motifs, DAYKKK (within the TPR2A domain) and a polyproline region (after the DP1 domain), were identified in mammalian STI1. Our data from in vitro interaction studies including surface plasmon resonance and high speed co-sedimentation assays suggested that both TPR1 and TPR2AB were required for the STI1-actin interaction, and peptides corresponding to either the DAYKKK or the polyproline motif, alone or in combination, could not block the STI1-actin interaction. Full length mSTI1 was shown to have ATPase activity and when combined with actin an increase in ATPase activity was seen. Ex vivo studies using STI1 knockdown shRNA HEK293T cells and non-targeting control shRNA HEK293T cells showed a change of F-actin morphology as well as reduction in levels of actin-binding proteins profilin, cofilin and tubulin in the STI1 knockdown cells. These data extend our understanding of the role of STI1 in regulating actin dynamics and may have implications for cell migration. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2015
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Date Issued: 2015

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The effects of extracellular and intracellular Hop on cell migration processes

- Contu, Lara

Authors: Contu, Lara
Date: 2014
Subjects: Heat shock proteins , Metastasis , Cancer Chemotherapy , Molecular chaperones , Cell migration
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/193961 , vital:45410
Description: The Hsp70/Hsp90-organising protein (Hop) is a 60 kDa co-chaperone that acts as an adaptor molecule, facilitating the transfer of client proteins between the Hsp70 and Hsp90 chaperone systems. Hop functions both intracellularly and extracellularly and has been implicated in many processes involved in cancer progression, including cell migration and invasion. Little is known about the mechanisms or domains by which extracellular Hop functions. In addition, little is known about the effects of Hop on signalling molecules involved in cell migration and invasion through regulation of actin dynamics. It was hypothesised that both extracellular and intracellular pools of Hop would regulate distinct cell migration processes by activation of cell signalling pathways or direct interactions with signalling intermediates. HS578T cells were treated with recombinant full length and truncated murine Hop proteins (overexpressed and purified in this study) to determine the effects of extracellular Hop and the independent domains on cell migration processes. Additionally, RNA interference (RNAi) techniques were used to determine the effect of Hop knockdown on cell migration related signalling intermediates and cell morphologies. A short hairpin RNA (shRNA) system for the stable knockdown of Hop was developed and used for a number of these studies. Treatment of HS578T cells with the TPR2A2B and TPR1 domains of Hop resulted in a significant decrease in cell migration and caused changes in the actin cytoskeleton and extracellular matrix proteins, gelatin and fibronectin. RhoC immunoprecipitated in a common complex with Hop and Hsp90. Hop knockdown reduced levels of actin and total RhoC, as well as active RhoC. In addition, knockdown of Hop resulted in a reduced migratory phenotype. We interpreted these data to indicate that intracellular Hop played a role in cell migration through regulation of RhoC activity, either through a direct interaction between Hop and RhoC, or an indirect interaction of RhoC with the Hsp90 multichaperone heterocomplex. Taken together, the data suggested that extracellular and intracellular Hop played distinct roles in extracellular and intracellular processes that lead to actin dynamics and cell migration. Understanding the mechanistic role of Hop in these processes is essential as it would aid in assessing the viability of Hop as a potential drug target for the treatment of metastatic cancers. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
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Date Issued: 2014

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